WO2019157608A1

WO2019157608A1 - Biological phosphorous solubiliser

Info

Publication number: WO2019157608A1
Application number: PCT/CL2019/050011
Authority: WO
Inventors: Eduardo Donoso Cuevas; Paulo Escobar Valdes; Gustavo Lobos Prats
Original assignee: Bio Insumos Nativa Spa
Priority date: 2018-02-15
Filing date: 2019-02-15
Publication date: 2019-08-22

Abstract

The present invention relates to an environmentally friendly biological phosphorous solubiliser (BPS) comprising a carrier microparticle and at least one microorganism, which solubilise phosphorous in different ways, wherein the microorganism is selected from fungi and bacteria and can belong to a group of organisms able to solubilise phosphorous contained in phosphorous sources of both organic and inorganic origin.

Description

BIOLOGICAL SOLUBILIZER OF PHOSPHORUS

DESCRIPTIVE MEMORY FIELD OF THE INVENTION

The present invention relates to an environmentally friendly phosphorus biological solubilizer (SBF), comprising a carrier microparticle and at least one microorganism that have different forms of phosphorus solubilization, where the microorganism is selected from fungi and bacteria, and where the microorganism can belong to a group of organisms capable of solubilizing phosphorus content, either in phosphorus sources of organic or inorganic origin. The SBF of the present invention allows the solubilization of phosphorus and, therefore, the increase in the availability of phosphorus, for the nutrition and growth of cultivated, ornamental, forestry or environmental restoration plants. Additionally, the present invention relates to formulations comprising the SBF. The invention also relates to an SBF comprising a carrier microparticle and a combination of microorganisms capable of solubilizing phosphorus.

BACKGROUND OF THE INVENTION

In agriculture, phosphorus is one of the main nutrients, which limits the growth of crops. Unlike nitrogen, there are no abundant atmospheric sources of phosphorus, which are bioavailable to plants (Ezawa et al., 2002). Phosphorus plant nutrition is involved in the development of roots, growth and thickness of stems, formation of flowers and fruits, maturity of the crop, fixation of nitrogen in legumes, quality of harvest and resistance to diseases. The dynamics of phosphorus in the soil is characterized by physical-chemical processes (adsorption and desorption), as well as biological (immobilization-mineralization). Large amounts of phosphorus applied to the soil as fertilizers, are integrated into the immobilized fraction through precipitation reactions, with highly reactive forms of Al ⁺³ and Fe ⁺³ in acidic soils, and Ca ⁺² in normal and calcareous soils (Gyaneshwar et al., 2002; Hao et al., 2002).

Worldwide, the efficiency of phosphorus-based fertilizers is around 10-25% (Isherword, 1998), and bioavailable phosphorus concentrations are very low, reaching levels of 1.0 mg kg ¹ soil (Goldstein, 1994 ).

Regarding the state of the art, the document US 2009308121 can be cited, where environmentally friendly compositions and methods are described to provide an increase in plant growth, where formulations comprising mixtures of microbial isolates are described . In particular, bacterial and fungal strains, isolated from a variety of soil types, rhizospheres and root nodules of leguminous plants, to provide plant growth, improve plant productivity, providing greater protection against plant pathogens, reducing the need for use of fertilizers containing nitrogen, solubilizing minerals, and making nutrients available to plants, for example: phosphate.

On the other hand, the invention request US 20120015806 Al describes a synergistic combination of microbial isolates that stimulate plant growth, which comprises a formulation that includes specific strains of bacteria, fungi and yeasts selected from Pseudomonas fluorescens MTCC 5525, Pseudomonas would be MTCC 5524, Bacillus polymyxa MTCC 5528, Bacillus subtilis MTCC 5527, Azaspirillum brasilense MTCC 5526, Rhizobium sp MTCC 5531, Azotobacter sp MTCC 5529, Trichoderma harzianum MTCC 5530 and Trichoderma viride MTCC 5532, Saccharomyces cerevisiae MTCC 5533. This mixture of microorganisms can also provide protection against plant pathogens. On the other hand, publication WO2013158900 Al, describes an effective association between mycorrhizal fungi and plant roots, in an environment rich in phosphorus. Another aspect of this reference is the germination of mycorrhizal propagules and the subsequent colonization of the roots by a molecular signal. Another aspect of this reference refers to the use of bacteria of the strain Bacillus amyloliquefaciens TJ1000 ATCC BAA-390 that produce phytase in environments highly rich in phosphorus, because the strain comprises the phyC gene. This publication also refers to the use of an isolated Trichoderma virens Gl-3 fungus (ATCC 58678), which produces phosphatase enzymes, capable of breaking bonds in compounds such as tricalcium phosphate and thus releasing calcium and phosphorus ions. Another document related to the present invention is CN publication 102433280 A, in which a phosphorus solubilizing microbial composition is described, composed of Pantoea ananatis, Pythium oligandrum, Trichoderma harzianum and Paecilomyces lilacinus organisms, which act as agents with high efficiency of phosphate solubilization.

As can be seen from the above, there is a great need for the industry to solve the problem of increasing the bioavailability of phosphorus for plants. The present invention provides an environmentally friendly SBF, which has an increased phosphorus solubilization from phosphorus from various sources, with respect to what is described in the state of the art.

BRIEF DESCRIPTION OF THE INVENTION

The present invention aims to provide a phosphorus solubilizer composition or an environmentally friendly phosphorus solubilizer (SBF), which has application in the field of agriculture, forestry, ornamental plants and wildlife, where the SBF it allows to increase the efficiency of the use of phosphorus fertilizers or to increase the availability of phosphorus, in naturally phosphorus-fixing soils, such as, for example, calcium-rich soils with alkaline pH, or in volcanic soils rich in aluminum and iron. In particular, the present invention describes a biological phosphorus solubilizer (SBF) comprising a carrier microparticle and at least one microorganism that have different forms of phosphorus solubilization, where the microorganism is selected from fungi and bacteria, and where the microorganism can belong to a group of organisms capable of solubilizing phosphorus content, either in phosphorus sources of organic or inorganic origin. The SBF of the present invention allows the solubilization of phosphorus and, therefore, the increase in the availability of phosphorus, for the nutrition and growth of cultivated, ornamental, forestry or environmental restoration plants.

Additionally, the present invention relates to formulations comprising the SBF.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A and IB: Photomicrographs showing a sample of fungal mycelium covering roots.

DETAILED DESCRIPTION OF THE INVENTION

The present invention aims to provide a phosphorus solubilizer composition or a phosphorus solubilizer product (SBF) which has application in the field of agriculture, forestry, ornamental plants and wildlife, where the SBF allows to increase the efficiency of use of phosphorus fertilizers or increase phosphorus availability, in naturally phosphorus-fixing soils, such as calcium-rich soils with alkaline pH, or in volcanic soils rich in aluminum and iron.

The SBF of the present invention comprises a microparticle that has a high cation exchange capacity, high pore density and high water and oxygen absorption. These characteristics allow the microparticle to establish a matrix, which serves as support and segregation of the different microorganisms adhered to it, so that they can solubilize phosphorus.

In a preferred embodiment, said microparticle can be of a material belonging to the group consisting of zeolite, graphite, leonardite, graphene, kaolinite, among others.

On the other hand, the SBF of the invention comprises at least one microorganism capable of solubilizing phosphorus contained in phosphorus sources of both organic and inorganic origin, wherein said microorganism is selected from fungi and bacteria. These microorganisms have the ability to colonize both roots and rhizosphere, which allows them to independently increase the natural efficiency of each of these organisms in the solubilization of phosphorus, an element that can be found both in fixing soils and in natural fertilizers and fertilizers synthetic Particularly, the microorganism can be selected from groups of microorganisms with phosphorus solubilization capacity, selected from fungi belonging to the Bionectriaceae family, such as Bionectria ochroleuca; to the Trichocomaceae family as Talaromyces amestolkiae; to the Hypocreaceae family, such as Trichoderma harzianun; and of bacteria belonging to the genus Bacillus, such as Bacillus licheniformis and Bacillus subtilis. More preferably the microorganisms of the present invention are selected from the group consisting of: Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca palqui strain NRRL 67323, Talaromyces amestolkiae strain Bacharys NRRL 67321, Trichoderma harzianun strain Gomorteka NRRL 67322, Bacillus licheniformis strain copihue NRRL B-67039 sub-treated all Bacill 50-tris, all treated Bcus1 Budapest

Said microorganisms with phosphorus solubilization capacity, according to the present invention are grouped as follows:

Group A:

Bionectria sp. strain Mitique NRRL 50824,

Bionectria ochroleuca strain palqui NRRL 67323, and

Talaromyces amestolkiae strain Bacharys NRRL 67321; Y

B Group:

Bacillus licheniformis strain copihue NRRL B-67023;

Bacillus subtilis strain N5 NRRL B-50391; Y

Trichoderma harzianun strain Gomorteka NRRL 67322,

where the microorganisms of these two groups allow phosphorus to be released from organic and inorganic phosphorus sources, for example, through phosphorus mineralization (phosphorus release from organic matter). In one embodiment of the invention, the SBF contains combinations of the microorganisms of groups A and B in a ratio from 3: 7 to 7: 3, of A: B.

The present invention demonstrates that the joint action of these components of the SBF, the microparticle and the microorganisms, allows the solubilization of phosphorus from various sources, resulting in phosphorus available for use by the plants. Likewise, the combined action of the microparticle and the microorganism (s) with different forms of solubilization, generate a considerable improvement, both in the field and under controlled conditions, of phosphorus solubilization with respect to the solubilization levels reported when using the o the microorganism (s) without the presence of the microparticle. This due to the cation exchange capacity of this microparticle.

In addition, the use of the combination of fungi with bacteria adds a greater capacity for soil exploration, based on the growth of mycelium of fungi, which allows them to cover a greater volume of soil, which in many cases follows the growth of roots and establishing a symbiotic interaction with them (See Fig. 1)

In turn, the present invention describes a formulation comprising the SBF. Said formulation can be used in the form of sprinkling, manual or mechanical dispersion, injection by irrigation systems, soaking, adhesion to seeds or roots of propagation material, etc., these characteristics being that they represent a greater flexibility of use compared to other alternatives known in the state of the art, such as the application of seed inoculant.

In a preferred embodiment, the formulations of the present invention may be in the form of a concentrated suspension, wettable powder, gel, concentrated paste, emulsifiable gel, oily suspension, dispersible powder, dispersible granules, effervescent tablets, pellets, without excluding other forms, including all the above formulations described herein in any of its conventional forms. Being able to serve as a carrier in the SBF of the invention, one or more carrier (s) selected from zeolite, water, leonardite or kaolinite.

In one embodiment of the present invention it refers to a biological phosphorus solubilizer (SBF) comprising a carrier microparticle and at least one microorganism that have different forms of phosphorus solubilization, where the microorganism is selected from fungi and bacteria, and where the fungal group is formed by Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL 67321, and the group of bacteria is formed by Bacillus licheniformis strain copapus NRRL Bc NR-BRL NRP-BRL NR5 NRP NRP-BRL NRP NR5 BRL NR-NRP NRP BRL NR5 NRP NR-BRL NRP BRL NR5 NRP NR-BRL NRC-NRRL NR-BRL NRP-NR65 BRL NR-NRP BRL-NRRL NR-NR65 50391; or any combination thereof.

In a second embodiment, the present invention relates to a biological phosphorus solubilizer (SBF) comprising a carrier microparticle and the At least two microorganisms that have different forms of phosphorus solubilization, where one microorganism is selected from group A and another microorganism is selected from group B, where Group A is formed by Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, and Talaromyces amestolkiae strain Bacharys NRRL 67321; and where Group B is formed by Bacillus licheniformis strain copihue NRRL B-67023 and Bacillus subtilis strain N5 NRRL B-50397 and the fungus Trichoderma harzianun strain Gomorteka NRRL 67322. In a third embodiment, the present invention relates to a biological solubilizer of Phosphorus (SBF) comprising a carrier microparticle and microorganisms that have different forms of phosphorus solubilization, where the microorganisms correspond to fungal and bacterial microorganisms, where the fungal group is formed by Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL 67321, and the group of bacteria is formed by Bacillus licheniformis strain copapus NRRL Bc NR-BRL NRP-BRL NR5 NRP NRP-BRL NRP NR5 BRL NR-NRP NRP BRL NR5 NRP NR-BRL NRP BRL NR5 NRP NR-BRL NRC-NRRL NR-BRL NRP-NR65 BRL NR-NRP BRL-NRRL NR-NR65 50391

In a preferred embodiment, said biological phosphorus solubilizer comprises a carrier, wherein the carrier is selected from zeolite, graphite, leonardite, graphene and kaolinite. In another preferred embodiment, the microorganisms of groups A and B of the SBF are in a ratio of 3: 7 to 7: 3, of A: B.

Additionally, in a preferred embodiment of the present invention, the bacteria of said SBF are in a spore suspension (10 ^s spores / ml) in equal proportion of each strain.

In another preferred embodiment, SBF fungi are found in a concentration of 10 ⁶ conidia / ml, in equal proportion of each fungus.

In a further embodiment, the invention relates to a formulation for increasing the availability of phosphorus comprising the SBF described above.

In a second embodiment, the formulation to increase phosphorus availability is in the form of a concentrated suspension, wettable powder, gel, concentrated paste, emulsifiable gel, oily suspension, dispersible powder, dispersible granules, effervescent tablets and pellet.

In a further embodiment, the present invention relates to a method of biological solubilization of phosphorus contained in organic and / or inorganic agricultural sources, which comprises applying the phosphorus SBF described previously, to the field of planting or planting crops and their surroundings. In another embodiment, the present invention describes the method for increasing the availability of phosphorus contained in an agricultural, organic and / or inorganic source, comprising applying the previously disclosed formulation, to the sowing or planting field of crops and its surroundings.

EXAMPLES

Example 1: Phosphorus Solubilization Analysis The determination of the activity of microorganisms was estimated by determining a solubilization halo formed around the colony in a solid medium.

The activity of the strains was analyzed below:

- Bionectria sp. strain Mitique NRRL 50824;

Bionectria ochroleuca strain palqui NRRL 67323;

Talaromyces amestolkiae strain Bacharys NRRL 67321;

Bacillus licheniformis strain copihue NRRL b-67023;

Bacillus subtilis strain N5 NRRL B-50391; Y

- Trichoderma harzianun strain Gomorteka NRRL 67322.

All such strains have been isolated and selected by Bio Insumos Nativa SpA, based on their potential use as a phosphorus solubilizer. Methodology

To carry out this analysis, two trials were set up: one in the middle of Pikovskaya and another where the phosphorus source was contributed by phosphoric rock. a) Plate solubilization in Pikovskaya medium

Phosphorus solubilizing strains were identified in Pikovskaya medium (calcium phosphate). Each treatment was replicated in different media. Halo formation was evaluated after one month and a relationship was made between the amount of phosphorus in the medium and the volume of transparency in the Petri dish. b) In vitro phosphoric rock solubilization test To obtain the phosphoric rock a commercial product was used. This was screened to obtain particles under 25 pm in diameter, subsequently a medium similar to Pikovskaya was prepared, but without calcium phosphate, instead 2.5 g / L of phosphoric rock was used. The results were collected after a month of planting in the middle.

Results a) Solubilization in plate in Pikovskaya medium In the Pikoyskava test, different levels of solubilization are observed, according to the strain, identifying two groups s: one consisting of B ionectrias and Talaromyces, which dramatically decrease the pH and the second group by B acillu s and Trichoderma, which achieve a slight decrease in pH

Table 1: Plate solubilization result in Pikovskaya medium

Similar to the previous test, two groups of solubilizers were observed, varying in relation to the pH changes achieved, also observing that fungi exhibit a better solubilization capacity with respect to bacteria, independent of changes in pH.

Table 2: Phosphoric rock solubilization result

conclusion

All strains exhibit phosphorus solubilizing activity, regardless of its origin, whether calcium phosphate or phosphoric rock.

The solubilization ranges were 16.5% to 43.7% for calcium phosphate and 46.7 to 65.6% for phosphoric rock. Example 2: Carrier and phosphorus solubilizing microorganisms

Carrier evaluation to increase the effect of phosphorus solubilizing microorganisms Using the soil incubation technique (mass and type of soil known and chemically and physically characterized), the rate was determined, dynamic and the nutrient availability of the product to be used was quantified, with different formulations of phosphorus solubilizing microorganisms.

Soil used:

The soil of the place is of volcanic origin, belonging to the Arrayán series, taxonomically classified as Melanoxerands. The initial chemical properties of the experiment (composite sample of soil collected from 0-20 cm) were:

Water pH 6.08;

organic matter 10.83%;

N available 8 ppm;

P available 6.5 ppm;

K available 248 ppm;

Exchangeable 4.07 cmol Ca;

Exchangeable Mg 0.94 cmol;

Exchangeable 0.04 cmol Na;

At exchangeable 0.08 cmol;

Aluminum saturation 1.46%;

Fe available 28.7 ppm;

Mn available 3.1 ppm;

Zn available 0.46 ppm;

Cu available 1.1 ppm; B available 0.57 ppm; Y

S available 38 ppm.

Its properties and limitations include the high percentage of organic matter, natural nitrogen (N) and potassium (K) delivery capacity, moderate effective depth (soil depth, where root growth is feasible), moderate concentration Ca, Mg, Zn and B, low phosphorus level and high phosphorus fixing capacity. In 5-liter polyethylene pots, 10 wheat seeds were sown at the center of each drawer, the seeds being applied at the same point.

The soil was analyzed before the treatments and after applying these, it was incubated in the greenhouse for 20 weeks, after that a 1 kg sample was extracted, eliminating the first and last 10 cm of depth, this sample was homogenized and analyzed in Regarding the availability of phosphorus.

Experimental design Completely random with 4x3 factorial arrangement where the factors were microorganisms (control, fungi, bacteria and combination of microorganisms) and carrier microparticle (water, zeolite, kaolinite).

Carrier control: Water, kaolinite and zeolite (without microorganisms). Bacteria mixture: it comprises equal parts of Bacillus licheniformis strain copihue NRRL B-67023 and Bacillus subtilis strain N5 NRRL B-50391. Mushroom mixture: includes equal parts of Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL 67321.

Combination of microorganisms: Mixture of fungi and bacteria described above. Water treatments consisted of the suspension of fungi or bacteria separately at a concentration of 10 ⁸ CFU / ml in equal proportions of both mixtures.

The treatments with zeolite and kaolinite carrying microparticles were generated as follows according to the microorganism used:

Bacteria: a spore suspension (10 ⁸ spores / ml) in equal proportion of each strain, were sprayed on zeolite, and then subjected to a drying process at 80 ° C for 15 minutes. - Fungi: A spore suspension in concentration (10 ⁸ conidia / ml) in the same proportion of each strain, was sprayed on previously sterilized zeolite, and then subjected to a drying process at 25 ° C for 24 hours under a flow chamber laminate. Mixture of phosphorus biological solubilizing microorganisms: combination of fungi and bacteria already described, the pre-formulated bacteria being applied by spraying a spore suspension (10 ⁸ spores / ml) in equal proportion of each strain, on zeolite, to then be subjected to a drying process at 80 ° C for 15 minutes, after cooling this zeolite to 30 ° C, a mixture of a conidide powder concentrate of the fungi of the invention was applied, at a concentration of 10 ⁶ conidia / ml (proportion of the specimens of the mushroom mixture: 1: 1: 1: 1), then allowed to dry in a laminar flow chamber for 24 hours at a temperature of 25 ° C.

Each treatment was applied in a dose of 5 g / L of water, sprinkling 300 ml per pot.

Results

When carrying out the analysis of variance, a significant effect of the treatment factors (P <0.0l), as well as the carrier microparticle factor (P <0.05) and the interaction of both factors already mentioned (P <0, 05).

As observed in the measurement of the 20th week of incubation, phosphorus solubilizing microorganisms have a greater capacity for phosphorus solubilization, especially in water and adsorbed on zeolite-carrying microparticles (SBF composition), while with kaolinite-carrying microparticles , no effect of the treatments is observed. Also with Zeolite carrying microparticles show a marked increase in the effect of SBF, the highest level of solubilization is achieved by the combination of fungal microorganisms and adsorbed bacteria on zeolite carrying microparticles.

Table 3. Effect on phosphorus solubilization (ppm), before and after different treatments s

* Equal dates, indicate absence of significant differences (Tukey HSD P <0.05) Conclusions

The SBF shows the highest level of solubilization, a synergistic effect being observed in the use of a formulation comprising zeolite carrying microparticles with the combination of microorganisms of the invention supported therein.

Example 3: Evaluation of the SBF on nutrient availability in soils incubated under controlled conditions, for three types of soils

Using the soil incubation technique (mass and type of soil known and chemically and physically characterized) the rate, dynamics and quantification of nutrient availability of the product to be used was determined.

The soils used were:

- Sandy soil with alkaline pH and high calcium content, phosphorus fixative;

- Clay loam soil with neutral pH; Y

- Andisol soil, of volcanic origin with high aluminum content, phosphorus fixative.

Each floor was characterized in terms of: SN available

'ό p available

After this, 500 g pots were placed, with each type of soil and with incubation times of 0, 8 and 16 weeks, in greenhouse conditions with temperatures between 12 and 28 ° C.

Experimental design Completely random with 3x4x3 factorial arrangement where the factors were soil type (sandy, andisol and clay loam so), incubation time (0, 4, 8 and 16 weeks) and fertilization (0, 25 and 50 ppm of P) in the form of a spherical rock.

Treatments:

Control without fertilization;

Control Phosphorus Fertilization 50 ppm;

SBF without fertilization;

SBF with phosphorus 25 ppm; Y

- SBF with phosphorus 50 ppm.

The SBF used corresponds to the combination of bacteria and fungus s. The bacteria were first pre-formulated, by spraying a spore suspension (10 ⁸ spores / ml) of Bacillus licheniformis strain copihue NRRL b-67023 and Bacillus subtilis strain N5 NRRL B -50391 in equal proportion of each strain, on zeolite carrying microparticles (with pores between 25 and 100 nm), then being subjected to a drying process at 80 ° C for 15 minutes, after cooling these zeolite carrying microparticles with adsorbed bacteria, at 30 ° C, it proceeded to apply a mixture of a powder concentrate of conidia of the fungi Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL, in a concentration of 10 ⁶ conidia / ml (1: 1: 1: 1 ratio), then let dry in laminar flow chamber for 24 hours at a temperature of 25 ° C. The resulting wettable powder was applied in doses of 1 kg / ha with 400 liters of water.

Each treatment was replicated 4 times, each replica composed of a pot. Results

Soil analyzes were performed at the time of mounting the test for each type of soil and then at 8 and 16 weeks of incubation. When carrying out the analysis of variance, a significant effect on phosphorus of all the factors is observed (Table 4), as well as the interactions, except in the treatment v / s type of soil and in the triple interaction.

In Table 4, the results of the analysis of variance are observed. For each type of soil separately, an effect on the phosphorus of the treatments, soil and incubation time, as well as the interactions of Treatment and time and soil with time (Table 5).

Table 4. Statistical effect of the different factors on the availability of Phosphorus under different treatments.

When observing the available phosphorus data (Table 5), we can observe that in all soils, in the absence of phosphorus fertilization, the application of the SBF generates an increase in the significant difference with the control treatment, the increase in availability being similar (fixative soil by Ca) or higher (fixative by Al or non-fixative) to fertilization at 25 ppm. Additionally, in all cases the combination of complete 50 ppm fertilization is significantly lower in P available in the treatments, which include fertilization and SBF, except in the non-fixing soil, but in this half of the fertilization dose plus the SBF achieves the same level of P available as 50 ppm fertilization. Table 5: Effect on phosphorus availability (ppm) under different treatments with microorganisms and phosphorus fertilization.

* Equal letters, indicate absence of significant differences (Tukey HSD P <0.05)

In all cases the use of SBF, in the absence of phosphorus fertilization, achieved a significant increase over the control. When combining fertilization with the SBF, an increase is observed with respect to fertilization alone, except in the higher dose of fertilization in non-fixative soil, since the SBF has an active solubilization of phosphorus, thereby improving the availability of phosphorus, since the solubilization rate would be higher than the fixation rate.

Conclusions

The SBF is capable of generating a solubilization of this nutrient, both in non-fixing and in fixing soils. In addition, the use of SBF in the absence of phosphorus fertilization, in fixing soils, achieves levels of available phosphorus, equal to or greater than those obtained by phosphorus fertilization, this being maximum at 16 weeks after applying the treatments.

Example 4: Evaluation of soil volume under the effect of SBF under controlled conditions

Using the soil incubation technique (mass and type of soil known and chemically-characterized) was determined the rate, dynamics and quantification of nutrient availability of the product to be used, at different distances from the point of application, with a reference culture .

Land used The soil of the place is of volcanic origin, belonging to the Arrayán series, taxonomically classified as Melanoxerands. The initial chemical properties of the experiment (composite sample of soil collected from 0-20 cm) were:

Water pH 6.08;

organic matter 10.83%;

N available 8 ppm;

P available 6.5 ppm;

K available 248 ppm;

Exchangeable 4.07 cmol Ca;

Exchangeable Mg 0.94 cmol;

Exchangeable 0.04 cmol Na;

At exchangeable 0.08 cmol;

Aluminum saturation 1.46%; Fe available 28.7 ppm;

Mn available 3.1 ppm;

Zn available 0.46 ppm;

Cu available 1.1 ppm;

- B available 0.57 ppm; Y

S available 38 ppm.

Its properties and limitations include the high percentage of organic matter, natural nitrogen (N) and potassium (K) delivery capacity, moderate effective depth (soil depth, where root growth is feasible), moderate concentration of Ca , Mg, Zn and B, low phosphorus level and high fixing capacity of this element. The soil was characterized in terms of:

Z Organic Matter

Z _P HZ CE

Z N available

Z p available In polyethylene drawers, l x l xl m long and deep, 10 wheat seeds were sown at the center of each drawer, being applied at the same point.

The soil was analyzed prior to the treatments and after applying this, it was incubated in the greenhouse for 20 weeks, after which a sample was extracted 5 cm in diameter at 30, 60 and 90 cm from the point of sowing and application of the treatments, eliminating the first and last 10 cm deep, this sample was homogenized and analyzed for the parameters already indicated. Subsequently, the plants were extracted and evaluated in terms of:

Height;

Dry weight ; Y

Root Volume Experimental design

Completely random with 3x4x3 factorial arrangement where the factors were distance (30, 60 and 90 cm from the point of application), where the treatments consisted of:

Control: Zeolite carrier microparticle without adsorbed microorganism

Bacteria mixture: Bacillus licheniformis strain copihue NRRL b-67023, Bacillus subtilis strain N5 NRRL B -5039 1 in equal proportion of each strain, on previously sterilized zeolite, to then be subjected to a drying process at 80 ° C for 15 minutes s.

Mushroom mixture: Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca palqui strain NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL 67321, in a concentration of 10 ⁶ conidia / ml (1: 1: 1: 1 ratio) in previously sterilized zeolite, then allowed to dry in zeolite, seeking adsorption in the pores in chamber laminar flow for 24 hours at a temperature of 25 ° C).

- SBF: combination of fungi and bacteria already described, the bacteria being pre-formulated by spraying a spore suspension (10 ⁸ spores / ml) in equal proportion of each strain, on zeolite, to then be subjected to a drying process at 80 ° C for 15 minutes, after cooling this zeolite to 30 ° C, a powder concentrate of conidia of the fungi was mixed, in a concentration of 10 ⁶ conidia / ml (1: 1: 1: 1 ratio) ), then let it dry in a laminar flow chamber for 24 hours at a temperature of 25 ° C. The resulting wettable powder was applied in doses of 1 kg / ha with 400 liters of water.

Each treatment was applied at a dose of 5 g in the planting hole of the 10 wheat seeds. Results

When performing the analysis of variance, a significant effect of the treatments (P <0.0l), as well as the distance (P <0.05) and the interaction of both (P <0.05) is observed. As observed in the measurement of the 20th week of incubation, at all distances from the point of inoculation, it can be seen that both in the SBF test (fungi + bacteria + carrier), as well as in the fungal test, there are a significant increase in phosphorus availability, the first being statistically greater than the effect achieved with fungi and bacteria separately. It is also observed that the test with the SBF, as well as with fungi, have an effect up to a distance of 90 cm from the point of application, unlike the test with bacteria that only has an effect up to 30 cm. As for pH associated with phosphorus solubilization, we see a decrease in treatments composed of fungi and bacteria independent of distance.

Table 6. Effect on phosphorus availability and changes in pH and organic matter (Mo), at different distances from the point of application of treatments with microorganisms.

When observing the performance data of the plants, we can observe that in height and dry weight, only the SBF showed a significant effect, while in terms of the volume of the roots the SBF behaved better, but without significant differences with the fungi in isolated form. Table 7. Effect on height, dry matter and root volume, under different treatments with microorganisms under controlled conditions.

Conclusions

The SBF is capable of generating both an increase in phosphorus availability, exceeding the intensity of solubilization and the area covered by the roots, from the point of application, to bacteria or fungi used in isolation.

Example 5: Field test

A field experiment was carried out with the use of the experimental phosphorus releasing product (SBF), on a PANDORA spring wheat crop (INIA).

This experiment was carried out in the “Santa Rosa” property of INIA Quilamapu, km 30 on the way to Cato, commune of Coihueco.

The soil of the place is of volcanic origin, belonging to the Arrayán series, taxonomically classified as Melanoxerands. The initial chemical properties of the experiment (composite sample of soil collected from 0-20 cm) were: water pH 6.08; organic matter 9, 83%;

N available 8 ppm;

P available 6.5 ppm;

K available 248 ppm;

Exchangeable 4.07 cmol Ca;

Exchangeable Mg 0.94 cmol;

Exchangeable 0.04 cmol Na;

At exchangeable 0.08 cmol;

Suration of aluminum 1, 46%;

Fe available 28.7 ppm;

Mn available 3.1 ppm;

Zn available 0.46 ppm;

Cu available 1.1 ppm;

B available 0.57 ppm;

S available 38 ppm.

Its properties and limitations include the high percentage of organic matter, natural nitrogen (N) and potassium (K) delivery capacity, moderate effective depth (soil depth, where root growth is feasible), moderate concentration of Ca , Mg, Zn and B, low phosphorus level and high fixing capacity of this element.

Treatments The fertilization treatments evaluated in the wheat crop were the following and its contribution of Nitrogen (N) is presented in table 1.

1. Control with conventional fertilization, with total nitrogen dose: phosphorus: potassium (N: R ₂ q ₅ : K ₂ 0) = 240: 120: 120 (should be 2: 1: 1), considering that the N was partialized 20% at planting, 45% at the beginning of the plant and 35% at the beginning of the planting.

2. SBF applied to the soil in doses of 50 kg / ha (prior to planting), excluding phosphorus fertilization. The dose of N and K and its management was the same used in the control.

3. SBF applied to the soil in doses of 50 kg / ha (prior to planting), with application to the seed in doses of 300 gr / 200 kg of seed (product agitated with the seed until homogeneous), excluding fertilization phosphorous The dose of N and K and its management were identical to those used in the control.

The microbial SBF used consisted of bacteria wettable powder, Bacillus licheniformis strain copihue NRRL b-67023 and Bacillus subtilis strain N5 NRRL B-50391, which were pre-formulated by spraying a spore suspension (10 ⁸ spores / ml) of Bacteria with the same proportion of each strain, on zeolite carrying microparticles, to then be subjected to a drying process at 80 ° C for 15 minutes, after cooling these zeolite carrying microparticles at 30 ° C with the adsorbed bacteria, proceeded to mix a concentrated powder of conidia from fungi Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL 67321, at a concentration of 10 ⁶ conidia / ml (ratio 1: 1: 1: 1), then let it dry in laminar flow chamber for 24 hours at a temperature of 25 ° C. Once dry, this powder was dissolved in sterile distilled water in a dose of 150 g / l.

Table 8: Nutrient Contribution via fertilization applied to the wheat crop with the different Treatments

Results and Discussion

Table 9 shows the grain yield, total dry matter production, harvest index, hectolitre weight and plant height at the time of harvest for grain. Table 10 shows the concentration and extraction of N, P and K in whole wheat plant at the time of harvest for grain.

Table 11 shows the chemical properties of soil at the time of harvest.

Table 9. Grain yield, total dry matter production, harvest index, hectolitre weight and height of wheat plants at the time of harvest for grain.

* Equal letters, indicate absence of significant differences (Tukey

HSD P <0.05)

The grain yield fluctuated between 71, 0 and 84, 1 qq / ha (Table 9) and was within the normal range obtained by the producers in the area, but below the yield s obtained in the breeding program PANDORA wheat (INIA), which is attributed in part to the limitations of effective soil depth (less than 60 cm), considering that this crop needs a depth of 90 cm to express its yield potential, and on the other hand to low ambient temperatures during spring.

No significant differences were obtained between treatments evaluated.

(p <0.05). At the same time, the parameter s total dry matter, crop index, hectolitre weight and plant height (Table 9) also did not show significant differences between treatments (p <0.05), and the values obtained were within the range normal for the Pandora-INIA variety in this growing area. Table 10. Concentration and Extraction of Nitrogen% (fertilizer use by the plant), Phosphorus and Potassium from wheat plants at the time of coarse grain.

* Equal letters indicate absence of significant differences (Tukey

HSD P <0.05)

The nutritional concentrations within the wheat plant (whole plant) and the extraction of N, P and K at the time of coarse grain for grain (Table 10) were presented within the normal range for spring wheat grown in this study area. Only significant difference in the concentration of N in whole plant and extraction of N by the whole wheat plant (Table 10), where the highest concentration and extraction of this element was achieved in the Conventional Fertilization Control treatment, which only surpassed the Phosphorus Release treatment to the seed (p <0.05).

Probably the highest pH obtained with the use of conventional fertilization (Table 11) is associated with the neutral reaction generated with the use of triple superphosphate (SFT) given its calcium content (20-22% CaO), however it attracts attention the effect obtained with the use of the SBF applied to the soil (higher dose than that used directly to the seed), which can be associated with its effect on the biological activity of the soil, and as a result in chain, to the release of compounds that neutralize the partial acidity generated during cultivation. This effect must be determined with more precise evaluations of both biological activity (soil respiration, phosphatase activity, mineralization) as well as the rate and dynamics of phosphorus release in the soil against the use of these products. Draws attention to the lower pH achieved with the use of phosphorus releasing to the soil, which may be associated with greater biological activity, which was not determined in this experiment. The highest concentration of available N and nitrates (Table 11) was achieved with the use of the SBF applied to the soil (higher dose than the application of this product to the seed) (p <0.05), whose dose of N was the same used in the other treatments. It should be noted that the concentration of available N obtained in this, unlike the other treatments that presented lower concentrations of this element. These results indicate that the Liberator application of phosphorus to the soil in the dose evaluated in this experiment (1 L / ha) has a positive effect on the increase in N available from the soil, therefore a reduction in the dose of N could be considered for subsequent experiments.

However, for the conditions of this experiment the increase in the concentration of available N did not translate into an increase in grain yield (Table 9).

In general, in all treatments a concentration of available phosphorus greater than the initial one (6.5 ppm) was achieved, and the greatest increases in concentration were achieved with the use of the SBF applied to the soil or to the seed, and with the SBF applied to the soil (Table 11). However, for the conditions of this experiment the increase in the concentration of available phosphorus did not translate into an increase in grain yield (Table 9).

The use of the SBF applied to the soil at a dose of 1 L / ha prior to planting improved the availability of N and phosphorus at the end of the wheat crop, with an acidification effect on the soil. These results suggest a positive effect on soil biomass responsible for the mineralization of N and phosphorus, as well as the solubilization of phosphorus, since the biological activity associated with the mineralization of organic matter generates organic acids that in turn reduce the pH of the soil, as has already been observed in previous soil incubation experiments in which nutrient-releasing microorganisms have been applied. Table 1 1. Soil parameters, under different treatments before and after their application.

* Equal letters, imply absence of significant differences (Tukey HSD P <0.05)

Conclusions

For the conditions of this experiment, the use of the SBF applied to the soil or to the seed in the cultivation of wheat of bread of spring habit as replacement of the phosphate fertilizer, allowed to obtain a similar grain yield, growth parameters and plant quality, such as also nutritional parameters within the plant. However, a positive effect was obtained with the use of the SBF applied to the soil prior to planting (1 L / ha) on the availability of Nitrogen and Phosphorus at the end of the crop, but also generated a reduction in pFI. The chemical properties of the soil only showed significant differences in the pH and in the concentrations of available nitrogen, nitrate and available phosphorus Table 11). BIOLOGICAL SOLUBILIZER OF PHOSPHORUS

The present invention relates to an environmentally friendly phosphorus biological solubilizer (SBF), comprising a carrier microparticle and at least one microorganism that have different forms of phosphorus solubilization, where the microorganism is selected from fungi and bacteria, and where the microorganism can belong to a group of organisms capable of solubilizing phosphorus content, either in phosphorus sources of organic or inorganic origin.

Claims

1. A biological phosphorus solubilizer (SBF), CHARACTERIZED because it comprises a carrier microparticle and at least one microorganism that have different forms of phosphorus solubilization, where the microorganism is selected from fungi and bacteria, and where the fungal group is formed by Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL 67321, and the group of bacteria is formed by Badilas licheniformis strain copihue NRRL Bpa-Nrus NRRL B -670 NRpa Bc-Nrus NRRL B -670 NRRL Bc-Nrus NRRL B -670 NRRL B -670 NRRL B -670 NRRL B -670 NRRL B -670 NRRL B -670 50391; or any combination of the same s.

2. A biological phosphorus solubilizer (SBF), CHARACTERIZED because it comprises a carrier microparticle and at least two microorganisms that have different forms of phosphorus solubilization, where one microorganism is selected from group A and another microorganism is selected from group B , where Group A is formed by Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, and Talaromyces amestolkiae strain Bacharys NRRL 67321; and where Group B is formed by Bacillus licheniformis strain copihue NRRL B -67023 and Bacillus subtilis strain N5 NRRL B -50391 and the fungus Trichoderma harzianun strain Gomorteka NRRL

67322

3. A biological phosphorus solubilizer (SBF), CHARACTERIZED because it comprises a carrier microparticle and microorganisms that have different forms of phosphorus solubilization, where the microorganisms correspond to fungal and bacterial microorganisms, where the fungal group is formed by Bionectria sp. strain Mitique NRRL 50824, Bionectria ochroleuca strain palqui NRRL 67323, Trichoderma harzianun strain Gomorteka NRRL 67322 and Talaromyces amestolkiae strain Bacharys NRRL 67321, and the group of bacteria is formed by Bacillus licheniformis strain cophue NRP B6 NR-Bc NR6 NR-Bc NR6 B-NRP NRP-BRL NR5 BRL NRP-NRP NR6 BRL NRP-NR6 NR-BRL NRP-NR6 BRL NRP-NR6 BRL NR5 BRLRL-NRP NRP-NR6 BRL NRP-NR60 BRLRL-NR60 BRLRL-NR60 50391

4. The biological phosphorus solubilizer according to any of claims 1-3, CHARACTERIZED in that the carrier is selected from zeolite, graphite, leonardite, graphene and kaolinite.

5. The biological phosphorus solubilizer according to claim 2, CHARACTERIZED because the microorganisms of the group s A and B are in a ratio of 3: 7 to 7: 3, of A: B.

6. The biological phosphorus solubilizer according to any of claims 1 -4, CHARACTERIZED because the bacteria are in a spore spension (10 ⁸ spores / ml) in equal proportion of each strain.

7. The biological phosphorus solubilizer according to any of claims 1 -4, CHARACTERIZED because the fungus s are in a concentration of 10 ⁶ conidia / ml, in equal proportion of each fungus.

8. Formulation to increase the availability of phosphorus, CHARACTERIZED because it comprises the SBF according to any of claims 1-7.

9. The formulation to increase the availability of phosphorus, CHARACTERIZED because it is in the form of a concentrated suspension, wettable powder, gel, concentrated paste, emulsifiable gel, oily suspension, dispersible powder, dispersible granules, effervescent tablets and pellet.

10. Method of biological solubilization of phosphorus contained in organic and / or inorganic agricultural sources, CHARACTERIZED because it comprises applying the biological phosphorus solubilizer according to any one of claims 1 to 7, to the field of planting or planting crops and their surroundings.

11. Method for increasing the availability of phosphorus contained in an agricultural, organic and / or inorganic source, CHARACTERIZED because it comprises applying the formulation according to any of claims 8 or 9, to the sowing or planting land of crops and its surroundings.