WO2019116232A1

WO2019116232A1 - Solid compositions comprising microorganisms with biocidal activity

Info

Publication number: WO2019116232A1
Application number: PCT/IB2018/059886
Authority: WO
Inventors: Diego Francisco CORTÉS ROJAS; Carlos ESPINEL CORREAL; Martha GÓMEZ
Original assignee: Corporación Colombiana De Investigación Agropecuaria-Agrosavia
Priority date: 2017-12-11
Filing date: 2018-12-11
Publication date: 2019-06-20
Also published as: CO2017012690A1; PE20210664A1; ECSP20038924A

Abstract

The invention relates to solid compositions in the form of wettable powder, granules, tablets or pellets, which comprise, as an active ingredient, a suitable amount of one or more microorganisms with probiotic, biocidal, fertilising and/or growth-promoting activity. The compositions of the invention comprise a suitable solid support that includes various excipients and adjuvants to maintain the stability of the microorganisms and giving the composition adequate fluidity, uniform particle size, rapid wettability and high disintegration, to facilitate the use thereof.

Description

SOLID COMPOSITIONS THAT UNDERSTAND MICROORGANISMS

WITH BIOCIDAL ACTIVITY

FIELD OF THE INVENTION

The present invention is found in the agricultural sector, particularly in the field of probiotic compositions, biocides, fertilizers, and plant growth promoters.

BACKGROUND OF THE INVENTION

Modern agriculture is based on the rational use of natural resources and the sustainable management of crops, mainly by reducing the use of highly toxic agrochemicals and replacing them with less polluting products, such as bioproducts developed from microorganisms. among which are biocides, biofertilizers and plant growth promoters. On the other hand, the use of biological products in the livestock market has been growing due to the prohibition of the use of antibiotics as growth promoters (Markets & Markets, 2014. Global Eubiotics Market Trends and Forecasts 2012-2019).

CA2485796 discloses a biopesticide composition comprising a combination of a fungus (Trichoderma) and a bacterium (Bacillus), together with a non-phytotoxic, non-bacteriostatic and non-bactericidal vehicle, which can be mixed with clay-based powders moj ables and dextrose, maltose dextrose or sucrose in granules or powder. The combination of microorganisms can also be mixed with seed coating agents such as calcium carbonate and calcium sulfate. The compositions described therein may be in the form of emulsions, aerosols, liquids, powders or other preparations by mixing with organic solvents, alcohols, natural and synthetic minerals, emulsifying agents (eg polyethylene oxide esters, alkyl sulfates, magnesium stearate ), dispersants (eg lignin, methylcellulose), colorants and binders (eg carboxymethyl cellulose, gum arabic and polyvinyl alcohol). US2015 / 0272129 discloses various solid pesticidal compositions, obtained by direct compression, comprising between 15% and 30% of a propagule (entomopathogenic fungi conidia) together with a water dispersible vehicle, dispersing agents (eg starch, carbonate calcium), binders (eg cellulose, xanthan gum), surfactants (eg tween, polyethylene glycol), disintegrants (citric acid) and a vehicle acceptable for agricultural use. The compositions described there present percentages of sedimentation higher than 70% and very long disintegration times.

US 2014/0274691 discloses solid compositions (granules or powders) for agricultural use, comprising biologically pure cultures of plant growth promoting bacteria (eg Bacillus sp, Paracoccus sp. And Enterobacter sp.) Together with an acceptable vehicle including dispersing agents, surfactants, additives, water, thickeners, composting formulation, oils, dyes, stabilizers, preservatives, polymers, coatings, among others. These formulations do not include disintegrating agents that promote the disintegration of aggregates in contact with water and do not report properties of wettability and suspensibility.

In the compositions that include microorganisms as active agents, it is necessary that they have the ability to adapt to adverse conditions, such as nutrient deficiency, direct exposure to UV light, rapid unexpected fluctuations in temperature and relative humidity, and also, compete effectively against other microorganisms, so there is a need to design and develop formulations that maintain the stability (viability, activity, physical and chemical characteristics) of the active agent and that at the same time guarantee the effectiveness and effectiveness of the bioproduct (CHIOU, AL WU , WS 2003. Formulation of Bacillus amyloliquefaciens B190 for Control of Lily Gray Mold (Botrytis elliptica), Journal of Phytopatology, 151: 13-18).

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to solid compositions comprising, as an active ingredient, an effective amount of one or more microorganisms with probiotic activity, biocide, fertilizer and / or plant growth promoter, together with formulation aids and a suitable vehicle, to thereby conform a stable delivery system. DETAILED DESCRIPTION OF THE INVENTION

The compositions of the invention comprise an active ingredient accompanied by an appropriate solid support that allows it to maintain its stability and confer to the compositions an adequate fluidity, a uniform size of the particles to facilitate its application in the field. Preferably, the solid compositions of the invention are in the form of a tablet, powder, granulate, dispersible granulate, pellet or wettable powder.

The active ingredient, in the compositions of the invention, corresponds to one or more microorganisms with probiotic activity, biocide, fertilizer and / or plant growth promoter. For purposes of the present invention, the biocidal activity of the active ingredient refers to the ability to kill living organisms, while the fertilizing activity refers to the ability to provide the soil with the necessary nutrients (eg, nitrogen, phosphorus) to maintain the plant growth, and growth promoting activity refers to the ability to stimulate, improve and facilitate the healthy development of plants. The probiotic activity, on the other hand, refers to the ability to positively favor the animals' microbiota and improve their quality of life.

The active ingredient of the compositions of the invention is an effective amount of one or more microorganisms selected from the group of: Lecanicillium sp., Beauveria sp., Isaria sp., Metarhizium sp., Hirsutella sp., Aschersonia sp., Trichoderma sp. , Gliocladium sp., Clonostachys sp., Pseudomonas sp., Bacillus sp. , Rhodotorula sp., Pichia sp., Rhizobium sp., Azotobacter sp., Azosphirillum sp., Herbaspirillum sp., Lactobacillus sp., Pediococcus sp., Streptomyces sp., Cryptococcus sp., Saccharomyces sp., Streptococcus sp., And Enterococcus sp, In a preferred embodiment, the active ingredient is a microorganism of the species Lecanicillium sp., Metarhizium sp., Trichoderma sp., Rhizobium sp., Bradirhizobium sp., Beauveria sp., Azotobacter sp., Lactobacillus sp., Pediococcus sp. ., Streptomyces sp., Cryptococcus sp., Saccharomyces sp., Streptococcus sp., And Enterococcus sp.

The microorganism or microorganisms that make up the active ingredient can be produced by fermentation or other known technique and be in the form of cells, spores, or in the form of conidia, defining the latter as asexual spores, characteristics of fungi Zygomycetes, Ascomycetes and some Basidiomycetes, which are relatively tolerant to temperature, stable under different environmental conditions and can be quantified and serve as units of measurement to evaluate parameters such as concentration and viability.

The term "effective amount" corresponds to an amount and / or concentration of the microorganism or microorganisms, sufficient to exhibit the indicated activity with an appropriate effectiveness and efficiency. The concentration of the microorganisms that make up the active ingredient is between lxlO ² and lxlO ¹² propagules / g, preferably between 1x10 ^s and lxlO ¹⁰ propagules / g, more preferably between lxlO ⁷ and lxlO ¹⁰ propagules / g, understood by propagules, the cells , conidia, spores or colony forming units (CFU).

In addition to the active ingredient, the solid compositions of the invention comprise adjuvants and excipients such as stabilizing agents, lubricants, binders, disintegrants, drying protectants, enhancers and diluents. The different components of the compositions according to the present invention are defined below:

Coadjuvants or Additives: They are defined as substances that favor the active ingredient to fulfill its function. They are added to improve certain properties of the composition such as increasing the coverage, penetration and absorption (e.g., by the plant), improving dispersion, suspending and helping to maintain the stability of the microorganisms that make up the active ingredient. Preferred additives include, inter alia, enzymatic extracts, plant extracts, essential oils, polysaccharides and proteins. The concentration of additives in the compositions of the invention may be between 0.0% and 20.0% (w / w), preferably between 7.0% and 15.0% (w / w), and more preferably between 5 , 0% and 10.0 (w / w).

Diluents: They are defined as liquid or solid agents that act as vehicles, allowing an adequate and effective distribution of the active ingredient. Preferred diluents may be mentioned, inter alia, starches (eg corn starch, cassava starch), cellulose derivatives (eg carboxymethylcellulose, cellulose) microcrystalline, hydroxypropyl-methyl cellulose), diatomaceous earth, skimmed milk and clays (eg kaolin, bentonites and sepiolites). The concentration of diluents in the compositions of the invention may be between 0.5% and 60.0% (w / w), preferably between 5.0% and 40.0% (w / w), and more preferably between 10%. , 0% and 30.0% (w / w).

Disintegrants: They are defined as agents that help the breakdown and disintegration of solid compositions. This group includes, among others, croscarmellose sodium, starch, pregelatinized starch, sodium starch glycolate, povidone, crospovidone, xanthan gum, guar gum, calcium silicate, lactose, alginic acid, cellulose, microcrystalline cellulose, sodium alginate, chitosan and silicon dioxide. The concentration of disintegrants in the compositions of the invention may be between 0.5% and 60.0% (w / w), preferably between 2.0% and 40.0% (w / w), and more preferably between 5 , 0% and 20.0% (w / w).

Binders: They are defined as agents that allow grouping or agglutinate powders during the preparation of a solid composition. Preferred binders include, but are not limited to, starch, sucrose, glucose, celluloses, gelatins, sorbitol, mannitol, dextrins, acrylates, alginates and gums (e.g. gum arabic, xanthan gum). The concentration of binders in the compositions of the invention may be between 0.0% and 15.0% (w / w), preferably between 2.0% and 12.0% (w / w), and more preferably between 5% and 20%. , 0% and 10.0% (w / w).

Drying protectors: They are defined as agents that act as a support material during dehydration and as a receptor during hydration, protecting the active ingredient. Preferred drying protectants include, but are not limited to, albumin, skimmed milk powder, whey protein, egg yolk, polyethylene glycol polyols, propylene glycol, sorbitol, mannitol, and sugars such as sucrose, glucose, trehalose, mannose, and sugar. fructose. The concentration of drying protectants in the compositions of the invention may be between 0.5% and 60.0% (w / w), preferably between 5.0% and 40.0% (w / w), and more preferably between 25.0% and 35.0 (w / w).

Lubricants: They are defined as agents that reduce inter-particular friction by improving flow properties and preventing compaction and adhesion. Among the preferred lubricants, mention may be made, among others, of magnesium stearate, monostearate of glyceryl, calcium stearate, palmitic acid, myristic acid, stearic acid, mineral oil and vegetable oil. The concentration of lubricants in the compositions of the invention may be between 0.0% and 15.0% (w / w), preferably between 0.5% and 10.0% (w / w), and more preferably between 1 , 0% and 5.0% (w / w).

In one embodiment of the invention, the solid compositions may be in the form of a dispersible granulate of high dispersibility, which, for purposes of the present invention, is understood as an asymmetric aggregate that rapidly disintegrates upon contact with water, generating in a time less than two minutes, a dispersion of particles of a size similar to that of the powder mixture with which it was made, allowing its application to be carried out more easily (eg with conventional field spraying equipment). The solid compositions of the invention can be kept in suspension in water or in an aqueous solution for the period of time required for their use.

In a further embodiment, the solid compositions of the invention may be in the form of a wettable powder, in which the active ingredient is dispersed in an inert solid material containing various excipients. The excipients used to formulate these compositions make it possible to neutralize the attractive forces between the particles, which improves the flow and the suspendability of the composition.

Additionally, excipients must be capable of altering the surface tension to prevent solids (e.g., wettable powder) from floating on the surface of the solvent. Other formulation aids, such as defoaming agents and emulsifiers, can be included to avoid excessive foaming during agitation and to facilitate wetting of the product and its subsequent uniform application. The concentration of the other formulation auxiliaries in the compositions of the invention may be between 0.0% and 60.0% (w / w), preferably between 2.0% and 40.0% (w / w), more preferably between 5.0% and 20.0 (w / w).

The following examples illustrate the invention, without the inventive concept being restricted thereto. EXAMPLES

Example 1. Obtaining Active Ingredients

1.1 Obtaining the active ingredient Trichoderma asnerellum

The microorganism Trichoderma asperellum was supplied by the germplasm bank of microorganisms with interest in Biological Control of Corpoica and kept cryopreserved in 10% glycerol until its recovery in Petri dishes with malt extract agar agar.

The conidia of Trichoderma asperellum produced by solid fermentation were separated wet by washing with Tween 80 solution (0.5% w / v) and centrifugation. The biomass obtained had a humidity percentage lower than 70% and a concentration of 2x10 ¹⁰ ULC / g.

1.2 Obtaining the active ingredient Beauveria bassiana

The microorganism Beauveria bassiana was supplied by the germplasm bank of microorganisms with interest in Biological Control of Corpoica and kept cryopreserved in 10% glycerol until it was recovered in Petri dishes with potato dextrose agar medium.

The Beauveria bassiana microorganism was produced by semisolid fermentation in a rice-based medium. The sporulated medium was dried and the conidia were separated via dry for its formulation. The dry biomass obtained had a humidity percentage lower than 6% and a concentration of lxlO ¹¹ CFU / g.

1.3 Obtaining the active ingredient Lecanicillium le canil

The Lecanicillium Lecanii strain was supplied by the germplasm bank of microorganisms interested in Corpoica Biological Control and kept cryopreserved in 10% glycerol until it was recovered in Petri dishes with potato dextrose agar medium. The conidia of the fungus Lecanicillium lecanii were produced by solid fermentation in a rice-based medium and the separation was carried out by wet washing with Tween 80 solution (0.5%) and centrifugation. The humid biomass obtained had a humidity percentage of 60% and a concentration of x 2x10 ¹⁰ CFU / g

Following this same methodology, other microorganisms of the species Trichoderma sp., Isaria sp., Metarhizium sp., Hirsutella sp., Aschersonia sp., Gliocladium sp., And Clonostachys sp.

1.4 Obtaining the active ingredient Bradyrhizobium japonicum

The microorganism Bradyrhizobium japonicum was supplied by the germplasm bank of microorganisms with interest Biofertilizer from Corpoica and kept cryopreserved in 10% glycerol until its recovery in Petri dishes agar yeast extract-mannitol.

The microorganism was produced by liquid fermentation in Erlenmeyers of lOOOmL with an effective work volume of 750 mL, using yeast-mannitol extract broth with final pH 6.6 ± 0.1. The culture media were inoculated with a suspension of the concentration strain lxlO ⁹ CFU / mL. The cultures were shaken at 150 rpm and incubated at 28 ± 2 ° C for seven days. The separation of the biomass was carried out by centrifugation. The humid biomass obtained had a humidity percentage of 70% and a concentration of lx10 ¹⁰ cells / g.

Following a similar liquid fermentation process, other microorganisms were obtained from the species Azotobacter sp., Lactobacillus sp., Pediococcus sp., Rhizobium sp., Streptomyces sp., Cryptococcus sp., Saccharomyces sp., Streptococcus sp., And Enterococcus sp. Example 2. Preparation of a solid composition of Trichoderma asverellum

2.1 Selection of excipients compatible with the active ingredient To select the most suitable excipients, samples of 0.05 grams of dried conidia of Trichoderma asperellum, obtained according to Example 1.1, were mixed with 0.05 grams of each of the indicated excipients in Table 1 and stored in glass vials capped with rubber stopper and agrafe. The vials were stored at 30 ° C for three weeks and, each week, germination was evaluated at 16 hours of incubation following the methodology described by Santos et al. (Santos, A. Cotes, A. Villamizar, L. 2012. Effect of the formulation on the useful life of biopesticides based on two Colombian isolates of Trichoderma koningiopsis Th003 and Trichoderma asperellum Th034. Iberoamerican Mycology Journal, Spain ISSN: 1130 -1406 ed: Elsevier, v.29 fasc.3, 150 - 156).

The stability results during the three weeks are presented in Table 1. All measurements were made in triplicate and the results were subjected to an ANOVA analysis of variance and a minimum significant difference comparison (SMD) (95%). The statistical analyzes were applied independently for each excipient.

Table 1.

* Results with the same letter do not show significant differences according to DMS (95%).

* Results with different letters show significant differences according to DMS (95%).

According to the results, dry conidia stored without excipients showed a significant loss (P <0.05) of the gemination after 3 weeks of storage at 30 ° C, with a reduction in germination of 18%. In contrast, the conidia showed no significant reduction in their ability to germinate, when stored for three weeks with the excipients: titanium dioxide, microcrystalline cellulose, sodium croscarmellose, pregelatinized starch, bentonite and Mg and Al silicate, which were considered compatible with the biomass and showed a stabilizing effect of the same in the storage conditions evaluated.

2.2 Preparation of dispersible granules of Trichoderma asperellum 4 granules (A, B, C and D) were prepared from conidia of Trichoderma asperellum, obtained according to Example 1.1. The processed granulates showed variations in the composition and dry base concentrations of each of the excipients (Table 2). Table 2

The wet biomass was mixed with the powdered excipients and the wet mixtures were granulated by extrusion through a No. 12 mesh. The granulates were dried in an oven with air flow at 25 ° C for 18 hours for further characterization, where the concentration of active ingredient was determined (by counting in Neubauer chamber), the percentage of humidity, particle size, percentage of germination at 24 hours of incubation, following the methodologies described by Santos et al (2012). The results are illustrated in Table 3. Table 3

The humidity in all the processed granulates, after 18 hours of drying, was less than 5.0%, a value considered adequate to maintain the viability of fungal-based bioproducts under storage conditions (Quiroga, I. et al. 2011. Stability of granulovirus-based formulations to control Teda solanivora (Lepidoptera: Gelechiidae) in the field Revista Colombiana de Entomología 37 (1): 27-35), as reported by Lawrie and cois (Lawrie, J .; J .: Greaves, M. 2001. Effects ofstorage on viability and efficacy of granular formulations of the microbial herbicides Alternaria alternata and Trematophoma lignicola, Biocontrol Science and Technology 11: 283-295), humidities below 5.0% allow microorganisms to they remain viable and virulent during their shelf life, due to the reduction of their metabolism. Example 3. Preparation of a solid composition (dispersible granulate) of Beauveria bassiana

8 granules (E, F, G, H, I, J, K and L) were prepared from pure and dry conidia of Beauveria bassiana with a humidity of 8.0%, obtained according to Example 1.2. The processed granulates presented variations in the composition and dry base concentrations of each of the excipients (Table 4).

Table 4

The conidia (active ingredient) were mixed with the powdered excipients and water was added. The wet mixes were granulated by extrusion through a number 12 mesh and the granulates were dried in an oven with air flow at 25 ° C for 18 hours for further characterization, where the concentration was determined (by counting in Neubauer chamber ), the percentage of humidity, particle size and germination at 24 hours of incubation following the methodologies described in Santos et al. (2012). The results are illustrated in Table 5. Table 5

The average humidity of the granules, after 18 hours of drying, was 4.6 ± 0.3% (<5.0%), value that is considered adequate to maintain the viability of these bioproducts under storage conditions. The percentage of germination of the 8 granulates was 100%, which indicates that the excipients used and the composition in the form of dispersible granules did not affect the viability of the conidia.

Example 4. Preparation of a solid composition (granules of direct application to the soil) of Beauveria bassiana

Four granules (M, N, O, P) were prepared from the dry and milled biomass of Beauveria bassiana. The milled biomass was mixed with the powdered excipients (mixtures of bentonite and talc) and a suspension of gelatin was added as binder. The wet mixes were granulated by extrusion through a mesh with an aperture of 1500 μm and the granulates were dried in an oven with air flow at 25 ° C for 18 hours for further characterization, where viability, pH in suspension was determined, percentage of humidity and porosity, following the methodologies described in Santos et al. (2012). The results are illustrated in Table 6.

Table 6

Example 5. Preparation of a solid composition of Lecanicillium lecanii

3 dispersible granules (Q, R, S) were prepared from the active ingredient Lecanicillium lecanii obtained according to Example 1.3 with a humidity of 57.8%. The processed granulates presented variations in the composition and in the dry base concentrations of each of the excipients (Table 7).

Table 7

The wet mixtures were granulated by extrusion through a No. 12 mesh and the granulates were dried in an oven with air flow at 25 ° C for 18 hours, for further characterization, where the concentration was determined (by counting in chamber de Neubauer), moisture content and germination at 24 hours of incubation, following the methodologies described by Santos et al. The results were subjected to an analysis of variance at 95% reliability and are shown in Table 8.

Table 8

The germination of the conidia, in all the granulates, exceeded 80.0% after 24 hours of incubation, which indicates that the excipients and the manufacturing process did not affect the viability of the microorganism. Humidity was found in an expected range between 3.0% and 5.0%.

Example 6. Preparation of a solid composition of Bradyrhizobium iavonicum

2 dispersible granules (T and U) were prepared from the active ingredient obtained according to Example 1.4 with a humidity of 80.0%. The processed granules presented variations in the composition and in the dry base concentrations of each of the excipients (Table 9).

Table 9

The wet mixtures were granulated by extrusion through a No. 12 mesh and the granulates were dried in an oven with air flow at 25 ° C for 24 hours, for further characterization, where the concentration was determined (by plate count). in yeast-mannitol extract agar medium) and moisture content. The granulates presented moisture percentages lower than 10.0%, and a concentration between 6 x 10 ⁸ and 1 x 10 ⁹ cells / g. Example 7. Preparation of a solid composition (movable powder) of Metarhizium anisoOliae

The wet biomass obtained after the centrifugation process was dried using the Niro AEROMATIC fluidised bed for 120 minutes at a temperature below 50 ° C.

Two powders were elaborated from the dried conidia of Metarhizium anisoplieae. The powders prepared (powder 1 and powder 2) showed variations in the composition and dry base concentrations of each of the excipients (Table 10).

Table 10

The dried conidia were mixed with the powdered excipients and subsequently the binder solution was added. The wet mixes were granulated by extrusion through a mesh with an aperture of 1500 μm and the granulates were dried in an oven with air flow at 25 ° C for 18 hours to subsequently grind them. Thus, a powder was obtained that was characterized by its viability, particle size, humidity, wettability and pH, following the methodologies described in Santos et al. (2012). The results are illustrated in Table 11.

Table 11

Example 8. Preparation of a solid composition (dispersible granulate) of Rliizobium sp.

Two granulates (V, W) were prepared from the Rhizobium biomass obtained according to Example 1.4. The processed granulates presented variations in the composition and in the dry base concentrations of each of the excipients (Table 12). The wet biomass was mixed with the powdered excipients, and the wet mixes were granulated by extrusion through a No. 12 mesh. The granulates were dried in an oven with air flow at 25 ° C for 3 hours.

Table 12

The V and W granules had a viability of 2.90 x 10 ⁸ and 1.29 x 10 ⁹ respectively. The humidity, for each one, was 3.53% and 6.99%.

Example 9. Disintegration test

The time or rate of disintegration of a solid composition depends on its chemical composition and other factors such as wetting, the solubility of the components of the formulation, the size and shape of the granules, the porosity (Helman J. (1982) Theoretical and practical pharmacology. (V) Ed. Continentals 1687-1728)), as well as disintegrating agents, binders and lubricants (Gennaro, AR (1995) Remington: The Science and Practice of Pharmacy, l9th Ed., Vol. II, Easton, PA, Mack Publishing Co ., pp. 1276 -1277).

The disintegration in water of the compositions obtained in Examples 2, 3 and 4 was determined, for which 100 mL of distilled water was poured into a 250 mL beaker and 5 grams of each of the granules were added. Once the granulate was wetted, the magnetic stirring was started and with a stopwatch the time (seconds) necessary for all the granules to disintegrate completely was counted (Voiggth, R., Bornschein, M. (1982) Treaty of Pharmaceutical Technology. Editorial Acribia, Spain, P. 297). Each measurement was made in triplicate.

Table 13 shows the disintegration times (seconds) of the granulates evaluated.

Table 13

All the granules presented a disintegration, in less than 120 seconds, higher than 80.0%, a value that exceeds the acceptance limit for dispersible products reported in the literature, which establishes that according to the CIPAC method Mtl74, at least 60% of the product must be completely dispersed in this period of time (Australian Pesticide and Veterinary Medicines Authority-APVMA, 2005. Guidelines for the Generation of Storage Stability Data of Agricultural Chemical).

The rapid and complete disintegration of the granules is directly related to an adequate concentration of the binding agents and the inclusion of disintegrants highly soluble in water, which generate the rapid collapse of the granular structure.

Example 10. Humidity Test

The wettability of the solid compositions depends on the number of intra or interarticular pores (Vargas, R. 2003. Preliminary evaluation of the method used in the determination of the photostability of fish food Mesoamerican Agronomy 14 (2): 193-199). The rapid wettability of the compositions, due to a high porosity of the granules, increases the permeability of the water in the granulate, thus facilitating wetting.

To determine the wettability or wettability of the solid compositions of Examples 2, 3 and 4, the methodology described by Voight et al. and cited by Quiroga et al. For this purpose, 100 mL of distilled water was poured into a 250 mL beaker and 5 g of the granulate were added without agitation. Then, the time (seconds) necessary for the entire product to be completely wetted was determined (Table 14).

Table 14

The times obtained, in all cases, did not exceed 40 seconds, suggesting rapid wettability of the particles, which facilitates their subsequent disintegration. Example 11. Insecticidal activity on Diatrea sacharallis

The insecticidal activity of a dispersible granulate based on Beauveria bassiana obtained according to Example 3 was determined under laboratory conditions. Initially, suspensions of the granulate adjusted to five concentrations were prepared, with which second instar larvae of Diatraea saccharalis were inoculated. by applying 2 pL on the back. Each larva was placed in a disposable container containing a grain of corn and placed in groups of 10 units, with three repetitions of each experimental unit, for a total of 30 larvae per treatment. There was an absolute witness in which the larvae were not subjected to any treatment. The larvae were incubated in a quarter of bioassays and, from day four after inoculation, the larvae were checked daily. The dead individuals were placed in a humid chamber at 25 ° C to confirm the fungal infection. The mortality results were subjected to a Probit analysis with the POLO Plus program. An average lethal concentration of 1.6 x 10 ⁶ conidia / mL and a lethal concentration of ninety CL90 (concentration at which 90% mortality of the larvae was reached) of 8.0 x 10 ⁷ conidia / mL was obtained. The results are shown in Table 15. Table 15

* Value of the statistic Subsequently the activity of the granulate was determined in semi-controlled conditions (in plant) by applying a suspension of the product to cane seedlings and performing artificial infestation with six eight-day-old D. sacharallis larvae (second instar). Seven days after the assembly of the test a destructive analysis of the plants was carried out. Live larvae were placed in a 0.5-ounce glass with a napkin and a grain of corn and transported to the laboratory where they were observed daily recording mortality.

The dead larvae were placed in a humid chamber for 7 days at 25 ° C to show sporulation. With the number of live larvae, the percentage of efficacy for each treatment was calculated using the Abbott formula. The dispersible granulate presented an efficiency of 80% to control larvae of D. saccharalis at a concentration of 4 x 10 ⁷ conidia / mL applied on cane plants.

Example 12. Insecticidal activity on white flies Trialeurodes vanorariarum and Bemisia tabaci.

To determine the efficacy of the granular formulation in the control of whitefly Trialeurodes vaporariorum in greenhouse tomato crops, tomato plants were infested with 30 adult insects using pincers cages located on the leaves. Nine days later, the nymphs of second instar were counted and the treatment was applied (dispersible granulate based on Lecanicillium Lecanii obtained according to Example 4). It was used as a chemical control (Imidacloprid) and an absolute control.

After fifteen days, the fourth instar and exuvia nymphs (evidence of emerged flies) were reviewed and, by counting the live and subtraction, the mortality was calculated by the action of the formulation. Nymphs of T. vaporariorum affected by the fungus were observed, characterized by changes in their color, shape, brightness and mycelial growth and / or sporulation.

The counts yielded mortality results of 74% and 73% for the dispersible granulate based on L. lecanii and for the insecticide Imidacloprid respectively, while with the absolute control a 13% mortality was obtained. To determine the efficacy of the granular formulation in the control of whitefly Bemisia tabaci in soybean crops, soybean plants were infested with 30 adult insects using pincers cages located on the leaves. Seven days later the second instar nymphs were counted and the treatment was applied (dispersible granulate based on Lecanicillium Lecanii obtained according to Example 4). A biological control (Vercani WP) and an absolute control were used.

After fifteen days, the fourth instar and exuvia nymphs (evidence of emerged flies) were reviewed and, by counting the live and subtraction, the mortality was calculated by the action of the formulation. Nymphs of B. tabaci affected by the fungus were observed, characterized by changes in their color, shape, brightness and mycelial growth and / or sporulation. The counts yielded mortality results of 68.7% and 61% for the dispersible granule based on L. lecanii and for the insecticide Vercani WP, respectively, while with the absolute control a 17% mortality was obtained.

Taking into account the same evaluation methodology, the efficacy of the formulation on Bemisia tabaci in cotton crops was determined (Rivera, F., Zuluaga, M. 2012. Use of the biopesticide based on Lecanicillium lecanii for the control of Bemisia tabaci in The cultivation of cotton In: Use of Lecanicillium lecanii for the control of whitefly Bemisia tabaci in cotton and eggplant Technical Bulletin - CORPOICA, Bogotá, pp. 59-76) and aubergine (Espinel, C., Zuluaga, M., Jiménez, N., Gómez, M. 2012. Use of the biopesticide based on Lecanicilliuem lecanii for the control of Bemisia tabaci in the eggplant culture In: Use of Lecanicillium lecanii for the control of whitefly Bemisia tabaci in cotton and eggplant. Technical Bulletin - CORPOICA, Bogotá, Pp 45-58).

The mortality for the dispersible granulate was 89.9% and 67.0% in cotton and eggplant, respectively.

Example 13. Insecticidal activity on the plains lobster Rhammatocerus schistocercoides

To determine the effectiveness of the wettable powder formulation in the control of the lobster Rhammatocerus schistocercoides, an assay was carried out under conditions sem i controlled from the field, using 1.2 m ² muslin cages to confine insect nymphs that were captured in the field (Gómez, M., Villamizar, L., Ebratt, E., Espinel, C., Cámmen, A ., Jiménez, Y., Cotes, A. 2000. Biological Control In: The Plains Lobster in Colombia, Boletín Técnico, Corpoica, Bogotá, Pp: 47-78). The concentrations of lxlO ⁶ and lxlO ⁷ conidia / mL were applied and rice was given as a feeding substrate. There was an absolute witness to whom no application was made.

The day after the application was made the daily record of mortality and the taking of dead individuals for analysis. The percentage of efficacy on the registered dead individuals was determined. The Schneider-Orelli formula was used (Zar 1999. Biostatistical Analysis, 4th Edition, Prentice Hall, New Jersey, page 663), efficiency percentage = ((bk) / (100-k)) x 100, where b equals the percentage of individuals killed in the treatment and k equals the percentage of individuals killed in the control, the results were subjected to parametric tests of ANOVA and Tukey.

After 17 days after application, significant differences were found in the efficacy of Bioinsectisol applied to the concentration of lxlO ⁸ conidia / mL in comparison with the concentration of lxlO ⁷ conidia / mL, which were 67.8 and 54 , 7%, respectively. The absolute witness behaved within the accepted range, registering a mortality of 11.7%.

Example 14. Insecticidal activity on potato white worm

In order to determine the insecticidal activity of the granulate, as a first measure, an experiment was carried out under greenhouse conditions in which lg / plant was applied to plants sown with Solanum phureja (Torres, L., Villamizar, L., López , J., Espinel, C., Gómez, M., Zuluaga, M., Valencia, C., García, M. Cotes, A., López, A. 2004. Technological development of a microbial insecticide. a microbial insecticide for the biological control of the white potato worm Bolentín Técnico, Corpoica, Bogotá, Pp: 25-52).

Each experimental unit was infested with 10 adults of Premnotrypes vorax (white potato worm) and mortality was monitored for 20 days. They had 3 replicas and an absolute witness. After this time, the mortality was 100% in the treatment with the granulate and 9% in the absolute control.

Subsequently, the effectiveness of this granulate was determined in experimental plots of 1,200 m ² , in which the bioproduct was applied at two concentrations, 0.5 and 1.0 g / plant.

(Espinel, C., Zuluaga, M., Villamizar, L., Lopez, J., Cotes, A., Lopez, A. 2004. Determination of doses and frequencies of application of the pre-formulated based on B. bassiana In the field: Development of a microbial insecticide for the biological control of the white potato worm, Boletín Técnico, Corpoica, Bogotá, Pág: 53-66).

The experimental design was randomized complete blocks with three replications. The application was made in three stages of the crop, at the time of sowing, in complete sprouting and in hilling. Taking into account the conditions of the crop, the percentage of protection against the attack of the white worm was determined by collecting all the tubers of each plot at the end of the crop cycle. The greatest protection was given with the granulate applied at a dose of 1.0 g / plant, with 73%, in contrast to the chemical control (Carbofuran), which exerted a protection of 38%.

Claims

A solid composition comprising, as an active ingredient, an effective amount of one or more microorganisms with biocidal activity, fertilizer and / or plant growth promoter; together with one or more additives, diluents, binders, disintegrants, drying protectors and lubricants.

2. The solid composition according to claim 1, wherein one or more microorganisms of the active ingredient are selected from the group consisting of: Lecanicillium sp., Beauveria sp., Isaria sp., Hirsutella sp., Aschersonia sp., Trichoderma sp. ., Gliocladium sp., Clonostachys sp., Pseudomonas sp., Bacillus sp., Rhodotorula sp., Pichia sp., Rhizobium sp., Azotobacter sp., Azosphirillum sp, Bradyrhizobium, and Metarhizium sp.

3. The solid composition according to claim 1, characterized in that it has the following composition: active ingredient in a concentration between 0.0 and 80.0% (w / w); additive in a concentration between 0.0 and 20.0% (w / w); diluent in a concentration between 10.0 and 60.0% (w / w); binder in a concentration between 0.0 and 10.0% (w / w); disintegrant in a concentration between 0.5 and 60.0% (w / w); Drying protector in a concentration between 0.5 and 60.0% (w / w) and lubricant in a concentration between 0.0 and 15.0% (w / w).

4. The solid composition according to claim 1, wherein the additive is selected from the group consisting of: enzymatic extracts, plant extracts, essential oils, polysaccharides and proteins.

5. The solid composition according to Claim 1, wherein the diluent is selected from the group consisting of: starches, cellulose derivatives and kaolin-type clays, bentonites and sepiolites.

6. The solid composition according to Claim 1, wherein the binder is selected from the group consisting of: starch, sucrose, glucose, celluloses, sorbitol, mannitol, dextrins, acrylates, alginates and gums.

The solid composition according to Claim 1, wherein the disintegrant is selected from the group consisting of: croscarmellose sodium, starch, pregelatinized starch, sodium starch glycolate, povidone, crospovidone, xanthan gum, guar gum, calcium silicate, lactose , alginic acid, cellulose, microcrystalline cellulose, sodium alginate, chitosan and silicon dioxide.

The solid composition according to Claim 1, wherein the drying protector is selected from the group consisting of: polyethylene glycol type polyols, propylene glycol, sorbitol, mannitol; sugars such as sucrose, glucose, trehalose, mannose, fructose; skim milk, egg yolk and albumin.

9. The solid composition according to claim 1, wherein the lubricant is selected from the group consisting of: magnesium stearate, glyceryl monoestearate, calcium stearate, palmitic acid, myristic acid, stearic acid, mineral oil and vegetable oil.

10. The solid composition according to claim 1, in the form of powder, tablet, pellets, wettable powder or dispersible granulate for foliar application, direct application to the soil, by dusting, by irrigation or by sprinkling.