Aqueous biocide formulation

Abstract

The present invention describes an aqueous biocidal formulation that comprises between 40% and 60% by mass of a biocidal agent of the genus Trichoderma and an aqueous solution of polyethylene glycol with a molar mass of between 50 and 1200Da, between 1 and 50% by mass. In addition, it describes methods for the protection of agricultural crops through the application of the developed formulation.

Description

TITLE “AQUEOUS BIOCIDE FORMULATION” FIELD OF THE TECHNOLOGY

[0001] The present invention is related to the control of pathogens of crops with agricultural interest, based on an aqueous microbial formulation capable of controlling the spread of said pathogens. STATE OF THE ART

[0002] Biocidal formulations based on microorganisms as biological control agents are recently under constant development in order to have an alternative to synthetic pest control agents that often have an adverse effect on the environment.

[0003] The international patent application WO2017210512 describes a liquid, not aqueous nor oily, formulation that comprises a microorganism and a liquid vehicle, where said vehicle can be a mixture of glycols, such as polyethylene glycol in a proportion of between 70 and 80% by mass and glycerol in a proportion of between 5 and 10% by mass.

[0004] US patent application US2022332658 describes formulations for biological control, said formulations containing Trichoderma strains, such as Trichoderma parareesei, Trichoderma virens, Trichoderma atroviride, Trichoderma asperellum. Said microorganisms may be part of a solid final formulation (where the vehicle may be sorbitol or kaolin), or they may be in an aqueous solution with cellulose as a vehicle and in an oily formulation with mineral oils as a vehicle.

[0005] Patent US10362786 describes an aqueous formulation comprising spores of Bacillus firmus I-1582, glycerin in a concentration of between 5 and 50% by mass, water, and between 0.5 and 20% by mass of a salt (sodium sulfate, sodium chloride, or zinc sulfate) that acts as a stabilizer.

[0006] In this way, the present invention comes to propose an alternative solution to the need for a greater variety of sustainable biocides with low environmental impact to replace or reduce the use of traditional pesticides, providing a formulation that provides great stability over time and is compatible with a wide variety of products for agricultural use.BRIEF DESCRIPTION OF THE FIGURES

[0007] Figure 1. Conidia count of Trichoderma afroharzianum Th2RI99 grown in mini- tanks at 24°C (A) and 30°C (B), at different storage times from formulation with different diluents.

[0008] Figure 2. CFU count of Trichoderma afroharzianum Th2RI99 grown in mini-tanks at 24°C (A) and 30°C (B), at different storage times from formulation with different diluents.

[0009] Figure 3. The graph shows the CFU [Colony Forming Units] count of Trichoderma afroharzianum Th2RI99 with PEG400 solution in triplicate over time, compared to control (Trichoderma afroharzianum Th2RI99 in the absence of PEG400).

[0010] Figure 4. The graph shows the % colonization of formulations (Trichoderma afroharzianum Th2RI99 + PEG400) with different storage times on seeds, compared to the control (Trichoderma afroharzianum Th2RI99 in the absence of PEG400).

[0011] Figure 5. Colony forming units of Trichoderma afroharzianum Th2RI99 in absence of PEG400 in direct mix in Jensen tubes 4 hours since beginning.

[0012] Figure 6. Colony forming units of Trichoderma afroharzianum Th2RI99 in absence of PEG400 on seeds by washing method from 0 to 90 days after treatment.

[0013] Figure 7. Trichoderma afroharzianum Th2RI99 in absence of PEG400 colonization on seeds in Petri dishes method from 0 to 90 days after treatment.

[0014] Figure 8. Trichoderma afroharzianum Th2RI99 in absence of PEG400 colonization on seeds in blotter test method from 0 to 90 days after treatment. BRIEF DESCRIPTION OF THE INVENTION

[0015] The present invention provides an aqueous biocidal formulation that comprises between 40% and 60% by mass of a biocidal agent of the genus Trichoderma and an aqueous solution of polyethylene glycol with a molar mass of between 50 and 1200Da between 1 and 50% by mass. Preferably, said biocidal agent is Trichoderma afroharzianum. More preferably, said biocidal agent is Trichoderma afroharzianum Th2RI99 deposit No. NRRL 67990.

[0016] In an embodiment aspect of the present invention, said polyethylene glycol has a molar mass of 400Da.

[0017] In an embodiment aspect of the present invention, said polyethylene glycol has a molar mass of 200Da.

[0018] In an embodiment aspect of the present invention, said polyethylene glycol has a molar mass of 600Da.

[0019] In an embodiment aspect of the present invention, said polyethylene glycol has a molar mass of 800Da.

[0020] In an embodiment aspect of the present invention, said polyethylene glycol has a molar mass of 1200Da.

[0021] The biocidal formulation of the present invention is characterized by maintaining at least 90% of the viability of the biocidal agent for more than 180 days.

[0022] The biocidal formulation of the present invention is characterized by maintaining at least 90% of the viability of the biocidal agent for more than 360 days.

[0023] The biocidal formulation of the present invention is characterized by maintaining at least 90% of the viability of the biocidal agent for more than 540 days.

[0024] The biocidal formulation of the present invention is characterized by maintaining at least 90% of the viability of the biocidal agent for more than 725 days.

[0025] An advantage of the present invention is its colonization capacity greater than 80% after at least 180 days of storage.

[0026] Another advantage of the present invention is its colonization capacity greater than 80% after at least 360 days of storage.

[0027] Another advantage of the present invention is its colonization capacity greater than 80% after at least 540 days of storage.

[0028] Another advantage of the present invention is its colonization capacity greater than 80% after at least 725 days of storage.

[0029] The biocide formulation has the capacity to maintain the concentration of the biocide agent between 1x105and 1x1010CFU / L for at least 180 days.

[0030] The biocide formulation has the capacity to maintain the concentration of the biocide agent between 1x105and 1x1010conidia / L for at least 180 days.

[0031] Another advantage of the biocidal formulation of the present invention is its ability to be compatible with a variety of products for agricultural use, such as fungicides, insecticides, growth promoters, and inorganic fertilizers. Thus, in another aspect of the present invention, the formulation also comprises at least one compound selected from the group comprised of: fungicides, insecticides, growth promoters, inorganic fertilizers, and their mixtures. Where said fungicide is selected from the set comprised of: Sedaxane (SDX), Prothioconazole (PTZ), Fludioxonil (FLD), and their mixtures. Where said insecticide is selected from the set comprised of:Teflutrine (TFT), Imidacloprid, Thiamethoxam, Fipronil, and their mixtures. Where said growth promoter is selected from the set comprised of: Pseudomonas fluorescens, Azospirillum brasiliense, Sinorhizobium meliloti, Rhizobium leguminosarum bv. Viciae, Mesorhizobium ciceri, Mesorhizobium loti, Bradyrhizobium spp., Rhizobium tropici, Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Rhizobium leguminosarum bv. Trifolii and their mixtures. Where said inorganic fertilizer is a metal oxide selected from the group comprised of: zinc oxide, magnesium oxide, potassium oxide, copper oxide, iron oxide (II and III), manganese oxide, calcium oxide, and their mixtures.

[0032] Another object of the present invention is a method for protecting agricultural crops from pathogenic microorganisms, which comprises: (i) preparing a solution of the aqueous biocide formulation which comprises between 40% and 60% by mass of a biocidal agent of the genus Trichoderma and an aqueous solution of polyethylene glycol with a molar mass of between 50 and 1200Da between 1 and 50% by mass; and (ii) applying one or more doses of said aqueous biocide formulation solution of at least 0.5ml / 100kg of seeds.

[0033] Preferably, said biocidal agent is Trichoderma afroharzianum. More preferably, said biocidal agent is Trichoderma afroharzianum Th2RI99 deposit No. NRRL 67990.

[0034] Preferably, said polyethylene glycol has a mass of between 200 and 600 Da. Preferably, said polyethylene glycol is 400Da.

[0035] In one embodiment, the application is made between 25 and 35 days prior to sowing.

[0036] Preferably, but without limiting the spectrum of protection of the present invention, the method allows control over pathogenic microorganisms, such as: Alternaria spp., Aspergillus spp., Bipolaris sorokiniana, Bipolaris spp., Cercospora kikuchii, Colletotrichum truncatum, Drechslera triticirepentis, Fusarium spp., Fusarium graminearum, Fusarium oxysporum, Fusarium tucumaniae, Fusarium virguliforme, Macrophomina phaseolina, Microdochium spp., Penicilium spp., Phomopsis spp., Puccinia triticina, Pythium debaryanum, Rhizoctonia solani, Rhizoctonia spp., Sclerotinia esclerotiorum, Tilletia leavis, and Ustilago tritici.DETAILED DESCRIPTION OF THE INVENTION Base Aqueous Formulation

[0037] The present invention provides an aqueous biocidal formulation containing a microbial biocidal agent, and an aqueous polyethylene glycol solution.

[0038] The biocide aqueous formulation of the present invention is useful to combat different pathogens that affect crops of agronomic interest. For example, the formulation of the present invention is useful for combating yellow spot in wheat, caused by Drechslera tritici-repentis, and wheat leaf rust caused by Puccinia triticina, managing to reduce the levels of said pathology in wheat. In wheat, it has also been shown that the present formulation is useful to combat pathogenic strains of Rhizoctonia spp., Fusarium graminearum, Bipolaris sorokiniana, Rhizoctonia solani, Tilletia leavis, and Ustilago tritici.

[0039] Likewise, the present biocidal aqueous formulation is an excellent treatment to combat Cercospora kikuchii, Fusarium spp., Macrophomina phaseolina, Phomopsis spp., Sclerotinia esclerotiorum, Pythium debaryanum, Rhizoctonia solani, important pathogens that affect soybean crops.

[0040] The formulation of the present invention is an excellent biocidal agent for the control of pathogens such as Macrophomina phaseolina, Alternaria spp., Colletotrichum truncatum, Fusarium tucumaniae, Fusarium graminearun, Fusarium virguliforme, Sclerotinia sclerotiorum.

[0041] The formulation of the present invention is an excellent biocidal agent for the control of rice pathogens such as Alternaria spp., Aspergillus spp., Bipolaris spp., Fusarium spp., Microdochium spp., and Penicilium spp.,

[0042] Within Argentine territory, there are pathogenic strains of Alternaria spp., mainly in productive areas in agricultural terms, and they can affect barley and wheat crops.

[0043] The formulation of the present invention is based on the use of a high molecular weight glycol, such as polyethylene glycol (PEG). The molar mass of polyethylene glycol can be between 50Da and 1200Da, for example 200, 400, 600, or 1200 daltons (Da). Preferably, the present invention utilizes 400 dalton polyethylene glycol (PEG400).

[0044] On the other hand, in the present invention, PEG is dissolved in water. The PEG400 : water ratios useful for the present invention can vary between 1:99 and 50:50.

[0045] Preferably, the concentrations of PEG400 in water are between 5% v / v and 50% v / v. Biocidal Agent

[0046] The present biocidal aqueous formulation uses a microbial agent as the biocidal agent. The use of a microorganism as a microbial agent is due to the need to have alternatives to the chemical agents of daily use in the agricultural industry. The microbial biocide agent has the advantage of being effective in controlling agricultural pests, without causing serious damage to the environment or worsening environmental pollution.

[0047] The microbial biocidal agent of the present invention belongs to the fungi kingdom. Preferably, the fungi that can exert a biocidal effect in the present formulation belong to the genus Trichoderma.

[0048] Even more preferably, the species of Trichoderma useful in the formulation of the present invention are selected from the group comprised of: Trichoderma afroharzianum, Trichoderma harzianum, Trichoderma atroviride, Trichoderma viridae, Trichoderma hamatum, Trichoderma koningiopsis, Trichoderma koningii, Trichoderma gamsii, Trichoderma asperellum and their mixtures.

[0049] In a preferred embodiment of the present invention, the microbial biocidal agent of the present invention is Trichoderma afroharzianum.

[0050] In a preferred embodiment of the present invention, the microbial biocidal agent of the present invention is Trichoderma afroharzianum Th2RI99 (Access No. NRRL 67990).

[0051] The microbial biocidal agent is mixed into the aqueous PEG formulation so as to form the aqueous biocidal formulation of the present invention. The ratios of biocidal agent: aqueous formulation can be between 20:80 and 60:40.

[0052] Preferably, the proportions are between 30:70 and 55:45. Even more preferably, the proportions are between 40:60 and 55:45. Even more preferably, useful ratios are between 45:55 and 55:45. Even more preferably, the ratio is 50:50. Viability and Efficacy of the Formulation

[0053] The viability of the formulation is based on the determination of the survival of the microorganism in the medium and the temperature in which it is found during a certain time.

[0054] In the field, that is, under conditions of use, both its application and the storage of the formulation are generally in a temperature range between 10 and 25°C, without the need to apply refrigeration or heating means.

[0055] Viability can be measured in days or months, is based on the colony-forming unit (CFU) count, and is related to the colonization capacity of the microorganism (colonization %). It has been seen that, in order to achieve an acceptable colonization of the biological agent in a seed, in this case of Trichoderma afroharzianum, the minimum concentration of viable CFU should be above 105CFU / ml of formulation at the time of formulation inoculation.

[0056] In an object of the present invention, the formulation maintains the CFU values above 1x106CFU / ml of formulation for at least 180 days, maintaining a % colonization greater than 90%. Preferably, the formulation maintains the CFU values of Trichoderma afroharzianum above 1x106CFU / ml of formulation for at least 540 days, maintaining a % colonization greater than 90%. Preferably, the formulation maintains the CFU values of Trichoderma afroharzianum above 1x106CFU / ml of formulation for at least 725 days, maintaining a % colonization greater than 80%. ^^^ ] In the present invention, the % viability is calculated as^^^^^ [0057^^^^^^^ 100. In an ^^^^^^^^ embodiment aspect of the invention, the % viability of the biocidal aqueous formulation, based on Trichoderma afroharzianum and PEG, is at least 90% for formulations with more than 180 days of storage. In another embodiment of the invention, the % viability of the biocidal aqueous formulation, based on Trichoderma afroharzianum and PEG, is at least 90% for formulations with more than 360 days of storage. In another embodiment of the invention, the % viability of the biocidal aqueous formulation, based on Trichoderma afroharzianum and PEG, is at least 90% for formulations with more than 540 days of storage. In another embodiment of the invention, the % viability of the aqueous biocidal formulation, based on Trichoderma afroharzianum and PEG, is at least 90% for formulations with more than 725 days of storage.

[0058] In another aspect of the invention, the % colonization (efficacy) of the aqueous biocidal formulation, based on Trichoderma afroharzianum and PEG, is at least 90% in formulations with 180 days of storage. In another aspect of the invention, the % colonization (efficacy) of the aqueous biocidal formulation, based onTrichoderma afroharzianum and PEG, is at least 90% in formulations with 360 days of storage. In another aspect of the invention, the % colonization (efficacy) of the aqueous biocidal formulation, based on Trichoderma afroharzianum and PEG, is at least 90% in formulations with 540 days of storage. In another aspect of the invention, the % colonization (efficacy) of the aqueous biocidal formulation, based on Trichoderma afroharzianum and PEG, is at least 80% in formulations with 725 days of storage. Application of the Aqueous Biocide Formulation

[0059] The application of the aqueous biocide formulation of the present invention can be applied in different ways depending on the pathogen to be controlled and its cycle. For example, it can be used in seed treatments to control pathogens such as Fusarium graminearum that lodge on the surface of the seed or in internal tissues and when they germinate, grow, and kill the seedling. The application on seed also allows to protect it in the first stages against other pathogens that live in the soil such as Rhizoctonia that, when germinating the seed, attack it and affect the implantation of the crops. Also, it can be used in applications directed to the sowing or planting furrow, through drip or gravity irrigation. This method includes the application of the aqueous biocide formulation in tanks or irrigation lines in order to obtain a dilution that reaches the seedlings when irrigated, thus occupying the rhizosphere zone and protecting it from attacks by pathogens such as Fusarium oxysporum.

[0060] Another method for the application of the aqueous biocide formulation of the present invention is via aerial spraying, in order to impact the canopy or the soil; in this way, plants can be protected from pathogens that cause damage to the leaves, such as Drechslera tritici-repentis, whose infection cycle begins with the arrival of the conidia that are in the soil on the leaves as a result from the splash of raindrops. When applying the biocide, it grows on the leaves, protecting them from the growth of Drechslera spores. Another case of spraying on the ground is to control Sclerotinia sclerotiorum. The aerial application allows the biocide to be carried over the sclerotia that are on the soil surface and, in this way, the biocide prevents these sclerotia from germinating and releasing ascospores that will later infect the soybean flowers, causing plant death.

[0061] In a preferred embodiment, for the application of the aqueous biocide formulation for seed treatments, between 0.5 ml and 600 ml per 100 kg of seeds are required, preferably between 100 ml and 600 ml per 100 kg of seed, in particular from 100 ml to 300 ml per 100 kg of seed. For its application to the seed furrow, between 50 ml and 10,000 ml / ha is required, preferably between 500 ml and 5,000 ml / ha, more preferably between 1,000 ml and 2,000 ml / ha. For its application by area spray, between 50 ml and 10,000 ml / ha is required, preferably between 500 ml and 5,000 ml / ha, more preferably between 1,000 ml and 2,000 ml / ha. Combination of the Formulation with Microbiological and Chemical Agents

[0062] Tests have been carried out where the compatibility of the biocide formulation of the present invention with insecticidal agents, fungicides, and with other microorganisms with the capacity to promote the growth of agronomic crops has been evaluated.

[0063] By compatibility, it is understood that the addition of a substance to the biocidal aqueous formulation of the present invention does not substantially affect the population of the biological agent, either by preventing the growth of the biocidal microorganism (manifesting itself in a decrease in the amount of colony-forming units), or preventing the formation of spores or conidia, or even negatively affecting the biocidal capacity of the microorganism.

[0064] In this way, a microbial biocide aqueous formulation has been obtained that is compatible with a wide variety of commercially available chemical substances that are used in the agricultural sector, such as: fungicides, insecticides, microorganisms-based growth promoters, among others.

[0065] In a preferred embodiment of the present invention, the microbial biocide aqueous formulation is compatible with fungicides, such as Sedaxane (SDX), Prothioconazole (PTZ), Fludioxonil (FLD).

[0066] In another preferred embodiment of the present invention, the microbial biocide aqueous formulation is compatible with insecticides, such as Teflutrine (TFT), Imidacloprid, Thiamethoxam, and Fipronil.

[0067] In another preferred embodiment of the present invention, the microbial biocide aqueous formulation is compatible with bacterial growth promoters such as: Rhizophos, particularly Pseudomonas fluorescens (PREMAX T®), and Rizospirillum, particularly Azospirillum brasiliense.

[0068] In another preferred embodiment of the present invention, the microbial biocide aqueous formulation is compatible with inorganic fertilizers, such as those based on minerals or metal oxides, such as zinc oxide, magnesium oxide, potassium oxide, copper oxide, iron (II and III) oxide, manganese oxide, calcium oxide, and their mixtures.

[0069] In another preferred embodiment of the present invention, the microbial biocide aqueous formulation is compatible with Rhizobia with the ability to fix nitrogen, such as: Sinorhizobium meliloti, Rhizobium leguminosarum bv. Viciae, Mesorhizobium ciceri, Mesorhizobium loti, Bradyrhizobium spp., Rhizobium tropici, Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Rhizobium leguminosarum bv. trifolii. EXAMPLES Example 1. Dose evaluation of polyethylene glycol 400 in the aqueous biocidal formulation of Trichoderma afroharzianum Th2RI99

[0070] T. afroharzianum strain Th2RI99 was grown in a reactor with 70 L of Trico1 culture medium. The culture was incubated at 30°C for 72 h, with 110 RPM and 200 LPM of induced sterile air. After incubation, the culture was transferred to another reactor with 700 L of the same culture medium, under the same growth conditions, for 96 hours.

[0071] The broth with Trichoderma afroharzianum grown in the reactor together with diluents that constituted different doses of polyethylene glycol 400 in the formulation (Table 1) was formulated in 20 L pyrex. The samples were packed in 500 mL balloon with 150 mL of the formulation and stored at 16°C. Quality controls were performed at different times: 0, 30, 120, 150, 180, 210, 240, 290, 360, 390, 450, 480, 540, 600, 650, and 725 days, and the following parameters were evaluated: concentration of conidia, CFU, pH, and appearance of the crop (presence / absence of biofilm, color, biomass, etc.). For each parameter evaluated at each time, two repetitions per treatment were performed. Conidia were counted using a Neubauer chamber, while serial dilutions were performed for the count of colony-forming units and subsequent seeding in Petri dishes with PDA [Potato Dextrose Agar] growth medium.Table 1. Treatments evaluated.

[0072] The preparation of polyethylene glycol 400 for each treatment was carried out prior to formulation, in 20 L pyrex, adding the amount of PEG400 and completing with sterile osmosis water.Table 2. Conidia per milliliter of formulation over time (days).

[0073] In terms of conidia per milliliter (Table 2), it was observed that all formulations containing polyethylene glycol 400 maintained values above 1x107conidia / mlwhile treatment with T. afroharzianum unformulated showed a drop of two orders of magnitude at 360 days (5x106conidia / ml at 360 days compared to 2.00x108conidia / ml at day 0). Among the different concentrations of PEG400, no substantial difference was observed between the values of conidia / ml up to 725 days, demonstrating that all the concentrations of PEG400 used had a protective and conservative effect on the resistance structures of T. afroharzianum.

[0074] To calculate the viability of the conidia, the average of all the formulations has been calculated, then the logarithm in base 10 is applied and the viability values at 180, 360, 540, and 725 days have been compared. For T. afroharzianum formulations with PEG400, the % viability were: 97.2%, 94.9%, 96.7%, and 93.2%, respectively. For the control (T. afroharzianum unformulated), the % viability were 94.5%, 80.7%, 75.0%, and 66%, respectively.Table 3. Colony-forming units per milliliter of formulation over time (days).

[0075] In the evaluation of colony-forming units (table 3), it was observed that the pure culture without formulating after 30 days of storage presented a drastic drop in CFU / ml, while all the formulations containing polyethylene glycol 400 were maintained in titles above 1x107CFU / ml up to 480 days. Then, in the highest doseof polyethylene glycol 400 (25%), a slight drop was seen in the order of 106that continued until 725 days; this small drop was also observed in the 20% dose of polyethylene glycol 400 after 600 days, while the 15%, 10%, and 5% doses remained in the order of 107up to 725 days.

[0076] To calculate the viability of the CFU, the average of all the formulations has been calculated, then the logarithm in base 10 is applied and the viability values at 180, 360, 540, and 725 days were compared. For T. afroharzianum formulations with PEG400, the % viability were: 95%, 90.5%, 92.1%, and 90%, respectively. For the control (T. afroharzianum unformulated), the % viability were 84.5%, 55.5%, 49.6%, and 53%, respectively.Table 4. pH in the formulations over time (days).

[0077] In terms of pH (table 4), variations are observed between the formulations, with the highest dose of polyethylene glycol 400 (25%) being the one that lowered the pH the most. The doses of 15%, 10%, and 5% were the most stable. However, in the case of fungi such as T. afroharzianum, these variations do not represent a problem since they have a wide pH range where they can grow and remain stable (pH between 4-9).

[0078] Formulations with added polyethylene glycol 400 in doses of 25%, 20%, 15%, 10%, and 5% made it possible to maintain stable structures of Trichoderma afroharzianum up to 725 days of storage in balloons, compared to the unformulated culture broth, which rapidly degenerated and decreased values.

[0079] Substantially, no toxicity from the polyethylene glycol 400 doses was observed in any case.

[0080] The balloons were visually observed with very good color, homogeneity, and without the formation of biofilms or aggregates in all the formulations. The unformulated pure culture broth formed aggregates quickly because it did not have any dispersing agent. Example 2. Formulation test with polyethylene glycols of different molar mass and other stabilizers.

[0081] Trichoderma afroharzianum Th2RI99 was grown in a 500 mL Erlenmeyer flask with 100 mL Tricho1 medium at 30°C and 200 rpm for 72 hours. Then the broth was formulated in 500 ml balloons with different additives: PEG400, PEG800, SURFOM ADJ 8099 (emulsifier for vegetable oils) and ULTRARIC PE 62 (ethylene and propylene oxide block copolymer). The treatments were: without additive (control), 0.1%, 0.5%, 1%, 5%, and final 10% of each additive, with the exception of ULTRARIC PE 62 where the treatments were: without ULTRARIC PE 62 (control), 0.25%, 0.5%, 0.75%, and final 1%. The concentration of Th2RI99 was evaluated by determining conidia / mL in the different treatments. Treatments Conidia count / mL Without PEG400 1.5x108(control)0.5% 1.43x1081% 1.91x1085% 1.98x10810% 1.58x108Table 5. Conidia count / mL of Th2RI99 grown and formulated with different concentrations of PEG800 (n=2). Treatments Average Without PEG800 2.03x108(control) 0.1% 2.41x1080.5% 2.17x1081% 3.08x1085% 3.16x10810% 3.08x108Table 6. Conidia count / ml of Th2RI99 grown and formulated with different concentrations of PEG400 (n=2). Treatments Average Without SURFORM 2.71x108ADJ 8099 (control) 0.1% 3.00x1080.5% 1.38x1085% _ 10% _ Table 7. Conidia count / ml of Th2RI99 grown and formulated with different concentrations of SURFORM ADJ 8099 (n=2).Treatments Conidia count / mL Without ULTRARIC 3.2x108PE 62 (control)Table 8. Conidia count / ml of Th2RI99 grown and formulated with different concentrations of ULTRARIC PE 62 (n=2).

[0082] The PEGs (PEG400 and PEG800, tables 5 and 6) did not affect the sporulation of Trichoderma afroharzianum Th2RI99.

[0083] No inhibition of growth and sporulation was observed when SURFORM ADJ 8099 was added up to 1% (table 7). A higher amount of conidia at 1% was recorded with this additive compared to the control. Higher concentrations inhibited Th2RI99.

[0084] A decrease in the concentration of conidia was observed when [ULTRARIC] PE 62 was added compared to the control (table 8). Example 3. Evaluation test of PEG400 as a diluent at different concentrations and storage temperatures.

[0085] T. afroharzianum Th2RI99 was grown in two 10 L reactors (mini-tanks), MC1 and MC2, with 8 L of the Trico1 culture medium. The culture was incubated at 30°C with constant shaking at 200 rpm for 72 hours and sterile induced air at a rate of 200 LPM. After incubation, the culture was formulated using a 1:1 ratio with different diluent concentrations (Table 1). The samples were packed in 500 ml balloons with 150 ml of the formulation and stored at 24 and 30°C.

[0086] Quality controls were performed at different times: 0, 15, 30, 60, 90, and 120 days, and the following parameters were evaluated: conidia concentration and CFU / ml. To determine the concentration of conidia / ml, a Neubauer chamber was used where the counts were made and serial dilutions and subsequent seeding in Petri disheswith potato dextrose agar culture medium were performed to determine the colony- forming units / ml (CFU / ml).Table 3. Different concentrations of additives used as diluents for the formulation of Trichoderma afroharzianum strain Th2RI99 grown in 10L reactors.

[0087] When the addition of PEG400 was evaluated, at different concentrations as a diluent in the formulation of Trichoderma afroharzianum Th2RI99, at both temperatures, it was observed that the concentration of conidia remained above 1x108conidia / ml during the 120 days of storage. On the contrary, in the treatment with Triton X100 + PVP, a decrease in the number of conidia was observed, after 30 days of storage, with values below 6x107conidia / ml (Figure 1).

[0088] Regarding the CFU count, at both temperatures, it was observed that the formulation with Triton + PVP was the one that presented the lowest counts, throughout the trial, while in the treatments with PEG400, the CFU count was higher (Figure 2). It should be noted that PEG at 30% showed higher levels of viable units than when it was added at 50%. Example 4. Test to determine stability of formulations over time and seed colonization efficiency

[0089] A production of Trichoderma afroharzianum Th2RI99 was carried out in 300L reactors. The formulation was made using the broths with 96 h of development in the fermenters with a ratio of 50% Trico1-broth and 50% PEG400 additive at 20%. 3 batches corresponding to said formulation and a batch of unformulated broth (in the absence of PEG400) were made as a control.

[0090] Subsequently, the formulation was packaged in BAGUIN balloons of 5 L capacity with 3L of formulation. They were stored in the dark at 20°C for 18 months.

[0091] Subsequently, the CFU of Trichoderma afroharzianum / mL of broth / packaged formula were evaluated at different storage times (0, 15, 30, and every 30 days, up to 540 days).

[0092] To evaluate the efficacy of seed treatment, wheat seeds of the Basilio variety were treated using the 3 formulations of Trichoderma afroharzianum Th2RI99 with PEG400 at 20% and compared with the broth with Trichoderma unformulated. Finally, the treatment volume was completed with osmosis water until a volume of 1000mL / 100kg of seeds was achieved. The balloons that were used corresponded to the storage times 0, 180, 260, and 540 days from their production.

[0093] Finally, the incidence percentages of Trichoderma afroharzianum Th2RI99 on the seeds were evaluated using the direct sowing methodology on plates with the PDA culture medium.

[0094] As observed in figure 3, the formulation of Trichoderma afroharzianum Th2RI99 with PEG400 at 20% in its three repetitions showed a convincingly superior curve than that of the broth with Trichoderma afroharzianum Th2RI99 without PEG (control), where at the end of the study (540 days), it reached CFU / ml concentration values of around 104, while the tested formulation maintained the CFU / ml values above of 106.

[0095] Regarding the efficacy of the formulation, figure 4 shows that colonization on seeds was high for those formulated with PEG400 even up to 540 days, where the CFU / ml were above 106, while a negative correlation was observed in the unformulated broth between the decrease in CFU / ml and colonization on seeds. At 540 days, the unformulated broth presented CFU / ml values in the order of 104, which represented 52% of seeds colonized with Trichoderma afroharzianum, while the formulated ones of Trichoderma afroharzianum strain Th2RI99 with PEG400 presented CFU / ml in the order of 106and colonization on seeds remained above 94%. Example 5. Compatibility test with insecticides for seed treatments.

[0096] Wheat seeds of the guava tree variety were treated with Formulation 2 (F2) of Example 1, Zinc Oxide (Status Zn from Rizobacter), Fipronil (Regent from Bayer), Thiamethoxan (Cruiser 35FS from Syngenta), Clothianidin (Poncho from Bayer), and imidachlorpid (Matrero 60 from Sumitomo). Recoveries of the microorganism Trichoderma afroharzianum were made at 0, 15, 30, 60, 90, and 120 days from the treatment, by the methodologies of washing seeds and direct sowing in Petri dishes with PDA. Mixtures were also made in Jensen tubes to determine the physicalcompatibility of the slurry and the colony-forming units / ml (CFU / ml) 2 and 6 hours after the mixture.Table 10. Treatments and dosage *The treatments were carried out at 1000 ml / 100 kg of seeds with osmosis water. **F49:1 is the pigment to differentiate treated seeds from untreated seeds.

[0097] The treatments were done simultaneously, that is, mixing the products involved in a treatment broth.

[0098] Subsequently, the determination of CFU / gram of seeds, the colonization percentage of Trichoderma afroharzianum on seeds (SD in plates) and the CFU / ml, and physical behavior of the slurry in Jensen tubes were performed.Table 11. Colony-forming units / ml of Trichoderma afroharzianum obtained from the product mixtures in Jensen tubes.

[0099] An excellent compatibility between the treatments was observed since the CFU / ml obtained in the mixtures did not differ from the unmixed F2 product, even after leaving the prepared broth for 6 hours.Table 12. Colonization percentage of Trichoderma afroharzianum on seeds over time, by direct sowing of the seeds in Petri dishes with PDA.

[0100] The treated seeds presented a high percentage of colonization from the day of treatment until 120 days later in storage at room temperature in the laboratory (20°C).Table 13. Colony-forming units of Trichoderma afroharzianum obtained by washing seeds over time (days).

[0101] Very high recoveries of viable colonies are observed even up to 120 days from the treatments. Example 6. Determination of the compatibility with chemical fungicides and insecticides used for seed treatment in wheat.

[0102] Seeds of wheat variety Bioceres 1008 were treated with the Formulation 1 of the example 1 under laboratory conditions. Two hundred grams of seeds were placed in plastic bags for each treatment. Products were applied using a slurry method and then each bag was agitated for 30 seconds to obtain a good cover of seeds.

[0103] Seeds treated were evaluated with following methods at 0, 15, 30, 45, 60, 90 days:

[0104] Deep Freezing Blotter test: To evaluate percentage of Trichoderma afroharzianum colonization on seeds (%), one hundred seeds of each treatment were placed on plastic box with cotton and osmosis water and incubated 24 hrs at 25 °C, then 24 hrs at - 20°C and, at the end, 7 days at 25 °C and 12 hours photoperiod with artificial lights.

[0105] Seeds treated on plates with culture media Potato dextrose agar (PDA): To evaluate percentage of Trichoderma colonization on seeds (%) one hundred seeds of each treatment were placed on Petri dishes with PDA culture media for seven days at 25 °C and darkness.

[0106] CFU / g of seeds by washing: To count the number of viable colonies of Trichoderma per gram of seeds, five grams of seeds of each treatment were placed in flasks with 45 ml of sterile water and shook for twelve minutes. After that, serial dilutions were done to quantify viable colonies.

[0107] Treatments mixed (Slurry) were evaluated with following methods:

[0108] Slurry in Jensen tubes: To evaluate behavior of Trichoderma afroharzianum in direct contact with chemical products (4 hrs and 24 hrs since mix), 10 microliters of each slurry were placed on a Petri dish with PDA.

[0109] Inhibitory concentration: The fungicides were mixed with the PDA medium to a final concentration of 50, 15, 10, 1, 0.1, 0.01 ppm and poured into petri dishes. An untreated check was included as reference, and four replicates per treatment were done. Each petri dish was inoculated with a mycelial disc (5 mm diameter) cut from freshly prepared source colonies of Trichoderma. After an incubation period of 3 days, at 25°C in the dark, for all 4 replicates the mycelium diameter was measured, including the agar disc, and the average was calculated.Table 14. Treatment description for seed applications.TFT: Teflutrine; SDX: Sedaxane; PTZ: Protioconazole; FLD: Fludioxonil; gai: grams of active ingredient *Volume of slurry was completed with water up to 1000 ml / 100 kg of seeds. **Seeds were treated with sequential method. Chemical products were applied first, and biological product was applied in second place. ***Seeds treated were stored at 20 °C.Table 15. Colony forming units (CFU) of Trichoderma in each slurry in Jensen tubes.Table 16. Diameter of colonies of Trichoderma afroharzianum with different concentration of chemical.Table 17. Colonization of Trichoderma afroharzianum on seeds in deep freezing blotter test (%).

[0110] The treatments where Prothioconazole was included were the most affected and showed low percentage of colonization. The rest of the chemical showed good compatibility even at double rates.Table 18. Colonization of Trichoderma afroharzianum on seeds in Petri dishes with PDA (%).

[0111] The percentage of colonization in PDA was higher than in the blotter test but Prothioconazole showed again an inhibitor effect on Trichoderma afroharzianum. The rest of the chemical showed good compatibility even at 2x rates.Table 19. Colony forming units of Trichoderma afroharzianum per gram of seeds by washing (CFU / g of seeds) *ND= not determined

[0112] The recovery of colonies was good for all treatments except for Prothioconazole as was observed in the other methodologies evaluated. Even mixtures with Sedaxane, Fludioxonil and Teflutrine show high values until 90 days after treatment.

[0113] Formulation 1 shows a very good compatibility with Sedaxane, Fludioxonil and Teflutrine until 90 days after seed treatment, even with mixtures of each chemical.

[0114] Formulation 1 did not show a good compatibility with Prothioconazole.

[0115] Formulation 1 had a good behavior in tank mixture at 4 and 24 hours for all the treatments.

[0116] The inhibitory concentration evaluation showed that treatments where Prothioconazole was included at different concentrations the diameter of colonies were reduced below 50 % of growth in comparison with control. For the rest of treatments, just Fludioxonil at 15 ppm and 10 ppm reduced the growth of colonies below 50 %, but this result does not indicate a bad compatibility since in the rest of evaluations Fludioxonil and Formulation 1 show a very good compatibility. Example 7. Compatibility between Formulation 1 and Azoxystrobin, fludioxonil, tebuconazole (Maxim Forte).

[0117] Seeds of wheat variety Bioceres Guayabo were treated with Formulation 1 of the Example 1 under laboratory conditions. Two hundred grams of seeds were placed in plastic bags for each treatment. Products were applied using slurry method and then each bag was agitated for 30 seconds to obtain a good cover of seeds.

[0118] Seeds treated were evaluated with following methods and evaluated at 0, 30, 60, 90 days: - Deep Freezing Blotter test: To evaluate percentage of Trichoderma afroharzianum colonization on seeds (%), one hundred seeds of each treatment were placed on plastic box with cotton and osmosis water and incubated 24 hrs at 25 °C, then 24 hrs at - 20°C and, at the end, 7 days at 25 °C and 12 hours photoperiod with artificial lights. - Seeds treated on plates with culture media Potato dextrose agar (PDA): To evaluate percentage of Trichoderma colonization on seeds (%) one hundred seeds of each treatment were placed on Petri dishes with PDA for seven days at 25 °C and darkness. - CFU / g of seeds by washing: To count the number of viable colonies of Trichoderma per gram of seeds, five grams of seeds of each treatment were placed in flasks with 45 ml of sterile water and shook for twelve minutes. After that, serial dilutions were done to quantify viable colonies.

[0119] Treatments mixed (Slurry) were evaluated with following methods: - Slurry in Jensen tubes: To evaluate behavior of Trichoderma in direct contact with chemical products (4 hrs since mix).Table 20. Treatments and rate applied on seeds.

[0120] Final volume was adjusted until 1000 ml / 100 kg of seeds with osmosis water.

[0121] Formulation 1 in mixture with Maxim Forte showed good compatibility in the slurry. Formulation 1 maintained colonies forming units in the same magnitude order when combined with Maxim Forte in rates of 0.5 and 1 ml / kg of seeds (Figure 5).

[0122] Formulation 1 applied on seeds with Maxim Forte presented good compatibility in terms of colonies forming units per gram of seeds by washing methodology, even until 90 days after treatment, where values of 2500 colonies per gram of seeds were achieved with lower rate of Formulation 1 and higher rate of Maxim Forte (Figure 6).

[0123] Formulation 1 applied on seeds with Maxim Forte presented good compatibility in deep freezing blotter test (Figure 8) and Petri dishes (Figure 7) methodologies until 90 days after treatment and in all rates tested. In both cases, the control shows no colonization.

[0124] Formulation 1 and Maxim Forte shower good compatibility under methodologies and rates tested. Example 8. Determination of the compatibility of the Formulation of Trichoderma afroharzianum with Bacillus simplex (Indigo30) and Zinc Oxide (Status Zn) for its use in wheat cultivation.

[0125] To determine compatibility, tests were carried out to determine the colony-forming units (CFU) from treated seeds and mixtures in Jensen tubes (slurry). The CFUs were determined by serial dilutions and subsequent seeding in Petri dishes with appropriate culture media for the growth of each microorganism. Potato dextrose agar (PDA) was used for the growth of Trichoderma afroharzianum and trypteinsoy agar (TSA) was used for Bacillus simplex. In the case of the determination of microorganisms in the combined treatment, the optimal culture medium for each microorganism was used and an inhibitor of the other was added.

[0126] In addition, in vitro confrontations between treated seeds and pathogens that affect the wheat crop were carried out to determine the control efficacy with the stacked products. For this test, treated seeds were placed on the periphery of a Petri dish with PDA culture medium and then a 5 mm disk in the center of the dish. They were incubated for 7 days and then evaluated by measuring the growth diameter of each pathogen. Comparing with each absolute control of the pathogens, the inhibition percentage was calculated. The wheat seed used was the 1008 variety from the company Bioceres seeds.Table 21. Results of quality controls of samples used in the test.

[0127] The values of Formulation 1 adjusted perfectly to those declared commercially. Regarding the values of Indigo30, they slightly exceeded the values detailed by Indigo for this lot (9.5x105). The sporulation percentage was higher than 100%.Table 22. Colony-forming units per gram of seeds, time 0. *zero time is 4 hours from seed treatment.Table 23. Colony-forming units per gram of seeds, 45 days from treatment.

[0128] As can be seen in tables 22 and 23, no negative effects of the tested combinations were observed after the treatment. The recoveries are within the acceptable ranges and in the same order of magnitude for all the treatments, except in the case of Formulation 1 + Indigo30 at 45 days from treatment, but the value is very close, the variation being attributable to experimental error.Table 24. Colony-forming units per milliliter of broth (slurry).

[0129] As can be seen in table 24, the microorganisms did not suffer alterations in their viability once they were combined in the treatment broth, both after 4 hours of preparing the broth and not after 24 hours of evaluation. In turn, no physical incompatibility problems were observed between the formulations, only after 24 hours the three products precipitated slightly, which required stirring to homogenize the broth before taking the sample.

[0130] As can be seen in table 25, both the treatment with Formulation 1 and the combinations (Formulation 1 + Indigo30 and Formulation 1 + Indigo30 + Status Zn) inhibited the pathogens to a greater extent than the chemical treatment with Metalaxyl-M and Difenoconazole. The uncombined Indigo30 treatment had a lower ability to inhibit pathogens compared to the treatment with Formulation 1 and its combinations, although, in the case of Fusarium and Bipolaris, it achieved greater inhibition than the chemical product.Table 25.

[0131] Formulation 1, Indigo30, and Status Zn are fully compatible for use in wheat seed treatments up to 45 days before sowing.

[0132] The treatment broth can be prepared with the 3 products at the same time.

[0133] The combination of Formulation 1 and Indigo30 made it possible to maintain the biofungicide capacity of Trichoderma afroharzianum vs. the tested pathogens. Example 9. Determination of the compatibility of the product Trichoderma afroharzianum Th2RI99 (Formulation 1) with commercial inoculants for soybean cultivation in seed treatment.

[0134] A soybean seed sample was treated with inoculants and Formulation 1 to determine the compatibility between them. For this, seed washings were carried out to determine the recovery of colonies of both microorganisms. Petri dishes with PDA- RB culture medium (potato dextrose agar with rose bengal) were used for recovery of Trichoderma afroharzianum colonies and plates with YMA culture medium (mannitol yeast agar) for recovery of Bradyrhizobium japonicum colonies. Theevaluation times were: 0, 24, 72 hours from treatment (HFT), 7, 14, 21, and 28 days from treatment (DFT).

[0135] In turn, blotter tests were performed to determine the colonization of Trichoderma afroharzianum. Two replicates per treatment were planted and incubated in the growth chamber at 25°C. The evaluation times were 1 and 7 days from treatment (DFT).

[0136] Finally, pots with seeds were made to determine nodulation. For this, pots were planted with Alpachiri soil and incubated in a growth chamber at 25°C for 35 days. They were removed and the roots were washed to determine the percentage of nodulation. The evaluation times were 0 and 7 days from treatment.

[0137] Treatments 1 Absolute control 2 Bradyrhizobium japonicum (Rizoliq Top HC): 200ml / 100kg 3 Bradyrhizobium japonicum (Rizoliq LLI HC) + carbohydrates (Premax): 250ml / 100kg + 50ml / 100kg 4 Bradyrhizobium japonicum (Signum HC): 200 ml / 100 kg 5 Trichoderma afroharzianum (Formulation 1) + carbohydrates (Premax T): 300 ml / 100 kg 6 Trichoderma afroharzianum (Formulation 1) + carbohydrates (Premax T) +Bradyrhizobium japonicum (Rizoliq Top HC): 300 ml / 100 kg + 100 ml / 100 kg + 200ml / 100kg 7 Trichoderma afroharzianum (Formulation 1) + carbohydrates (Premax T) +Bradyrhizobium japonicum (Rizoliq LLI HC): 300 ml / 100 kg + 100 ml / 100 kg + 250 ml / 100kg 8 Trichoderma afroharzianum (Formulation 1) + carbohydrates (Premax T) +Bradyrhizobium japonicum (Signum HC): 300 ml / 100 kg + 100 ml / 100 kg + 200 ml / 100 kgTable 26. Results of CFU / seed of Trichoderma afroharzianum by washing method. 0, 1, 3, 7, 14, 21, and 28 days from treatment. * Values were transformed for statistical evaluation to LOG10 ** Comparisons were made between treatments for the same time.Table 27. Results of CFU / seed of Bradyrhizobium japonicum by washing method.0, 1, 3, 7 days from the treatment.

[0138] Evaluation scale of response to nodulation with bacteria on seeds.Table 28. Results of colonization percentage of Trichoderma afroharzianum on soybean seeds in standard blotter test. *DFT: days from treatment

[0139] Formulation 1 was compatible with Rizoliq Top, Rizoliq LLI, and Signum inoculants.

[0140] Trichoderma afroharzianum Th2RI99 values were obtained on soybean seeds from 98 to 100% in terms of colonization in the standard blotter test methodology up to 28 days after treatment.

[0141] The inoculants mixed with Formulation 1 showed values in the same range of response up to 7 days after treatment in the methodology for recovering viable colonies of Bradyrhizobium japonicum by washing.

[0142] 100 viable colonies of Trichoderma afroharzianum Th2RI99 per seed were obtained after 28 days of storage using the washing technique, which guarantees high colonization and correlates with the results obtained in the standard blotter test. Example 10. Evaluation of Formulation 1 as seed therapy in wheat crop (Triticum aestivum L.).

[0143] A treatment based on Trichoderma afroharzianum Th2RI99, Formulation 1 from Example 1, strain was evaluated adding a witness without seed treatment and a commercial witness used in the market (Table 29).

[0144] A network of five field trials was installed. The design was randomized complete blocks, with 4 repetitions, where the experimental unit consisted of 6 furrows of 1.5 m long, planted 19 cm between furrows. The sowing was done manually, placing 75 seeds per furrow, equivalent to 50 seeds / linear m.Table 29: List of evaluated treatments *A dye was added to identify the treated seed.

[0145] At 15 days post-sowing, the number of emerged plants in the three central furrows was determined, counting in turn the number of plants that showed symptoms of leaf spots in the plumule.

[0146] Subsequently, at 30-, 45- and 60-days post-sowing, the number of stems with symptoms was determined in 3 sampling stations of 10 stems each, located in the 3 central furrows. In turn, 10 main stems were taken randomly, and the severity of leaf spots was evaluated on each leaf of the main stem. Therefore, incidence % and severity % in each plot were determined. In each evaluation, samples were collected to be analyzed in the laboratory and to confirm the identity of the existing disease or diseases.

[0147] The response variables were subjected to variance analysis using Infostat®, and when the null hypothesis was rejected, the means were separated by Fisher’s test (P = 0.05).Table 30: Information of each Experiment InstalledTable 31: Fungal load of the used seed of each cultivar. Percentage of seed with sporulation of each of the three main pathogenic genera detected in seed.

[0148] In all the experiments there were conditions for the wheat yellow spot, caused by Drechslera tritici-repentis, which allowed a very good assessment of Formulation 1 performance against this disease.

[0149] Results obtained in each experiment and in joint analysis of the five experiments are presented. In general terms, Formulation 1 was able to significantly reduce the levels of disease at the beginning of stem elongation (Z30). The positive effect of Formulation 1 was observed in all experiments, which indicates the consistency of the treatment under the evaluated conditions (Table 32).Table 32: Summary table of implantation, and of health readings carried out at each moment (see table 30). Incidence and severity correspond to yellow spot caused by Drechslera tritici- repentis. Note: The P-value and the coefficient of variation correspond to the ANOVA. When the null hypothesis was rejected, separation of means was performed by Fisher’s LSD (P = 0.05).

[0150] According to the results obtained, average of the five experiments, a significant reduction in disease levels for Formulation 1 was observed at the beginning of tillering (approx. Z21), and although Formulation 1 and the commercial witness managed to significantly reduce the levels of yellow spot in Z30, Formulation 1 had a significantly lower level of disease than the commercial witness. Example 11. Wheat’s pathogen control using Formulation 1.

[0151] Four varieties of Bioceres seeds were selected: BioINTA1006, Bioceres 1008, Timbó and Basilio. 200 grams of seeds of each variety were treated by the slurry method.

[0152] For pathogen’s evaluation, the seeds were sown in Blotter tests with freezing on paper (ISTA, 2017). Cotnyl No. 5 plastic trays, vallot paper and cotton were used for the Blotters. The trays were prepared by placing 9 grams of cotton and a sheet of vallot paper per tray, and they were moistened with 40 ml of osmosis water. Two hundred seeds per tray were sown and incubated in a growth chamber with a controlled temperature of 25°C +-1° C and a photoperiod of 12 hours, for 24 hours. Next, they were incubated in a freezer at -20°C for 24 hours. Finally, they were incubated again in a chamber at 25°C +-1 and a photoperiod of 12 hours for 7 days.The pathogen evaluation was carried out by observing each seed under a binocular magnifying glass (20X-40X). After determining the presence of each fungus in the seeds, the percentage of incidence on each genus was calculated. Taking on account these results, the control percentage of each genus was also calculated from the treatments.

[0153] The same treated seeds were subjected to germination power analysis in the Rizobacter seed laboratory. Four hundred seeds per tray were sown in Cotnyl No. 5 plastic trays, vallot paper and cotton and incubated in a growth chamber with a controlled temperature of 20 °C+-1° C and darkness for 8 days. After incubation, seeds were evaluated and counted as germinated and not germinated. Results were expressed as the percentage of seeds germinated per treatment (ISTA, 2017).Table 33. Treatments and doses applied to seeds. *Formulation 1 is a biological fungicide based on Trichoderma afroharzianum Th2RI99 strain (2x108spores per ml). ANOVA and 5% LSD mean test were applied to the results obtained using the Infostat statistical tool.Table 34. Pathogen incidence (%) of variety 1006. Total fungal load.Table 35. Fusarium spp. percentage control in variety 1006.Table 36. Pathogen incidence (%) of variety 1008. Total fungal load.Table 37. Fusarium spp. and Bipolaris spp. percentage control in variety 1008.Table 38. Pathogen incidence (%) in Basilio variety. Total fungal load.Table 39. Fusarium spp. and Bipolaris spp. percentage control in Basilio variety.Table 40. Pathogens incidence (%) in Timbó variety. Total fungal load.Table 41. Fusarium spp. percentage control in Timbó variety.Table 42. Germination power of four varieties of evaluated wheat. *Values marked with the same letters are not statistically different.

[0154] In the evaluation of germination power of the varieties, statistical differences were only found between the treatments applied to the Basilio variety, where Formulation 1 and the chemical product differed from the untreated witness. In the remaining varieties, no statistical differences were found, but the treatment with Formulation 1 had the same or higher performance than the treatment with the chemical product. Example 12. Evaluation of seed treatments for the control of Rhizoctonia solani in soybean.

[0155] The test was carried out in an experimental field of the EEA-INTA Marcos Juárez, province of Córdoba (32º 43’ 32” S, 62º 05’ 03” W), in a plot under direct seeding on soybean stubble. The formulation used was Formulation 1 of example 1. The sowing density was 38.4 seeds per m2. The soil is dark, deep, and well drained fromthe almost flat hills, with a silty loam texture. The aptitude for use is Class I (IP 90), with a wide aptitude for crops, fodder, and pastures. Through soil analysis (0-20), it was determined that it presents a pH in water (1:2:5) of 5.4, an electrical conductivity (1:2:5) mS / cm / 25 ºC of 0.14, a content of 2.9% of organic matter, 0.146% total nitrogen, 122 ppm nitrate, and 35 ppm phosphorus.

[0156] The treatments evaluated in the trial were the combinations of 8 seed treatments and two inoculation levels of Rhizoctonia solani, with and without inoculation (except T1, which was not inoculated in any case) (Table 43):Table 43 – Seed treatments evaluated and grams of inoculum per plot at each inoculation level.

[0157] Each combination of seed treatment by inoculation level had four repetitions, consisting of a sub-plot of two rows with a spacing of 0.52 m and three meters long (surface area of 3.12 m2) (experimental unit). The experimental design was divided plots, with the seed treatment as the main plot (8 levels: T1 to T8) and the inoculation with R. solani as a sub-plot (2 levels: without and with inoculation).

[0158] The seed with the corresponding treatments was provided by INDEAR (Instituto de Agrobiotecnología Rosario [Rosario Institute of Agrobiotechnology]) (table 43). For the inoculation of the plots with R. solani, 15 g of inoculum per plot (2.5 g / m of furrow) were used, placed in the furrow together with the soybean seed in the sowing of the test. Rice grains colonized by the pathogen were used as inoculum. For its preparation, 150 grams of rice with 90 ml of sterile distilled water were placed in 500 ml Erlenmeyer flasks that were autoclaved for 20 minutes at 121°C for two consecutive days. Subsequently, a piece of the edge of a colony of R. solani of 7 days of growth in potato glucose agar was added. The vials were kept in the dark for 10 days at 25°C, after which the inoculum was removed, allowed to air at room temperature, and stored in a refrigerator (5°C) until use.

[0159] At 7, 15, and 21 days after emergence (DAE), in phenological stages (PS) V1 (Nov / 19 / 20), V2 (Nov / 27 / 20) and V3 (Dec / 03 / 20) respectively, in each sub-plot (in the 3 m of the two rows), the number of emerged plants (No. of PL), the number of dead plants, the number of diseased plants, and vigor were recorded. Vigor was estimated visually on a scale from 1 (lower vigor) to 10 (greater vigor), taking as reference for grade 5 the sub-plot corresponding to T1 without inoculation of R. solani in the first repetition. At 21 DAE, five plants were extracted from each plot and the incidence of plants with symptoms of R. solani in roots was determined (Incidence = No. of plants with symptoms / 5*100). In PS R6, the number of plants with symptoms of root and stem diseases was recorded.

[0160] Five days after PS R8, the trial was harvested with a two-row Wintersteiger thresher. The yield of each plot, the Hº content and the Thousand Kernel Weight (TKW) were recorded, and the yield was calculated in Kg / ha corrected to 13.5% Hº.

[0161] Analysis of variance was performed to evaluate the effect of seed treatments, inoculation with R. solani, and the Treatment*Inoculation interaction on the number of plants and vigor at 7, 14, and 21 DAE, on the performance, and on the TKW, and the means were compared with the LSD test (α=5%) with the Infostat software.

[0162] The ANAVA detected significant effects of seed treatments (p<0.1), inoculation with R. solani (p<0.05) and treatment*inoculation interaction (p<0.05) on the number of plants emerged at 7, 14, and 21 DAE and on the incidence of plants with symptoms of R. solani in roots at 21 DAE. In the case of vigor, the effect of inoculation (p<0.05) at 7, 14, and 21 DAE and of the interaction (p<0.1) at 7 and 14 DAE were significant, but not the effect of the treatments. In the case of yield, there was a significant effect of the inoculation (p<0.05), but not of the treatments or of the interaction, and there were no significant effects of any of the factors on the TKW (Table 44).

[0163] In the treatments without inoculation of R. solani, T3 presented the highest number of PL, differing significantly from T1 at 7 DAA, from T1 and T2 at 14 DAE, and from T1, T2, and T7 at 21 DAE. The rest of the treatments presented intermediate values without significantly differentiating between them or the treatments mentioned above. In the treatments with R. solani inoculation, in general, T1, T8, and T3 presented the highest number of PL, and the T2 and T6 had the lowest. For the same seed treatment, the number of PL was lower with the inoculation with R. solani compared to the treatments without inoculation, except in the case of T1 and T8, which presented similar values in both situations. The number of PL of T3 and T8 with R. solani inoculation did not differ significantly from the number of PL of T1 and T2 without inoculation (Tables 45, 46, and 47). Table 44 - Summary of the ANAVA of the variables analyzed. FreedoVariable Source of variation mpDegreesvalueSeed treatment 7 2.04 0.0976 No. PL 7Inoculation 1 90.81DAE Treatment*Inoculatio 7 2.85 0.027 n Treatment 7 5.65 0.0009 No. PL 15Inoculation 1 197.51<0.000DAE1Treatment*Inoculatio7 8.<0.000n591Treatment 7 5.18 0.0015 No. PL 21 Inoculation 1 2.61 0.0139 DAE Treatment*Inoculatio1<0.000n72.711Treatment 7 1.79 0.1432 Inoculati<0.000Vigor 7 DAEon 1 34.81Treatment*Inoculatio2 0.0999 n Treatment 7 1.5 0.2199 Inoculation 1 83.59<0.000Vigor 15 DAE1Treatment*Inoculatio0.0913 n Treatment 7 1.49 0.2254 1 DAEInocul<0.000Vigor 2ation 1 50.11 1Treatment*Inoculatio n 7 1.48 0.2247 Treatment 7 4.2 0.0049 Incidence ofInoculation 1 199.<0.000R. solani in831roots 21 DAE Treatment*Inoculatio 7 5.82 0.0006 n Treatment 7 0.8 0.5993 Inoculation 1 20.03 0.0002 Yield (kg / ha) Treatment*Inoculatio n 7 1.68 0.1628 Treatment 7 0.73 0.6466 TKW Inoculation 1 0.33 0.5723 Treatment*Inoculatio 7 1.11 0.3899 n Table 45– Average number of plants / plot at 7 DAE. Difference % Seed Wit Difference (difference / Wit Treatment hout Inoculated with inoculation* R. solani (without inoculation- hout No. *+ inoculated) inoculation*10 0) T1 81.5D E71.0B C D10.5 12.9 T2 89.9D E F43.0A46.9 52.2 T3 107.5F71.8C D35.8 33.3 T4 89.0D E F49.3A B39.8 44.7 T5 98.5E F52.8A B C45.8 46.4 T6 88.5D E F39.5A49.0 55.4 T7 87.5D E F42.5A45.0 51.4 T8 91.8D E F90.8D E F1.0 1.1 Average 91.8 57.6 34.2 37.2 *Different letters indicate significant differences between combinations of seed treatments and inoculation level of R. solani (LSD, α=5%). +All treatments were inoculated with R. solani, except T1. NS: no significant differences were detected between treatmentsTable 46 - Average number of plants / plot at 15 DAE. Difference Difference % Seed Treatment Without Inoculated with R. (without (difference / With inoculation* solani*+ inoculation- out No. inoculated) inoculation*100) T1100.8C D95.7C D5.1 5.0 T299.0C D56.0A43.0 43.5 T3113.0E89.8C23.3 20.6 T4103.8D E73.0B30.8 29.6 T5107.0D E73.8B33.3 31.1 T6105.0D E57.8A47.3 45.0 T7101.5D E62.8A B38.8 38.2 T8104.5D E106.0D E-1.5 -1.4Average104.3 76.8 27.5 26.4 *Different letters indicate significant differences between combinations of seed treatments and inoculation level of R. solani (LSD, α=5%). +All treatments were inoculated with R. solani, except T1. NS: no significant differences were detected between treatments Table 47 - Average number of plants / plot at 21 DAE. Seed Difference Difference % Without Inoculated with R. (without (difference / With Treatment No. inoculation* solani*+ inoculation- out inoculated) inoculation*100) T1100.0C D95.7C D4.3 4.3 T2101.5C D56.5A45.0 44.3 T3116.8E95.5C21.3 18.2 T4105.5C D E73.3B32.3 30.6 T5108.0D E74.8B33.3 30.8 T6105.3C D E57.5A47.8 45.4 T7102.3C D66.0A B36.3 35.5 T8107.0C D E107.0C D E0.0 0.0Average105.8 78.3 27.5 26.1 *Different letters indicate significant differences between combinations of seed treatments and inoculation level of R. solani (LSD, α=5%). +All treatments were inoculated with R. solani, except T1. NS: no significant differences were detected between treatments

[0164] The vigor in T1 and T8 was similar with and without inoculation of R. solani at 7 and 15 DAE, and with T3 at 7 DAE, while, for the rest of the seed treatments, it was lower with the inoculation of the pathogen in all the evaluations (Tables 48, 49, and 50).

[0165] The incidence of plants with symptoms of R solani in roots 21 DAE was higher in the inoculated treatments compared to the ones without inoculation, except in the case of T1. There were no differences in this variable between the seed treatments without inoculation, while with inoculation of the pathogen, the incidence in roots was lower in T8, and higher in T3, compared to the rest of the treatments (Table 51). Table 48 – Average vigor at 7 DAE.Difference % Seed Without Inoculated with R. (without (difference / Wi Treatment thout No. inoculation* solani*+ inoculation- inoculation*1 inoculated)T1 5.0C D4.3B C D0.7 13.4 T2 4.9C D3.5A B1.4 29.1 T3 5.8D4.5B C D1.3 21.7 T4 5.3D3.8A B C1.5 28.6 T5 5.8D3.8A B C2.0 34.8 T6 5.3D2.8A2.5 47.6 T7 5.5D3.5A B2.0 36.4 T8 5.0C D5.5D-0.5 -10.0 Average 5.3 3.9 1.4 25.2 *Different letters indicate significant differences between combinations of seed treatments and inoculation level of R. solani (LSD, α=5%). +All treatments were inoculated with R. solani, except T1. NS: no significant differences were detected between treatmentsTable 49 – Average vigor at 15 DAE. Difference % Seed Difference (difference / Wi Without Inoculated with R. (without Treatment thout No. inoculation* solani*+ inoculation- inoculation*1 inoculated)T1 5.5C D E4.7B C0.8 15.1 T2 5.5C D E4.0A B1.5 27.5 T3 5.5C D E4.3A B1.3 22.7 T4 6.0D E4.3A B1.8 29.2 T5 6.3E4.8B C1.5 24.0 T6 5.3C D3.8A1.5 28.6 T7 5.8D E4.3A B1.5 26.1 T8 5.8D E5.8D E0.0 0.0 Average 5.7 4.5 1.2 21.6 *Different letters indicate significant differences between combinations of seed treatments and inoculation level of R. solani (LSD, α=5%). +All treatments were inoculated with R. solani, except T1. NS: no significant differences were detected between treatments Table 50 – Average vigor at 21 DAE. Difference % Seed Difference (difference / Wi Without Inoculated with R. (without TreatmentNthout InS NSNo. oculating solani+ inoculation- inoculation*1 inoculated)T1 5.3 4.7 0.6 11.0 T2 5.0 4.3 0.8 15.7 T3 5.8 5.3 0.5 8.7 T4 5.8 4.8 1.0 17.4 T5 5.8 4.8 1.0 17.4 T6 5.0 4.3 0.8 15.0 T7 5.8 4.8 1.0 17.4 T8 5.5 5.5 0.0 0.0Average*5.5A4.8B0.7 12.8 *Different letters indicate significant differences between inoculation levels of R. solani (LSD, α=5%). +All treatments were inoculated with R. solani, except T1. NS: no significant differences were detected between treatments Table 51 – Average Incidence of number of plants with symptoms of R. solani in roots 21 DAE. Seed Treatment No.Without inoculation* Inoculated with R. solani*+T1 5.0A0.0AT2 9.5A65.0C DT3 5.0A70.0DT4 0.0A50.0CT5 0.0A50.0CT6 0.0A50.0CT7 5.0A50.0CT8 5.0A30.0BAverage 3.7 45.6 *Different letters indicate significant differences between combinations of seed treatments and inoculation level of R. solani (LSD, α=5%). +All treatments were inoculated with R. solani, except T1. NS: no significant differences were detected between treatments

[0166] The overall yield was lower in the treatments with R. solani inoculation compared to the treatments without inoculation. Although a significant effect of the seed treatment*inoculation interaction was not detected, there was a decrease in yield with T2, T4, T5, T6, and T7 of more than 1100 kg / ha in the inoculated plots compared to the ones without inoculation, while the decrease with T1 was only 9 kg / ha, approximately 600 kg / ha with T3, and with T8, the yield was higher in the inoculated plots compared to the ones without inoculation (Table 52). There were no differences in TKW between seed treatments or between inoculation levels (Table 53), which would indicate that the yield differences were mainly due to differences in the number of grains / m2associated with a higher number of PL / sub-plot.Table 52 – Average yield (Kg / ha)Difference % Seed (difference / Wit Without Inoculated with (without Treatment oculationNhout InSRNSNo. . solani+ inoculation- inoculation*100 inoculated)T1 4768.0 4759.0 9.0 0.2 T2 4680.5 3554.0 1126.5 24.1 T3 4825.0 4228.8 596.3 12.4 T4 5177.3 4052.8 1124.5 21.7 T5 5303.8 4069.0 1234.8 23.3 T6 5126.5 3567.5 1559.0 30.4 T7 5001.0 3807.0 1194.0 23.9 T8 4476.3 4864.0 -387.8 -8.7 Average* 4919.8A4112.8B807.0 15.9 *Different letters indicate significant differences between inoculation levels of R. solani (LSD, α=5%). + All treatments were inoculated with 15 g of R. solani inoculum / plot, except T1, which was not inoculated. NS: no significant differences were detected between treatmentsTable 53 – Average Thousand Kernel Weight (g) Difference % Difference Seed (difference / W Without Inoculated with R. (without Treatment it oculationNhout InSsolani+NSinoculation- No. inoculation*1 inoculated)T1 174.8 179.3 -4.5 -2.6 T2 173.0 172.0 1.0 0.6 T3 175.2 174.0 1.2 0.7 T4 169.0 174.1 -5.1 -3.0 T5 173.1 171.7 1.4 0.8 T6 169.1 171.5 -2.4 -1.4 T7 172.9 171.7 1.3 0.7 T8 171.6 171.2 0.4 0.3 AverageNS172.3 173.2 -0.8 -0.5 *Different letters indicate significant differences between combinations of seed treatments with and without R. solani inoculation (LSD, α=5%). + All treatments were inoculated with 15 g of R. solani inoculum / plot, except T1, which was not inoculated.NS: no significant differences were detected between treatments

Claims

CLAIMS 1. An aqueous biocide formulation characterized in that it comprises between 40% and 60% by mass of a biocidal agent of the genus Trichoderma and an aqueous solution of polyethylene glycol with a molar mass of between 50 and 1200Da between 1 and 50% by mass.

2. The aqueous biocidal formulation of claim 1 characterized in that said biocidal agent is Trichoderma afroharzianum.

3. The aqueous biocidal formulation of claim 2 characterized in that said biocidal agent is Trichoderma afroharzianum Th2RI99 deposit No. NRRL 67990.

4. The biocidal formulation of claims 1 to 3 characterized in that said polyethylene glycol has a molar mass of 400Da.

5. The biocidal formulation of claims 1 to 3 characterized in that said polyethylene glycol has a molar mass of 200Da.

6. The biocidal formulation of claims 1 to 3 characterized in that said polyethylene glycol has a molar mass of 600Da.

7. The biocidal formulation of claims 1 to 3 characterized in that said polyethylene glycol has a molar mass of 800Da.

8. The biocidal formulation of claims 1 to 3 characterized in that said polyethylene glycol has a molar mass of 1200Da.

9. The biocidal formulation of claim 1, characterized in that it maintains at least 90% of the viability of the biocidal agent for more than 180 days.

10. The biocidal formulation of claim 1, characterized in that it maintains at least 90% of the viability of the biocidal agent for more than 360 days.

11. The biocidal formulation of claim 1, characterized in that it maintains at least12. The biocidal formulation of claim 1, characterized in that it maintains at least 90% of the viability of the biocidal agent for more than 725 days.

13. The biocidal formulation of claim 1, characterized in that it comprises a % colonization greater than 80%, after at least 180 days of storage.

14. The biocidal formulation of claim 1, characterized in that it comprises a % colonization greater than 80%, after at least 360 days of storage.

15. The biocidal formulation of claim 1, characterized in that it comprises a % colonization greater than 80%, after at least 540 days of storage.

16. The biocidal formulation of claim 1, characterized in that it comprises a % colonization greater than 80%, after at least 725 days of storage.

17. The biocidal formulation of claim 1, characterized in that it comprises Trichoderma afroharzianum Th2RI99 deposit No. NRRL 67990 between 1x105and 1x1010CFU / L for at least 180 days.

18. The biocidal formulation of claim 1, characterized in that it comprises conidia of Trichoderma afroharzianum Th2RI99 deposit No. NRRL 67990 between 1x105and 1x1010conidia per L, for at least 180 days.

19. The biocidal formulation of claim 1, characterized in that it also comprises at least one compound selected from the group comprised of: fungicides, insecticides, growth promoters, inorganic fertilizers, and their mixtures.

20. The biocide formulation of claim 19, characterized in that said fungicide is selected from the group comprised of: Sedaxane (SDX), Prothioconazole (PTZ), Fludioxonil (FLD), and their mixtures.

21. The biocide formulation of claim 19, characterized in that said insecticide is selected from the group comprised of: Teflutrine (TFT), Imidacloprid, Thiamethoxam, Fipronil, and their mixtures.

22. The biocide formulation of claim 19, characterized in that said growth promoter is selected from the group comprised of: Pseudomonas fluorescens, Azospirillum brasiliense, Sinorhizobium meliloti, Rhizobium leguminosarumbv. Viciae, Mesorhizobium ciceri, Mesorhizobium loti, Bradyrhizobium spp., Rhizobium tropici, Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Rhizobium leguminosarum bv. Trifolii and their mixtures.

23. The biocidal formulation of claim 19, characterized in that said inorganic fertilizer is metal oxide.

24. The biocidal formulation of claim 23, characterized in that said metal oxide is selected from the group comprising: zinc oxide, magnesium oxide, potassium oxide, copper oxide, iron (II and III) oxide, manganese oxide, calcium, and their mixtures.

25. A method for protecting agricultural crops from pathogenic microorganisms, characterized in that it comprises: i. preparing a solution of aqueous biocide formulation which comprises between 40% and 60% by mass of a biocidal agent of the genus Trichoderma and an aqueous solution of polyethylene glycol with a molar mass of between 50 and 1200Da between 1 and 50% by mass; ii. applying one or more doses of said aqueous biocide formulation solution of at least 0.5ml / 100kg of seeds.

26. The method of claim 25, wherein said biocidal agent is Trichoderma afroharzianum.

27. The method of claim 25, characterized in that said biocidal agent is Trichoderma afroharzianum Th2RI99 deposit No. NRRL 67990.

28. The method of claim 25, characterized in that said polyethylene glycol has a molar mass of 400Da.

29. The method of claim 25, characterized in that said polyethylene glycol has a molar mass of 200Da.

30. The method of claim 25, characterized in that said polyethylene glycol has a molar mass of 600Da.

31. The method of claim 25, characterized in that said polyethylene glycol has a molar mass of 800Da.

32. The method of claim 25, characterized in that said polyethylene glycol has a molar mass of 1200Da.

33. The method of claim 25, characterized in that the application is made between 25 and 35 days prior to sowing.

34. The method of claim 25, characterized in that said pathogenic microorganisms comprise: Alternaria spp., Aspergillus spp., Bipolaris sorokiniana, Bipolaris spp., Cercospora kikuchii, Colletotrichum truncatum, Drechslera triticirepentis, Fusarium spp., Fusarium graminearum, Fusarium oxysporum, Fusarium tucumaniae, Fusarium virguliforme, Macrophomina phaseolina, Microdochium spp., Penicilium spp., Phomopsis spp., Puccinia triticina, Pythium debaryanum, Rhizoctonia solani, Rhizoctonia spp., Sclerotinia esclerotiorum, Tilletia leavis, and Ustilago tritici.

35. Use of an aqueous biocide formulation solution which comprises between 40% and 60% by mass of a biocidal agent of the genus Trichoderma and an aqueous solution of polyethylene glycol with a molar mass of between 50 and 1200Da between 1 and 50% by mass to protect agricultural crops from pathogenic microorganisms.

36. The use of claim 35, wherein said biocidal agent is Trichoderma afroharzianum.

37. The use of claim 35, characterized in that said biocidal agent is Trichoderma afroharzianum Th2RI99 deposit No. NRRL 67990.

38. The use of claim 35, characterized in that said polyethylene glycol has a molar mass of 400Da.

39. The use of claim 35, characterized in that said polyethylene glycol has a molar mass of 200Da.

40. The use of claim 35, characterized in that said polyethylene glycol has a molar mass of 600Da.

41. The use of claim 35, characterized in that said polyethylene glycol has a molar mass of 800Da.

42. The use of claim 35, characterized in that said polyethylene glycol has a molar mass of 1200Da.

43. The use of claim 35, characterized in that said pathogenic microorganisms comprise: Alternaria spp., Aspergillus spp., Bipolaris sorokiniana, Bipolaris spp., Cercospora kikuchii, Colletotrichum truncatum, Drechslera triticirepentis, Fusarium spp., Fusarium graminearum, Fusarium oxysporum, Fusarium tucumaniae, Fusarium virguliforme, Macrophomina phaseolina, Microdochium spp., Penicilium spp., Phomopsis spp., Puccinia triticina, Pythium debaryanum, Rhizoctonia solani, Rhizoctonia spp., Sclerotinia esclerotiorum, Tilletia leavis, and Ustilago tritici.

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