WO2017089641A1 - Plant-biostimulating compositions comprising microorganism strains - Google Patents

Plant-biostimulating compositions comprising microorganism strains Download PDF

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Publication number
WO2017089641A1
WO2017089641A1 PCT/ES2016/070839 ES2016070839W WO2017089641A1 WO 2017089641 A1 WO2017089641 A1 WO 2017089641A1 ES 2016070839 W ES2016070839 W ES 2016070839W WO 2017089641 A1 WO2017089641 A1 WO 2017089641A1
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cect
plant
plants
composition
fluorescens
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PCT/ES2016/070839
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Spanish (es)
French (fr)
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José Ignacio HORCHE TRUEBA
Francisco Javier APARICIO ADARO
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Biobab R&D, S.L
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Publication of WO2017089641A1 publication Critical patent/WO2017089641A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Definitions

  • the present invention relates to bacterial strains promoting plant growth, for use as biostimulants, applicable to different substrates in agricultural crops.
  • the European Biostimulants Industry Council (EBIC) and DG Enterprise Fertilisers Working Group defined the term biostimulant on June 14, 2012, updated in 2015: "Plant biostimulants contain substance (s) and / or micro-organisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance / benefit nutr ⁇ ent uptake, nutr ⁇ ent efficiency, tolerance to abiotic stress, and crop quality.
  • Plant biostimulants contain a substance or substances and / or microorganisms whose function, when applied to plants or the rhizosphere, is to stimulate natural processes that increase / benefit efficiency in nutrient intake, tolerance to abiotic stress and the quality of the harvest).
  • the differentiating effect of the invention is based on the action of microorganisms, especially plant growth promoting bacteria (Plant Growth-Promoting Rhizobacteria or PGPR) that produce a biostimulant effect on plants when applied to them in compositions containing said microorganisms
  • PGPRs are microorganisms that develop in the rhizosphere and stimulate the growth and development of plants (Kloepper et al., 1980).
  • the rhizosphere is the layer of soil that surrounds the roots of plants and constitutes an ecosystem with a high concentration of microorganisms that are attracted by root exudates that constitute its main source of food (Lugtenberg & Kamilova, 2009). Plants release up to 40% of the carbon compounds that they assimilate through the roots and microorganisms of the rhizosphere metabolize these compounds, excreting others that in turn are used by plants (Kang et al., 2010). Thus, an interaction is established between the microorganisms and the roots of the plants in which both are mutually affected by the molecules secreted by the other. This interaction can be neutral; harmful (in the case of pathogens) or beneficial, as in the case of PGPRs (Antoun & Prévost, 2006).
  • the PGPR designation includes strains of the genera Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Pseudomonas and Serratia, among others (Choudhary, 2012). These bacteria are distinguished within the abundant microbial community of the rhizosphere because they are able to colonize the surface of the roots efficiently, multiply and promote plant growth (Ahemad & Kribet, 2014). Thanks to these mechanisms, the application of PGPR in the rhizosphere has a favorable effect on the development and growth of cultivated plants. This field of research has led to the development of numerous commercial products based on PGPR and, consequently, the use of microbial inocula in agriculture has increased considerably in recent decades (Hayat et al, 2010).
  • systemic induced tolerance (IST) has been proposed to refer to physical and chemical changes induced in plants by PGPRs, which result in an increase in tolerance to abiotic stress similar to the mechanisms activated in responses of induced systemic resistance (ISR) against pathogens (Yang et al., 2009).
  • PGPR inoculation can reduce salinity stress in different plant species and also relieve stress caused by extreme temperatures (Choudhary, 2012).
  • the tests carried out inoculating different species of crops with PGPR to increase tolerance to cold conditions suggest that bacteria could increase the concentration of sugars, proline and anthocyanins, among other metabolites, in plant tissues, favoring acclimatization of the plants (Dimpka et al., 2009).
  • the world population is expected to reach 9 million people by 2050, and to cope with the growing demand for food, agricultural production would have to increase by 70% (Coleman-Derr & Tringe, 2014).
  • the availability of land suitable for cultivation is limited and, with the effect of climate change, an increase in drought, salinity and other abiotic stress factors that could reduce agricultural production and threaten global food security is expected. (Grover et al., 2011). Therefore, the use of microbial inoculums is a biological, economic, simple and short-term solution for the management of abiotic stress in crops and to improve the efficiency of nutrient use.
  • the present invention relates to biostimulant compositions comprising microorganisms for use in agriculture.
  • the present invention relates to a plant biostimulant composition comprising the strain Pseudomonas fluorescens CECT 9015.
  • said composition further comprises at least a second microorganism selected from the genera Pseudomonas, Bacillus, Arthrobacter, Tr ⁇ choderma or a combination thereof.
  • the second microorganism of the genus Pseudomonas present in the composition is polished Pseudomonas.
  • the plant growth promoting biostimulant composition described above comprises, in addition to strain CECT 9015, at least one other strain selected from: Bacillus subtilis CECT 9016, Pseudomonas putida CECT 901 1, Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Tr ⁇ choderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, or combinations thereof.
  • the composition described in the present invention is characterized by being in solid or liquid form.
  • composition described in the present invention may further comprise, in addition, at least one coformulant.
  • the coformulant is selected from the group consisting of: fertilizers, fertilizers composed of the elements nitrogen, phosphorus and / or potassium and their double or triple combinations, fertilizer products, polysorbates associated with fatty acids, asparagine, mannitol, organic acids, CAS medium, nutritive medium for the cultivation of bacteria, macro chelating substances and nutritional microelements, algae or their extracts and yeasts.
  • the present invention relates to a method for stimulating cultivated plants which consists in applying in them, any of the compositions described above.
  • the stimulating effect on the crop to which the composition described above is applied is measured by any of the parameters, or by a combination thereof, selected from among those consisting of: increased production without reducing the size or size of the fruit , increase in the number of fruits maintaining their quality, increase in plant and root mass, increase in photosynthetic activity of vegetables in adverse conditions, increase in the availability, absorption and content of potassium (K) and / or iron (Fe), and / or phosphorus (P), and / or nitrogen (N), and / or other nutrients such as magnesium (Mg), zinc (Zn) or boron (B), by mechanisms which increase the solubility of the element, and / or its availability, and / or its absorption through the membrane.
  • K potassium
  • Fe iron
  • P phosphorus
  • N nitrogen
  • Mg magnesium
  • Zn zinc
  • B boron
  • the method for stimulating the cultivated plants of the present invention by the composition described above is preferably applied in liquid form and in hydroponic culture or in soil cultivation.
  • the composition of the invention is applied, either as a solid powder or as a liquid, in both cases, which dissolves in the irrigation water.
  • the present invention relates to the use of the compositions described above comprising Pseudomonas fluorescens CECT 9015 for the manufacture of plant biostimulants.
  • said use of the strain Pseudomonas fluorescens CECT 9015 to manufacture plant biostimulants can be used with at least a second microorganism selected from the genera Pseudomonas, Bacillus, Arthrobacter, Tr ⁇ choderma, or a combination thereof.
  • the second microorganism is Pseudomonas putida.
  • the use of Pseudomonas fluorescens CECT 9015 for the manufacture of the compositions described above comprises at least one strain selected from: Bacillus subtilis CECT 9016, Pseudomonas putida CECT 901 1, Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018 , Tr ⁇ choderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, or combinations thereof.
  • FIGURES Figure 1 Production test of plate siderophores.
  • the strain BB17B (Pseudomonas fluorescens CECT 9015), is capable of growing in CAS medium and producing siderophores, resulting in a yellow / orange color shift in the growth zone of the colony.
  • FIG. 1 Potassium mobilization assay in vitro. Hydrolysis halos of strain BB17B (Pseudomonas fluorescens CECT 9015) and of the combination of strains BB17B and BB17F (Pseudomonas putida CECT 901 1). It is observed that the combination of the two strains has a greater effect than the BB17B strain alone. The combination of both strains has a synergistic one, favoring the absorption of potassium.
  • FIG. 3 Phosphorus solubilization test in potted cucumber plants according to each treatment (Control and strain P. fluorescens CECT 9015 (BB17B) in the presence of soluble (Ps) or insoluble (Pi) phosphorus).
  • B. Net photosynthesis The columns represent the average of 17-21 plants per treatment.
  • C Phosphorus content in leaf.
  • the columns represent the phosphorus content (mg) in the total foliar tissue (g) of all the plants corresponding to each treatment.
  • FIG. 4 Photosynthetic parameters measured in the phosphorus solubilization and absorption test plants corresponding to each treatment (Control and strain P. fluorescens CECT 9015 (BB17B) in the presence of soluble or insoluble phosphorus).
  • A. Fo is the fluorescence emission under soft light representative of the state of the photosystem;
  • B. Fv / Fm indicates the maximum potential capacity to channel the energy to photosynthesis that Photosystem II has;
  • C. 0PSII is the actual capacity of Photosystem II and
  • NPQ is the amount of energy that dissipates Photosystem II.
  • the bars on the columns represent the standard error and the different letters (a, b and c) denote values with statistically significant differences according to the Tukey test (P ⁇ 0.05).
  • compositions employed are P. fluorescens CECT 9015 strain, the combination of P. fluorescens CECT 9015 and P. putida CECT 9011 strains, and the control strain. Black columns correspond to normal irrigation conditions, white columns correspond to saline stress conditions and gray columns correspond to drought conditions. Each column represents the average of 5 plants per treatment and the bars represent the standard error.
  • FIG. 6 Effect of biostimulant compositions on plant photosynthesis under conditions of saline stress.
  • the compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P fluorescens CECT 9015 + P put CECT 9011, both at 10 8 CFU / g + amino acids 0.2%), M4 (P fluorescens CECT 9015 + P. putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T.
  • A. Fo is the fluorescence emission under soft light representative of the state of the photosystem;
  • C. PPSII is the actual capacity of Photosystem II and
  • NPQ is the amount of energy dissipated by Photosystem II. The bars on the columns they represent the standard error and the different letters (a, b and c) denote values with statistically significant differences according to the LSD test (P ⁇ 0.05).
  • FIG. 7 Water potential in tomato plants treated with different compositions under salinity conditions (500 mM).
  • the compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P fluorescens CECT 9015 + P. polished CECT 9011, both at 10 8 CFU / g + amino acids 0.2%), M4 (P. fluorescens CECT 9015 + P. putida CECT 9011 + B. subtilis CECT
  • FIG. 8 Proline concentration (mg / g plant) of tomato plants treated with different compositions under salinity conditions (500 mM).
  • the compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P. fluorescens CECT 9015 + P. putida CECT 901 1, both at 10 8 CFU / g + amino acids 0.2%), M4 (P fluorescens CECT 9015 + P putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT
  • Figure 9 Root development of two celery plants taken at random from each plot.
  • Figure 10 Cumulative production per tomato plant in the soil test. The data represent the total production of the plot to date, divided by the total number of plants in the plot (3157) for each of the treatments: IM1 (P fluorescens CECT 9015 and the coformulants S. cerevisiae, humic acids and amino acids 2 kg / ha) represented by squares, IM2 (IM1 + P put CECT 901 1 2 kg / ha) represented by triangles, and the Witness (represented by rhombuses). B. Distribution of production in the three plots by size and category. Each column represents the total kg of tomato collected per plant of each of the calibers (GG, G, Mg, Mp, MMg, MMp and MMM).
  • the white section corresponds to the fruits of Extra X category, and the area grated to the rest.
  • the black section corresponds to the fruits of Extra X category, and the area grated to the rest.
  • the gray section corresponds to the fruits of Extra X category and the grated area to the rest.
  • FIG. 11 Cumulative production per tomato plant in the hydroponic rock wool substrate test. The data represent the total production of the plot to date, divided by the total number of plants in the plot, for each of the treatments: IM3 (P. fluorescens CECT 9015 and the coformulants S. cerevisiae, humic acids and amino acids 200 g / ha) represented by asterisks and the Witness represented by rhombuses.
  • IM3 P. fluorescens CECT 9015 and the coformulants S. cerevisiae, humic acids and amino acids 200 g / ha
  • B Distribution of production by size and category. Each group of columns represents the total kg of tomato collected per plant of each of the calibers (GG, G, Mg, Mp, MMg, MMp, and MMM).
  • Figure 13 Effect of composition T1 (10 8 cfu / g of B. subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015 and S. cerevisiae, 40% w / w total organic carbon, 41% humic acids , 19% fulvic acids, 6.5% w / w total nitrogen, 4.5% w / w total potassium and 11% w / w free amino acids) in avocado plants.
  • the number of applications correspond to the numbers 1, 2 and 3 on the X axis.
  • the data of the plot treated with the composition T1 is represented by rhombuses and the data of the control plot by squares. A.
  • Figure 14 Number of fruits per avocado tree. The columns represent the average of 10 trees in each plot and the bars represent the standard error after treatment with T1 (black column) or the Witness (white column).
  • Figure 15. Photosynthetic parameters measured in the tomato field test. The compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P. fluorescens CECT 9015 + P put CECT 9016, both at 10 8 CFU / g + amino acids 0.2%), M4 (P fluorescens CECT 9015 + P putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B.
  • M1 Control: 0.2% amino acid solution
  • M2 P fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%)
  • M3 P. fluorescens CECT 9015 + P put CECT 9016, both
  • A. Fo is the fluorescence emission under soft light representative of the state of the photosystem;
  • B. Fv / Fm indicates the maximum potential capacity to channel the energy to photosynthesis that Photosystem II has;
  • C. 0PSII is the actual capacity of Photosystem II and D.
  • NPQ is the amount of energy that dissipates Photosystem II.
  • FIG. 1 Bioactive compounds (flavonols) in tomato leaf after each of the crops treated with the compositions of the previous example, measured as Equivalent mg of catechin in 100 mg of fresh weight.
  • Fertilizer product product used in agriculture or gardening that facilitates the growth of plants, increases their yield and improves the quality of the crops or that, by their specific action, modifies the fertility of the soil or its physical, chemical or biological characteristics. Fertilizers include fertilizers, whose main function is to provide nutrients to plants (Royal Decree of the Kingdom of Spain 506/2013).
  • Biofertilizer Biological product that contains live microorganisms that, when applied to seeds, plant surfaces or crops, promote their natural process of nutrition, increasing the capacity of the plant for the absorption of nutrients, stimulating growth and protecting plants against to pathogens
  • Biostimulant composition or stimulant composition for the purposes of the present invention, it is defined as that biofertilizer composition produced in the plant in which an increase in production is applied by 10-45% without reducing the size or size of the fruit, and / or an increase in the number of fruits by 1 1-40% while maintaining their quality, and / or an increase in plant and root mass by 5-35%, and / or an increase in the photosynthetic activity of vegetables in adverse conditions by 4-30%, and / or an increase in the availability, absorption and content of potassium (K) by 2-21%, and / or iron (Fe) by 11-100%, and / or phosphorus (P) in 6-40%, and / or nitrogen (N) in 5-25%, and / or other nutrients such as magnesium (Mg), zinc (Zn) or boron ( B).
  • K potassium
  • Fe iron
  • P phosphorus
  • N nitrogen
  • Co-formulant A substance or preparation that is used or is intended to be used in an additional product to the active substance. In the present invention it refers to any compound other than PGPR microorganisms present in the composition.
  • Nutrient chemical element essential for plant life and plant growth.
  • C carbon
  • O oxygen
  • H hydrogen
  • the nutrient elements are classified into: main nutrients, secondary nutrients and micronutrients
  • Primary macroelements or main nutrients Chemical compounds that organisms absorb in large quantities and therefore constitute their main nutrients. They correspond to nitrogen (N), phosphorus (P) and potassium (K).
  • Secondary macroelements or secondary nutrients Chemical compounds that constitute the secondary nutrients of plants. They correspond to calcium (Ca), magnesium (Mg), sodium (Na) and sulfur (S).
  • CAS medium Bacterial culture medium that incorporates the compound chromium azurol S (CAS) for the detection of siderophores (Alexander & Zuberer, 1991).
  • Micronutrients Chemical compounds essential for plant growth in small quantities. They correspond to boron (B), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo) and zinc (Zn).
  • B boron
  • Co cobalt
  • Cu copper
  • Fe iron
  • Mn manganese
  • Mo molybdenum
  • Zn zinc
  • - Siderophore organic molecules capable of binding to Fe 3+ cations and transporting them to the bacterial cell wall, where they are reduced to Fe 2+ cations that can be absorbed by both bacteria and plants.
  • control samples are compositions that lack microorganisms.
  • the controls are cultures treated with control compositions that lack microorganisms.
  • the present invention relates to biostimulant compositions of plants comprising microorganisms.
  • the microbial strains that are included in the biostimulant compositions of the present invention are described below.
  • the microorganisms of the compositions of the present invention favor root development, produce siderophores, organic acids and phosphatases that make iron and phosphorus available to the plant, and emit substances that activate the metabolism of the plant favoring the development of leaf buds and florals, increasing the number of flowers and leaves and, consequently, causing an increase in production.
  • the differentiating effect of the biostimulant compositions of the invention is based on the action of plant growth promoting microorganisms such as PGPR bacteria, fungi and / or yeasts of the Saccharomyces genus. For each composition a specific strain combination is selected, depending on the desired effect on the culture.
  • the microbial strains present in the composition of the invention belong to the genera Pseudomonas, Bacillus, Arthrobacter and Trichoderma.
  • the composition comprises strain CECT 9015 of Pseudomonas fluorescens.
  • this composition may comprise at least a second strain selected from the genera Pseudomonas, Bacillus, Arthrobacter, Trichoderma, or a combination thereof.
  • This second strain of the genus Pseudomonas can be a strain of the species Pseudomonas putida.
  • the strain of Pseudomonas putida present in the composition, together with Pseudomonas fluorescens CECT 9015 is strain CECT 9011.
  • a strain is preferably selected of the species Bacillus licheniformis, such as strain CECT 9018, a strain of the species Bacillus subtillis, such as strain CECT 9016, a strain of the species Bacillus amyloliquefaciens, such as strain CECT 9017, or a combination thereof.
  • a strain of the species Arthrobacter is preferably selected, such as strain CECT 7170.
  • a strain of the species Trichoderma without thereby limiting the present invention, preferably select a strain of the species Trichoderma harzianum, such as strain CECT 20946.
  • the object of the present invention is a method of biostimulating the cultivated plants which consists in applying the compositions described above in them.
  • strain Pseudomonas fluorescens CECT 9015 for the manufacture of plant biostimulant compositions, such as biofertilizers, is an object of the present invention.
  • the use of Pseudomonas fluorescens CECT 9015 in combination with at least a second microorganism selected from the genera Pseudomonas, Bacillus, Arthrobacter, Trichoderma or a combination thereof for the manufacture of plant biostimulants is part of the invention.
  • the second microorganism that is used in the manufacture of biostimulants is Pseudomonas putida.
  • the strain of Pseudomonas putida present in the biostimulant is strain CECT 9011.
  • the strain of the genus Bacillus is preferably selected from the species Bacillus licheniformis, such as strain CECT 9018, of the species Bacillus subtilis, such as strain CECT 9016, of the species Bacillus amyloliquefaciens, such as strain CECT 9017 or a combination thereof.
  • compositions of the present invention may be in solid or liquid form.
  • the biostimulating composition in solid form comprising bacterial strains of the invention at a minimum concentration of April 10 to September 10 CFU / g of total product.
  • the biostimulant composition in liquid form comprises the bacterial strains of the invention at a minimum concentration of 10 4 CFU / ml of total product.
  • the compositions of the invention are applied to the soil or the aerial part of the plant so that the microorganisms present in said compositions are established in the root system or on the surface of the plant. When applying these compositions with water, the microorganisms recover their activity quickly and begin to reproduce in the rhizosphere or philosophy, significantly increasing its concentration in areas located about 2-8 cm from the apical end of the roots in the case of the rhizosphere.
  • the microorganisms form microcolonies on the surface of the epidermis of the main root, particularly in the areas of union between the main root and the lateral roots, because in them there is usually a high concentration of root exudates and they end up constituting a biofilm that ends up covering much of the surface of the plant's root system as a protective layer.
  • a mutualistic relationship with the plant is established.
  • the object of the present invention is also a method for fertilizing crops which consists in applying any of the above-described compositions therein.
  • the application of said compositions can be in liquid or solid form and in hydroponic culture or soil cultivation.
  • the biostimulant effect on the crop to which the compositions described herein are applied is measured by any of the parameters, or by a combination thereof, selected from among those consisting of: increased production without reducing the size or size of the fruit, increase in the number of fruits maintaining their quality, increase in plant and root mass, increase in photosynthetic activity of vegetables in adverse conditions, increase in availability, absorption and content of potassium (K), and / or of iron (Fe), and / or of phosphorus (P), and / or of nitrogen (N), and / or of other nutrients by mechanisms that increase the solubility of the element, and / or its availability, and / or its absorption through the membrane.
  • K potassium
  • Fe iron
  • P phosphorus
  • N nitrogen
  • the biostimulant effect on the culture to which the compositions described herein are applied is measured by any of the parameters, or by a combination thereof, selected from among those consisting of: increased production by 10-45% without reducing the size or size of the fruit, increasing the number of fruits by 11-40% while maintaining their quality, increasing the plant and root mass by 5-35%, increasing of the photosynthetic activity of vegetables in adverse conditions in a
  • the biostimulant compositions comprising the strain P. fluorescens CECT 9015 described herein produce an increase in production. of the harvest, increase of the number of fruits by plant, increase of the index of maturity of the fruit, lower acidity of the fruit and in addition they are able to increase the concentration of flavonols in leaf.
  • photosynthesis is the process by which plants transform inorganic matter to organic matter thanks to the energy provided by light.
  • the photosynthetic activity of a plant is therefore critical for plants and crops. Light, therefore, influences crop development. Although we must bear in mind that the quantity, quality and duration of the light depends on the conditions of the crops and the photosynthetic efficiency of the plant depends on the resources it has to increase its activity.
  • Photosynthesis is, therefore, key in the development of plants and will determine their productivity, fruit size, plant development and nutrient absorption. When evaluating the effect of the compositions on plants, it is necessary to interpret together the effects on growth, development of the root system, nutrient absorption ... in the light of photosynthesis in said plant.
  • the greater growth of a plant may not be important if, for example, such growth is due to the increase in cell size (instead of the number of cells), if that plant is inefficiently using the resources to grow instead of, for example, increasing root development under conditions of lack of nutrients. Therefore, the effects produced by the compositions indicated herein should be interpreted as a whole since it is in the light of photosynthetic efficiency in the framework in which the parameters measured for the actual development of the plant make sense.
  • Pseudomonas fluorescens BB17B (deposit code CECT 9015) is a bacterium with a large negative wall isolated from soil and belonging to the gamma class of the phylum Proteobacteria. It has a cane shape, is mobile thanks to the presence of one or several polar flagella and does not form spores. It has a chemoganotrophic metabolism and strictly aerobic. Solubilizes phosphates, produces siderophores and has ACC-deaminase activity.
  • Pseudomonas putlda BB17F (deposit code CECT 901 1) is a bacterium with a large negative wall isolated from soil and belonging to the gamma class of the phylum Proteobacteria. It has a cane shape, is mobile thanks to the presence of one or several polar flagella and does not form spores. It has a chemoganotrophic metabolism and strictly aerobic. Solubilizes phosphates and produces siderophores.
  • Bacillus licheniformis BB02L (deposit code CECT 9018) is a large positive bacterium belonging to the phylum Firmicutes, mobile and shaped like a cane. It has a mostly aerobic metabolism, although sometimes it can be anaerobic. It forms a resistance endospora with an ellipsoidal shape and is capable of developing up to 55 ° C. It can produce plate siderophores.
  • Bacillus subtilis BB02H (deposit code CECT 9016) is a large positive bacterium belonging to the phylum Firmicutes, mobile and shaped like a cane. It has a mostly aerobic metabolism and forms an endospora of resistance with an ellipsoidal shape. It can degrade ACC and solubilize plate phosphates.
  • Bacillus amyloliquefaciens BB02N (deposit code CECT 9017) is a large positive bacterium belonging to the phylum Firmicutes, mobile and shaped like a cane. It has a mostly anaerobic metabolism and forms an endospora of resistance with an ellipsoidal shape. It can solubilize phosphates in plaque.
  • Trichoderma harzianum BB21A (deposit code CECT 20946) is a fungus from the Ascomycota division, order Hypocreales, of asexual reproduction through conidia.
  • the conidiophores of the fungus are very branched, each branch forming a right angle to the previous level, and each set of branches has a pyramidal shape. It has positive phosphate solubilization activity in plaque.
  • Arthrobacter oxydans BB01A (deposit code CECT 7170) is a microorganism of the Gram + bacteria group, Arthrobacter genus, plant growth stimulant in saline stress environments. This strain has been isolated from the rhizosphere of Pinus pinaster Aitón and Pinus pinea (L), and from the mycosphere of the mycorrhizal fungus associated with both Lactarius deliciosus (Fries) SF Gray, in nutritive agar (PCA), and has been characterized from the morphological, biochemical and genetic point of view.
  • Example 1 Manufacturing process of the biostimulant composition of the invention 1.1 Reproduction of microorganisms
  • a small volume of suspension of the pure culture of each strain is taken, and it is inoculated under sterile conditions in a medium with the nutrients and pH that most favor its reproduction.
  • Each strain requires a specific nutritional medium for its growth that is incubated in the optimal conditions of temperature and aeration for a period of time between 24 and 72 hours.
  • the liquid medium contains a suitable carbon source, glucose being the best, although other compounds such as starch, sucrose and molasses are also used. In addition, an adequate source of nitrogen in the form of amino acids is essential. Likewise, the presence of sources of K, P, Mg, S, Ca, Cl, Zn, Fe, Mn in the form of mineral salts is required. All these elements, together with sterile water, are deposited in fermenters of different capacity according to the amount of biofertilizer that is desired to be produced. The reproduced and / or preserved microorganisms are added to this liquid medium and the pH of the medium is adjusted. The temperature, stirring and aeration parameters are then programmed, and the fermentation is carried out for 24-72 hours, until a desired concentration of CFU / ml is obtained.
  • a suitable carbon source glucose being the best, although other compounds such as starch, sucrose and molasses are also used.
  • an adequate source of nitrogen in the form of amino acids is essential.
  • the microorganisms are stable in the culture medium itself for a period of 6 months without the need for a stabilization or cold preservation treatment.
  • the culture medium obtained in the previous point can be mixed with other components, such as humic acids or amino acids, which improve the synergistic effect of the product on the plants.
  • the solid product formulation process that is, the mixture of lyophilized microorganisms with coformulants, is carried out in two phases to guarantee the highest quality and homogeneity:
  • composition obtained in point (I) is homogenized with the coformulants in the proportions established for each composition, in a larger volume mixer.
  • Compositions in solid form are packaged with the help of an automatic packaging machine in bags of variable material, depending on the purpose to which the product is intended.
  • the product intended for export or intensive horticultural crops is packaged in a complex material that isolates from light and moisture, or other material with the same properties that allows its conservation at room temperature. After being properly labeled, the product is presented to consumers in the form of opaque bags with a capacity of between 0.1 and 25 kg.
  • compositions in liquid form are dosed with the aid of a liquid packing machine in bottles of opaque plastic material with a capacity of between 1 and 5 liters.
  • Example 2 Fertilization method by application of the compositions of the invention
  • compositions of the invention have been designed for possible application in a wide range of crops, admitting the possibility of adding small variations in their composition that are better suited to the specific techniques and needs of certain crops. Its effectiveness has been proven by the R&D department of the authors of the present invention, in collaboration with farmers in commercial conditions, as well as universities and independent consultants in Spain, the United States, Chile, Mexico, Peru and Egypt.
  • the compositions of the invention can be applied in intensive horticultural crops under greenhouse and outdoors. They can be applied, for example, but not limited to tomato, celery, lettuce and cucumber, pepper, melon, watermelon, zucchini, squash or other horticultural crops. They are also indicated for crops of berries or shrub species such as strawberry, raspberry, blackberry or blueberry.
  • compositions of the invention improve the availability of water and nutrients, can also be used in citrus crops (orange, mandarin), fruit crops such as table grapes, peaches and other rosaceae (apple, pear, apricot, plum, cherry, etc.), or crops Tropical like avocado.
  • the compositions can also be applied to extensive cereal crops such as wheat, barley or corn.
  • crops that could benefit from its application would be flower and ornamental crops, industrial crops such as potatoes or beets, and typically Mediterranean crops such as olive trees.
  • the recommended use dose is between 0.1 kg / ha and 2 kg / ha.
  • the recommended use dose is between 0.5 and 2 l / ha.
  • the recommended employment dose for compositions in solid form is between 0.2 kg / ha and 2 kg / ha, and the recommended employment dose for compositions in liquid form is between 1 and 2 l / ha .
  • the number and timing of applications will vary according to the type of crop, as indicated below. In general, for all crops that begin with the transplant from the seedbed it is recommended to perform the first application at the time of transplantation, ideally with the first irrigation. Thus, it will be possible to favor the root development and the correct establishment of the crop. It is recommended to renew the applications once a month or coinciding with times of special energy demand such as: the departure of winter lethargy, budding, flowering or fruit set and fruit formation; up to an approximate total of 2-4 applications per crop cycle. The final number of applications will depend on the crop and the duration of the cycle. It is recommended to repeat the application of the biostimulant composition approximately every 30 days, to ensure that the level of microbial population is optimal to produce all the effects sought in the crop. 2.3 Method of application a) Biostimulant composition in solid form
  • the solid form composition is a soluble powder applied to the soil or culture substrate that must be diluted in the irrigation water and applied by the irrigation system. It is also possible to apply by sprinkler irrigation, but in that case it is advisable to prolong the irrigation a bit so that the excess water washes the product of the leaf surface and is deposited in the soil, which is the natural means of development of microorganisms
  • the product can be applied directly to the leaf surface with a wetting agent and to remain in contact with the leaves of the plant.
  • the product is completely soluble in water and perfectly compatible with irrigation systems, where it has been proven that no clogs filters or valves. It can be applied both to the soil and to any crop substrate: perlite, peat, coconut fiber, rock wool, etc.
  • the strains it contains are reproduced with the help of coformulants present in the composition, colonizing the roots of the crop.
  • the microorganism community is established on the surface of the roots and the interaction with the plant begins.
  • compositions of the invention are based on the action of live microorganisms, it is necessary to take a series of precautions that ensure a good development of the microbial community.
  • the product should be applied with non-chlorinated waters or that have not received a treatment of purification for human consumption, since these treatments can affect the beneficial PGPR strains, reducing their viability. For this same reason, it is also recommended to avoid the application of substances with a very low (acidic) or very high (basic) pH, and reduce as far as possible the application of phytosanitary products such as fungicides or insecticides that may have side effects About microorganisms b) Biostimulant composition of the invention in liquid form
  • biostimulant composition in liquid form, it is recommended to follow the same indications as in the solid product, with the proviso that it is not necessary for the product to rest before distribution through the irrigation system, since the microorganisms are They are in an active state.
  • Fresh biomass It is the weight of the plant extracted directly from its substrate. A granataria scale is used and measured in grams. The fresh weight of the whole plant or parts of it, such as roots or leaves, can be measured.
  • Dry biomass It is the weight of the plant extracted directly from its substrate, after drying in an oven at 60 ° C for two days. A granataria scale is used and measured in grams. You can measure the dry weight of the whole plant or parts of it, such as roots or leaves.
  • Root length The root length is determined with a previous rule separating the roots of the stem, and is measured in centimeters (cm). There is a direct relationship between the roots and the development of the aerial part of the plant: the greater the root mass, the greater the thickness of the stem, the better development of the leaves and increasing the caliber of the fruits.
  • Plant height To determine the height of the aerial part of the plant, each plant is measured from the birth zone to the last leaf or apical meristem. It is measured in centimeters (cm).
  • Photosynthesis is the most important process that occurs in plants. It distributes the energy collected by determining the development of the plants, producing different effects according to the needs of the organism at all times: increasing the reserves, increasing the secondary metabolism of the plant, increasing its size ... The photosynthetic capacity of the plants must therefore interpreted globally.
  • Net photosynthesis the photosynthetic capacity of plants is assessed from the process of fixation of CO2 in the Calvin Cycle. It corresponds to the difference between the amount of CO2 fixed and the amount of CO2 emitted by breathing.
  • An indirect method of measuring plant photosynthesis is by detecting the fluorescence emitted by photosystem II. In this document photosynthesis is measured from the fluorescence emitted with a fluorimeter.
  • Fluorescence emission of photosystem II (FSII): The following parameters are calculated to determine the status of FSII:
  • Fo corresponds to the minimum level of fluorescence, obtained when soft light falls on the sheet. All the pigments of the FS antennas are open (adapted to the dark). In conditions of stress, Fo's novel increases.
  • Fm maximum fluorescence. It is the novel of fluorescence after applying high intensity light to the sheet, when the antennas are closed.
  • Fv variable fluorescence.
  • the maximum potential capacity of FSII (Fv / Fm): corresponds to the ratio between variable fluorescence and maximum fluorescence.
  • Photochemical quenching corresponds to the real capacity of the FSII to channel the light energy received by the sheet into the photosynthetic process and produce glucose.
  • Non-photochemical quenching represents the amount of energy that dissipates the FSII in the form of heat, and therefore is not used in the photosynthetic process and will not have a beneficial effect on the plant.
  • Chlorophyll content Chlorophylls are inserted into the membranes of doroplast thylakoids. They are attached to the membrane by a phytoid residue and are associated with other pigments (forming the antennas) and proteins forming the photosystems.
  • a portable chlorophyll meter is used, such as SPAD-502 , Minolta, which determines the relative amount of chlorophyll present by measuring the absorption of the leaf in two regions of wavelength; in the red and near infrared regions.Using these two transmissions the meter calculates the numerical value SPAD that is proportional to the amount of chlorophyll present in the leaf.
  • Degrees Brix measure the amount of sucrose present in the leaf of the plant or in a fruit, which allows to determine the state of its maturation. They are determined as the total ratio of sucrose dissolved in a liquid. It is an indicator of an increase in the Calvin cycle of the plant, since by increasing the production of glucose-3-phosphate, the plant uses it well to increase the content in starch (and ° Bx) or in glycolysis ( and favor the growth of the plant). By assessing the Brix grades, the amount of sugars that are being transformed after photosynthesis is analyzed. The higher the sugar level within the plant tissue, the stronger the plant will be and the more it will produce. Brix degrees are measured by using a refractometer.
  • Fruit size The size of the fruits is measured according to their size. To determine the size, a universal fruit caliber is used whose loop design allows a measurement accurate. Fruit sizes are classified in the categories GG, G, M, MM and MMM according to their size, according to Table 1:
  • Table 1 Types of caliber according to the diameter of the fruit.
  • Fruits are classified into categories depending on their quality. For example, tomatoes are classified in an extra category, which corresponds to tomatoes that have firm flesh, have a characteristic shape, appearance and development of the variety and do not present defects that affect the general appearance of the product, its quality, preservation or presentation in the container
  • the first category corresponds to fruits of good quality, which do not present cracks or apparent "green backs," but may present defects such as: slight malformations and developmental defects, slight color defects, slight bruises or slight defects in the epidermis.
  • Cumulative production per plant It is determined by weighing or counting the total fruits of each plot to be analyzed by the total number of plants analyzed.
  • Nutritional analysis The mineral elements of a soil, necessary for plant feeding can be found in many different forms. Not all of them are suitable to be absorbed by the roots. It is necessary that the macro- and microelements present in the soil be available so that the plant can absorb them through its roots. They are determined in dry foliar matter.
  • Nitrogen Dumas (mg / kg): It consists of the transformation of all forms of nitrogen present in the plant into N gas by calcination. It is determined by thermal conductivity (Sweeney and Rexroad, 1987).
  • Available calcium (meq / 100 g): It is measured by an atomic absorption spectrophotometer (Perkin Elmer 2280, Norwalk, Connecticut. USA).
  • Available magnesium (meq / 100 g): Measured with an atomic absorption spectrophotometer (Perkin Elmer 2280, Norwalk, Connecticut. USA).
  • DTPA Iron
  • Effective cation exchange capacity (meq / 100 g): It is determined by the method of ammonium acetate pH7 1 N (normal), Kjeldahl distillation and volumetry. Oxidizable organic matter (%). It is determined using the volumetric technique of the Walkley and Black method (1974). This technique is based on a wet combustion of organic matter with a mixture of potassium dichromate and sulfuric acid. The value that indicates the degree of accumulation of organic matter on a horizon and is used to differentiate organic soils from minerals. As of 1998 it is referred to as Organic Carbon.
  • Leaf damage This parameter is measured after a crop has been subjected to some type of stress (salt, drought ). Damage is assessed by visual observation according to the following scale: 0: healthy leaves; 1: slight chlorosis; 2: severe chlorosis and / or wrinkle; 3: necrosis and / or severe wrinkling; 4: widespread necrosis.
  • Phenolic compounds have an antioxidant effect on plants and are synthesized as secondary metabolites of the plant. Depending on the compound, it can intervene in the defense of the plant to pathogens, provide mechanical support to the plant, attract pollinators or fruit dispersers, or act as allopathic agents. They are quantitatively determined with the Folin-Ciocalteau reagent (Sigma-Aldrich, St Louis, MO) by colorimetry (Xu 2007) with modifications, using gallic acid as a reference (Sigma-Aldrich, St Louis, MO).
  • a 1 ml aliquot of the extract is mixed with 0.25 ml of Folin-Ciocalteu 2 N reagent and 0.75 ml of a 20% solution of Na2C03. The mixture is kept 30 minutes at room temperature and then the absorbance at 760 nm is measured in a UV-Visible spectrophotometer (Biomate 5). A calibration curve is made with gallic acid and the content of phenolic compounds is obtained.
  • Water potential This parameter is determined by a pressure chamber which gives a measure of the negative hydrostatic pressure that occurs in the xylem of an intact plant due to the evaporation of water from the tissue by perspiration and resistance to water movement from the ground to the fabric (Scholander, 1965).
  • Proline content This parameter is measured by absorbance at 520 nm, after mixing with the ethanolic extract from leaf samples and preparation of a reagent (Irygoyen 1992).
  • Root system development This parameter is measured by visual observation once the plant has been removed from its substrate (pot or field crop). It is evaluated according to the following scale: 0: damaged roots; 1: healthy roots, but not very developed; 2: abundant and strong roots; 3: very abundant and strong roots. In addition, you can also measure the percentage of new roots (identified by their light color, not suberized) with respect to the total roots. Vigor and degree of development of the plants: It is determined visually by analyzing the foliar surface and shadow cast on the ground, cluster density.
  • Example 3 Effect of the biostimulant compositions of the invention.
  • biostimulant compositions of the invention comprising microbial strains are capable of increasing the availability, absorption and / or plant content of certain nutritional elements such as phosphorus, iron and / or potassium.
  • the siderophores production assay was carried out by inoculating in CAS medium (Chrome Azurol S) 10 ⁇ 90 of the P. fluorescens CECT 9015 strain previously grown in liquid medium with sufficient growth (24h at 28 ° C under vigorous stirring). The test is considered positive if the bacterium releases the siderophores capable of sequestering iron, which is bound to the blue CAS dye. When iron is released from CAS, there is a turn to yellow-orange (hydroxy) or pink (catechol).
  • Figure 1 shows that the strain P. fluorescens CECT 9015 (BB17B) is able to grow in this medium and produce siderophores, resulting in a color shift in the growth zone.
  • the production of siderophores indicates that the P. fluorescens CECT 9015 strain is capable of solubilizing the iron present in the soil (Fe 3+ ) to Fe 2+ , capable of being absorbed by the plant.
  • putida CECT 901 1 (BB17F) strains has a greater effect than the P. fluorescens CECT 9015 strain alone, indicating that this strain combination has a synergistic effect, which will favor the absorption of potassium by the plant.
  • compositions of the invention have the ability to solubilize insoluble phosphorus and make it available to the plant, fast developing hybrid cucumber was grown on a cocopeat substrate in 350 cm 3 seedbeds. 21 plants were treated for each of the biofertilizing compositions:
  • a first application of 3 ml of each composition was made 13 days after germination and a second application was made 10 days after the first application (both at a concentration of the microorganism 10 8 CFU / ml).
  • biometric parameters dry weight
  • photosynthetic parameters photosystem II fluorescence emission, CO2 fixation, chlorophyll content
  • macro and micronutrient content were analyzed.
  • Figure 3A shows the dry weight of the plants at the end of the experiment. An increase in weight of the aerial part of the cucumber plants inoculated with the strain P. fluorescens CECT 9015 (BB17B) with respect to the control is observed, both by adding PS and Pl (Table 4).
  • Figure 3B reflects a higher photosynthetic capacity of plants inoculated and treated with soluble phosphate, which is in accordance with the highest growth achieved with this treatment.
  • Figure 3C represents the amount of sheet phosphorus, where it is clearly observed that P. fluorescens strain CECT 9015 (BB17B) improves the phosphorus content of the sheet, regardless of the type of phosphorus used.
  • the effect of bacteria with insoluble phosphorus is significant, if we compare it with the non-inoculated control, clearly improving the nutrition of this element in deficit conditions. It is interesting to note how the increase is more significant in cases where it is added to the insoluble phosphorus medium. This indicates that the presence of the P. fluorescens CECT 9015 strain favors the solubilization of this element, which translates into better root development and plant growth.
  • Figure 4 shows the photosynthetic efficiency data of the plant.
  • Figure 4A shows the Fo parameter, which is an indicator of stress in the plant at the level of photosystem II.
  • the plants treated with P. fluorescens CECT 9015 (BB17B) and soluble phosphorus have a lower level of stress than other treatments.
  • Figure 4B shows that potential photosynthesis (Fv / Fm) increases in plants treated with P. fluorescens CECT 9015 and soluble phosphorus with respect to the control.
  • Figure 4C represents the real photosynthesis of the plant, in which it is seen how the addition of the P. fluorescens CECT 9015 strain, in the presence of soluble phosphorus, has much more photosynthetic efficiency than the control.
  • Figure 4D shows that plants treated with the composition comprising P. fluorescens CECT 9015 have less heat dissipation than their respective control. All this, taken together, indicates that photosynthesis is more efficient and the performance of these plants will be better.
  • Analyzes in adverse conditions such as abiotic stress or drought are important when assessing the effect of the compositions on crops, because they allow determining the real effect of the compositions on the plant. Under controlled conditions on many occasions no differences are observed between controls and biostimulant compositions because the plant has access to all metabolic, defensive resources ... that allow its development.
  • the objective of this test is to evaluate the biological effectiveness of three compositions comprising microorganisms in a corn crop (Zea mays L.), against simulated abiotic stress conditions (drought and salinity) in a greenhouse on peat pots. Three compositions were tested and compared with a control (water), each with 5 repetitions, under 3 growth conditions (optimal irrigation, salinity and drought), up to a total of 12 treatments and 60 plants.
  • the composition of the biostimulants tested is detailed in Table 6.
  • Treatment Product to be evaluated cfu dose / ml / pot
  • compositions were applied by root 15 days after sowing and one week after the first application. Drought stress was caused by stopping irrigation and salinity stress by applying 20 ml of a saline solution at 25 mM NaCI. Both stress conditions began two weeks after the second application, and lasted for 5 days.
  • foliar damage was evaluated by visual observation based on a scale of 0 to 4 for the degree of damage (0: healthy leaves; 1: slight chlorosis; 2: severe chlorosis and / or wrinkling; 3: necrosis and / or severe wrinkling; 4: widespread necrosis.) and root system development by visual observation on a scale of 0 to 3 (0: damaged roots; 1: healthy but not very developed roots; 2: abundant and strong roots; 3: very abundant and strong roots In addition, you can also measure the percentage of new roots (identified by their light color, not submerged) with respect to the total roots.
  • P fluorescens CECT 378 is a commercial Pseudomonas fluorescens strain, available in the state of the art, used herein as a positive control to assess the biostimulant capacity of the strain of the invention, CECT 9015.
  • the photosynthetic parameters analyzed indicate that the composition M2, which comprises P fluorescens CECT 9015 has a lower Fo than the composition comprising P fluorescens CECT 378, indicating that they suffer less stress (Figure 6A).
  • Figure 6D shows that all treatments, except M5 (P. fluorescens CECT 378), increase the energy dissipation (NPQ) in salt-treated plants, which means a dissipation of the Excess energy that decreases the formation of free radicals and therefore keeps the plant healthier.
  • compositions comprising the P. fluorescens CECT 9015 strain, especially the strain alone and combined with P. putida CECT 901 1, protect the plant against saline stress by other mechanisms, in addition to increasing the proline content.
  • the protective effect of P. fluorescens CECT 9015 is superior to that of other strains of the same species, which demonstrates its characteristic differentiating stimulating effect of the strain, and not of the species to which it belongs. 3.3 Field / natural efficacy tests.
  • the composition is composed of: 10 8 CFU / g Bacillus amyloliquefaciens CECT 9017, 10 8 CFU / g Bacillus licheniformis CECT 9018, 5.1x10 2 CFU / g Pseudomonas fluorescens CECT 9015, and the co-formulants alginic acid 12% w / w, Total nitrogen (N) 4.7% w / w, phosphorus (P 2 0 5 ) 0.2% w / w potassium (K 2 0) 10% w / w.
  • T2 100 g / ha of biostimulant composition diluted in 1000 liters of water
  • T3 150 g / ha of biostimulant composition diluted in 1000 liters of water
  • T4 200 g / ha of biostimulant composition diluted in 1000 liters of water
  • the height of the plant, the days of flowering, the number of flowers, the number and weight of fruits per plant, the characteristics of the fruits, the yield of the plant and the percentage of deformed fruits of the plant were evaluated.
  • the number of days to flowering was quantified taking as a starting point the moment in which 50% of the total plants in the experimental lot presented flowers.
  • Table 8 shows a decrease in the days needed to reach flowering in the strawberry crop treated with the composition, and where the earliest plants were those treated with doses of 150 (T3) and 200 g / ha (T4 ) in 1000 L of water that required only 56.50 days, while the witness that was required the largest number of days with 58.75.
  • the total number of flowers present per plant was quantified by visual observation, in each of the 5 randomly selected plants per useful plot.
  • the results presented in Table 9 demonstrate the highly significant statistical differences between the treatments with the composition and the control, achieving the highest flower production treatment with the dose of 200 g / ha in 1000 L of water (T4) with an average of 27.75 flowers per plant, which represents a superior efficiency with respect to the control of 63.06% since the witness only presented an average of 10.25 flowers per plant.
  • the total number of fruits present per plant was quantified by visual observation in each of the 5 randomly selected plants per useful plot and the weight of the total fruits of each plant was determined with the help of a granatary scale.
  • the number of fruits presented significant statistical differences as shown in Table 10, where the treatment with the composition at a dose of 200 g / ha in 1000 L of water (T4) which was the which obtained the highest amount of fruits per plant with an average of 25.25, while the control only reached an average production of 9.00 fruits; T4 treatment has a greater efficiency 64.36% higher than the control.
  • Table 10 Fruits per plant
  • a random sample of five fruits was taken from the five randomly selected strawberry plants per useful plot and macerated to determine the pH with the help of a portable potentiometer and the Brix grades were determined with a refractometer, as described in section 2.6 of this report.
  • Table 12 shows that fruit quality also showed statistical differences with respect to the control in relation to the pH and ° Brix of the fruits.
  • the experiment is proposed with the objective of verifying the biological effectiveness of a biofertilizing composition comprising bacterial strains.
  • lettuce was grown Roman type and "Colosus" variety on a loamy clay soil with 20 to 45% silt, and between 15 and 25% clay.
  • Four treatments (T1, T2, T3 and T4) were performed on a total area of 16 m 2 , divided into experimental units of 1 m 2 . In each useful plot, five randomly selected plants were grown.
  • the composition of the biofertilizer of the assay is Pseudomonas fluorescens CECT 9015 (5 x 10 3 cfu / g), Pseudomonas putida CECT 9011 (5 x 10 3 cfu / g), Saccharomyces cerevisiae (6.2 x 10 7 cfu / g), total humic extract (20% w / w), humic acids (10% w / w), fulvic acids (10% w / w) and free amino acids (15% w / w).
  • composition was applied to the plants via fertirrigation on the day of the transplant, 30 days after the first application and 60 days after the transplant.
  • Three concentrations of the composition were handled (T2: 0.5 kg / ha; T3: 0.75 kg / ha; T4: 1.0 kg / ha) that were compared with a control to which only water was applied (T1).
  • Table 15 shows that the application of the composition at its highest dose shows significant differences with respect to the control with a height of 20.42 cm versus 13.82 cm, which represents a product efficacy of 20.69%.
  • Table 16 shows the fresh biomass (g) of the plants according to the treatment used.
  • the control has an average of 1 17,103 g while the biofertilizer treatment at a dose of 1.0 Kg / ha has the highest amount of fresh biomass with an average of 164.10 which represents an effectiveness of 28.64% higher than the control.
  • Table 17 shows the dry biomass (g) of the plants according to the treatment used.
  • the control has an average of 3.44 g while the biofertilizer treatment at a dose of 1.0 Kg / ha has the highest amount of dry biomass with an average of 5.49 g, which represents an effectiveness of 37.34% higher than the control.
  • Table 18 shows the root length of the plants according to the treatment used.
  • the control has a length of 7.42 while the treatment with the biofertilizer at doses of 0.75 Kg / ha and 1.0 Kg / ha has a length of 11.86 and 12.45 cm respectively.
  • the efficiency of these treatments is 37.44 and 40.40% respectively higher than the control.
  • T4 1.0 Kg / ha 12.45 A 40.40 Table 19 shows the yield of the plants according to the treatment used.
  • the T4 treatment (1.0 Kg / ha) has a yield of 14,052.3 and an efficiency compared to the control (T1) of 28.64%.
  • composition comprising the strain Pseudomonas fluorescens CECT 9015 promotes height growth, increased biomass (both fresh and dry), root length, increases yield and absorption of nitrogen, phosphorus and Potassium in growing plants. 3.3.3 Production increase test on celery cultivation
  • the experiment is proposed with the objective of checking the biological effectiveness of a composition comprising P. fluorescens CECT 9015 strain on a celery crop (Apium graveolens L).
  • a composition comprising P. fluorescens CECT 9015 strain on a celery crop (Apium graveolens L).
  • the plants were cultivated in furrows with two crop lines separated by a drip irrigation pipe. In one plot the product will be applied and in the other the control.
  • composition used in this test is: P. fluorescens CECT 9015 (5 x 10 8 cfu / g), Bacillus amyloliquefaciens CECT 9017 (5 x 10 8 cfu / g), B. subtilis CECT 9016 (5 x 10 8 cfu / g ), Trichoderma harzianum CECT 20946 (5 x 10 8 cfu / g) and as co-formulants a protein hydrolyzate of vegetable origin, Saccharomyces cerevisiae, humic acids and fulvic acids.
  • the composition was applied to the plants via drip irrigation 15 days (first application) and 45 days (second application) after transplantation at a concentration of 2.0 kg / ha.
  • the control consisted of the usual farm management protocol, without the addition of the biostimulant / biofertilizer composition of the present invention.
  • Each treatment was evaluated: root growth and fresh biomass (45 days after transplantation) as described in section 2.4 of this report.
  • a clear difference between the size of the root ball of the treated plants was observed, with a greater number of root hairs (Figure 9).
  • 5 plants are randomly selected in the control plot and 5 plants in the treated plot.
  • the 5 selected plants of each of the plots were weighed and it was observed that the average weight of the celery plants of the plot treated with the composition (T1) is 29% greater than the control plot (To), and there are also greater homogeneity of weights (Table 21).
  • the Mariana hybrid tomato variety grafted onto the Arnold variety pattern was cultivated using the Dutch-type pick-up technique.
  • the test was carried out in parallel on soil and on inert substrate with fertirrigation.
  • soil two compositions IM1 and IM2
  • IM1 and IM2 were applied to two sectors of a plot (3157 plants), and compared with a Witness sector of the same dimensions.
  • 2 sectors were used, one in rock wool and the other in coconut fiber, to which the IM3 composition was applied, and a rock wool sector was used as a Witness in which the usual treatment of the farm without application of the biostimulant composition of the present invention.
  • the product IM1 contains P. fluorescens CECT 9015 and the coformulants S.
  • the product IM2 contains the formulation IM 1 and also P. putida CECT 9011.
  • the product IM3 contains P. fluorescens CECT 9015 and S. cerevisiae formulated on a protein hydrolyzate of vegetable origin.
  • the IM3 product is applied at a dose of 200 g / ha, while the IM1 and IM2 products are applied at a dose of 2 kg / ha.
  • 6 applications of each of the products were made, approximately once a month with the following application protocol:
  • the powder product is diluted in 100-200 liters of unchlorinated water
  • the product is left standing in the water for at least 30 minutes so that the lyophilized microorganisms rehydrate correctly and recover their activity.
  • the product is applied by injecting it to the drip irrigation system, in order to reach the ground.
  • Figure 10A shows the cumulative production.
  • the data clearly indicate that the treatment with the microbial inoculum IM2 (represented by triangles in the graph) increases the cumulative production at the end of the test by 42.41% with respect to the control, while the plot treated with the inoculum IM1 (represented by squares in the graph) initially shows lower production than the control, and subsequently exceeds it with 6.02% more accumulated production at the end of the test.
  • the results indicate that the cumulative production of the plants treated with the combination of P. fluorescens CECT 9015 and P. putida CECT 9011 strains is greater than the production with plants treated with the composition comprising P. fluorescens CECT 9015, which in turn It is higher than the production in untreated plants.
  • FIG. 10B shows classification by caliber and categories of cumulative production in each plot of the soil test.
  • the application of the IM2 composition increases the production with respect to the Witness and the treatment with IM1, favoring in a balanced way an increase in tomato production in all sizes. It is also appreciated that the majority (80.1%) of the production of the plot treated with IM2 is classified as Extra Category, the one with the highest economic value.
  • the cumulative production data of the hydroponic culture are represented in Figure 11 A.
  • the treatment with the biostimulant composition IM3 (represented by crosses in the graph) increases the accumulated production by 20.03% with respect to the witness (represented by rhombuses in the graph).
  • the distribution by categories of the accumulated production until the end of the test shows that in rock wool the percentage of tomatoes of Extra category in the sector treated with IM3 is similar to the Witness.
  • the distribution by categories indicates that with the IM3 composition 1, 47 have been obtained kg of Extra Category tomatoes more per plant than in the Witness plot, which will revert to greater benefits for the farmer.
  • compositions comprise P fluorescens CECT 9015 have a biostimulant and biofertilizing effect on a commercial tomato crop.
  • the application of the IM2 biostimulant composition significantly increases the accumulated production compared to the Witness (42.41%), predominantly tomatoes of Extra category (82.69%) and caliber Mp.
  • the results of the rock wool test indicate that the IM3 microbial inoculum (P fluorescens CECT 9015) significantly increases (20%) the accumulated production with respect to the control, also predominantly tomatoes of Extra category (84%) and Mp caliber.
  • T1 is a composition composed of the following microorganisms and coformulants: Microbial inoculum (10 8 CFU / g) consisting of Bacillus subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015, and coformulants S. cerevisiae, 40% p / p total organic carbon, 41% humic acids, 19% fulvic acids, 6.5% w / w total nitrogen, 4.5% w / w total potassium (K2O) and 1 1% w / w free amino acids. Three applications of 2 kg / ha were made.
  • Microbial inoculum (10 8 CFU / g) consisting of Bacillus subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015, and coformulants S. cerevisiae, 40% p /
  • the first application was at the time of the root flash of late summer, the second about 40-60 days after the pruning of production (beginning of spring root growth, and the third before the "pint" or envero of the Grape, at which time the fruit softens, increasing sugars and decreasing acidity, changes color and increases in size.
  • the roots of the plants were evaluated by calicatas of 1 m 2 of surface in two lines of the treated plot and two of the control plot.
  • the surface of each calicata was divided into 100 quadrants of 10 m 2 , and the number of quadrants that have roots was counted, and among these, those that have new, light, non-sub-rooted new roots.
  • Table 22 it is observed that in the plot treated with the composition T1 there is a higher percentage of quadrants with roots, and more new roots, which shows that the product stimulates root development.
  • the percentage represents the number of quadrants on the surface of 1 m 2 in which the presence of roots (total roots) and the presence of new roots were detected.
  • LINE 41 - To 54 19 To assess the dry weight of the roots, a cage was installed in the treated and control sectors. The cages are installed in the middle of each crop stop and between plants with the same phenological characteristics. The area occupied by each plant was divided into 4 quadrants, and in one of them (the same in all the plants) the cage was installed before the application of the composition. These cages were removed at the end of the trial (post harvest) to compare the development and fresh root weight. The T1 composition significantly increases the development of roots with respect to the control, increasing the weight of roots within the cage placed more than double. The data of the treated part is the average of two cages installed ( Figure 12).
  • composition comprising the strain P. fluorescens CECT 9015 promotes root development, producing a 50% increase in root surface area compared to the control and stimulates the development of new roots to values more than double in the part treated with respect to untreated culture.
  • T1 root stimulation, nutrient absorption and improvement of the quantity and quality of the harvest of an avocado crop Hass avocado variety was grown on 10-year-old trees. The test was carried out by applying two treatments (To: control and T1: composition described in example 3.3.5). For application in cultivation, T1 is dissolved in water and injected into the irrigation system. The treatment consisted of three applications of T1 at a dose of 2 kg / ha at the beginning of flowering, 30 days after the first application and 30 days after the second application (1, 2 and 3).
  • the soil was analyzed by taking a sample as close as possible to the roots, in the layer with the highest concentration of roots at different times of the test (before the application of the T1 composition, 15 days after the application, 30 days after application and at the end of the test).
  • Parameters such as electrical conductivity, pH, available phosphorus, active limestone, Dumas nitrogen, oxidizable organic matter, effective ion exchange capacity, physical properties (content in sand, clay, silt and grain size), C / N ratio, calcium, were evaluated.
  • magnesium, potassium, sodium and zinc available the distribution of these compounds and the relationship between them, calcium, magnesium, potassium, sodium and exchange bases, boron, copper (DTPA) and iron (DTPA).
  • composition T1 comprising P. fluorescens CECT 9015 increases the availability of nutrients in the soil, in particular nitrogen and potassium, increases root development, both in surface and weight and favors an increase in the number of fruits per tree 3.3.7 Test of evaluation of the biological effectiveness of a microbial inoculant in tomato cultivation.
  • the test was carried out in a tomato crop (Solanum lycopersicum var. Toro) outdoors in Yechar (Mu ⁇ a). 6 different compositions (M1, M2, M3, M4, M5 and M6) were used and 4 applications of each treatment were made via irrigation during the crop cycle, with 20 plants per repetition, in the root zone of the plants. Applications took place at intervals of approximately 15 days.
  • the sowing density was 0.5 x 2 m, 10,000 plants per ha outdoors and drip irrigation (4 l / h). It was applied at a dose of 250 liters per hectare, so the dose per plant and application was 25 ml / plant.
  • M6 P fluorescens CECT 378 + P. putida MTCC 5670 + amino acids at 0.2%.
  • the P. fluorescens CECT 378 and P putida MTCC 5670 strains are commercial strains, available in the state of the art, used herein as positive controls to assess the biostimulant capacity of the compositions of the invention.
  • the treatment that produced the greatest number of fruits was M2 (44.8 fruits / plant), while M6 was the least effective treatment (36.4 fruits / plant).
  • the maturity index was measured as the quotient Brix /% of titratable acidity. This value was obtained by measuring the volume displaced by titration with burette (25 ml) and 0.1 N NaOH.
  • the acidity evaluation in tomato juice was quantified by an acid-base titration, where the acids present in the juice were neutralized with a basic solution of sodium hydroxide. A neutralization reaction of the acid with sodium hydroxide occurs.
  • Table 27 Fruit acidity.
  • the fruits of the culture treated with the compositions containing the strain P fluorescens CECT 9015 strains are less acidic than the rest of the fruits.
  • the compositions comprising the commercial strain of P. fluorescens (M5 and M6) are more acidic, demonstrating the best effect of the claimed strain against others of the same species.
  • FIG. 16A shows the phenol content in tomato leaves measured as mg / gallic acid equivalents per 100 grams of fresh weight.
  • Figure 16B shows the flavonial content of leaves, measured as mg equivalent of catechin per 100 grams of fresh weight.
  • Figure 16A shows that compositions M2 and M5 increase the concentration of phenols in tomato leaves, while composition M2 is the one that contains the most flavonols, which confirms once more than the strain P.
  • fluorescens CECT 9015 has a better effect on plants than other strains of the same species. Flavonols, in addition to antioxidants, have a very important effect on the defense of the plant, so it is concluded that the strain P.
  • fluorescens CECT 9015 is the one with the greatest defensive potential.
  • the effects of the combination of the P. fluorescens CECT 9015 and P. putida CECT 9011 strains are also better than the combination of the respective strains of the same species described in the state of the art (P. fluorescens CECT 378 and P. MTCC putida).
  • Figure 17 shows the phenols (Figure 17) and flavonols (Figure 18) in tomato fruits in each of the three crops. While there are no differences in the phenol contents in the compositions used in this test, the content of flavonols after harvest 3 is much greater in the compositions M3 and M4. In the 0.15 mg of catechins equivalent observed after the third harvest, it represents 5 times more flavonols in these fruits, which represents an increase in their quality. From these results it is concluded that the plants to which compositions comprising the strain P.
  • fluorescens CECT 9015 are applied, alone or in combination with other microorganisms have better defensive potential, better production, their fruits ripen before and are less acidic, and They contain bioactive beneficial to the health of the consumer, which gives this strain great value, compared to other strains of the same species, which do not produce these stimulating effects on plants.
  • Plant-rhizobacteria interactions alleviate abiotic stress conditions. Plant, Cell & Environment, 32, 1682-1694.
  • Rhizosphere bacteria help plants tolérate abiotic stress. Trends in Plant Science, 14 (), 1-4.

Abstract

The present invention relates to microbial strains that promote plant growth and can be combined with co-formulants, for use as biostimulants that can be applied to various substrates in agricultural crops. According to the invention, the biostimulants are characterised in that they comprise Pseudomonas fluorescens CECT 9015 bacterium, and can also comprise other microorganisms selected from the genera Pseudomonas, Bacillus, Arthrobacter, and Trichoderma, or a combination thereof. The effect on the crop to which the composition is applied is characterised by one of the following indicators or combinations thereof: increased yield without a reduction in the size or quality of the fruit; increased number of fruits, maintaining the quality thereof; increased plant and root mass of the plant; increased photosynthetic activity of the plants in adverse conditions; and increased availability, absorption and content of nutrients in the plant.

Description

COMPOSICIONES BIOESTIMULANTES DE PLANTAS QUE COMPRENDEN CEPAS DE  BIO-STIMULATING COMPOSITIONS OF PLANTS THAT INCLUDE STATES OF
MICROORGANISMOS  MICROORGANISMS
CAMPO TÉCNICO DE LA INVENCIÓN TECHNICAL FIELD OF THE INVENTION
Biotecnología aplicada a la agricultura. Biotechnology applied to agriculture.
ANTECEDENTES BACKGROUND
La presente invención se refiere a cepas bacterianas promotoras del crecimiento vegetal, para su uso como bioestimulantes, aplicables a distintos sustratos en cultivos agrícolas. El European Biostimulants Industry Council (EBIC) y el DG Enterprise Fertilisers Working Group definieron el término bioestimulante el 14 de junio de 2012, actualizado en 2015: "Plant biostimulants contain substance(s) and/or micro-organisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance/benefit nutríent uptake, nutríent efficiency, tolerance to abiotic stress, and crop quality." (Los bioestimulantes de plantas contienen una sustancia o sustancias y/o microorganismos cuya función, cuando se aplican a plantas o a la rizosfera, es la de estimular los procesos naturales que incrementen/beneficien la eficiencia en la toma de nutrientes, la tolerancia a estrés abiótico y la calidad de la cosecha). El efecto diferenciador de la invención está basado en la acción de los microorganismos, especialmente de las bacterias promotoras del crecimiento vegetal (Plant Growth-Promoting Rhizobacteria o PGPR) que producen un efecto bioestimulante en plantas al ser aplicadas sobre las mismas en composiciones que contienen dichos microorganismos. Las PGPR son microorganismos que se desarrollan en la rizosfera y estimulan el crecimiento y desarrollo de las plantas (Kloepper et al., 1980).  The present invention relates to bacterial strains promoting plant growth, for use as biostimulants, applicable to different substrates in agricultural crops. The European Biostimulants Industry Council (EBIC) and DG Enterprise Fertilisers Working Group defined the term biostimulant on June 14, 2012, updated in 2015: "Plant biostimulants contain substance (s) and / or micro-organisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance / benefit nutríent uptake, nutríent efficiency, tolerance to abiotic stress, and crop quality. " (Plant biostimulants contain a substance or substances and / or microorganisms whose function, when applied to plants or the rhizosphere, is to stimulate natural processes that increase / benefit efficiency in nutrient intake, tolerance to abiotic stress and the quality of the harvest). The differentiating effect of the invention is based on the action of microorganisms, especially plant growth promoting bacteria (Plant Growth-Promoting Rhizobacteria or PGPR) that produce a biostimulant effect on plants when applied to them in compositions containing said microorganisms PGPRs are microorganisms that develop in the rhizosphere and stimulate the growth and development of plants (Kloepper et al., 1980).
Durante las últimas dos décadas el consumo de fertilizantes químicos ha aumentado a un ritmo constante de un 3% anual, empleándose a nivel mundial aproximadamente 100 millones de toneladas al año de fertilizantes nitrogenados y unos 90 millones de toneladas de fertilizantes fosfatados (Kang et al., 2010). Su uso continuado para incrementar la fertilidad del suelo supone una amenaza para el medio ambiente dado que puede causar daños como la lixiviación de nitratos hacia los acuíferos, lavado superficial del fósforo y nitrógeno y la eutrofización de los ecosistemas acuáticos, con el no deseable aumento de la concentración de nitrógeno en el agua que puede llegar a ser consumida por animales y seres humanos (Townsend & Howarth, 2013; Vaccari, 2013). Para mantener la productividad agrícola y preservar el medio ambiente, muchos científicos defienden que es necesario desarrollar estrategias de manejo integrado de la fertilización que incorporen el uso de microorganismos para mejorar la absorción de nutrientes y elevar la eficiencia de los fertilizantes, con lo que no sería necesario emplear cantidades tan altas de fertilizantes químicos que aporten al suelo elementos como fósforo o nitrógeno (Adesemoye & Kloepper, 2009). During the last two decades, the consumption of chemical fertilizers has increased at a constant rate of 3% per year, with approximately 100 million tons per year of nitrogen fertilizers and about 90 million tons of phosphate fertilizers being used worldwide (Kang et al. , 2010). Its continued use to increase soil fertility poses a threat to the environment since it can cause damage such as leaching of nitrates to aquifers, superficial washing of phosphorus and nitrogen and eutrophication of aquatic ecosystems, with the undesirable increase in the concentration of nitrogen in the water that can be consumed by animals and humans (Townsend & Howarth, 2013; Vaccari, 2013). To maintain agricultural productivity and preserve the environment, many scientists argue that it is necessary to develop integrated fertilization management strategies that incorporate the use of microorganisms to improve nutrient absorption and increase fertilizer efficiency, so that it would be necessary to use such high amounts of chemical fertilizers that contribute to the soil elements such as phosphorus or nitrogen (Adesemoye & Kloepper, 2009).
La rizosfera es la capa del suelo que rodea las raíces de las plantas y constituye un ecosistema con una elevada concentración de microorganismos que se establecen atraídos por los exudados radiculares que constituyen su principal fuente de alimento (Lugtenberg & Kamilova, 2009). Las plantas liberan hasta un 40% de los compuestos carbonados que asimilan a través de las raíces y los microorganismos de la rizosfera metabolizan estos compuestos, excretando otros que a su vez son aprovechados por las plantas (Kang et al., 2010). Así, se establece una interacción entre los microorganismos y las raíces de las plantas en la que ambos se ven afectados mutuamente por las moléculas secretadas por el otro. Esta interacción puede ser neutra; perjudicial (en el caso de los patógenos) o beneficiosa, como en el caso de las PGPRs (Antoun & Prévost, 2006). The rhizosphere is the layer of soil that surrounds the roots of plants and constitutes an ecosystem with a high concentration of microorganisms that are attracted by root exudates that constitute its main source of food (Lugtenberg & Kamilova, 2009). Plants release up to 40% of the carbon compounds that they assimilate through the roots and microorganisms of the rhizosphere metabolize these compounds, excreting others that in turn are used by plants (Kang et al., 2010). Thus, an interaction is established between the microorganisms and the roots of the plants in which both are mutually affected by the molecules secreted by the other. This interaction can be neutral; harmful (in the case of pathogens) or beneficial, as in the case of PGPRs (Antoun & Prévost, 2006).
La denominación PGPR incluye cepas de los géneros Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Pseudomonas y Serratia, entre otros (Choudhary, 2012). Estas bacterias se distinguen dentro de la abundante comunidad microbiana de la rizosfera porque son capaces de colonizar la superficie de las raíces de forma eficiente, se multiplican y promueven el crecimiento vegetal (Ahemad & Kribet, 2014). Gracias a estos mecanismos, la aplicación de PGPR en la rizosfera tiene un efecto favorable sobre el desarrollo y crecimiento de las plantas cultivadas. Este campo de investigación ha derivado en el desarrollo de numerosos productos comerciales a base de PGPR y, en consecuencia, el uso de inóculos microbianos en la agricultura ha aumentado considerablemente en las últimas décadas (Hayat et al, 2010). The PGPR designation includes strains of the genera Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Pseudomonas and Serratia, among others (Choudhary, 2012). These bacteria are distinguished within the abundant microbial community of the rhizosphere because they are able to colonize the surface of the roots efficiently, multiply and promote plant growth (Ahemad & Kribet, 2014). Thanks to these mechanisms, the application of PGPR in the rhizosphere has a favorable effect on the development and growth of cultivated plants. This field of research has led to the development of numerous commercial products based on PGPR and, consequently, the use of microbial inocula in agriculture has increased considerably in recent decades (Hayat et al, 2010).
La mayor parte de los suelos cultivados del mundo contienen importantes reservas de fósforo orgánico e inorgánico, pero una gran proporción está inmovilizada y es inaccesible para las plantas, que sólo pueden absorber este compuesto en las formas solubles aniónicas H2PO4" y HPO42" (Vessey, 2003). Además, entre un 75 y un 90% de estos aniones reaccionan con cationes metálicos como el hierro, el aluminio y el calcio, formando sales que precipitan o quedan adsorbidas en el propio suelo, fuera del alcance y de las posibilidades de asimilación de las plantas. El hierro es otro nutriente esencial para las plantas, que en su forma oxidada (Fe3+) frecuentemente precipita en forma de óxidos de hierro insolubles, que las plantas no pueden absorber. Se ha visto que algunas PGPR producen sus propios sideróforos bacterianos (Vessey, 2003). En los últimos años, con la búsqueda de soluciones para aumentar la tolerancia de los cultivos al estrés abiótico, se ha visto que comunidades microbianas asociadas a las plantas son capaces de conferir tolerancia al estrés abiótico mediante una serie de mecanismos como el aumento de la disponibilidad de nutrientes, la estimulación del desarrollo radicular a pesar del estrés y la reducción en la síntesis de etileno por acción de la enzima ACC desaminasa (Dimpka et al., 2009). Numerosos ensayos han demostrado que la inoculación con PGPR favorece cambios físicos y químicos en las plantas que mejoran la tolerancia al estrés abiótico. Recientemente se ha propuesto el término "tolerancia sistémica inducida" (IST) para referirse a los cambios físicos y químicos inducidos en las plantas por las PGPR, que resultan en un aumento de tolerancia al estrés abiótico de forma similar a los mecanismos activados en las respuestas de resistencia sistémica inducida (ISR) frente a patógenos (Yang et al., 2009). Most of the world's cultivated soils contain important reserves of organic and inorganic phosphorus, but a large proportion is immobilized and inaccessible to plants, which can only absorb this compound in the anionic soluble forms H2PO4 " and HPO4 2" (Vessey , 2003). In addition, between 75 and 90% of these anions react with metal cations such as iron, aluminum and calcium, forming salts that precipitate or remain adsorbed on the soil itself, outside the scope and possibilities of assimilation of plants . Iron is another essential nutrient for plants, which in its oxidized form (Fe 3+ ) frequently precipitates in the form of insoluble iron oxides, which plants cannot absorb. It has been seen that some PGPRs produce their own bacterial siderophores (Vessey, 2003). In recent years, with the search for solutions to increase the tolerance of crops to abiotic stress, it has been seen that microbial communities associated with plants are able to confer tolerance to abiotic stress through a series of mechanisms such as increased nutrient availability, stimulation of root development despite stress and reduction in ethylene synthesis by the action of ACC enzyme deaminase (Dimpka et al., 2009). Numerous trials have shown that PGPR inoculation favors physical and chemical changes in plants that improve tolerance to abiotic stress. Recently the term "systemic induced tolerance" (IST) has been proposed to refer to physical and chemical changes induced in plants by PGPRs, which result in an increase in tolerance to abiotic stress similar to the mechanisms activated in responses of induced systemic resistance (ISR) against pathogens (Yang et al., 2009).
La inoculación con PGPR puede reducir el estrés por salinidad en distintas especies de plantas y también aliviar el estrés causado por temperaturas extremas (Choudhary, 2012). Por otro lado, los ensayos realizados inoculando distintas especies de cultivos con PGPR para aumentar la tolerancia a condiciones de frío sugieren que las bacterias podrían aumentar la concentración de azúcares, prolina y antocianinas, entre otros metabolitos, en los tejidos vegetales, favoreciendo la aclimatación de las plantas (Dimpka et al., 2009). PGPR inoculation can reduce salinity stress in different plant species and also relieve stress caused by extreme temperatures (Choudhary, 2012). On the other hand, the tests carried out inoculating different species of crops with PGPR to increase tolerance to cold conditions suggest that bacteria could increase the concentration of sugars, proline and anthocyanins, among other metabolites, in plant tissues, favoring acclimatization of the plants (Dimpka et al., 2009).
Se prevé que la población mundial alcance la cifra de 9 millones de personas en 2050, y para hacer frente a la demanda creciente de alimentos, la producción agrícola tendría que aumentar en un 70% (Coleman-Derr & Tringe, 2014). Sin embargo, la disponibilidad de tierras aptas para el cultivo es limitada y, con el efecto del cambio climático, se prevé un aumento de la sequía, la salinidad y otros factores de estrés abiótico que podrían reducir la producción agrícola y amenazar la seguridad alimentaria global (Grover et al., 2011). Por ello, el uso de inóculos microbianos es una solución biológica, económica, simple y a corto plazo, para la gestión del estrés abiótico en los cultivos y para mejorar la eficiencia del uso de nutrientes. La presente invención se refiere a composiciones bioestimulantes que comprenden microorganismos para su uso en agricultura. Las composiciones existentes en el estado del arte empleadas en agricultura se indican en la Tabla 2 del documento Bashan et al., 2014. Además, los autores de dicho documento subrayan que los inóculos deben ser fáciles de usar, compatibles con las prácticas habituales de la finca, resistentes al almacenamiento, capaces de funcionar bajo distintas condiciones de suelo y de clima, capaces de que las bacterias sobrevivan durante el tiempo necesario para ejercer un efecto beneficioso sobre la planta, con resultados reproducibles en condiciones de campo y seguros para los seres humanos, los animales y el medio ambiente. Las composiciones descritas en la presente memoria cumplen con todos los puntos descritos anteriormente. DESCRIPCIÓN DE LA INVENCIÓN The world population is expected to reach 9 million people by 2050, and to cope with the growing demand for food, agricultural production would have to increase by 70% (Coleman-Derr & Tringe, 2014). However, the availability of land suitable for cultivation is limited and, with the effect of climate change, an increase in drought, salinity and other abiotic stress factors that could reduce agricultural production and threaten global food security is expected. (Grover et al., 2011). Therefore, the use of microbial inoculums is a biological, economic, simple and short-term solution for the management of abiotic stress in crops and to improve the efficiency of nutrient use. The present invention relates to biostimulant compositions comprising microorganisms for use in agriculture. The existing compositions in the state of the art used in agriculture are indicated in Table 2 of the document Bashan et al., 2014. In addition, the authors of said document stress that the inoculums should be easy to use, compatible with the usual practices of the farm, resistant to storage, capable of operating under different soil and weather conditions, capable of bacteria surviving for the time necessary to exert a beneficial effect on the plant, with reproducible results in field conditions and safe for humans , animals and the environment. The compositions described herein comply with all the points described above. DESCRIPTION OF THE INVENTION
BREVE DESCRIPCIÓN DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
La presente invención se refiere a una composición bioestimulante de plantas que comprende la cepa Pseudomonas fluorescens CECT 9015. The present invention relates to a plant biostimulant composition comprising the strain Pseudomonas fluorescens CECT 9015.
En una realización preferida, dicha composición además comprende al menos un segundo microorganismo seleccionado entre los géneros Pseudomonas, Bacillus, Arthrobacter, Tríchoderma o una combinación de los mismos. En una realización más preferida, el segundo microorganismo del género Pseudomonas presente en la composición es Pseudomonas pulida. In a preferred embodiment, said composition further comprises at least a second microorganism selected from the genera Pseudomonas, Bacillus, Arthrobacter, Tríchoderma or a combination thereof. In a more preferred embodiment, the second microorganism of the genus Pseudomonas present in the composition is polished Pseudomonas.
En una realización más preferida, la composición bioestimulante promotora del crecimiento de las plantas descrita anteriormente, comprende, además de la cepa CECT 9015, al menos otra cepa seleccionada entre: Bacillus subtilis CECT 9016, Pseudomonas putida CECT 901 1 , Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Tríchoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, o combinaciones de las mismas. La composición descrita en la presente invención se caracteriza por estar en forma sólida o líquida. In a more preferred embodiment, the plant growth promoting biostimulant composition described above comprises, in addition to strain CECT 9015, at least one other strain selected from: Bacillus subtilis CECT 9016, Pseudomonas putida CECT 901 1, Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Tríchoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, or combinations thereof. The composition described in the present invention is characterized by being in solid or liquid form.
La composición descrita en la presente invención puede además comprender, adicionalmente, al menos un coformulante. El coformulante se selecciona del grupo que consiste en: abonos, abonos compuestos por los elementos nitrógeno, fósforo y/o potasio y sus combinaciones dobles o triples, productos fertilizantes, polisorbatos asociados a ácidos grasos, asparagina, manitol, ácidos orgánicos, medio CAS, medio nutritivo para el cultivo de bacterias, sustancias quelantes de macro y microelementos nutricionales, algas o sus extractos y levaduras.  The composition described in the present invention may further comprise, in addition, at least one coformulant. The coformulant is selected from the group consisting of: fertilizers, fertilizers composed of the elements nitrogen, phosphorus and / or potassium and their double or triple combinations, fertilizer products, polysorbates associated with fatty acids, asparagine, mannitol, organic acids, CAS medium, nutritive medium for the cultivation of bacteria, macro chelating substances and nutritional microelements, algae or their extracts and yeasts.
Además, la presente invención se refiere a un método para estimular las plantas cultivadas que consiste en aplicar en las mismas, cualquiera de las composiciones descritas anteriormente. El efecto estimulante sobre el cultivo al que se aplica la composición descrita anteriormente, se mide por cualquiera de los parámetros, o por una combinación de los mismos, seleccionados entre los que consisten en: incremento de la producción sin reducir el tamaño o calibre del fruto, incremento del número de frutos manteniendo la calidad de los mismos, incremento de la masa vegetal y radicular, incremento de la actividad fotosintética de los vegetales en condiciones adversas, incremento de la disponibilidad, absorción y contenido de potasio (K) y/o de hierro (Fe), y/o de fósforo (P), y/o de nitrógeno (N), y/o de otros nutrientes como el magnesio (Mg), el zinc (Zn) o el boro (B), por mecanismos que incrementan la solubilidad del elemento, y/o su disponibilidad, y/o su absorción a través de la membrana. El método para estimular las plantas cultivadas de la presente invención mediante la composición descrita anteriormente se aplica preferentemente en forma líquida y en cultivo hidropónico o en cultivo sobre suelo. Preferentemente, la composición de la invención, se aplica, bien como un polvo sólido o bien como un líquido, en ambos casos, que se disuelve en el agua de riego. In addition, the present invention relates to a method for stimulating cultivated plants which consists in applying in them, any of the compositions described above. The stimulating effect on the crop to which the composition described above is applied, is measured by any of the parameters, or by a combination thereof, selected from among those consisting of: increased production without reducing the size or size of the fruit , increase in the number of fruits maintaining their quality, increase in plant and root mass, increase in photosynthetic activity of vegetables in adverse conditions, increase in the availability, absorption and content of potassium (K) and / or iron (Fe), and / or phosphorus (P), and / or nitrogen (N), and / or other nutrients such as magnesium (Mg), zinc (Zn) or boron (B), by mechanisms which increase the solubility of the element, and / or its availability, and / or its absorption through the membrane. The method for stimulating the cultivated plants of the present invention by the composition described above is preferably applied in liquid form and in hydroponic culture or in soil cultivation. Preferably, the composition of the invention is applied, either as a solid powder or as a liquid, in both cases, which dissolves in the irrigation water.
Además, la presente invención se refiere al uso de las composiciones descritas anteriormente que comprenden Pseudomonas fluorescens CECT 9015 para la fabricación de bioestimulantes de plantas.  In addition, the present invention relates to the use of the compositions described above comprising Pseudomonas fluorescens CECT 9015 for the manufacture of plant biostimulants.
Adicionalmente, dicho uso de la cepa Pseudomonas fluorescens CECT 9015 para fabricar bioestimulantes de plantas se puede emplear con al menos un segundo microorganismo seleccionado entre los géneros Pseudomonas, Bacillus, Arthrobacter, Tríchoderma, o una combinación de los mismos. En una realización preferida, el segundo microorganismo es Pseudomonas putida. En una realización más preferida, el uso de Pseudomonas fluorescens CECT 9015 para la fabricación de las composiciones descritas anteriormente, comprende al menos una cepa seleccionada entre: Bacillus subtilis CECT 9016, Pseudomonas putida CECT 901 1 , Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Tríchoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, o combinaciones de las mismas.  Additionally, said use of the strain Pseudomonas fluorescens CECT 9015 to manufacture plant biostimulants can be used with at least a second microorganism selected from the genera Pseudomonas, Bacillus, Arthrobacter, Tríchoderma, or a combination thereof. In a preferred embodiment, the second microorganism is Pseudomonas putida. In a more preferred embodiment, the use of Pseudomonas fluorescens CECT 9015 for the manufacture of the compositions described above comprises at least one strain selected from: Bacillus subtilis CECT 9016, Pseudomonas putida CECT 901 1, Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018 , Tríchoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, or combinations thereof.
DESCRIPCIÓN DE LAS FIGURAS Figura 1. Ensayo de producción de sideróforos en placa. La cepa BB17B (Pseudomonas fluorescens CECT 9015), es capaz de crecer en medio CAS y producir sideróforos, dando lugar a un viraje de color a amarillo/naranja en la zona de crecimiento de la colonia. DESCRIPTION OF THE FIGURES Figure 1. Production test of plate siderophores. The strain BB17B (Pseudomonas fluorescens CECT 9015), is capable of growing in CAS medium and producing siderophores, resulting in a yellow / orange color shift in the growth zone of the colony.
Figura 2. Ensayo de movilización de potasio in vitro. Halos de hidrólisis de la cepa BB17B (Pseudomonas fluorescens CECT 9015) y de la combinación de las cepas BB17B y BB17F (Pseudomonas putida CECT 901 1). Se observa que la combinación de las dos cepas tiene mayor efecto que la cepa BB17B sola. La combinación de ambas cepas tiene un sinérgico, favoreciendo la absorción de potasio. Figure 2. Potassium mobilization assay in vitro. Hydrolysis halos of strain BB17B (Pseudomonas fluorescens CECT 9015) and of the combination of strains BB17B and BB17F (Pseudomonas putida CECT 901 1). It is observed that the combination of the two strains has a greater effect than the BB17B strain alone. The combination of both strains has a synergistic one, favoring the absorption of potassium.
Figura 3. Ensayo de solubilización de fósforo en plantas de pepino cultivadas en maceta según cada tratamiento (Control y cepa P. fluorescens CECT 9015 (BB17B) en presencia de fósforo soluble (Ps) o insoluble (Pi)). A. Peso seco (g) de las plantas de pepino. Las columnas representan la media de 17-21 plantas por tratamiento. Las barras sobre las columnas representan el error estándar y las diferentes letras (a y b) denotan valores con diferencias estadísticamente significativas según el test de Tukey (P≤0,05) El peso de la parte aérea corresponde a las barras blancas, mientras que el peso de la parte radical corresponde a las barras negras. B. Fotosíntesis neta. Las columnas representan la media de 17-21 plantas por tratamiento. Las barras sobre las columnas representan el error estándar, y las diferentes letras (a y b) denotan valores con diferencias estadísticamente significativas según el test de Tukey (P≤0,05). C. Contenido de fósforo en hoja. Las columnas representan el contenido de fósforo (mg) en el total de tejido foliar (g) de todas las plantas correspondientes a cada tratamiento. Figure 3. Phosphorus solubilization test in potted cucumber plants according to each treatment (Control and strain P. fluorescens CECT 9015 (BB17B) in the presence of soluble (Ps) or insoluble (Pi) phosphorus). A. Dry weight (g) of cucumber plants. The columns represent the average of 17-21 plants per treatment. The bars on the columns represent the standard error and the different letters (a and b) denote values with statistically significant differences according to the Tukey test (P≤0.05). The weight of the aerial part corresponds to the white bars, while the weight of the radical part corresponds to the black bars. B. Net photosynthesis. The columns represent the average of 17-21 plants per treatment. The bars on the columns represent the standard error, and the different letters (a and b) denote values with statistically significant differences according to the Tukey test (P≤0.05). C. Phosphorus content in leaf. The columns represent the phosphorus content (mg) in the total foliar tissue (g) of all the plants corresponding to each treatment.
Figura 4. Parámetros fotosintéticos medidos en el ensayo de solubilización y absorción de fósforo plantas correspondientes a cada tratamiento (Control y cepa P. fluorescens CECT 9015 (BB17B) en presencia de fósforo soluble o insoluble). A. Fo es la emisión de fluorescencia bajo luz suave representativo del estado del fotosistema; B. Fv/Fm indica la capacidad potencial máxima de canalizar la energía a la fotosíntesis que tiene el Fotosistema II; C. 0PSII es la capacidad real del Fotosistema II y D. NPQ es la cantidad de energía que disipa el Fotosistema II. Las barras sobre las columnas representan el error estándar y las diferentes letras (a, b y c) denotan valores con diferencias estadísticamente significativas según el test de Tukey (P≤0,05). Figura 5. Efecto de las composiciones bioestimulantes en el incremento de la tolerancia al estrés abiótico: Condiciones de alta salinidad y sequía. Las composiciones empleadas son la cepa P. fluorescens CECT 9015, la combinación de las cepas P. fluorescens CECT 9015 y P. putida CECT 9011 , y el testigo. Las columnas de color negro corresponden a las condiciones de riego normal, las columnas de color blanco corresponden a las condiciones de estrés salino y las de color gris corresponden a las condiciones de sequía. Cada columna representa la media de 5 plantas por tratamiento y las barras representan el error estándar. A. Daños en el sistema foliar de las plantas en el momento de la cosecha. B. Desarrollo del sistema radicular de las plantas en el momento de la cosecha. Figure 4. Photosynthetic parameters measured in the phosphorus solubilization and absorption test plants corresponding to each treatment (Control and strain P. fluorescens CECT 9015 (BB17B) in the presence of soluble or insoluble phosphorus). A. Fo is the fluorescence emission under soft light representative of the state of the photosystem; B. Fv / Fm indicates the maximum potential capacity to channel the energy to photosynthesis that Photosystem II has; C. 0PSII is the actual capacity of Photosystem II and D. NPQ is the amount of energy that dissipates Photosystem II. The bars on the columns represent the standard error and the different letters (a, b and c) denote values with statistically significant differences according to the Tukey test (P≤0.05). Figure 5. Effect of biostimulant compositions on increasing tolerance to abiotic stress: Conditions of high salinity and drought. The compositions employed are P. fluorescens CECT 9015 strain, the combination of P. fluorescens CECT 9015 and P. putida CECT 9011 strains, and the control strain. Black columns correspond to normal irrigation conditions, white columns correspond to saline stress conditions and gray columns correspond to drought conditions. Each column represents the average of 5 plants per treatment and the bars represent the standard error. A. Damage to the leaf system of plants at the time of harvest. B. Development of the root system of plants at the time of harvest.
Figura 6. Efecto de las composiciones bioestimulantes en la fotosíntesis de las plantas en condiciones de estrés salino. Las composiciones ensayadas son: M1 (Control: Solución de aminoácidos 0.2%), M2 (P. fluorescens CECT 9015 108 UFC/g + aminoácidos 0.2%), M3 (P fluorescens CECT 9015 + P putida CECT 9011 , ambas a 108 UFC/g+ aminoácidos 0.2%), M4 (P fluorescens CECT 9015 + P. putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, todas a 108 UFC/g + aminoácidos 0.2%) y M5 (P. fluorescens CECT 378 + aminoácidos 0.2%). A. Fo es la emisión de fluorescencia bajo luz suave representativo del estado del fotosistema; B. Fv/Fm indica la capacidad potencial máxima de canalizar la energía a la fotosíntesis que tiene el Fotosistema II; C. (PPSII es la capacidad real del Fotosistema II y D. NPQ es la cantidad de energía que disipa el Fotosistema II. Las barras sobre las columnas representan el error estándar y las diferentes letras (a, b y c) denotan valores con diferencias estadísticamente significativas según el test de LSD (P≤0,05). Figure 6. Effect of biostimulant compositions on plant photosynthesis under conditions of saline stress. The compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P fluorescens CECT 9015 + P put CECT 9011, both at 10 8 CFU / g + amino acids 0.2%), M4 (P fluorescens CECT 9015 + P. putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, all at 10 8 CFU / g + 0.2% amino acids) and M5 (P. fluorescens CECT 378 + 0.2% amino acids). A. Fo is the fluorescence emission under soft light representative of the state of the photosystem; B. Fv / Fm indicates the maximum potential capacity to channel the energy to photosynthesis that Photosystem II has; C. ( PPSII is the actual capacity of Photosystem II and D. NPQ is the amount of energy dissipated by Photosystem II. The bars on the columns they represent the standard error and the different letters (a, b and c) denote values with statistically significant differences according to the LSD test (P≤0.05).
Figura 7. Potencial hídrico en las plantas de tomate tratadas con diferentes composiciones en condiciones de salinidad (500 mM). Las composiciones ensayadas son: M1 (Control: Solución de aminoácidos 0.2%), M2 (P. fluorescens CECT 9015 108 UFC/g + aminoácidos 0.2%), M3 (P fluorescens CECT 9015 + P. pulida CECT 9011 , ambas a 108 UFC/g+ aminoácidos 0.2%), M4 (P. fluorescens CECT 9015 + P. putida CECT 9011 + B. subtilis CECTFigure 7. Water potential in tomato plants treated with different compositions under salinity conditions (500 mM). The compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P fluorescens CECT 9015 + P. polished CECT 9011, both at 10 8 CFU / g + amino acids 0.2%), M4 (P. fluorescens CECT 9015 + P. putida CECT 9011 + B. subtilis CECT
9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, todas a 108 UFC/g + aminoácidos 0.2%) y M5 (P. fluorescens CECT 378 + aminoácidos 0.2%). Las barras sobre las columnas representan el error estándar y las diferentes letras (a, b, c y d) denotan valores con diferencias estadísticamente significativas según el test de LSD (P≤0,05). 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, all at 10 8 CFU / g + amino acids 0.2%) and M5 (P. fluorescens CECT 378 + amino acids 0.2% ). The bars on the columns represent the standard error and the different letters (a, b, c and d) denote values with statistically significant differences according to the LSD test (P≤0.05).
Figura 8. Concentración de prolina (mg/g planta) de las plantas de tomate tratadas con diferentes composiciones en condiciones de salinidad (500 mM). Las composiciones ensayadas son: M1 (Control: Solución de aminoácidos 0.2%), M2 (P. fluorescens CECT 9015 108 UFC/g + aminoácidos 0.2%), M3 (P. fluorescens CECT 9015 + P. putida CECT 901 1 , ambas a 108 UFC/g+ aminoácidos 0.2%), M4 (P fluorescens CECT 9015 + P putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECTFigure 8. Proline concentration (mg / g plant) of tomato plants treated with different compositions under salinity conditions (500 mM). The compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P. fluorescens CECT 9015 + P. putida CECT 901 1, both at 10 8 CFU / g + amino acids 0.2%), M4 (P fluorescens CECT 9015 + P putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT
9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, todas a 108 UFC/g + aminoácidos 0.2%) y M5 (P fluorescens CECT 378 + aminoácidos 0.2%). Las barras sobre las columnas representan el error estándar y las diferentes letras (a, b, c, d y e) denotan valores con diferencias estadísticamente significativas según el test de LSD (P≤0,05). 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, all at 10 8 CFU / g + amino acids 0.2%) and M5 (P fluorescens CECT 378 + amino acids 0.2%). The bars on the columns represent the standard error and the different letters (a, b, c, dye) denote values with statistically significant differences according to the LSD test (P≤0.05).
Figura 9. Desarrollo radicular de dos plantas de apio tomadas al azar de cada parcela. Las raíces de la planta tratada con la composición que comprende la cepa P. fluorescens BB17B (CECT 9015) (derecha), tienen mayor longitud y superficie de absorción de nutrientes que las del testigo (izquierda). Figure 9. Root development of two celery plants taken at random from each plot. The roots of the plant treated with the composition comprising the strain P. fluorescens BB17B (CECT 9015) (right), have a greater length and surface area of nutrient absorption than those of the control (left).
Figura 10. A. Producción acumulada por planta de tomate en el ensayo de suelo. Los datos representan la producción total de la parcela hasta cada fecha, dividida entre el número total de plantas de la parcela (3157) para cada uno de los tratamientos: IM1 (P fluorescens CECT 9015 y los coformulantes S. cerevisiae, ácidos húmicos y aminoácidos 2 kg/ha) representado por cuadrados, IM2 (IM1 + P putida CECT 901 1 2 kg/ha) representado por triángulos, y el Testigo (representado por rombos). B. Distribución de la producción en las tres parcelas por calibres y categorías. Cada columna representa el total de kg de tomate recolectados por planta de cada uno de los calibres (GG, G, Mg, Mp, MMg, MMp y MMM). Para la composición IM1 , el tramo blanco corresponde a los frutos de categoría Extra X, y la zona rallada al resto. Para la composición IM2, el tramo de color negro corresponde con los frutos de categoría Extra X, y la zona rallada al resto. Para la composición Testigo, el tramo gris corresponde a los frutos de categoría Extra X y la zona rallada al resto. Figure 10. A. Cumulative production per tomato plant in the soil test. The data represent the total production of the plot to date, divided by the total number of plants in the plot (3157) for each of the treatments: IM1 (P fluorescens CECT 9015 and the coformulants S. cerevisiae, humic acids and amino acids 2 kg / ha) represented by squares, IM2 (IM1 + P put CECT 901 1 2 kg / ha) represented by triangles, and the Witness (represented by rhombuses). B. Distribution of production in the three plots by size and category. Each column represents the total kg of tomato collected per plant of each of the calibers (GG, G, Mg, Mp, MMg, MMp and MMM). For the IM1 composition, the white section corresponds to the fruits of Extra X category, and the area grated to the rest. For the IM2 composition, the black section corresponds to the fruits of Extra X category, and the area grated to the rest. For the Witness composition, the gray section corresponds to the fruits of Extra X category and the grated area to the rest.
Figura 11. A. Producción acumulada por planta de tomate en el ensayo sobre sustrato hidropónico de lana de roca. Los datos representan la producción total de la parcela hasta cada fecha, dividida entre el número total de plantas de la parcela, para cada uno de los tratamientos: IM3 (P. fluorescens CECT 9015 y los coformulantes S. cerevisiae, ácidos húmicos y aminoácidos 200 g/ha) representado por asteriscos y el Testigo representado por rombos. B. Distribución de la producción por calibres y categorías. Cada grupo de columnas representa el total de kg de tomate recolectados por planta de cada uno de los calibres (GG, G, Mg, Mp, MMg, MMp, y MMM). Para la composición IM3, el tramo de color negro corresponde con los frutos de categoría Extra X, y la zona blanca al resto. Para la composición Testigo, el tramo gris claro corresponde a los frutos de categoría Extra X y la zona gris oscuro al resto. Figura 12. Peso de raíces (g) de una planta representativa muestreada al azar en la parcela tratada con la composición T1 (a la izquierda, compuesta por 108 ufc/g de B. subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015 y S. cerevisiae, 40% p/p de carbono orgánico total, 41 % ácidos húmicos, 19% ácidos fúlvicos, 6.5% p/p nitrógeno total, 4.5% p/p potasio total y 11 % p/p aminoácidos libres) y en la parcela testigo To sin tratamiento (derecha, columna blanca). Figure 11. A. Cumulative production per tomato plant in the hydroponic rock wool substrate test. The data represent the total production of the plot to date, divided by the total number of plants in the plot, for each of the treatments: IM3 (P. fluorescens CECT 9015 and the coformulants S. cerevisiae, humic acids and amino acids 200 g / ha) represented by asterisks and the Witness represented by rhombuses. B. Distribution of production by size and category. Each group of columns represents the total kg of tomato collected per plant of each of the calibers (GG, G, Mg, Mp, MMg, MMp, and MMM). For the IM3 composition, the black section corresponds to the fruits of Extra X category, and the white zone to the rest. For the Witness composition, the light gray section corresponds to the fruits of Extra X category and the dark gray area to the rest. Figure 12. Root weight (g) of a randomly sampled representative plant on the plot treated with the composition T1 (on the left, composed of 10 8 cfu / g of B. subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas CECT 9015 and S. cerevisiae fluorescens, 40% w / w total organic carbon, 41% humic acids, 19% fulvic acids, 6.5% w / w total nitrogen, 4.5% w / w total potassium and 11% w / w amino acids free) and in the control plot To without treatment (right, white column).
Figura 13. Efecto de la composición T1 (108 ufc/g de B. subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015 y S. cerevisiae, 40% p/p de carbono orgánico total, 41 % ácidos húmicos, 19% ácidos fúlvicos, 6.5% p/p nitrógeno total, 4.5% p/p potasio total y 11 % p/p aminoácidos libres) en plantas de aguacate. El número de aplicaciones corresponden a los números 1 , 2 y 3 en el eje X. Los datos de la parcela tratada con la composición T1 se representan mediante rombos y los datos de la parcela testigo mediante cuadrados. A. Fósforo disponible (mg/kg). B. Nitrógeno Dumas (mg/kg). C. Potasio disponible (meq/100g). D. Calcio disponible (meq/100g). E. Magnesio disponible (meq/100g). F. Hierro (DPTA) (mg/kg). G. Capacidad de intercambio catiónico efectiva (meq/100 g). H. Materia orgánica oxidable (%). Figure 13. Effect of composition T1 (10 8 cfu / g of B. subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015 and S. cerevisiae, 40% w / w total organic carbon, 41% humic acids , 19% fulvic acids, 6.5% w / w total nitrogen, 4.5% w / w total potassium and 11% w / w free amino acids) in avocado plants. The number of applications correspond to the numbers 1, 2 and 3 on the X axis. The data of the plot treated with the composition T1 is represented by rhombuses and the data of the control plot by squares. A. Available phosphorus (mg / kg). B. Nitrogen Dumas (mg / kg). C. Available potassium (meq / 100g). D. Calcium available (meq / 100g). E. Available magnesium (meq / 100g). F. Iron (DPTA) (mg / kg). G. Effective cation exchange capacity (meq / 100 g). H. Oxidizable organic matter (%).
Figura 14. Número de frutos por árbol de aguacate. Las columnas representan la media de 10 árboles en cada parcela y las barras representan el error estándar después del tratamiento con T1 (columna de color negro) o el Testigo (columna de color blanco). Figura 15. Parámetros fotosintéticos medidos en el ensayo de tomate en campo. Las composiciones ensayadas son: M1 (Control: Solución de aminoácidos 0.2%), M2 (P fluorescens CECT 9015 108 UFC/g + aminoácidos 0.2%), M3 (P. fluorescens CECT 9015 + P putida CECT 9016, ambas a 108 UFC/g+ aminoácidos 0.2%), M4 (P fluorescens CECT 9015 + P putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, todas a 108 UFC/g + aminoácidos 0.2%), M5 (P fluorescens CECT 378 + aminoácidos 0.2%) y M6 (P fluorescens CECT 378 + P. putida MTCC 5670 + aminoácidos a 0.2%). A. Fo es la emisión de fluorescencia bajo luz suave representativo del estado del fotosistema; B. Fv/Fm indica la capacidad potencial máxima de canalizar la energía a la fotosíntesis que tiene el Fotosistema II; C. 0PSII es la capacidad real del Fotosistema II y D. NPQ es la cantidad de energía que disipa el Fotosistema II. Las barras sobre las columnas representan el error estándar y las diferentes letras (a, b, c y d) denotan valores con diferencias estadísticamente significativas según el test de LSD (P≤0,05). Figura 16. Compuestos bioactivos en hoja de tomate en plantas tratadas con las composiciones del ejemplo anterior. A. Fenoles (Equivalentes mg de Ácido gálico en 100 g de peso fresco). B. Flavonoles (Equivalentes mg de catequina en 100 mg de peso fresco). Las barras sobre las columnas representan el error estándar y las diferentes letras (a, b, c, d, f y g) denotan valores con diferencias estadísticamente significativas según el test de LSD (P≤0,05). Figure 14. Number of fruits per avocado tree. The columns represent the average of 10 trees in each plot and the bars represent the standard error after treatment with T1 (black column) or the Witness (white column). Figure 15. Photosynthetic parameters measured in the tomato field test. The compositions tested are: M1 (Control: 0.2% amino acid solution), M2 (P fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%), M3 (P. fluorescens CECT 9015 + P put CECT 9016, both at 10 8 CFU / g + amino acids 0.2%), M4 (P fluorescens CECT 9015 + P putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946 , all at 10 8 CFU / g + amino acids 0.2%), M5 (P fluorescens CECT 378 + amino acids 0.2%) and M6 (P fluorescens CECT 378 + P. putida MTCC 5670 + amino acids at 0.2%). A. Fo is the fluorescence emission under soft light representative of the state of the photosystem; B. Fv / Fm indicates the maximum potential capacity to channel the energy to photosynthesis that Photosystem II has; C. 0PSII is the actual capacity of Photosystem II and D. NPQ is the amount of energy that dissipates Photosystem II. The bars on the columns represent the standard error and the different letters (a, b, c and d) denote values with statistically significant differences according to the LSD test (P≤0.05). Figure 16. Bioactive compounds in tomato leaf in plants treated with the compositions of the previous example. A. Phenols (Equivalent mg of Gallic acid in 100 g of fresh weight). B. Flavonols (Equivalent mg of catechin in 100 mg of fresh weight). The bars on the columns represent the standard error and the different letters (a, b, c, d, f and g) denote values with statistically significant differences according to the LSD test (P≤0.05).
Figura 17. Compuestos bioactivos (fenoles) en hoja de tomate después de cada una de las cosechas tratadas con las composiciones del ejemplo anterior, medidos como Equivalentes mg de Ácido gálico en 100 g de peso fresco. Figure 17. Bioactive compounds (phenols) in tomato leaf after each of the crops treated with the compositions of the previous example, measured as Equivalent mg of Gallic acid in 100 g of fresh weight.
Figura 18. Compuestos bioactivos (flavonoles) en hoja de tomate después de cada una de las cosechas tratadas con las composiciones del ejemplo anterior, medidos como Equivalentes mg de catequina en 100 mg de peso fresco. Figure 18. Bioactive compounds (flavonols) in tomato leaf after each of the crops treated with the compositions of the previous example, measured as Equivalent mg of catechin in 100 mg of fresh weight.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN 1. Definiciones A efectos de la presente invención se hacen constar los siguientes términos: DETAILED DESCRIPTION OF THE INVENTION 1. Definitions For the purposes of the present invention the following terms are stated:
- Producto fertilizante: producto utilizado en agricultura o jardinería que facilita el crecimiento de las plantas, aumenta su rendimiento y mejora la calidad de las cosechas o que, por su acción específica, modifica la fertilidad del suelo o sus características físicas, químicas o biológicas. Los fertilizantes incluyen abonos, cuya función principal es proporcionar elementos nutrientes a las plantas (Real Decreto del Reino de España 506/2013). - Fertilizer product: product used in agriculture or gardening that facilitates the growth of plants, increases their yield and improves the quality of the crops or that, by their specific action, modifies the fertility of the soil or its physical, chemical or biological characteristics. Fertilizers include fertilizers, whose main function is to provide nutrients to plants (Royal Decree of the Kingdom of Spain 506/2013).
Biofertilizante: Producto biológico que contiene microorganismos vivos que, cuando se aplican a semillas, superficies de plantas o cultivos, promueven su proceso natural de nutrición, aumentando la capacidad de la planta para la absorción de nutrientes, estimulando el crecimiento y protegiendo a las plantas frente a patógenos.  Biofertilizer: Biological product that contains live microorganisms that, when applied to seeds, plant surfaces or crops, promote their natural process of nutrition, increasing the capacity of the plant for the absorption of nutrients, stimulating growth and protecting plants against to pathogens
Composición bioestimulante o composición estimulante: a efectos de la presente invención, se define como aquella composición biofertilizante que produce en la planta en la cual se aplica un incremento de la producción en un 10-45% sin reducir el tamaño o calibre del fruto, y/o un incremento del número de frutos en un 1 1-40% manteniendo su calidad, y/o un incremento de la masa vegetal y radicular en un 5-35%, y/o un incremento de la actividad fotosintética de los vegetales en condiciones adversas en un 4-30%, y/o un incremento de la disponibilidad, absorción y contenido de potasio (K) en un 2-21 %, y/o de hierro (Fe) en un 11-100%, y/o de fósforo (P) en un 6-40%, y/o de nitrógeno (N) en un 5-25%, y/o de otros nutrientes como el magnesio (Mg), el zinc (Zn) o el boro (B). Biostimulant composition or stimulant composition: for the purposes of the present invention, it is defined as that biofertilizer composition produced in the plant in which an increase in production is applied by 10-45% without reducing the size or size of the fruit, and / or an increase in the number of fruits by 1 1-40% while maintaining their quality, and / or an increase in plant and root mass by 5-35%, and / or an increase in the photosynthetic activity of vegetables in adverse conditions by 4-30%, and / or an increase in the availability, absorption and content of potassium (K) by 2-21%, and / or iron (Fe) by 11-100%, and / or phosphorus (P) in 6-40%, and / or nitrogen (N) in 5-25%, and / or other nutrients such as magnesium (Mg), zinc (Zn) or boron ( B).
Coformulante: Sustancia o preparado que se usa o está destinado a usarse en un producto adicional al principio activo. En la presente invención se refiere a cualquier compuesto distinto a los microorganismos PGPR presente en la composición.  Co-formulant: A substance or preparation that is used or is intended to be used in an additional product to the active substance. In the present invention it refers to any compound other than PGPR microorganisms present in the composition.
Nutriente: elemento químico esencial para la vida vegetal y el crecimiento de las plantas. Además del carbono (C), el oxígeno (O) y el hidrógeno (H), procedentes especialmente del aire y del agua, los elementos nutrientes se clasifican en: nutrientes principales, nutrientes secundarios y micronutrientes Nutrient: chemical element essential for plant life and plant growth. In addition to carbon (C), oxygen (O) and hydrogen (H), especially from air and water, the nutrient elements are classified into: main nutrients, secondary nutrients and micronutrients
Macroelementos primarios o nutrientes principales: Compuestos químicos que los organismos absorben en grandes cantidades y por lo tanto constituyen sus nutrientes principales. Corresponden al nitrógeno (N), fósforo (P) y potasio (K).  Primary macroelements or main nutrients: Chemical compounds that organisms absorb in large quantities and therefore constitute their main nutrients. They correspond to nitrogen (N), phosphorus (P) and potassium (K).
Macroelementos secundarios o nutrientes secundarios: Compuestos químicos que constituyen los nutrientes secundarios de las plantas. Corresponden al calcio (Ca), magnesio (Mg), sodio (Na) y azufre (S). Secondary macroelements or secondary nutrients: Chemical compounds that constitute the secondary nutrients of plants. They correspond to calcium (Ca), magnesium (Mg), sodium (Na) and sulfur (S).
Medio CAS: Medio de cultivo bacteriano que incorpora el compuesto cromo azurol S (CAS) para la detección de sideróforos (Alexander & Zuberer, 1991).  CAS medium: Bacterial culture medium that incorporates the compound chromium azurol S (CAS) for the detection of siderophores (Alexander & Zuberer, 1991).
Micronutrientes: Compuestos químicos esenciales para el crecimiento de las plantas en pequeñas cantidades. Corresponden al boro (B), cobalto (Co), cobre (Cu), hierro (Fe), manganeso (Mn), molibdeno (Mo) y zinc (Zn). - Sideróforo: moléculas orgánicas capaces de unirse a los cationes Fe3+ y transportarlos hasta la pared celular bacteriana, donde estos se reducen a cationes Fe2+ que pueden ser absorbidos tanto por las bacterias como por las plantas. Micronutrients: Chemical compounds essential for plant growth in small quantities. They correspond to boron (B), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo) and zinc (Zn). - Siderophore: organic molecules capable of binding to Fe 3+ cations and transporting them to the bacterial cell wall, where they are reduced to Fe 2+ cations that can be absorbed by both bacteria and plants.
- Incremento en la producción de un cultivo: Mayor producción de un cultivo por unidad de superficie o planta debido a la mejora del aprovechamiento de la planta de los recursos disponibles.  - Increase in the production of a crop: Increased production of a crop per unit of area or plant due to the improvement of the use of the plant of the available resources.
- Control: A efectos de la presente invención, las muestras control son composiciones que carecen de microorganismos.  - Control: For the purposes of the present invention, control samples are compositions that lack microorganisms.
- Testigo: A efectos de la presente invención, los testigos son cultivos tratados con composiciones control que carecen de microorganismos.  - Witness: For the purposes of the present invention, the controls are cultures treated with control compositions that lack microorganisms.
La presente invención se refiere a composiciones bioestimulantes de plantas que comprenden microorganismos. The present invention relates to biostimulant compositions of plants comprising microorganisms.
2. Cepas 2. Strains
Las cepas microbianas que se incluyen en las composiciones bioestimulantes de la presente invención se describen a continuación. Los microorganismos de las composiciones de la presente invención favorecen el desarrollo radicular, producen sideróforos, ácidos orgánicos y fosfatasas que ponen el hierro y el fósforo a disposición de la planta, y emiten sustancias que activan el metabolismo de la planta favoreciendo el desarrollo de yemas foliares y florales, aumentando el número de flores y hojas y, en consecuencia, provocando un incremento de la producción. El efecto diferenciador de las composiciones bioestimulantes de la invención se fundamenta en la acción de microorganismos promotores del crecimiento vegetal tales como bacterias PGPR, hongos y/o levaduras del género Saccharomyces. Para cada composición se selecciona una combinación de cepas específica, dependiendo del efecto deseado en el cultivo. Las cepas microbianas presentes en la composición de la invención pertenecen a los géneros Pseudomonas, Bacillus, Arthrobacter y Trichoderma. The microbial strains that are included in the biostimulant compositions of the present invention are described below. The microorganisms of the compositions of the present invention favor root development, produce siderophores, organic acids and phosphatases that make iron and phosphorus available to the plant, and emit substances that activate the metabolism of the plant favoring the development of leaf buds and florals, increasing the number of flowers and leaves and, consequently, causing an increase in production. The differentiating effect of the biostimulant compositions of the invention is based on the action of plant growth promoting microorganisms such as PGPR bacteria, fungi and / or yeasts of the Saccharomyces genus. For each composition a specific strain combination is selected, depending on the desired effect on the culture. The microbial strains present in the composition of the invention belong to the genera Pseudomonas, Bacillus, Arthrobacter and Trichoderma.
En la presente invención, la composición comprende la cepa CECT 9015 de Pseudomonas fluorescens. Además, esta composición puede comprender al menos una segunda cepa seleccionada de entre los géneros Pseudomonas, Bacillus, Arthrobacter, Trichoderma, o una combinación de los mismos. Esta segunda cepa del género Pseudomonas, puede ser una cepa de la especie Pseudomonas putida. En una realización más preferida, la cepa de Pseudomonas putida presente en la composición, junto a Pseudomonas fluorescens CECT 9015, es la cepa CECT 9011. Como ejemplo de cepa del género Bacillus, sin limitar por ello la presente invención, se selecciona preferentemente una cepa de la especie Bacillus licheniformis, como la cepa CECT 9018, una cepa de la especie Bacillus subtillis, como la cepa CECT 9016, una cepa de la especie Bacillus amyloliquefaciens, como la cepa CECT 9017, o una combinación de las mismas. Como ejemplo de cepa del género Arthrobacter, sin limitar por ello la presente invención, se selecciona preferentemente una cepa de la especie Arthrobacter oxydans, como la cepa CECT 7170. Como ejemplo de cepa del género Trichoderma, sin limitar por ello la presente invención, se selecciona preferentemente una cepa de la especie Trichoderma harzianum, como la cepa CECT 20946. In the present invention, the composition comprises strain CECT 9015 of Pseudomonas fluorescens. In addition, this composition may comprise at least a second strain selected from the genera Pseudomonas, Bacillus, Arthrobacter, Trichoderma, or a combination thereof. This second strain of the genus Pseudomonas, can be a strain of the species Pseudomonas putida. In a more preferred embodiment, the strain of Pseudomonas putida present in the composition, together with Pseudomonas fluorescens CECT 9015, is strain CECT 9011. As an example of strain of the genus Bacillus, without thereby limiting the present invention, a strain is preferably selected of the species Bacillus licheniformis, such as strain CECT 9018, a strain of the species Bacillus subtillis, such as strain CECT 9016, a strain of the species Bacillus amyloliquefaciens, such as strain CECT 9017, or a combination thereof. As an example of strain of the genus Arthrobacter, without limiting the present invention, a strain of the species Arthrobacter oxydans is preferably selected, such as strain CECT 7170. As an example of strain of the genus Trichoderma, without thereby limiting the present invention, preferably select a strain of the species Trichoderma harzianum, such as strain CECT 20946.
Es objeto de la presente invención un método para bioestimular las plantas cultivadas que consiste en aplicar en las mismas las composiciones descritas anteriormente. The object of the present invention is a method of biostimulating the cultivated plants which consists in applying the compositions described above in them.
Además, es objeto de la presente invención, el uso de la cepa Pseudomonas fluorescens CECT 9015 para la fabricación de composiciones bioestimulantes de plantas, tales como biofertilizantes. Adicionalmente, es parte de la invención el uso de Pseudomonas fluorescens CECT 9015 en combinación con al menos un segundo microorganismo seleccionado entre los géneros Pseudomonas, Bacillus, Arthrobacter, Trichoderma o una combinación de los mismos para la fabricación de bioestimulantes de plantas. En una realización de la invención, el segundo microorganismo que se utiliza en la fabricación de bioestimulantes es Pseudomonas putida. En una realización más preferida, la cepa de Pseudomonas putida presente en el bioestimulante es la cepa CECT 9011. En otra realización, la cepa del género Bacillus se selecciona preferentemente de la especie Bacillus licheniformis, como por ejemplo la cepa CECT 9018, de la especie Bacillus subtilis, como por ejemplo la cepa CECT 9016, de la especie Bacillus amyloliquefaciens, como por ejemplo la cepa CECT 9017 o una combinación de las mismas. Furthermore, the use of the strain Pseudomonas fluorescens CECT 9015 for the manufacture of plant biostimulant compositions, such as biofertilizers, is an object of the present invention. Additionally, the use of Pseudomonas fluorescens CECT 9015 in combination with at least a second microorganism selected from the genera Pseudomonas, Bacillus, Arthrobacter, Trichoderma or a combination thereof for the manufacture of plant biostimulants is part of the invention. In one embodiment of the invention, the second microorganism that is used in the manufacture of biostimulants is Pseudomonas putida. In a more preferred embodiment, the strain of Pseudomonas putida present in the biostimulant is strain CECT 9011. In another embodiment, the strain of the genus Bacillus is preferably selected from the species Bacillus licheniformis, such as strain CECT 9018, of the species Bacillus subtilis, such as strain CECT 9016, of the species Bacillus amyloliquefaciens, such as strain CECT 9017 or a combination thereof.
3. Composiciones bioestimulantes de la invención 3. Biostimulant compositions of the invention
Como se ha indicado anteriormente, las composiciones de la presente invención pueden estar en forma sólida o líquida. As indicated above, the compositions of the present invention may be in solid or liquid form.
En una realización de la invención, la composición bioestimulante en forma sólida comprende las cepas bacterianas de la invención a una concentración mínima de 104- 109 UFC/g de producto total. In one embodiment of the invention, the biostimulating composition in solid form comprising bacterial strains of the invention at a minimum concentration of April 10 to September 10 CFU / g of total product.
En otra realización de la invención, la composición bioestimulante en forma líquida comprende las cepas bacterianas de la invención a una concentración mínima de 104 UFC/ml de producto total. Las composiciones de la invención se aplican al suelo o la parte aérea de la planta para que los microorganismos presentes en dichas composiciones se establezcan en el sistema radicular o en la superficie de la planta. Al aplicar dichas composiciones con agua, los microorganismos recuperan su actividad de forma rápida y comienzan a reproducirse en la rizosfera o filosfera, aumentando significativamente su concentración en las zonas situadas a unos 2-8 cm del extremo apical de las raíces en el caso de la rizosfera. En 2 o 3 días, los microorganismos forman microcolonias sobre la superficie de la epidermis de la raíz principal, particularmente en las zonas de unión entre la raíz principal y las raíces laterales, debido a que en ellas suele haber una alta concentración de exudados radiculares y acaban constituyendo un biofilm que termina por cubrir gran parte de la superficie del sistema radicular de la planta como una capa protectora. (Fan et al., 201 1 ; Gamalero et al., 2004). Una vez establecidos los microorganismos en el sistema radicular de las plantas cultivadas, se establece una relación mutualista con la planta. Es objeto también de la presente invención un método para fertilizar cultivos que consiste en aplicar en los mismos cualesquiera de las composiciones anteriormente descritas. La aplicación de dichas composiciones puede ser en forma líquida o sólida y en cultivo hidropónico o cultivo en suelo. In another embodiment of the invention, the biostimulant composition in liquid form comprises the bacterial strains of the invention at a minimum concentration of 10 4 CFU / ml of total product. The compositions of the invention are applied to the soil or the aerial part of the plant so that the microorganisms present in said compositions are established in the root system or on the surface of the plant. When applying these compositions with water, the microorganisms recover their activity quickly and begin to reproduce in the rhizosphere or philosophy, significantly increasing its concentration in areas located about 2-8 cm from the apical end of the roots in the case of the rhizosphere. In 2 or 3 days, the microorganisms form microcolonies on the surface of the epidermis of the main root, particularly in the areas of union between the main root and the lateral roots, because in them there is usually a high concentration of root exudates and they end up constituting a biofilm that ends up covering much of the surface of the plant's root system as a protective layer. (Fan et al., 201 1; Gamalero et al., 2004). Once the microorganisms are established in the root system of the cultivated plants, a mutualistic relationship with the plant is established. The object of the present invention is also a method for fertilizing crops which consists in applying any of the above-described compositions therein. The application of said compositions can be in liquid or solid form and in hydroponic culture or soil cultivation.
El efecto bioestimulante sobre el cultivo al que se aplican las composiciones descritas en esta memoria se mide por cualquiera de los parámetros, o por una combinación de los mismos, seleccionados entre los que consisten en: incremento de la producción sin reducir el tamaño o calibre del fruto, incremento del número de frutos manteniendo la calidad de los mismos, incremento de la masa vegetal y radicular, incremento de la actividad fotosintética de los vegetales en condiciones adversas, incremento de la disponibilidad, absorción y contenido de potasio (K), y/o de hierro (Fe), y/o de fósforo (P), y/o de nitrógeno (N), y/o de otros nutrientes por mecanismos que incrementan la solubilidad del elemento, y/o su disponibilidad, y/o su absorción a través de la membrana. En una realización más preferida de la invención, el efecto bioestimulante sobre el cultivo al que se aplican las composiciones descritas en esta memoria se mide por cualquiera de los parámetros, o por una combinación de los mismos, seleccionados entre los que consisten en: incremento de la producción en un 10-45% sin reducir el tamaño o calibre del fruto, incremento del número de frutos en un 11-40% manteniendo la calidad de los mismos, incremento de la masa vegetal y radicular en un 5- 35%, incremento de la actividad fotosintética de los vegetales en condiciones adversas en unThe biostimulant effect on the crop to which the compositions described herein are applied is measured by any of the parameters, or by a combination thereof, selected from among those consisting of: increased production without reducing the size or size of the fruit, increase in the number of fruits maintaining their quality, increase in plant and root mass, increase in photosynthetic activity of vegetables in adverse conditions, increase in availability, absorption and content of potassium (K), and / or of iron (Fe), and / or of phosphorus (P), and / or of nitrogen (N), and / or of other nutrients by mechanisms that increase the solubility of the element, and / or its availability, and / or its absorption through the membrane. In a more preferred embodiment of the invention, the biostimulant effect on the culture to which the compositions described herein are applied is measured by any of the parameters, or by a combination thereof, selected from among those consisting of: increased production by 10-45% without reducing the size or size of the fruit, increasing the number of fruits by 11-40% while maintaining their quality, increasing the plant and root mass by 5-35%, increasing of the photosynthetic activity of vegetables in adverse conditions in a
4- 30%, incremento de la disponibilidad, absorción y contenido de potasio (K) en un 2-21 %, y/o de hierro (Fe) en un 1 1-100%, y/o de fósforo (P) en un 6-40%, y/o de nitrógeno (N) en un4- 30%, increased availability, absorption and content of potassium (K) by 2-21%, and / or iron (Fe) by 1 1-100%, and / or phosphorus (P) in 6-40%, and / or nitrogen (N) in a
5- 25%, y/o de otros nutrientes como el magnesio (Mg), el zinc (Zn) o el boro (B) por mecanismos que incrementan la solubilidad del elemento, y/o su disponibilidad, y/o su absorción a través de la membrana. 5- 25%, and / or other nutrients such as magnesium (Mg), zinc (Zn) or boron (B) by mechanisms that increase the element's solubility, and / or its availability, and / or its absorption at across the membrane
En una realización preferida, las composiciones bioestimulantes que comprenden la cepa P. fluorescens CECT 9015 descritas en esta memoria producen un incremento de la producción de la cosecha, incremento del número de frutos por planta, incremento del índice de madurez del fruto, menor acidez del fruto y además son capaces de incrementar la concentración de flavonoles en hoja. In a preferred embodiment, the biostimulant compositions comprising the strain P. fluorescens CECT 9015 described herein produce an increase in production. of the harvest, increase of the number of fruits by plant, increase of the index of maturity of the fruit, lower acidity of the fruit and in addition they are able to increase the concentration of flavonols in leaf.
De forma general, la fotosíntesis es el proceso por el cual las plantas transforman la materia inorgánica a materia orgánica gracias a la energía que aporta la luz. La actividad fotosintética de una planta por lo tanto es crítica para las plantas y los cultivos. La luz, por lo tanto, influye en el desarrollo de los cultivos. Aunque hay que tener en cuenta que la cantidad, calidad y duración de la luz depende de las condiciones de los cultivos y la eficiencia fotosintética de la planta depende de los recursos que ésta tenga para incrementar su actividad. La fotosíntesis es, por lo tanto, clave en el desarrollo de las plantas y va a determinar su productividad, el tamaño de los frutos, el desarrollo de la planta y la absorción de los nutrientes. A la hora de evaluar el efecto de las composiciones en las plantas es necesario interpretar de forma conjunta los efectos en el crecimiento, desarrollo del sistema radicular, absorción de nutrientes... a la luz de la fotosíntesis en dicha planta. Por ejemplo, el mayor crecimiento de una planta puede no ser importante si, por ejemplo, dicho crecimiento es debido al incremento en el tamaño de células (en vez de en el número de células), si esa planta está utilizando ineficientemente los recursos para crecer en vez de, por ejemplo, incrementar el desarrollo radicular en condiciones de falta de nutrientes. Por ello, los efectos producidos por las composiciones indicadas en el presente documento deben interpretarse en su conjunto dado que es a la luz de la eficiencia fotosintética en el marco en el cual tienen sentido los parámetros medidos para el desarrollo real de la planta.  In general, photosynthesis is the process by which plants transform inorganic matter to organic matter thanks to the energy provided by light. The photosynthetic activity of a plant is therefore critical for plants and crops. Light, therefore, influences crop development. Although we must bear in mind that the quantity, quality and duration of the light depends on the conditions of the crops and the photosynthetic efficiency of the plant depends on the resources it has to increase its activity. Photosynthesis is, therefore, key in the development of plants and will determine their productivity, fruit size, plant development and nutrient absorption. When evaluating the effect of the compositions on plants, it is necessary to interpret together the effects on growth, development of the root system, nutrient absorption ... in the light of photosynthesis in said plant. For example, the greater growth of a plant may not be important if, for example, such growth is due to the increase in cell size (instead of the number of cells), if that plant is inefficiently using the resources to grow instead of, for example, increasing root development under conditions of lack of nutrients. Therefore, the effects produced by the compositions indicated herein should be interpreted as a whole since it is in the light of photosynthetic efficiency in the framework in which the parameters measured for the actual development of the plant make sense.
Es importante destacar que, aunque se ha descrito en el estado del arte que las PGPRs estimulan las plantas, no todas las cepas bacterianas tienen los mismos efectos. En este documento se describen composiciones que comprenden la cepa BB17B (CECT 9015) de Pseudomonas fluorescens, dado que se ha comprobado que tiene un efecto significativamente mayor que otras cepas de la misma especie y otras PGPRs, en la estimulación de plantas.  Importantly, although it has been described in the state of the art that PGPRs stimulate plants, not all bacterial strains have the same effects. In this document, compositions comprising strain BB17B (CECT 9015) of Pseudomonas fluorescens are described, since it has been shown to have a significantly greater effect than other strains of the same species and other PGPRs, in plant stimulation.
DEPÓSITO DE MICROORGANISMOS BAJO EL TRATADO DE BUDAPEST DEPOSIT OF MICROORGANISMS UNDER THE BUDAPEST TREATY
Los microorganismos utilizados en la presente invención fueron depositados en la Colección Española de Cultivos Tipo (CECT) sita en el Edificio de Investigación de la Universidad de Valencia, Campus Burjassot, Burjassot 46100 (Valencia, España). Se ha comprobado la identidad de todas las cepas con su especie correspondiente mediante análisis del RNA ribosomal 16S. Pseudomonas fluorescens BB17B (código de depósito CECT 9015) es una bacteria con pared gran negativa aislada de suelo y perteneciente a la clase gamma del phylum Proteobacteria. Tiene forma de bastón, es móvil gracias a la presencia de uno o varios flagelos polares y no forma esporas. Tiene un metabolismo quimiorganotrofo y estrictamente aerobio. Solubiliza fosfatos, produce sideróforos y tiene actividad ACC-desaminasa. The microorganisms used in the present invention were deposited in the Spanish Type Culture Collection (CECT) located in the Research Building of the University of Valencia, Burjassot Campus, Burjassot 46100 (Valencia, Spain). The identity of all strains with their corresponding species has been verified by analysis of 16S ribosomal RNA. Pseudomonas fluorescens BB17B (deposit code CECT 9015) is a bacterium with a large negative wall isolated from soil and belonging to the gamma class of the phylum Proteobacteria. It has a cane shape, is mobile thanks to the presence of one or several polar flagella and does not form spores. It has a chemoganotrophic metabolism and strictly aerobic. Solubilizes phosphates, produces siderophores and has ACC-deaminase activity.
Pseudomonas putlda BB17F (código de depósito CECT 901 1) es una bacteria con pared gran negativa aislada de suelo y perteneciente a la clase gamma del phylum Proteobacteria. Tiene forma de bastón, es móvil gracias a la presencia de uno o varios flagelos polares y no forma esporas. Tiene un metabolismo quimiorganotrofo y estrictamente aerobio. Solubiliza fosfatos y produce sideróforos. Pseudomonas putlda BB17F (deposit code CECT 901 1) is a bacterium with a large negative wall isolated from soil and belonging to the gamma class of the phylum Proteobacteria. It has a cane shape, is mobile thanks to the presence of one or several polar flagella and does not form spores. It has a chemoganotrophic metabolism and strictly aerobic. Solubilizes phosphates and produces siderophores.
Bacillus licheniformis BB02L (código de depósito CECT 9018) es una bacteria gran positiva perteneciente al phylum Firmicutes, móvil y con forma de bastón. Tiene un metabolismo mayoritariamente aerobio, aunque en ocasiones puede ser anaerobio. Forma una endospora de resistencia con forma elipsoidal y es capaz de desarrollarse hasta 55°C. Puede producir sideróforos en placa. Bacillus licheniformis BB02L (deposit code CECT 9018) is a large positive bacterium belonging to the phylum Firmicutes, mobile and shaped like a cane. It has a mostly aerobic metabolism, although sometimes it can be anaerobic. It forms a resistance endospora with an ellipsoidal shape and is capable of developing up to 55 ° C. It can produce plate siderophores.
Bacillus subtilis BB02H (código de depósito CECT 9016) es una bacteria gran positiva perteneciente al phylum Firmicutes, móvil y con forma de bastón. Tiene un metabolismo mayoritariamente aerobio y forma una endospora de resistencia con forma elipsoidal. Puede degradar ACC y solubilizar fosfatos en placa. Bacillus amyloliquefaciens BB02N (código de depósito CECT 9017) es una bacteria gran positiva perteneciente al phylum Firmicutes, móvil y con forma de bastón. Tiene un metabolismo mayoritariamente anaerobio y forma una endospora de resistencia con forma elipsoidal. Puede solubilizar fosfatos en placa. Bacillus subtilis BB02H (deposit code CECT 9016) is a large positive bacterium belonging to the phylum Firmicutes, mobile and shaped like a cane. It has a mostly aerobic metabolism and forms an endospora of resistance with an ellipsoidal shape. It can degrade ACC and solubilize plate phosphates. Bacillus amyloliquefaciens BB02N (deposit code CECT 9017) is a large positive bacterium belonging to the phylum Firmicutes, mobile and shaped like a cane. It has a mostly anaerobic metabolism and forms an endospora of resistance with an ellipsoidal shape. It can solubilize phosphates in plaque.
Trichoderma harzianum BB21A (código de depósito CECT 20946) es un hongo de la división Ascomycota, orden Hypocreales, de reproducción asexual mediante conidias. Los conidióforos del hongo están muy ramificados, formando cada ramificación un ángulo recto con el nivel anterior, y cada conjunto de ramificaciones tiene forma piramidal. Tiene actividad positiva de solubilización de fosfatos en placa. Trichoderma harzianum BB21A (deposit code CECT 20946) is a fungus from the Ascomycota division, order Hypocreales, of asexual reproduction through conidia. The conidiophores of the fungus are very branched, each branch forming a right angle to the previous level, and each set of branches has a pyramidal shape. It has positive phosphate solubilization activity in plaque.
Arthrobacter oxydans BB01A (código de depósito CECT 7170) es un microorganismo del grupo de las bacterias Gram +, genero Arthrobacter, estimulante del crecimiento vegetal en ambientes de estrés salino. Esta cepa ha sido aislada a partir de la rizosfera de Pinus pinaster Aitón y Pinus pinea (L), y de la micosfera del hongo micorrizógeno asociado a ambos Lactarius deliciosus (Fríes) S.F. Gray, en agar nutritivo (PCA), y ha sido caracterizada desde el punto de vista morfológico, bioquímico y genético. EJEMPLOS Arthrobacter oxydans BB01A (deposit code CECT 7170) is a microorganism of the Gram + bacteria group, Arthrobacter genus, plant growth stimulant in saline stress environments. This strain has been isolated from the rhizosphere of Pinus pinaster Aitón and Pinus pinea (L), and from the mycosphere of the mycorrhizal fungus associated with both Lactarius deliciosus (Fries) SF Gray, in nutritive agar (PCA), and has been characterized from the morphological, biochemical and genetic point of view. EXAMPLES
Los ejemplos que se detallan a continuación tienen como objetivo ilustrar la invención sin limitar el alcance de la misma. The examples detailed below are intended to illustrate the invention without limiting the scope thereof.
Ejemplo 1. Proceso de fabricación de la composición bioestimulante de la invención 1.1 Reproducción de microorganismos Example 1. Manufacturing process of the biostimulant composition of the invention 1.1 Reproduction of microorganisms
Se toma un pequeño volumen de suspensión del cultivo puro de cada cepa, y se inocula en condiciones de esterilidad en un medio con los nutrientes y pH que más favorecen su reproducción. Cada cepa requiere de un medio nutricional específico para su crecimiento que se incuba en las condiciones óptimas de temperatura y aireación durante un periodo de tiempo de entre 24 y 72 horas. A small volume of suspension of the pure culture of each strain is taken, and it is inoculated under sterile conditions in a medium with the nutrients and pH that most favor its reproduction. Each strain requires a specific nutritional medium for its growth that is incubated in the optimal conditions of temperature and aeration for a period of time between 24 and 72 hours.
El medio líquido contiene una fuente de carbono adecuada, siendo la mejor la glucosa, aunque también se utilizan otros compuestos, tales como almidón, sacarosa y melazas. Además, es esencial una fuente adecuada de nitrógeno en forma de aminoácidos. Así mismo se requiere la presencia de fuentes de K, P, Mg, S, Ca, Cl, Zn, Fe, Mn en forma de sales minerales. Todos estos elementos, junto con agua estéril, se depositan en fermentadores de distinta capacidad según la cantidad de biofertilizante que se desea producir. Los microorganismos reproducidos y/o conservados se añaden a este medio líquido y se ajusta el pH del medio. A continuación, se programan los parámetros de temperatura, agitación y aireación, y la fermentación se lleva a cabo durante 24-72 horas, hasta obtener una concentración deseada de UFC/ml. The liquid medium contains a suitable carbon source, glucose being the best, although other compounds such as starch, sucrose and molasses are also used. In addition, an adequate source of nitrogen in the form of amino acids is essential. Likewise, the presence of sources of K, P, Mg, S, Ca, Cl, Zn, Fe, Mn in the form of mineral salts is required. All these elements, together with sterile water, are deposited in fermenters of different capacity according to the amount of biofertilizer that is desired to be produced. The reproduced and / or preserved microorganisms are added to this liquid medium and the pH of the medium is adjusted. The temperature, stirring and aeration parameters are then programmed, and the fermentation is carried out for 24-72 hours, until a desired concentration of CFU / ml is obtained.
1.2 Estabilización y formulación de microorganismos para el producto líquido 1.2 Stabilization and formulation of microorganisms for the liquid product
Los microorganismos son estables en el propio medio de cultivo durante un periodo de 6 meses sin necesidad de un tratamiento de estabilización ni de conservación en frío. No obstante, para la formulación de las composiciones de la invención en forma líquida se puede mezclar el medio de cultivo obtenido en el punto anterior con otros componentes, como ácidos húmicos o aminoácidos, que mejoren de forma el efecto sinérgico del producto sobre las plantas.  The microorganisms are stable in the culture medium itself for a period of 6 months without the need for a stabilization or cold preservation treatment. However, for the formulation of the compositions of the invention in liquid form, the culture medium obtained in the previous point can be mixed with other components, such as humic acids or amino acids, which improve the synergistic effect of the product on the plants.
1.3 Liofilización de microorganismos para el producto sólido 1.3 Lyophilization of microorganisms for the solid product
Cuando ya se ha alcanzado la fase de reproducción adecuada en el punto 1.2, se aislan los microorganismos del medio de cultivo y se someten a un proceso de liofilización o congelación en seco. 1.4 Formulación del producto sólido When the appropriate reproduction phase in point 1.2 has already been reached, the microorganisms are isolated from the culture medium and subjected to a dry freeze-drying or freeze-drying process. 1.4 Formulation of the solid product
El proceso de formulación del producto sólido, es decir, la mezcla de los microorganismos liofilizados con los coformulantes, se realiza en dos fases para garantizar la máxima calidad y homogeneidad:  The solid product formulation process, that is, the mixture of lyophilized microorganisms with coformulants, is carried out in two phases to guarantee the highest quality and homogeneity:
I. Se añade en un mezclador la cantidad total de microorganismos liofilizados que contendrá el producto junto con los coformulantes.  I. The total amount of lyophilized microorganisms that the product will contain together with the co-formulants is added in a mixer.
II. Se homogeniza la composición obtenida en el punto (I) con los coformulantes en las proporciones establecidas para cada composición, en un mezclador de mayor volumen. Las composiciones en forma sólida se envasan con ayuda de una máquina envasadora automática en bolsas de material variable, dependiendo del fin al que se destine el producto. El producto destinado a la exportación o a cultivos hortícolas intensivos se envasa en un material complejo que aisla de la luz y la humedad, u otro material con iguales propiedades que permita su conservación a temperatura ambiente. Tras ser etiquetadas convenientemente, el producto se presenta a los consumidores en forma de bolsas opacas con una capacidad de entre 0, 1 y 25 kg.  II. The composition obtained in point (I) is homogenized with the coformulants in the proportions established for each composition, in a larger volume mixer. Compositions in solid form are packaged with the help of an automatic packaging machine in bags of variable material, depending on the purpose to which the product is intended. The product intended for export or intensive horticultural crops is packaged in a complex material that isolates from light and moisture, or other material with the same properties that allows its conservation at room temperature. After being properly labeled, the product is presented to consumers in the form of opaque bags with a capacity of between 0.1 and 25 kg.
Las composiciones en forma líquida se dosifican con ayuda de una envasadora de líquidos en botellas de material plástico opaco con una capacidad de entre 1 y 5 litros. Ejemplo 2. Método de fertilización mediante aplicación de las composiciones de la invención The compositions in liquid form are dosed with the aid of a liquid packing machine in bottles of opaque plastic material with a capacity of between 1 and 5 liters. Example 2. Fertilization method by application of the compositions of the invention
2.1 Tipos de cultivos 2.1 Types of crops
Las composiciones de la invención han sido diseñadas para su posible aplicación en una amplia gama de cultivos, admitiendo la posibilidad de añadir pequeñas variaciones en su composición que se adapten mejor a las técnicas y necesidades específicas de determinados cultivos. Su eficacia ha sido probada por el departamento de l+D de los autores de la presente invención, en colaboración con agricultores en condiciones comerciales, así como universidades y consultores independientes en España, Estados Unidos, Chile, México, Perú y Egipto. Las composiciones de la invención pueden aplicarse en cultivos hortícolas intensivos bajo invernadero y al aire libre. Se pueden aplicar, por ejemplo, pero sin limitarse, a cultivos de tomate, apio, lechuga y pepino, pimiento, melón, sandía, calabacín, calabaza u otros cultivos hortícolas. También están indicadas para cultivos de bayas o especies arbustivas como la fresa, frambuesa, mora o arándano. Dado que las composiciones de la invención mejoran la disponibilidad de agua y nutrientes, se pueden utilizar también en cultivos de cítricos (naranjo, mandarino), cultivos frutales como la uva de mesa, el melocotonero y otras rosáceas (manzano, peral, albaricoquero, ciruelo, cerezo, etc.), o cultivos tropicales como el aguacate. Las composiciones también pueden aplicarse a cultivos cereales extensivos como el trigo, la cebada o el maíz. Otros cultivos que podrían beneficiarse de su aplicación serían cultivos de flor y ornamentales, cultivos industriales como la patata o la remolacha y cultivos típicamente mediterráneos como el olivo. The compositions of the invention have been designed for possible application in a wide range of crops, admitting the possibility of adding small variations in their composition that are better suited to the specific techniques and needs of certain crops. Its effectiveness has been proven by the R&D department of the authors of the present invention, in collaboration with farmers in commercial conditions, as well as universities and independent consultants in Spain, the United States, Chile, Mexico, Peru and Egypt. The compositions of the invention can be applied in intensive horticultural crops under greenhouse and outdoors. They can be applied, for example, but not limited to tomato, celery, lettuce and cucumber, pepper, melon, watermelon, zucchini, squash or other horticultural crops. They are also indicated for crops of berries or shrub species such as strawberry, raspberry, blackberry or blueberry. Since the compositions of the invention improve the availability of water and nutrients, can also be used in citrus crops (orange, mandarin), fruit crops such as table grapes, peaches and other rosaceae (apple, pear, apricot, plum, cherry, etc.), or crops Tropical like avocado. The compositions can also be applied to extensive cereal crops such as wheat, barley or corn. Other crops that could benefit from its application would be flower and ornamental crops, industrial crops such as potatoes or beets, and typically Mediterranean crops such as olive trees.
2.2 Dosis de empleo 2.2 Dose of employment
En el caso de las composiciones en forma sólida, la dosis de empleo recomendada es entre 0, 1 kg/ha y 2 kg/ha. Para las composiciones en forma líquida, la dosis de empleo recomendada es entre 0,5 y 2 l/ha. En una realización preferida, la dosis de empleo recomendada para las composiciones en forma sólida es entre 0,2 kg/ha y 2 kg/ha, y la dosis de empleo recomendada para las composiciones en forma líquida es entre 1 y 2 l/ha. In the case of compositions in solid form, the recommended use dose is between 0.1 kg / ha and 2 kg / ha. For compositions in liquid form, the recommended use dose is between 0.5 and 2 l / ha. In a preferred embodiment, the recommended employment dose for compositions in solid form is between 0.2 kg / ha and 2 kg / ha, and the recommended employment dose for compositions in liquid form is between 1 and 2 l / ha .
El número y el momento de las aplicaciones variarán según el tipo de cultivo, tal y como se indica a continuación. De forma general, para todos los cultivos que comiencen con el trasplante desde semillero se recomienda realizar la primera aplicación en el momento del trasplante, idealmente con el primer riego. Así se logrará favorecer el desarrollo radicular y el correcto establecimiento del cultivo. Se recomienda renovar las aplicaciones una vez al mes o coincidiendo con momentos de especial demanda energética como son: la salida del letargo invernal, la brotación, la floración o el cuajado y formación de los frutos; hasta un total aproximado de 2-4 aplicaciones por ciclo del cultivo. El número final de aplicaciones dependerá del cultivo y de la duración del ciclo. Se recomienda repetir la aplicación de la composición bioestimulante aproximadamente cada 30 días, para asegurarse de que el nivel de población microbiana es óptimo para producir todos los efectos buscados en el cultivo. 2.3 Método de aplicación a) Composición bioestimulante en forma sólida The number and timing of applications will vary according to the type of crop, as indicated below. In general, for all crops that begin with the transplant from the seedbed it is recommended to perform the first application at the time of transplantation, ideally with the first irrigation. Thus, it will be possible to favor the root development and the correct establishment of the crop. It is recommended to renew the applications once a month or coinciding with times of special energy demand such as: the departure of winter lethargy, budding, flowering or fruit set and fruit formation; up to an approximate total of 2-4 applications per crop cycle. The final number of applications will depend on the crop and the duration of the cycle. It is recommended to repeat the application of the biostimulant composition approximately every 30 days, to ensure that the level of microbial population is optimal to produce all the effects sought in the crop. 2.3 Method of application a) Biostimulant composition in solid form
La composición en forma sólida es un polvo soluble de aplicación al suelo o sustrato de cultivo que debe diluirse en el agua de riego y aplicarse mediante el sistema de riego. También es posible la aplicación mediante riego por aspersión, pero en ese caso es recomendable prolongar un poco el riego para que el exceso de agua lave el producto de la superficie foliar y se deposite en el suelo, que es el medio natural de desarrollo de los microorganismos. El producto puede aplicarse directamente en la superficie foliar con un mojante y que permanezca en contacto con las hojas de la planta. El producto es completamente soluble en agua y perfectamente compatible con los sistemas de riego, donde se ha comprobado que no obstruye filtros ni válvulas. Se puede aplicar tanto al suelo como a cualquier sustrato de cultivo: perlita, turba, fibra de coco, lana de roca, etc. The solid form composition is a soluble powder applied to the soil or culture substrate that must be diluted in the irrigation water and applied by the irrigation system. It is also possible to apply by sprinkler irrigation, but in that case it is advisable to prolong the irrigation a bit so that the excess water washes the product of the leaf surface and is deposited in the soil, which is the natural means of development of microorganisms The product can be applied directly to the leaf surface with a wetting agent and to remain in contact with the leaves of the plant. The product is completely soluble in water and perfectly compatible with irrigation systems, where it has been proven that no clogs filters or valves. It can be applied both to the soil and to any crop substrate: perlite, peat, coconut fiber, rock wool, etc.
De forma general, se recomienda seguir el siguiente protocolo de aplicación: In general, it is recommended to follow the following application protocol:
1. Disolver la dosis recomendada de producto en un volumen de agua (100-200 litros) que permita que la concentración final de la disolución sea aproximadamente del 1 % (~ 10 kg en 1000 litros).  1. Dissolve the recommended dose of product in a volume of water (100-200 liters) that allows the final concentration of the solution to be approximately 1% (~ 10 kg in 1000 liters).
2. Dejar reposar la disolución entre 30 minutos y 8 horas (una noche), para permitir que los microorganismos liofilizados contenidos en el producto se rehidraten y recuperen su actividad biológica.  2. Let the solution stand for 30 minutes to 8 hours (one night), to allow the lyophilized microorganisms contained in the product to rehydrate and recover their biological activity.
3. Aplicar el producto al suelo mediante el sistema de riego, preferiblemente riego localizado, a la dosis recomendada de 0,2 - 2 kg/ha. 3. Apply the product to the soil through the irrigation system, preferably localized irrigation, at the recommended dose of 0.2 - 2 kg / ha.
Una vez se aplica el producto al suelo, las cepas que éste contiene se reproducen con ayuda de los coformulantes presentes en la composición, colonizando las raíces del cultivo. La comunidad de microorganismos se establece sobre la superficie de las raíces y comienza la interacción con la planta. Once the product is applied to the soil, the strains it contains are reproduced with the help of coformulants present in the composition, colonizing the roots of the crop. The microorganism community is established on the surface of the roots and the interaction with the plant begins.
Debido a que el efecto de las composiciones de la invención se basa en la acción de microorganismos vivos, es necesario tomar una serie de precauciones que aseguren un buen desarrollo de la comunidad microbiana. El producto se debe aplicar con aguas no cloradas o que no hayan recibido un tratamiento de potabilización para consumo humano, ya que estos tratamientos pueden afectar a las cepas beneficiosas de PGPR, reduciendo su viabilidad. Por esta misma razón, también se recomienda evitar la aplicación de sustancias con un pH muy bajo (ácido) o muy elevado (básico), y reducir en la medida de lo posible la aplicación de productos fitosanitarios como fungicidas o insecticidas que puedan tener efectos secundarios sobre los microorganismos. b) Composición bioestimulante de la invención en forma líquida Because the effect of the compositions of the invention is based on the action of live microorganisms, it is necessary to take a series of precautions that ensure a good development of the microbial community. The product should be applied with non-chlorinated waters or that have not received a treatment of purification for human consumption, since these treatments can affect the beneficial PGPR strains, reducing their viability. For this same reason, it is also recommended to avoid the application of substances with a very low (acidic) or very high (basic) pH, and reduce as far as possible the application of phytosanitary products such as fungicides or insecticides that may have side effects About microorganisms b) Biostimulant composition of the invention in liquid form
En el caso de la composición bioestimulante en forma líquida, se recomienda seguir las mismas indicaciones que en el producto sólido, con la salvedad de que no es necesario que el producto repose antes de su distribución mediante el sistema de riego, dado que los microorganismos se encuentran en estado activo. 2.4 Medida de los parámetros de bioestimulación  In the case of the biostimulant composition in liquid form, it is recommended to follow the same indications as in the solid product, with the proviso that it is not necessary for the product to rest before distribution through the irrigation system, since the microorganisms are They are in an active state. 2.4 Measurement of biostimulation parameters
Los sistemas y dispositivos de medida de todos los parámetros, descritos a continuación, es conocida por cualquier experto medio en el campo de la agrobiología, ingeniería agrónoma o agricultores. Los parámetros analizados en el presente documento se miden tal y como se indica a continuación: The measurement systems and devices of all parameters, described below, are known by any expert in the field of agrobiology, agronomic engineering or farmers. The parameters analyzed in this document are measured as follows:
Biomasa fresca: Es el peso de la planta extraída directamente de su sustrato. Se utiliza una balanza granataria y se mide en gramos. Se puede medir el peso fresco de la planta completa o de partes de ella, como las raíces o las hojas.  Fresh biomass: It is the weight of the plant extracted directly from its substrate. A granataria scale is used and measured in grams. The fresh weight of the whole plant or parts of it, such as roots or leaves, can be measured.
Biomasa seca: Es el peso de la planta extraída directamente de su sustrato, después de secarse en un horno a 60°C durante dos días. Se utiliza una balanza granataria y se mide en gramos. Se puede medir el peso seco de la planta completa o de partes de ella, como las raíces o las hojas.  Dry biomass: It is the weight of the plant extracted directly from its substrate, after drying in an oven at 60 ° C for two days. A granataria scale is used and measured in grams. You can measure the dry weight of the whole plant or parts of it, such as roots or leaves.
Longitud de la raíz: La longitud de la raíz se determina con una regla previa separación de las raíces del tallo, y se mide en centímetros (cm). Hay una relación directa entre las raíces y el desarrollo de la parte aérea de la planta: a mayor masa radicular, mayor grosor del tallo, mejor desarrollo de las hojas y aumentando el calibre de los frutos. Root length: The root length is determined with a previous rule separating the roots of the stem, and is measured in centimeters (cm). There is a direct relationship between the roots and the development of the aerial part of the plant: the greater the root mass, the greater the thickness of the stem, the better development of the leaves and increasing the caliber of the fruits.
Altura de la planta: Para determinar la altura de la parte aérea de la planta se mide cada planta desde la zona de nacimiento hasta la última hoja o meristemo apical. Se mide en centímetros (cm).  Plant height: To determine the height of the aerial part of the plant, each plant is measured from the birth zone to the last leaf or apical meristem. It is measured in centimeters (cm).
La fotosíntesis es el proceso más importante que ocurre en las plantas. Distribuye la energía captada determinando el desarrollo de las plantas, produciendo diferentes efectos de acuerdo con las necesidades del organismo en cada momento: incrementando las reservas, aumentando el metabolismo secundario de la planta, aumentando su tamaño... La capacidad fotosintética de las plantas debe por lo tanto interpretarse de forma global. Algunos de los parámetros que se utilizan en el presente documento para determinar la actividad fotosintética son:  Photosynthesis is the most important process that occurs in plants. It distributes the energy collected by determining the development of the plants, producing different effects according to the needs of the organism at all times: increasing the reserves, increasing the secondary metabolism of the plant, increasing its size ... The photosynthetic capacity of the plants must therefore interpreted globally. Some of the parameters used in this document to determine photosynthetic activity are:
Fotosíntesis neta: la capacidad fotosintética de las plantas se valora a partir del proceso de fijación del CO2 en el Ciclo de Calvin. Corresponde a la diferencia entre la cantidad de CO2 fijada y la cantidad de CO2 emitida por la respiración. Un método indirecto de medir la fotosíntesis de las plantas es mediante la detección de la fluorescencia emitida por el fotosistema II. En el presente documento la fotosíntesis se mide a partir de la fluorescencia emitida con un fluorímetro.  Net photosynthesis: the photosynthetic capacity of plants is assessed from the process of fixation of CO2 in the Calvin Cycle. It corresponds to the difference between the amount of CO2 fixed and the amount of CO2 emitted by breathing. An indirect method of measuring plant photosynthesis is by detecting the fluorescence emitted by photosystem II. In this document photosynthesis is measured from the fluorescence emitted with a fluorimeter.
Emisión de fluorescencia del fotosistema II (FSII): Para determinar el estado del FSII se calculan los siguientes parámetros: Fluorescence emission of photosystem II (FSII): The following parameters are calculated to determine the status of FSII:
Fo: corresponde al mínimo nivel de fluorescencia, obtenido al incidir luz suave sobre la hoja. Todos los pigmentos de las antenas del FS están abiertas (adaptadas a la oscuridad). En condiciones de estrés, el novel de Fo aumenta. Fm: fluorescencia máxima. Es el novel de fluorescencia después de aplicar luz de alta intensidad a la hoja, cuando las antenas están cerradas. Fo: corresponds to the minimum level of fluorescence, obtained when soft light falls on the sheet. All the pigments of the FS antennas are open (adapted to the dark). In conditions of stress, Fo's novel increases. Fm: maximum fluorescence. It is the novel of fluorescence after applying high intensity light to the sheet, when the antennas are closed.
Fv: fluorescencia variable. Fm-Fo  Fv: variable fluorescence. Fm-Fo
La capacidad potencial máxima del FSII (Fv/Fm): corresponde al cociente entre la fluorescencia variable y la fluorescencia máxima.  The maximum potential capacity of FSII (Fv / Fm): corresponds to the ratio between variable fluorescence and maximum fluorescence.
Quenching fotoquímico (OPSII): corresponde a la capacidad real del FSII para canalizar la energía lumínica recibida por la hoja hacia el proceso fotosintético y producir glucosa.  Photochemical quenching (OPSII): corresponds to the real capacity of the FSII to channel the light energy received by the sheet into the photosynthetic process and produce glucose.
Quenching no fotoquímico (NPQ): representa la cantidad de energía que disipa el FSII en forma de calor, y que por lo tanto no se usa en el proceso fotosintético y no va a tener efecto beneficioso en la planta.  Non-photochemical quenching (NPQ): represents the amount of energy that dissipates the FSII in the form of heat, and therefore is not used in the photosynthetic process and will not have a beneficial effect on the plant.
Contenido en clorofilas: Las clorofilas se encuentran insertadas en las membranas de los tilacoides del doroplasto. Se andan a la membrana por un resto fitoí y se asocian a otros pigmentos (formando las antenas} y a proteínas formando los fotosístemas. Para la determinación de las clorofilas presentes en la hoja se utiliza un medidor de clorofila portátil, como por ejemplo SPAD-502, Minolta, que determina la cantidad relativa de clorofila presente mediante la medición de la absorción de la hoja en dos regiones de longitud de onda; en las regiones roja y cercanas a infrarroja. Utilizando estas dos transmisiones el medidor calcula el valor numérico SPAD que es proporcional a la cantidad de clorofila presente en la hoja.  Chlorophyll content: Chlorophylls are inserted into the membranes of doroplast thylakoids. They are attached to the membrane by a phytoid residue and are associated with other pigments (forming the antennas) and proteins forming the photosystems. For the determination of the chlorophylls present in the leaf a portable chlorophyll meter is used, such as SPAD-502 , Minolta, which determines the relative amount of chlorophyll present by measuring the absorption of the leaf in two regions of wavelength; in the red and near infrared regions.Using these two transmissions the meter calculates the numerical value SPAD that is proportional to the amount of chlorophyll present in the leaf.
Grados Brix (°Bx): miden la cantidad de sacarosa presente en la hoja de la planta o en un fruto, lo que permite determinar el estado de su maduración. Se determinan como el cociente total de sacarosa disuelta en un líquido. Es un indicador de un incremento en el ciclo de Calvin de la planta, dado que al aumentar la producción de glucosa-3-fosfato, la planta lo usa bien para incrementar el contenido en almidón (y los °Bx) o en la glicolisis (y favorecer el crecimiento de la planta). Mediante la valoración de los grados Brix se analiza la cantidad de azúcares que están siendo transformados tras la fotosíntesis. Cuanto mayor es el nivel d azúcar dentro del tejido d la planta, más fuerte será la planta y más producirá. Los grados Brix se miden mediante la utilización de un refractómetro.  Degrees Brix (° Bx): measure the amount of sucrose present in the leaf of the plant or in a fruit, which allows to determine the state of its maturation. They are determined as the total ratio of sucrose dissolved in a liquid. It is an indicator of an increase in the Calvin cycle of the plant, since by increasing the production of glucose-3-phosphate, the plant uses it well to increase the content in starch (and ° Bx) or in glycolysis ( and favor the growth of the plant). By assessing the Brix grades, the amount of sugars that are being transformed after photosynthesis is analyzed. The higher the sugar level within the plant tissue, the stronger the plant will be and the more it will produce. Brix degrees are measured by using a refractometer.
Rendimiento en planta: Se determina mediante la fórmula Abbot: mide la eficacia del producto en relación al control. Eficacia= (Ca-Ta)x100/Ca, donde Ta son los valores obtenidos en el control o testigo y Ca son los valores obtenidos en los tratamientos. Se mide en kilogramos (kg) por unidad de área (ha).  Plant performance: Determined by the Abbot formula: measures the effectiveness of the product in relation to the control. Efficacy = (Ca-Ta) x100 / Ca, where Ta are the values obtained in the control or control and Ca are the values obtained in the treatments. It is measured in kilograms (kg) per unit area (ha).
Tamaño del fruto: El tamaño de los frutos se mide según su calibre. Para determinar el tamaño se emplea un Calibre universal para fruta cuyo diseño de lazo permite una medición precisa. Los calibres del fruto se clasifican en las categorías GG, G, M, MM y MMM según su tamaño, de acuerdo a la Tabla 1 : Fruit size: The size of the fruits is measured according to their size. To determine the size, a universal fruit caliber is used whose loop design allows a measurement accurate. Fruit sizes are classified in the categories GG, G, M, MM and MMM according to their size, according to Table 1:
Tabla 1 : Tipos de calibre de acuerdo con el diámetro del fruto. Table 1: Types of caliber according to the diameter of the fruit.
Calibre Diámetro mínimo Diámetro máximo Caliber Minimum diameter Maximum diameter
MMM 40 47 MMM 40 47
MM MMp 47 52  MM MMp 47 52
MMg 53 57  MMg 53 57
M Mp 57 62  M Mp 57 62
Mg 63 67  Mg 63 67
G 67 82  G 67 82
GG 82 102 Categoría del fruto: Los frutos se clasifican en categorías dependiendo su calidad. Por ejemplo, los tomates se clasifican en categoría extra, que corresponde a tomates que tienen la pulpa firme, presentan forma, aspecto y desarrollo característicos de la variedad y no presentan defectos que afecten al aspecto general del producto, su calidad, conservación o presentación en el envase. La categoría primer corresponde con frutos de buena calidad, que no presentan grietas ni "dorso verde" aparente, pero pueden presentar defectos como: ligeras malformaciones y defectos de desarrollo, ligeros defectos de coloración, ligeras magulladuras o ligeros defectos en la epidermis.  GG 82 102 Fruit category: Fruits are classified into categories depending on their quality. For example, tomatoes are classified in an extra category, which corresponds to tomatoes that have firm flesh, have a characteristic shape, appearance and development of the variety and do not present defects that affect the general appearance of the product, its quality, preservation or presentation in the container The first category corresponds to fruits of good quality, which do not present cracks or apparent "green backs," but may present defects such as: slight malformations and developmental defects, slight color defects, slight bruises or slight defects in the epidermis.
Producción acumulada por planta: Se determina pesando o contando los frutos totales de cada parcela a analizar por el número totales de plantas analizadas.  Cumulative production per plant: It is determined by weighing or counting the total fruits of each plot to be analyzed by the total number of plants analyzed.
Análisis nutrimental: Los elementos minerales de un suelo, necesarios para la alimentación de las plantas pueden encontrase en muy diversas formas. No todas ellas son aptas para ser absorbidas por las raíces. Es necesario que los macro- y microelementos presentes en el suelo estén disponibles para que la planta pueda absorberlos a través de sus raíces. Se determinan en materia seca foliar. Nutritional analysis: The mineral elements of a soil, necessary for plant feeding can be found in many different forms. Not all of them are suitable to be absorbed by the roots. It is necessary that the macro- and microelements present in the soil be available so that the plant can absorb them through its roots. They are determined in dry foliar matter.
- Fósforo disponible (mg/kg): se mide mediante un espectrofotómetro de luz visible (Thermo Scientific Genesys 20, Verona Road, USA).  - Available phosphorus (mg / kg): it is measured by a visible light spectrophotometer (Thermo Scientific Genesys 20, Verona Road, USA).
Nitrógeno Dumas (mg/kg): Consiste en la transformación de todas las formas de nitrógeno presentes en la planta en N gaseoso por calcinación. Se determina por conductividad térmica (Sweeney y Rexroad, 1987).  Nitrogen Dumas (mg / kg): It consists of the transformation of all forms of nitrogen present in the plant into N gas by calcination. It is determined by thermal conductivity (Sweeney and Rexroad, 1987).
- Potasio disponible (meq/100 g): Se mide con un fotómetro de emisión de llama (Jenway PFP7, Burlington, NJ. United States).  - Available potassium (meq / 100 g): Measured with a flame emission photometer (Jenway PFP7, Burlington, NJ. United States).
Calcio disponible (meq/100 g): Se mide mediante un espectrofotómetro de absorción atómica (Perkin Elmer 2280, Norwalk, Connecticut. USA). Magnesio disponible (meq/100 g): Se mide con un espectrofotómetro de absorción atómica (Perkin Elmer 2280, Norwalk, Connecticut. USA). Available calcium (meq / 100 g): It is measured by an atomic absorption spectrophotometer (Perkin Elmer 2280, Norwalk, Connecticut. USA). Available magnesium (meq / 100 g): Measured with an atomic absorption spectrophotometer (Perkin Elmer 2280, Norwalk, Connecticut. USA).
Hierro (DTPA) (mg/kg): Se mide con un espectrofotómetro de absorción atómica (GBC modelo 905 AA, Australia), previa calcinación a 520°C durante 15 h y extracción con ácido clorhídrico 1 N . La concentración de hierro soluble se mide por el método descrito por Koseoglu y Acikgoz (1995).  Iron (DTPA) (mg / kg): Measured with an atomic absorption spectrophotometer (GBC model 905 AA, Australia), after calcination at 520 ° C for 15 h and extraction with 1 N hydrochloric acid. The concentration of soluble iron is measured by the method described by Koseoglu and Acikgoz (1995).
Capacidad de intercambio catiónico efectiva (meq/100 g): Se determina mediante el método del acetato de amonio pH7 1 N (normal), destilación Kjeldahl y volumetría. Materia orgánica oxidable (%). Se determina utilizando la técnica volumétrica del método de Walkley y Black (1974). Esta técnica se basa en una combustión húmeda de la materia orgánica con una mezcla de dicromato de potasio y ácido sulfúrico. El valor que indica el grado de acumulación de materia orgánica en un horizonte y se utiliza para diferenciar los suelos orgánicos de los minerales. A partir de 1998 se refiere como Carbono Orgánico.  Effective cation exchange capacity (meq / 100 g): It is determined by the method of ammonium acetate pH7 1 N (normal), Kjeldahl distillation and volumetry. Oxidizable organic matter (%). It is determined using the volumetric technique of the Walkley and Black method (1974). This technique is based on a wet combustion of organic matter with a mixture of potassium dichromate and sulfuric acid. The value that indicates the degree of accumulation of organic matter on a horizon and is used to differentiate organic soils from minerals. As of 1998 it is referred to as Organic Carbon.
Daño foliar: Este parámetro se mide después de que un cultivo haya sido sometido a algún tipo de estrés (sal, sequía... ). El daño se evalúa mediante observación visual de acuerdo con la siguiente escala: 0: hojas sanas; 1 : ligera clorosis; 2: clorosis grave y/o arrugamiento; 3: necrosis y/o arrugamiento severo; 4: necrosis muy extendida. Leaf damage: This parameter is measured after a crop has been subjected to some type of stress (salt, drought ...). Damage is assessed by visual observation according to the following scale: 0: healthy leaves; 1: slight chlorosis; 2: severe chlorosis and / or wrinkle; 3: necrosis and / or severe wrinkling; 4: widespread necrosis.
Contenido de compuestos fenólicos (mg Ác. Gálico/100 g peso fresco): los compuestos fenólicos tienen efecto antioxidante en las plantas y se sintetizan como metabolitos secundarios de la planta. Dependiendo del compuesto, puede intervenir en la defensa de la planta a patógenos, proveer soporte mecánico a la planta, atraer polinizadores o dispersores de frutos, o actuar como agentes aleopáticos. Se determinan cuantitativamente con el reactivo de Folin-Ciocalteau (Sigma-Aldrich, St Louis, MO) por colorimetría (Xu 2007) con modificaciones, utilizando ácido gálico como referencia (Sigma-Aldrich, St Louis, MO). Una alícuota de 1 mi del extracto se mezcla con 0.25 mi del reactivo Folin-Ciocalteu 2 N y 0.75 mi de una solución al 20% de Na2C03. La mezcla se mantiene 30 minutos a temperatura ambiente y a continuación se mide la absorbancia a 760 nm en un espectrofotómetro UV- Visible (Biomate 5). Se hace una curva de calibración con ácido gálico y se obtiene el contenido de compuestos fenólicos.  Content of phenolic compounds (mg Gallic Acid / 100 g fresh weight): Phenolic compounds have an antioxidant effect on plants and are synthesized as secondary metabolites of the plant. Depending on the compound, it can intervene in the defense of the plant to pathogens, provide mechanical support to the plant, attract pollinators or fruit dispersers, or act as allopathic agents. They are quantitatively determined with the Folin-Ciocalteau reagent (Sigma-Aldrich, St Louis, MO) by colorimetry (Xu 2007) with modifications, using gallic acid as a reference (Sigma-Aldrich, St Louis, MO). A 1 ml aliquot of the extract is mixed with 0.25 ml of Folin-Ciocalteu 2 N reagent and 0.75 ml of a 20% solution of Na2C03. The mixture is kept 30 minutes at room temperature and then the absorbance at 760 nm is measured in a UV-Visible spectrophotometer (Biomate 5). A calibration curve is made with gallic acid and the content of phenolic compounds is obtained.
Potencial hídrico: Este parámetro se determina mediante una cámara de presión la cual da una medida de la presión hidrostática negativa que se produce en el xilema de una planta intacta debido a la evaporación de agua desde el tejido por transpiración y a las resistencias al movimiento del agua desde el suelo hasta el tejido (Scholander, 1965). Contenido en prolina: Este parámetro se mide mediante absorbancia a 520 nm, tras realizar una mezcla con el extracto etanólico procedente de muestras de hojas y preparación de un reactivo (Irygoyen 1992). Water potential: This parameter is determined by a pressure chamber which gives a measure of the negative hydrostatic pressure that occurs in the xylem of an intact plant due to the evaporation of water from the tissue by perspiration and resistance to water movement from the ground to the fabric (Scholander, 1965). Proline content: This parameter is measured by absorbance at 520 nm, after mixing with the ethanolic extract from leaf samples and preparation of a reagent (Irygoyen 1992).
Desarrollo del sistema radicular: Este parámetro se mide por observación visual una vez la planta ha sido arrancada de su sustrato (maceta o cultivo en campo). Se evalúa de acuerdo con la siguiente escala: 0: raíces dañadas; 1 : raíces sanas, pero no muy desarrolladas; 2: raíces abundantes y fuertes; 3: raíces muy abundantes y fuertes. Además, también se puede medir el porcentaje de raíces nuevas (identificadas por su color claro, no suberizadas) respecto a las raíces totales. Vigor y grado de desarrollo de las plantas: Se determina visualmente analizando la superficie foliar y sombra proyectada en el suelo, densidad de racimos.  Root system development: This parameter is measured by visual observation once the plant has been removed from its substrate (pot or field crop). It is evaluated according to the following scale: 0: damaged roots; 1: healthy roots, but not very developed; 2: abundant and strong roots; 3: very abundant and strong roots. In addition, you can also measure the percentage of new roots (identified by their light color, not suberized) with respect to the total roots. Vigor and degree of development of the plants: It is determined visually by analyzing the foliar surface and shadow cast on the ground, cluster density.
2. 5 Análisis estadístico 2. 5 Statistical analysis
Para determinar las diferencias entre las composiciones utilizadas en los ejemplos detallados a continuación, se llevaron a cabo los análisis estadísticos ANOVA, Test de Tukey y LSD. To determine the differences between the compositions used in the examples detailed below, the ANOVA, Tukey Test and LSD statistical analyzes were carried out.
Ejemplo 3. Efecto de las composiciones bioestimulantes de la invención. Example 3. Effect of the biostimulant compositions of the invention.
Los ensayos mostrados a continuación muestran como las composiciones bioestimulantes de la invención que comprenden cepas microbianas son capaces de incrementar la disponibilidad, absorción y/o contenido en la planta de determinados elementos nutricionales como el fósforo, hierro y/o potasio. The tests shown below show how the biostimulant compositions of the invention comprising microbial strains are capable of increasing the availability, absorption and / or plant content of certain nutritional elements such as phosphorus, iron and / or potassium.
En la Tabla 2 se detallan las cepas, a modo de ejemplo, sin que por ello sea necesario limitarse a dichas cepas, los microorganismos empleados en las composiciones bioestimulantes de los ensayos presentados en esta memoria. In Table 2, the strains are detailed, by way of example, without it being necessary to limit said strains, the microorganisms used in the biostimulant compositions of the tests presented herein.
Tabla 2: Cepas empleadas en los ejemplos.  Table 2: Strains used in the examples.
Código de la cepa Microorganismo Strain code Microorganism
CECT 9015 Pseudomonas fluorescens  CECT 9015 Pseudomonas fluorescens
CECT 901 1 Pseudomonas pulida  CECT 901 1 Polished Pseudomonas
CECT20946 Tríchoderma harzianum  CECT20946 Tríchoderma harzianum
CECT 9016 Bacillus subtilis  CECT 9016 Bacillus subtilis
CECT 9017 Bacillus amyloliquefaciens  CECT 9017 Bacillus amyloliquefaciens
CECT 9018 Bacillus licheniformis  CECT 9018 Bacillus licheniformis
CECT 7170 Arthrobacter oxydans  CECT 7170 Arthrobacter oxydans
CECT 378 Pseudomonas fluorescens  CECT 378 Pseudomonas fluorescens
MTCC 5670 Pseudomonas pulida 3.1 Ensayos en placa Petri MTCC 5670 Pseudomonas polished 3.1 Petri dish tests
3.1.1 Ensayo de producción de sideróforos en placa  3.1.1 Production test of plate siderophores
El ensayo de producción de sideróforos se llevó a cabo inoculando en medio CAS (Chrome Azurol S) 10 μΙ_ de la cepa P. fluorescens CECT 9015 cultivada previamente en medio líquido con suficiente crecimiento (24h a 28°C en agitación enérgica). La prueba se considera positiva si la bacteria libera al medio sideróforos capaces de secuestrar el hierro, que se encuentra ligado al colorante azul CAS. Cuando el hierro se libera del CAS, se produce un viraje a amarillo-naranja (hidroximato) o a rosa (catecol).  The siderophores production assay was carried out by inoculating in CAS medium (Chrome Azurol S) 10 μ 90 of the P. fluorescens CECT 9015 strain previously grown in liquid medium with sufficient growth (24h at 28 ° C under vigorous stirring). The test is considered positive if the bacterium releases the siderophores capable of sequestering iron, which is bound to the blue CAS dye. When iron is released from CAS, there is a turn to yellow-orange (hydroxy) or pink (catechol).
La Figura 1 muestra que la cepa P. fluorescens CECT 9015 (BB17B) es capaz de crecer en este medio y producir sideróforos, dando lugar a un viraje de color en la zona de crecimiento. La producción de sideróforos indica que la cepa P. fluorescens CECT 9015 es capaz de solubilizar el hierro presente en el suelo (Fe3+) a Fe2+, capaz de ser absorbido por la planta.Figure 1 shows that the strain P. fluorescens CECT 9015 (BB17B) is able to grow in this medium and produce siderophores, resulting in a color shift in the growth zone. The production of siderophores indicates that the P. fluorescens CECT 9015 strain is capable of solubilizing the iron present in the soil (Fe 3+ ) to Fe 2+ , capable of being absorbed by the plant.
3.1.2 Ensayo de absorción de potasio en placa 3.1.2 Potassium absorption plate test
Para evaluar la capacidad que tiene la cepa P. fluorescens CECT 9015 de solubilizar potasio, se sembraron 10 de un cultivo de esta cepa y la combinación de cepas P. fluorescens CECT 9015 (BB17B) y P. putida CECT 901 1 (BB17F) en placas de medio Aleksandrov modificado, que contiene caolín como fuente insoluble de potasio. Si la bacteria es capaz de solubilizar potasio, se producirá un halo de hidrólisis en el medio La Figura 2 muestra los halos de hidrólisis de las cepas y la Tabla 3 muestra el tamaño de dichos halos. Se observa que la combinación de las cepas P. fluorescens CECT 9015 (BB17B) y P. putida CECT 901 1 (BB17F) tiene mayor efecto que la cepa P. fluorescens CECT 9015 sola, indicando que esta combinación de cepas tiene un efecto sinérgico, que favorecerá la absorción de potasio por parte de la planta.  To assess the ability of P. fluorescens CECT 9015 strain to solubilize potassium, 10 of a culture of this strain and the combination of P. fluorescens CECT 9015 (BB17B) and P. putida CECT 901 1 (BB17F) strains were seeded Modified Aleksandrov medium plates, containing kaolin as an insoluble source of potassium. If the bacterium is capable of solubilizing potassium, a hydrolysis halo will occur in the medium. Figure 2 shows the hydrolysis halos of the strains and Table 3 shows the size of these halos. It is observed that the combination of the P. fluorescens CECT 9015 (BB17B) and P. putida CECT 901 1 (BB17F) strains has a greater effect than the P. fluorescens CECT 9015 strain alone, indicating that this strain combination has a synergistic effect, which will favor the absorption of potassium by the plant.
Tabla 3: Diámetro de los halos de hidrólisis en ensayo de absorción de potasio en placa  Table 3: Diameter of hydrolysis halos in potassium absorption plate test
CEPA Medida halo de hidrólisis (mm) CEPA Hydrolysis halo measurement (mm)
BB17B 2,0  BB17B 2.0
BB17F 1 ,5  BB17F 1, 5
BB17B+BB17F 2,6  BB17B + BB17F 2.6
Control negativo 0  Negative control 0
3.2 Ensayos de eficacia en planta en condiciones controladas 3.2 Efficiency tests in the plant under controlled conditions
3.2.1 Ensayo de solubilización de fósforo en maceta con pepino  3.2.1 Potassium phosphorus solubilization test with cucumber
Para comprobar si las composiciones de la invención tienen la capacidad de solubilizar fósforo insoluble y ponerlo a disposición de la planta, se cultivó pepino híbrido de rápido desarrollo sobre un sustrato cocopeat en semilleros de 350 cm3. Se trataron 21 plantas para cada una de las composiciones biofertilizantes: To check if the compositions of the invention have the ability to solubilize insoluble phosphorus and make it available to the plant, fast developing hybrid cucumber was grown on a cocopeat substrate in 350 cm 3 seedbeds. 21 plants were treated for each of the biofertilizing compositions:
Control con solución Hoagland completa (Hoagland, 1938) con fósforo soluble (PS) - Control con solución Hoagland y fósforo insoluble (Pl) Ca3(P04)2 Control with complete Hoagland solution (Hoagland, 1938) with soluble phosphorus (PS) - Control with Hoagland solution and insoluble phosphorus (Pl) Ca3 (P0 4 ) 2
- Cepa P. fluorescens CECT 9015 + Hoagland +PS  - P. fluorescens strain CECT 9015 + Hoagland + PS
- Cepa P. fluorescens CECT 9015 + Hoagland + Pl  - P. fluorescens strain CECT 9015 + Hoagland + Pl
Se realizó una primera aplicación de 3 mi de cada composición 13 días después de la germinación y una segunda aplicación fue realizada a los 10 días de la primera aplicación (ambas a una concentración del microorganismo 108 UFC/ml). A continuación, se analizaron parámetros biométricos (peso seco), fotosintéticos (emisión de fluorescencia del Fotosistema II, fijación de CO2, contenido en clorofilas) y contenido en macro y micronutrientes en hoja. La Figura 3A muestra el peso seco de las plantas al final del experimento. Se observa un incremento de peso de la parte aérea de las plantas de pepino inoculadas con la cepa P. fluorescens CECT 9015 (BB17B) con respecto al control, tanto al añadir PS como Pl (Tabla 4). La figura 3B refleja una mayor capacidad fotosintética de las plantas inoculadas y tratadas con fosfato soluble, que está de acuerdo con el mayor crecimiento alcanzado con este tratamiento. La Figura 3C representa la cantidad de fósforo en hoja, donde claramente se observa que la cepa P. fluorescens CECT 9015 (BB17B) mejora el contenido en fósforo de la hoja, independientemente del tipo de fósforo utilizado. Es significativo el efecto que tienen las bacterias con fósforo insoluble, si lo comparamos con el control no inoculado, mejorando claramente la nutrición de este elemento en condiciones de déficit. Es interesante observar cómo el incremento es más significativo en los casos en los que se añade al medio fósforo insoluble. Esto indica que la presencia de la cepa P. fluorescens CECT 9015 favorece la solubilización de este elemento, que se traduce en mejor desarrollo radicular y crecimiento de la planta. A first application of 3 ml of each composition was made 13 days after germination and a second application was made 10 days after the first application (both at a concentration of the microorganism 10 8 CFU / ml). Next, biometric parameters (dry weight), photosynthetic parameters (photosystem II fluorescence emission, CO2 fixation, chlorophyll content) and macro and micronutrient content were analyzed. Figure 3A shows the dry weight of the plants at the end of the experiment. An increase in weight of the aerial part of the cucumber plants inoculated with the strain P. fluorescens CECT 9015 (BB17B) with respect to the control is observed, both by adding PS and Pl (Table 4). Figure 3B reflects a higher photosynthetic capacity of plants inoculated and treated with soluble phosphate, which is in accordance with the highest growth achieved with this treatment. Figure 3C represents the amount of sheet phosphorus, where it is clearly observed that P. fluorescens strain CECT 9015 (BB17B) improves the phosphorus content of the sheet, regardless of the type of phosphorus used. The effect of bacteria with insoluble phosphorus is significant, if we compare it with the non-inoculated control, clearly improving the nutrition of this element in deficit conditions. It is interesting to note how the increase is more significant in cases where it is added to the insoluble phosphorus medium. This indicates that the presence of the P. fluorescens CECT 9015 strain favors the solubilization of this element, which translates into better root development and plant growth.
Tabla 4: Peso de la parte aérea de las plantas de pepino Table 4: Weight of the aerial part of cucumber plants
Tratamiento Peso parte aérea (g) Treatment Weight aerial part (g)
Control PS 0.518 ± 0.018  PS control 0.518 ± 0.018
Control Pl 0.417 ± 0.019  Control Pl 0.417 ± 0.019
BB17B PS (CECT 9015) 0.545 ± 0.016  BB17B PS (CECT 9015) 0.545 ± 0.016
BB17B Pl (CECT 9015) 0.51 1 ± 0.020  BB17B Pl (CECT 9015) 0.51 1 ± 0.020
La Figura 4 muestra los datos de eficiencia fotosintética de la planta. La Figura 4A muestra el parámetro Fo, que es indicador de estrés en la planta a nivel del fotosistema II. Las plantas tratadas con P. fluorescens CECT 9015 (BB17B) y con fósforo soluble presentan un menor nivel de estrés que el resto de tratamientos. En la Figura 4B se muestra que la fotosíntesis potencial (Fv/Fm) aumenta en las plantas tratadas con P. fluorescens CECT 9015 y con fósforo soluble respecto al control. La Figura 4C representa la fotosíntesis real de la planta, en la que se ve cómo la adición de la cepa P. fluorescens CECT 9015, en presencia de fósforo soluble, tiene mucha más eficiencia fotosintética que el control. Por último, la Figura 4D muestra que las plantas tratadas con la composición que comprende P. fluorescens CECT 9015 tienen menor disipación de calor que su respectivo control. Todo esto, considerado en conjunto, indica que la fotosíntesis es más eficiente y el rendimiento de estas plantas será mejor. Figure 4 shows the photosynthetic efficiency data of the plant. Figure 4A shows the Fo parameter, which is an indicator of stress in the plant at the level of photosystem II. The plants treated with P. fluorescens CECT 9015 (BB17B) and soluble phosphorus have a lower level of stress than other treatments. Figure 4B shows that potential photosynthesis (Fv / Fm) increases in plants treated with P. fluorescens CECT 9015 and soluble phosphorus with respect to the control. Figure 4C represents the real photosynthesis of the plant, in which it is seen how the addition of the P. fluorescens CECT 9015 strain, in the presence of soluble phosphorus, has much more photosynthetic efficiency than the control. Finally, Figure 4D shows that plants treated with the composition comprising P. fluorescens CECT 9015 have less heat dissipation than their respective control. All this, taken together, indicates that photosynthesis is more efficient and the performance of these plants will be better.
Tabla 5: Contenido de nutrientes de las plantas de pepino del ensayo Table 5: Nutrient content of test cucumber plants
B Ca Cu Fe Mg Mn Na Zn  B Ca Cu Fe Mg Mn Na Zn
(mg/Kg) (%) (mg/Kg) (mg/Kg) (%) (mg/Kg) (mg/Kg) (mg/Kg) (mg / kg) (%) (mg / kg) (mg / kg) (%) (mg / kg) (mg / kg) (mg / kg)
Control PS 91^5 0^56 1^66 58,78 0,728 20,77 3ΪΪ6 69^4 PS control 91 ^ 5 0 ^ 56 1 ^ 66 58.78 0.728 20.77 3ΪΪ6 69 ^ 4
Control Pi 16,05 0,44 0,387 53,29 0,515 19,324 2267 49,1  Pi control 16.05 0.44 0.387 53.29 0.515 19.324 2267 49.1
CECT 9015 PS 17,07 0,51 0,73 54,77 0,66 19,92 2959 61 ,6  CECT 9015 PS 17.07 0.51 0.73 54.77 0.66 19.92 2959 61.6
CECT 9015 Pi 57.07 0.57 0.98 66.94 0.75 21 .57 3133 69  CECT 9015 Pi 57.07 0.57 0.98 66.94 0.75 21 .57 3133 69
Los resultados expuestos en la Tabla 5 demuestran la eficacia de los tratamientos con bacterias en situaciones de déficit de fosforo (Pi). En esta situación, las bacterias mejoran de forma acusada la nutrición en fosforo, y de manera sinérgica, la absorción del resto de los nutrientes en las plantas en las que se ha aplicado la composición que comprende la cepa P. fluorescens CECT 9015. The results presented in Table 5 demonstrate the efficacy of treatments with bacteria in situations of phosphorus deficit (Pi). In this situation, the bacteria significantly improve the phosphorus nutrition, and synergistically, the absorption of the rest of the nutrients in the plants in which the composition comprising the strain P. fluorescens CECT 9015 has been applied.
3.2.2 Estudio de evaluación de la efectividad biológica de tres inóculos microbianos frente a estrés abiótico en el cultivo de maíz (Zea mavs L.) 3.2.2 Evaluation study of the biological effectiveness of three microbial inoculums against abiotic stress in the corn crop (Zea mavs L.)
Los análisis en condiciones adversas tales como el estrés abiótico o la sequía son importantes a la hora de valorar el efecto de las composiciones en los cultivos, porque permiten determinar el efecto real de las composiciones en la planta. En condiciones controladas en muchas ocasiones no se observan diferencias entre los controles y las composiciones bioestimulantes debido a que la planta tiene acceso a todos los recursos metabólicos, defensivos... que permiten su desarrollo. El objetivo de este ensayo es evaluar la efectividad biológica de tres composiciones que comprenden microorganismos en un cultivo de maíz (Zea mays L.), frente a condiciones de estrés abiótico (sequía y salinidad) simuladas en invernadero sobre macetas con turba. Se ensayaron 3 composiciones que se compararon con un control (agua), cada una con 5 repeticiones, bajo 3 condiciones de crecimiento (riego óptimo, salinidad y sequía), hasta un total de 12 tratamientos y 60 plantas. La composición de los bioestimulantes ensayados se detalla en la Tabla 6. Analyzes in adverse conditions such as abiotic stress or drought are important when assessing the effect of the compositions on crops, because they allow determining the real effect of the compositions on the plant. Under controlled conditions on many occasions no differences are observed between controls and biostimulant compositions because the plant has access to all metabolic, defensive resources ... that allow its development. The objective of this test is to evaluate the biological effectiveness of three compositions comprising microorganisms in a corn crop (Zea mays L.), against simulated abiotic stress conditions (drought and salinity) in a greenhouse on peat pots. Three compositions were tested and compared with a control (water), each with 5 repetitions, under 3 growth conditions (optimal irrigation, salinity and drought), up to a total of 12 treatments and 60 plants. The composition of the biostimulants tested is detailed in Table 6.
Tabla 6: Tratamientos Table 6: Treatments
Tratamiento Producto a evaluar Dosis ufc/ml/maceta Treatment Product to be evaluated cfu dose / ml / pot
T1 TESTIGO Control con agua  T1 WITNESS Control with water
Control con solución salina Saline control
T2 TESTIGO CON SAL T2 WITNESS WITH SALT
25 mM de NaCI  25 mM NaCI
Testigo sometido a sequía Witness subjected to drought
T3 TESTIGO CON SEQUIA T3 WITNESS WITH DROUGHT
durante 5 días  For 5 days
T4 CECT 9015 con agua 5 x 108 ufc/ml T4 CECT 9015 with water 5 x 10 8 cfu / ml
T5 CECT 9015 con Sal 5 x 108 ufc/ml T5 CECT 9015 with Salt 5 x 10 8 cfu / ml
T6 CECT 9015 con Sequía 5 x 108 ufc/ml T6 CECT 9015 with Drought 5 x 10 8 cfu / ml
Las composiciones se aplicaron vía radicular 15 días después de la siembra y una semana después de la primera aplicación. El estrés por sequía se causó deteniendo el riego y el estrés por salinidad aplicando 20 mi de una solución salina a 25 mM de NaCI. Ambas condiciones de estrés comenzaron dos semanas después de la segunda aplicación, y se prolongaron durante 5 días. The compositions were applied by root 15 days after sowing and one week after the first application. Drought stress was caused by stopping irrigation and salinity stress by applying 20 ml of a saline solution at 25 mM NaCI. Both stress conditions began two weeks after the second application, and lasted for 5 days.
De cada tratamiento se evaluó el daño foliar mediante observación visual basándose en una escala de 0 a 4 para el grado de daño (0: hojas sanas; 1 : ligera clorosis; 2: clorosis grave y/o arrugamiento; 3: necrosis y/o arrugamiento severo; 4: necrosis muy extendida.) y el desarrollo sistema radicular por observación visual según una escala de 0 a 3 (0: raíces dañadas; 1 : raíces sanas, pero no muy desarrolladas; 2: raíces abundantes y fuertes; 3: raíces muy abundantes y fuertes. Además, también se puede medir el porcentaje de raíces nuevas (identificadas por su color claro, no suberizadas) respecto a las raíces totales. From each treatment the foliar damage was evaluated by visual observation based on a scale of 0 to 4 for the degree of damage (0: healthy leaves; 1: slight chlorosis; 2: severe chlorosis and / or wrinkling; 3: necrosis and / or severe wrinkling; 4: widespread necrosis.) and root system development by visual observation on a scale of 0 to 3 (0: damaged roots; 1: healthy but not very developed roots; 2: abundant and strong roots; 3: very abundant and strong roots In addition, you can also measure the percentage of new roots (identified by their light color, not submerged) with respect to the total roots.
Las variables se sometieron, sin transformar, a un análisis de varianza para determinar si existen diferencias significativas entre los grupos analizados (ANOVA, α = 0.05). Posteriormente los datos se sometieron a una prueba de comparación múltiple para ordenar las diferencias entre los tratamientos bajo estudio (Tukey, P≤ 0.05). Como se observa en la Figura 5A, las plantas inoculadas con la composición que comprende la cepa P. fluorescens CECT 9015 presentan menos daño foliar tanto en condiciones normales, como en las condiciones adversas de salinidad y sequía. En el caso del desarrollo radicular (Figura 5B), se observa que ya en condiciones óptimas, las plantas inoculadas con las composiciones que comprendes la cepa P. fluorescens CECT 9015 tienen mejor desarrollo que el testigo. Este resultado se ve acentuado en condiciones adversas, en las que se ve cómo las plantas inoculadas con las composiciones estimulantes tienen mejor desarrollo radicular que las plantas testigo. Entre las dos composiciones ensayadas, se observa un efecto sinérgico en la combinación de cepas P. fluorescens CECT 9015 y Arthrobacter oxydans, mejorando el desarrollo radicular de las plantas significativamente en todas las condiciones ensayadas. 3.2.3 Ensayo de evaluación de la efectividad biológica de un inoculante microbiano en protección frente a estrés salino en Tomate. The variables were subjected, without transforming, to an analysis of variance to determine if there are significant differences between the groups analyzed (ANOVA, α = 0.05). Subsequently, the data were subjected to a multiple comparison test to order the differences between the treatments under study (Tukey, P≤ 0.05). As seen in Figure 5A, plants inoculated with the composition comprising strain P. fluorescens CECT 9015 have less foliar damage both in normal conditions and in adverse salinity and drought conditions. In the case of root development (Figure 5B), it is observed that already under optimal conditions, the plants inoculated with the compositions comprising the strain P. fluorescens CECT 9015 have better development than the control. This result is accentuated in adverse conditions, in which it is seen how plants inoculated with stimulating compositions have better development root that witness plants. Between the two compositions tested, a synergistic effect is observed in the combination of P. fluorescens CECT 9015 and Arthrobacter oxydans strains, improving the root development of the plants significantly in all the conditions tested. 3.2.3 Test of evaluation of the biological effectiveness of a microbial inoculant in protection against saline stress in Tomato.
El ensayo se llevó a cabo en plantas de tomate (Solanum lycopersicum var. Toro) en invernadero. Se utilizaron 6 composiciones diferentes (M1-M6), que consisten en:  The test was carried out in tomato plants (Solanum lycopersicum var. Toro) in a greenhouse. 6 different compositions (M1-M6) were used, which consist of:
M1 : Control. Solución de aminoácidos 0.2%.  M1: Control. 0.2% amino acid solution.
- M2: P. fluorescens CECT 9015 108 UFC/g + aminoácidos 0.2%. - M2: P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%.
- M3: P. fluorescens CECT 9015 + P. putida CECT 9016, ambas a 108 UFC/g+ aminoácidos 0.2%. - M3: P. fluorescens CECT 9015 + P. putida CECT 9016, both at 10 8 CFU / g + amino acids 0.2%.
- M4: P. fluorescens CECT 9015 + P. putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, todas a 108 UFC/g + aminoácidos 0.2%. - M4: P. fluorescens CECT 9015 + P. putida CECT 901 1 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, all at 10 8 CFU / g + amino acids 0.2%.
- M5: P. fluorescens CECT 378 + aminoácidos 0.2%.  - M5: P. fluorescens CECT 378 + amino acids 0.2%.
P fluorescens CECT 378 es una cepa de Pseudomonas fluorescens comercial, disponible en el estado de la técnica, utilizada en el presente documento como control positivo para valorar la capacidad bioestimulante de la cepa de la invención, CECT 9015. P fluorescens CECT 378 is a commercial Pseudomonas fluorescens strain, available in the state of the art, used herein as a positive control to assess the biostimulant capacity of the strain of the invention, CECT 9015.
Se realizaron 4 aplicaciones de cada tratamiento vía riego durante el ciclo de cultivo, con 28 plantas por repetición. Las plantas crecieron en macetas rellenas con sustrato PROJAR y se aplicaron las composiciones, con intervalos de aproximadamente 15 días, en la que los microorganismos se encuentran a una densidad de 108 ufc/ml. Se aplicó un riego con sal a las 4 semanas de la primera aplicación para inducir estrés salino a las plantas, y al cabo de una semana se midió el potencial hídrico mediante cámara Scholander y se cosechó el experimento. Four applications of each treatment were made via irrigation during the crop cycle, with 28 plants per repetition. The plants grew in pots filled with PROJAR substrate and the compositions were applied, with intervals of approximately 15 days, in which the microorganisms are at a density of 10 8 cfu / ml. A salt irrigation was applied at 4 weeks after the first application to induce saline stress to the plants, and after one week the water potential was measured by means of the Scholander chamber and the experiment was harvested.
Los parámetros fotosintéticos analizados indican que la composición M2, que comprende P fluorescens CECT 9015 tienen un menor Fo que la composición que comprende P fluorescens CECT 378, lo que indica que sufren menos estrés (Figura 6A). Aunque los datos de capacidad potencial del fotosistema II, la disipación energética (NPQ) ni de quenching fotoquímico no difieren significativamente entre los 5 tratamientos analizados, la Figura 6D muestra que todos los tratamientos, excepto M5 (P. fluorescens CECT 378), aumentan la disipación energética (NPQ) en las plantas tratadas con sal, lo que supone una disipación del exceso de energía que disminuye la formación de radicales libres y, por tanto, mantiene la planta más saludable. The photosynthetic parameters analyzed indicate that the composition M2, which comprises P fluorescens CECT 9015 has a lower Fo than the composition comprising P fluorescens CECT 378, indicating that they suffer less stress (Figure 6A). Although the potential capacity data of photosystem II, energy dissipation (NPQ) and photochemical quenching do not differ significantly between the 5 treatments analyzed, Figure 6D shows that all treatments, except M5 (P. fluorescens CECT 378), increase the energy dissipation (NPQ) in salt-treated plants, which means a dissipation of the Excess energy that decreases the formation of free radicals and therefore keeps the plant healthier.
El análisis del potencial hídrico de las plantas tratadas con las diferentes composiciones muestra cómo los tratamientos con P. fluorescens CECT 9015 (M2) y la combinación de P. fluorescens CECT 9015 y P. putida CECT 901 1 (M3), aumenta el potencial hídrico en las plantas de forma muy acusada, mientras que las otras combinaciones lo aumentan en menor medida (Figura 7). El incremento del potencial hídrico de P. fluorescens CECT 9015 es significativamente mayor que el incremento de otra P. fluorescens (M5), lo que indica que el efecto que tiene la cepa sobre la planta no depende de la especie bacteriana en sí misma, sino que es una característica intrínseca de la cepa. Este efecto en el potencial hídrico va asociado a un aumento significativo de Prolina en las composiciones que comprenden P. fluorescens CECT 9015, y es especialmente importante (el doble) en el tratamiento combinado de las cepas P. fluorescens CECT 9015 y P. putida CECT 9011 (M3) (Figura 8). The analysis of the water potential of the plants treated with the different compositions shows how treatments with P. fluorescens CECT 9015 (M2) and the combination of P. fluorescens CECT 9015 and P. putida CECT 901 1 (M3), increase the water potential in plants very strongly, while the other combinations increase it to a lesser extent (Figure 7). The increase in the water potential of P. fluorescens CECT 9015 is significantly greater than the increase in another P. fluorescens (M5), which indicates that the effect that the strain has on the plant does not depend on the bacterial species itself, but which is an intrinsic characteristic of the strain. This effect on water potential is associated with a significant increase in Proline in compositions comprising P. fluorescens CECT 9015, and is especially important (double) in the combined treatment of P. fluorescens CECT 9015 and P. putida CECT strains 9011 (M3) (Figure 8).
Por lo tanto, las composiciones que comprenden la cepa P. fluorescens CECT 9015, especialmente la cepa sola y combinada con P. putida CECT 901 1 , protegen a la planta frente a estrés salino por otros mecanismos, además de incrementando el contenido en prolina. El efecto protector de P. fluorescens CECT 9015 es superior al de otras cepas de la misma especie, lo que demuestra su efecto estimulante diferenciador característico de la cepa, y no de la especie a la que pertenece. 3.3 Ensayos de eficacia en campo/naturales. Therefore, the compositions comprising the P. fluorescens CECT 9015 strain, especially the strain alone and combined with P. putida CECT 901 1, protect the plant against saline stress by other mechanisms, in addition to increasing the proline content. The protective effect of P. fluorescens CECT 9015 is superior to that of other strains of the same species, which demonstrates its characteristic differentiating stimulating effect of the strain, and not of the species to which it belongs. 3.3 Field / natural efficacy tests.
3.3.1 Ensayo de evaluación de la efectividad biológica de un inoculante microbiano en el cultivo de fresa (Fragaria x ananassa).  3.3.1 Test of evaluation of the biological effectiveness of a microbial inoculant in strawberry cultivation (Fragaria x ananassa).
Se cultivó cultivo de fresa (Fragaria x ananassa) variedad Camarosa sobre un suelo franco- arcilloso con un 20 a 45% de limo, y entre 15 y 25% de arcilla. El ensayo se llevó a cabo de aplicando una composición a cuatro concentraciones diferentes (T1 , T2, T3 y T4) por cuadriplicado en parcelas de 1.5 m2. La composición está compuesta por: 108 UFC/g Bacillus amyloliquefaciens CECT 9017, 108 UFC/g Bacillus licheniformis CECT 9018, 5.1x102 UFC/g Pseudomonas fluorescens CECT 9015, y los coformulantes ácido algínico 12% p/p, Nitrógeno total (N) 4.7% p/p, fósforo (P205) 0.2% p/p y potasio (K20) 10% p/p. Se realizaron tres aplicaciones foliares con un aspersor motorizado Robín RS 450, equipada con boquillas de cono hueco, previamente calibrada, a fin de determinar el volumen adecuado de caldo de aplicación por hectárea. Las aplicaciones se hicieron a una distancia aproximada de 30 cm del objetivo y en ángulo que cubra a la planta completa. Las aplicaciones tuvieron lugar sobre los cultivos al inicio de la floración, tres semanas después de la primera aplicación (en la formación de los frutos) y tres semanas después de la segunda aplicación (en el pico de producción de la planta). La concentración de la composición en cada tratamiento fue: Strawberry cultivation (Fragaria x ananassa) Camarosa variety was grown on a loamy soil with 20 to 45% silt, and between 15 and 25% clay. The test was carried out by applying a composition at four different concentrations (T1, T2, T3 and T4) in quadruplicate in plots of 1.5 m 2 . The composition is composed of: 10 8 CFU / g Bacillus amyloliquefaciens CECT 9017, 10 8 CFU / g Bacillus licheniformis CECT 9018, 5.1x10 2 CFU / g Pseudomonas fluorescens CECT 9015, and the co-formulants alginic acid 12% w / w, Total nitrogen (N) 4.7% w / w, phosphorus (P 2 0 5 ) 0.2% w / w potassium (K 2 0) 10% w / w. Three foliar applications were made with a Robín RS 450 motorized sprinkler, equipped with hollow cone nozzles, previously calibrated, in order to determine the appropriate volume of application broth per hectare. Applications were made at an approximate distance of 30 cm from the target and at an angle that covers the entire floor. Applications took place on the crops at the beginning of flowering, three weeks after the first application (in the fruit formation) and three weeks after the second application (at the peak of plant production). The concentration of the composition in each treatment was:
T1 : Parcela testigo sin tratamiento T1: Control plot without treatment
T2: 100 g/ha de composición bioestimulante diluida en 1000 litros de agua  T2: 100 g / ha of biostimulant composition diluted in 1000 liters of water
T3: 150 g/ha de composición bioestimulante diluida en 1000 litros de agua T3: 150 g / ha of biostimulant composition diluted in 1000 liters of water
T4: 200 g/ha de composición bioestimulante diluida en 1000 litros de agua T4: 200 g / ha of biostimulant composition diluted in 1000 liters of water
De cada tratamiento se evaluó: la altura de la planta, los días a floración, el número de flores, el número y peso de frutos por planta, las características de los frutos, el rendimiento de la planta y el porcentaje de frutos deformes de la composición. Las variables se sometieron, sin transformar, a un análisis de varianza para determinar si al menos un tratamiento era diferente de los demás (ANOVA, a=0.05). Posteriormente los datos se sometieron a una prueba de comparación múltiple para ordenar la efectividad biológica de los tratamientos bajo estudio. For each treatment, the height of the plant, the days of flowering, the number of flowers, the number and weight of fruits per plant, the characteristics of the fruits, the yield of the plant and the percentage of deformed fruits of the plant were evaluated. composition. The variables were subjected, without transforming, to an analysis of variance to determine if at least one treatment was different from the others (ANOVA, a = 0.05). Subsequently, the data were subjected to a multiple comparison test to order the biological effectiveness of the treatments under study.
De acuerdo a la prueba de separación de medias, existen diferencias estadísticas entre los tratamientos, donde se observa que el tratamiento T4 fue el que presenta la mayor altura de planta con una media de 41.25 cm, lo que se refleja en la eficacia del producto frente al testigo que fue de 34.55 % superior a este (Tabla 7). According to the means separation test, there are statistical differences between the treatments, where it is observed that the T4 treatment was the one with the highest plant height with an average of 41.25 cm, which is reflected in the efficacy of the product versus to the witness who was 34.55% higher than this (Table 7).
Tabla 7: Altura de planta Table 7: Plant height
Figure imgf000032_0001
Se cuantificó el número de días a floración tomando como punto de partida el momento en el que el 50% del total de las plantas del lote experimental presentaron flores.
Figure imgf000032_0001
The number of days to flowering was quantified taking as a starting point the moment in which 50% of the total plants in the experimental lot presented flowers.
La Tabla 8 muestra una disminución de los días necesarios para llegar a floración en el cultivo de fresa tratados con la composición, y donde las plantas más precoces fueron las que se trataron con las dosis de 150 (T3) y 200 g/ha (T4) en 1000 L de agua que requirieron de únicamente 56.50 días, mientras que el testigo que fue la requirió del mayor número de días con 58.75. Tabla 8: Días a floración Table 8 shows a decrease in the days needed to reach flowering in the strawberry crop treated with the composition, and where the earliest plants were those treated with doses of 150 (T3) and 200 g / ha (T4 ) in 1000 L of water that required only 56.50 days, while the witness that was required the largest number of days with 58.75. Table 8: Days to flowering
Figure imgf000033_0001
Figure imgf000033_0001
Se cuantificó por observación visual el número total de flores presentes por planta, en cada una de las 5 plantas seleccionadas al azar por parcela útil. Los resultados presentados en la Tabla 9 demuestran las diferencias estadísticas altamente significativas entre los tratamientos con la composición y el testigo, logrando la mayor producción de flores el tratamiento con la dosis de 200 g/ha en 1000 L de agua (T4) con una media de 27.75 flores por planta, lo que representa una eficacia superior con respecto al testigo del 63.06% ya que el testigo solo presento un promedio de 10.25 flores por planta. The total number of flowers present per plant was quantified by visual observation, in each of the 5 randomly selected plants per useful plot. The results presented in Table 9 demonstrate the highly significant statistical differences between the treatments with the composition and the control, achieving the highest flower production treatment with the dose of 200 g / ha in 1000 L of water (T4) with an average of 27.75 flowers per plant, which represents a superior efficiency with respect to the control of 63.06% since the witness only presented an average of 10.25 flowers per plant.
Tabla 9: Flores por planta Table 9: Flowers by plant
Figure imgf000033_0002
Figure imgf000033_0002
Se cuantificó por observación visual el número total de frutos presentes por planta, en cada una de las 5 plantas seleccionadas al azar por parcela útil y se determinó con ayuda de una balanza granataria el peso de los frutos totales de cada una de las plantas. Al igual que para la variable flores por planta, el número de frutos presento diferencias estadísticas significativas como se muestra en la Tabla 10, donde el tratamiento con la composición a dosis de 200 g/ha en 1000 L de agua (T4) que fue el que obtuvo la mayor cantidad de frutos por planta con una media de 25.25, mientras que el testigo únicamente alcanzo una producción media de 9.00 frutos; el tratamiento T4 tiene una eficacia mayor 64.36% superior al testigo. Tabla 10: Frutos por planta The total number of fruits present per plant was quantified by visual observation in each of the 5 randomly selected plants per useful plot and the weight of the total fruits of each plant was determined with the help of a granatary scale. As for the variable flowers per plant, the number of fruits presented significant statistical differences as shown in Table 10, where the treatment with the composition at a dose of 200 g / ha in 1000 L of water (T4) which was the which obtained the highest amount of fruits per plant with an average of 25.25, while the control only reached an average production of 9.00 fruits; T4 treatment has a greater efficiency 64.36% higher than the control. Table 10: Fruits per plant
Figure imgf000034_0001
Figure imgf000034_0001
Se tomó una muestra aleatoria de cinco frutos de las cinco plantas de fresa seleccionadas al azar por parcela útil y se maceraron para determinar el pH con la ayuda de un potenciómetro portátil y los grados Brix se determinaron con un refractó metro, tal y como está descrito en el apartado 2.6 de esta memoria. A random sample of five fruits was taken from the five randomly selected strawberry plants per useful plot and macerated to determine the pH with the help of a portable potentiometer and the Brix grades were determined with a refractometer, as described in section 2.6 of this report.
De acuerdo con el análisis de varianza presente en la Tabla 11 para la variable peso de frutos se observa que se existen diferencias estadísticas significativas entre los cuatro tratamientos, siendo el mejor tratamiento la composición T4 (200 g/ha en 1000 L de agua), con un peso medio de fruto de 12.60 g, y una eficacia del 36.43 % superior al testigo (T1) que únicamente logro un rendimiento de 8.01 g. According to the analysis of variance present in Table 11 for the variable weight of fruits, it is observed that there are significant statistical differences between the four treatments, the best treatment being the T4 composition (200 g / ha in 1000 L of water), with an average fruit weight of 12.60 g, and an efficiency of 36.43% higher than the control (T1) that only achieved a yield of 8.01 g.
Tabla 11 : Peso de fruto en planta de fresa Table 11: Fruit weight in strawberry plant
Figure imgf000034_0002
Figure imgf000034_0002
Además, en la Tabla 12 se muestra que calidad del fruto también presentó diferencias estadísticas respecto al testigo en relación al pH y °Brix de los frutos. In addition, Table 12 shows that fruit quality also showed statistical differences with respect to the control in relation to the pH and ° Brix of the fruits.
Tabla 12: pH y grados brix del fruto Table 12: pH and brix degrees of the fruit
Figure imgf000034_0003
Figure imgf000034_0003
De acuerdo a la prueba de separación de medias de la Tabla 13, se observaron diferencias estadísticamente significativas entre los cuatro tratamientos, observando que la composición T4 tiene el mayor rendimiento, 318.04 g/planta, logrando una eficacia del producto superior al testigo (T1) en un 72.65 %, dado que este solo alcanzó una producción media de 86.98 g/planta. According to the mean separation test of Table 13, statistically significant differences were observed between the four treatments, observing that the T4 composition has the highest yield, 318.04 g / plant, achieving a product efficiency greater than control (T1) in 72.65%, since this only reached an average production of 86.98 g / plant.
Tabla 13: Rendimiento de planta de fresa Table 13: Strawberry plant yield
Figure imgf000035_0001
De las mismas plantas seleccionadas para evaluar las variables anteriores, se cuantificó por observación visual el número total de frutos deformes presentes.
Figure imgf000035_0001
From the same plants selected to evaluate the above variables, the total number of deformed fruits present was quantified by visual observation.
De acuerdo a los datos presentes en la Tabla 14, se observan diferencias estadísticas altamente significativas entre los cuatro grupos de tratamiento, obteniendo una disminución significativa de frutos deformes con la aplicación de la composición T4 (200 g/ha en 1000 L de agua), que redujo la presencia de los frutos deformes hasta un 0.25% mientras que el testigo presenta un 8.25% de dichos frutos. According to the data present in Table 14, highly significant statistical differences are observed between the four treatment groups, obtaining a significant decrease in fruits deformed with the application of the T4 composition (200 g / ha in 1000 L of water), which reduced the presence of deformed fruits up to 0.25% while the control shows 8.25% of said fruits.
Tabla 14: Porcentaje de frutos deformes del estudio Table 14: Percentage of deformed fruits of the study
Figure imgf000035_0002
Figure imgf000035_0002
En base en los resultados presentados en este ejemplo se concluye que la aplicación de una composición que comprende la cepa Pseudomonas fluorescens CECT 9015 induce en las plantas de fresa una entrada a floración y a fructificación más precoz. Además, la dosis 200 g/ha en 1000 L de agua mostró ser estadísticamente el mejor tratamiento, ya que indujo una mejor respuesta en la producción del cultivo de fresa lo cual se vio reflejado en la mayoría de las variables evaluadas, aunque se recomienda el uso la composición en cualquiera de las dosis ensayadas, ya que mostraron una mayor efectividad respecto al testigo, incrementando el rendimiento y las variables de importancia para el productor.  Based on the results presented in this example, it is concluded that the application of a composition comprising the Pseudomonas fluorescens CECT 9015 strain induces earlier flowering and fruiting entry in strawberry plants. In addition, the 200 g / ha dose in 1000 L of water was statistically the best treatment, since it induced a better response in the production of the strawberry crop which was reflected in most of the variables evaluated, although the I use the composition in any of the doses tested, since they showed greater effectiveness with respect to the control, increasing the yield and the variables of importance for the producer.
3.3.2. Ensayo de biofertilización en suelo con lechuga 3.3.2. Biofertilization test on soil with lettuce
El experimento se plantea con el objetivo de comprobar la efectividad biológica de una composición biofertilizante que comprende cepas bacterianas. Para ello se cultivó lechuga de tipo romana y variedad "Colosus" sobre un suelo franco-arcilloso con un 20 a 45% de limo, y entre 15 y 25% de arcilla. Se realizaron cuatro tratamientos (T1 , T2, T3 y T4) en una superficie total de 16 m2, divididas en unidades experimentales de 1 m2. En cada parcela útil se cultivaron cinco plantas seleccionadas al azar. La composición del biofertilizante del ensayo es Pseudomonas fluorescens CECT 9015 (5 x 103 ufc/g), Pseudomonas putida CECT 9011 (5 x 103 ufc/g), Saccharomyces cerevisiae (6.2 x 107 ufc/g), extracto húmico total (20% p/p), ácidos húmicos (10% p/p), ácidos fúlvicos (10% p/p) y aminoácidos libres (15% p/p). The experiment is proposed with the objective of verifying the biological effectiveness of a biofertilizing composition comprising bacterial strains. For this, lettuce was grown Roman type and "Colosus" variety on a loamy clay soil with 20 to 45% silt, and between 15 and 25% clay. Four treatments (T1, T2, T3 and T4) were performed on a total area of 16 m 2 , divided into experimental units of 1 m 2 . In each useful plot, five randomly selected plants were grown. The composition of the biofertilizer of the assay is Pseudomonas fluorescens CECT 9015 (5 x 10 3 cfu / g), Pseudomonas putida CECT 9011 (5 x 10 3 cfu / g), Saccharomyces cerevisiae (6.2 x 10 7 cfu / g), total humic extract (20% w / w), humic acids (10% w / w), fulvic acids (10% w / w) and free amino acids (15% w / w).
La composición se aplicó a las plantas vía fertirrigación el día del trasplante, 30 días después de la primera aplicación y 60 días después del trasplante. Se manejaron tres concentraciones de la composición (T2: 0.5 kg/ha; T3: 0.75 kg/ha; T4: 1.0 kg/ha) que se compararon con un testigo al que sólo se aplicó agua (T1). The composition was applied to the plants via fertirrigation on the day of the transplant, 30 days after the first application and 60 days after the transplant. Three concentrations of the composition were handled (T2: 0.5 kg / ha; T3: 0.75 kg / ha; T4: 1.0 kg / ha) that were compared with a control to which only water was applied (T1).
De cada tratamiento se evaluó: la altura de planta (Tabla 15), la biomasa fresca (Tabla 16) la biomasa seca (Tabla 17), la longitud de raíz de cinco plantas al azar por parcela útil en el momento de la cosecha (Tabla 18), el rendimiento (Tabla 19) el análisis nutrimental a partir de la materia seca (se realizó un análisis químico de los elementos Nitrógeno, fósforo y potasio) en una digestión húmeda (Tabla 20), de acuerdo con la metodología descrita en el apartado 2.4 de esta memoria. For each treatment, the plant height (Table 15), the fresh biomass (Table 16), the dry biomass (Table 17), the root length of five random plants per useful plot at the time of harvest (Table) were evaluated 18), the yield (Table 19) the nutritional analysis from the dry matter (a chemical analysis of the elements Nitrogen, phosphorus and potassium) was performed in a wet digestion (Table 20), according to the methodology described in the Section 2.4 of this report.
Las variables se sometieron, sin transformar, a un análisis de varianza para determinar si al menos un tratamiento es diferente de los demás (ANOVA, a=0.05). Posteriormente los datos se sometieron a una prueba de comparación múltiple para ordenar la efectividad biológica de los tratamientos bajo estudio. Letras diferentes denotan diferencias estadísticamente significativas según el test de Tukey. The variables were subjected, without transforming, to an analysis of variance to determine if at least one treatment is different from the others (ANOVA, a = 0.05). Subsequently, the data were subjected to a multiple comparison test to order the biological effectiveness of the treatments under study. Different letters denote statistically significant differences according to the Tukey test.
En la Tabla 15 se observa que la aplicación de la composición en su dosis más alta presenta diferencias significativas respecto al testigo con una altura de 20.42 cm frente a 13.82 cm, lo que representa una eficacia del producto del 20.69%. Table 15 shows that the application of the composition at its highest dose shows significant differences with respect to the control with a height of 20.42 cm versus 13.82 cm, which represents a product efficacy of 20.69%.
Tabla 15: Altura de planta (cm) Table 15: Plant height (cm)
Tukey Tukey
Tratamiento Dosis Altura (cm) % Eficacia  Treatment Dose Height (cm)% Efficacy
(a=0.05)  (a = 0.05)
T1 agua 13.82 C 0.00  T1 water 13.82 C 0.00
T2 0.5 Kg/ha 15.82 B 15.72  T2 0.5 Kg / ha 15.82 B 15.72
T3 0.75Kg/ha 17.45 B 19.58  T3 0.75Kg / ha 17.45 B 19.58
T4 1.0 Kg/ha 20.42 A 20.69 En la Tabla 16 se muestra la biomasa fresca (g) de las plantas según el tratamiento empleado. El testigo presenta una media de 1 17.103 g mientras que el tratamiento con el biofertilizante a dosis de 1.0 Kg/ha presenta la mayor cantidad de biomasa fresca con una media de 164.10 lo que representa una efectividad del 28.64% superior al testigo. Tabla 16: Biomasa fresca (g) T4 1.0 Kg / ha 20.42 A 20.69 Table 16 shows the fresh biomass (g) of the plants according to the treatment used. The control has an average of 1 17,103 g while the biofertilizer treatment at a dose of 1.0 Kg / ha has the highest amount of fresh biomass with an average of 164.10 which represents an effectiveness of 28.64% higher than the control. Table 16: Fresh biomass (g)
Tratamiento Dosis Biomasa Tukey % eficacia  Treatment Dosage Biomass Tukey% efficacy
fresca (g) (a=0.05)  fresh (g) (a = 0.05)
T1 agua 117.103 D 0.00  T1 water 117.103 D 0.00
T2 0.5 Kg/ha 129.068 C 9.27  T2 0.5 Kg / ha 129.068 C 9.27
T3 0.75Kg/ha 145.300 B 19.41  T3 0.75Kg / ha 145,300 B 19.41
T4 1.0 Kg/ha 164.100 A 28.64  T4 1.0 Kg / ha 164.100 A 28.64
En la Tabla 17 se muestra la biomasa seca (g) de las plantas según el tratamiento empleado. El testigo presenta una media de 3.44 g mientras que el tratamiento con el biofertilizante a dosis de 1.0 Kg/ha presenta la mayor cantidad de biomasa seca con una media de 5.49 g lo que representa una efectividad del 37.34% superior al testigo. Table 17 shows the dry biomass (g) of the plants according to the treatment used. The control has an average of 3.44 g while the biofertilizer treatment at a dose of 1.0 Kg / ha has the highest amount of dry biomass with an average of 5.49 g, which represents an effectiveness of 37.34% higher than the control.
Tabla 17: Biomasa seca (g) Table 17: Dry biomass (g)
Biomasa Tukey Tukey Biomass
Tratamiento Dosis % eficacia  Treatment Dose% efficacy
seca (g) (a=0.05)  dry (g) (a = 0.05)
T1 agua 3.44 D 0.00  T1 water 3.44 D 0.00
T2 0.5 Kg/ha 4.26 C 19.25 T2 0.5 Kg / ha 4.26 C 19.25
T3 0.75Kg/ha 4.90 B 29.80T3 0.75Kg / ha 4.90 B 29.80
T4 1.0 Kg/ha 5.49 A 37.34 T4 1.0 Kg / ha 5.49 A 37.34
En la Tabla 18 se muestra la longitud de la raíz de las plantas según el tratamiento empleado. El testigo presenta una longitud de 7.42 mientras que el tratamiento con el biofertilizante a dosis de 0.75 Kg/ha y 1.0 Kg/ha presentan una longitud de 11.86 y 12.45 cm respectivamente. La eficiencia de estos tratamientos es de un 37.44 y un 40.40% respectivamente superior al testigo. Table 18 shows the root length of the plants according to the treatment used. The control has a length of 7.42 while the treatment with the biofertilizer at doses of 0.75 Kg / ha and 1.0 Kg / ha has a length of 11.86 and 12.45 cm respectively. The efficiency of these treatments is 37.44 and 40.40% respectively higher than the control.
Tabla 18: Longitud de raíz. Table 18: Root length.
Longitud de Tukey Tukey Length
Tratamiento Dosis % eficacia  Treatment Dose% efficacy
raíz (cm) (a=0.05)  root (cm) (a = 0.05)
T1 agua 7.42 B 0.00  T1 water 7.42 B 0.00
T2 0.5 Kg/ha 9.98 BA 25.65  T2 0.5 Kg / ha 9.98 BA 25.65
T3 0.75Kg/ha 11 .86 A 37.44  T3 0.75Kg / ha 11 .86 TO 37.44
T4 1.0 Kg/ha 12.45 A 40.40 En la Tabla 19 se muestra el rendimiento de las plantas según el tratamiento empleado. El tratamiento T4 (1.0 Kg/ha) presenta un rendimiento de 14.052,3 y una eficiencia en comparación con el testigo (T1) del 28.64%. T4 1.0 Kg / ha 12.45 A 40.40 Table 19 shows the yield of the plants according to the treatment used. The T4 treatment (1.0 Kg / ha) has a yield of 14,052.3 and an efficiency compared to the control (T1) of 28.64%.
Tabla 19: Rendimiento en planta de lechuga Table 19: Yield in lettuce plant
Rendimiento Tukey  Tukey performance
Tratamiento Dosis % eficacia  Treatment Dose% efficacy
(Kg/ha) (a=0.05)  (Kg / ha) (a = 0.05)
T1 agua 14,052.3 D 0.00  T1 water 14,052.3 D 0.00
T2 0.5 Kg/ha 15,488.0 C 9.27  T2 0.5 Kg / ha 15,488.0 C 9.27
T3 0.75Kg/ha 17,436.0 B 19.41  T3 0.75Kg / ha 17,436.0 B 19.41
T4 1.0 Kg/ha 19,692.0 A 28.64  T4 1.0 Kg / ha 19,692.0 A 28.64
El análisis nutrimental de la planta de lechuga, tal y como se observa en la tabla 20, hay diferencias estadísticas significativas entre los tratamientos y el testigo, para la concentración del nitrógeno, fósforo y potasio, donde la aplicación del biofertilizante en la dosis de 1.0 Kg/ha fueron más altas para estos elementos. The nutritional analysis of the lettuce plant, as observed in table 20, there are significant statistical differences between the treatments and the control, for the concentration of nitrogen, phosphorus and potassium, where the application of the biofertilizer in the dose of 1.0 Kg / ha were higher for these elements.
Tabla 20: Análisis nutrimental de planta de lechuga Table 20: Nutritional analysis of lettuce plant
Tratamiento Dosis N P K  Dose Treatment N P K
T1 agua 1.55 C 0.65 B 5.60 C  T1 water 1.55 C 0.65 B 5.60 C
T2 0.5 Kg/ha 2.67 B 0.71 B 6.22 B  T2 0.5 Kg / ha 2.67 B 0.71 B 6.22 B
T3 0.75Kg/ha 3.25 B 0.78 B 6.60 BA  T3 0.75Kg / ha 3.25 B 0.78 B 6.60 BA
T4 1.0 Kg/ha 4.02 A 0.91 A 6.77 A  T4 1.0 Kg / ha 4.02 A 0.91 A 6.77 A
De este ensayo se concluye que la composición que comprende la cepa Pseudomonas fluorescens CECT 9015 promueve el crecimiento en altura, aumento de la biomasa (tanto fresca como seca), la longitud de la raíz, incrementa el rendimiento y la absorción de nitrógeno, fósforo y potasio en plantas en cultivo. 3.3.3 Ensayo de incremento de producción sobre cultivo de apio From this test it is concluded that the composition comprising the strain Pseudomonas fluorescens CECT 9015 promotes height growth, increased biomass (both fresh and dry), root length, increases yield and absorption of nitrogen, phosphorus and Potassium in growing plants. 3.3.3 Production increase test on celery cultivation
El experimento se plantea con el objetivo de comprobar la efectividad biológica de una composición que comprende la cepa P. fluorescens CECT 9015 sobre un cultivo de apio (Apium graveolens L). Para ello se cultivaron las plantas en surcos con dos líneas de cultivo separadas por una tubería de riego por goteo. En una parcela se aplicará el producto y en la otra el control.  The experiment is proposed with the objective of checking the biological effectiveness of a composition comprising P. fluorescens CECT 9015 strain on a celery crop (Apium graveolens L). For this, the plants were cultivated in furrows with two crop lines separated by a drip irrigation pipe. In one plot the product will be applied and in the other the control.
La composición usada en este ensayo es: P. fluorescens CECT 9015 (5 x 108 ufc/g), Bacillus amyloliquefaciens CECT 9017 (5 x 108 ufc/g), B. subtilis CECT 9016 (5 x 108 ufc/g), Trichoderma harzianum CECT 20946 (5 x 108 ufc/g) y como coformulantes un hidrolizado proteico de origen vegetal, Saccharomyces cerevisiae, ácidos húmicos y ácidos fúlvicos. La composición se aplicó a las plantas vía riego por goteo 15 días (primera aplicación) y 45 días (segunda aplicación) después del trasplante a una concentración de 2.0 kg/ha. El control consistió en el protocolo de manejo habitual de la finca, sin la adición de la composición bioestimulante/biofertilizante de la presente invención. De cada tratamiento se evaluó: el crecimiento de las raíces y la biomasa fresca (45 días después del trasplante) tal y como está descrito en el apartado 2.4 de esta memoria. A los 15 días de la primera aplicación del producto se observó ya una clara diferencia entre el tamaño del cepellón de las plantas tratadas, con mayor número de pelos radiculares (Figura 9). En el momento de la recolección de las plantas se seleccionan al azar 5 plantas en la parcela testigo y 5 plantas en la parcela tratada. Se pesaron las 5 plantas seleccionadas de cada una de las parcelas y se observó que el peso medio de las plantas de apio de la parcela tratada con la composición (T1) es un 29% mayor que la parcela testigo (To), y además hay mayor homogeneidad de pesos (Tabla 21). The composition used in this test is: P. fluorescens CECT 9015 (5 x 10 8 cfu / g), Bacillus amyloliquefaciens CECT 9017 (5 x 10 8 cfu / g), B. subtilis CECT 9016 (5 x 10 8 cfu / g ), Trichoderma harzianum CECT 20946 (5 x 10 8 cfu / g) and as co-formulants a protein hydrolyzate of vegetable origin, Saccharomyces cerevisiae, humic acids and fulvic acids. The composition was applied to the plants via drip irrigation 15 days (first application) and 45 days (second application) after transplantation at a concentration of 2.0 kg / ha. The control consisted of the usual farm management protocol, without the addition of the biostimulant / biofertilizer composition of the present invention. Each treatment was evaluated: root growth and fresh biomass (45 days after transplantation) as described in section 2.4 of this report. At 15 days after the first application of the product, a clear difference between the size of the root ball of the treated plants was observed, with a greater number of root hairs (Figure 9). At the time of plant collection, 5 plants are randomly selected in the control plot and 5 plants in the treated plot. The 5 selected plants of each of the plots were weighed and it was observed that the average weight of the celery plants of the plot treated with the composition (T1) is 29% greater than the control plot (To), and there are also greater homogeneity of weights (Table 21).
Tabla 21 : Peso fresco de las plantas de apio Table 21: Fresh weight of celery plants
Plantas  Plants
T1 To  T1 To
analizadas  analyzed
1 1 ,47 1 ,77  1 1, 47 1, 77
2 1 ,90 1 ,04  2 1, 90 1, 04
3 1 ,95 1 ,61  3 1, 95 1, 61
4 1 ,73 1 ,70  4 1, 73 1, 70
5 2, 14 1 ,00  5 2, 14 1, 00
Media 1 ,84 1 ,42  Mean 1, 84 1, 42
Desviación  Deviation
0,25 0,37  0.25 0.37
estándar  standard
Error estándar 0, 11 0,17  Standard error 0.11 0.17
Incremento (%) 29,07 3.3.4 Ensayo de incremento de producción sobre cultivo de tomate  Increase (%) 29.07 3.3.4 Production increase test on tomato crop
Se cultivó la variedad de tomate híbrido Mariana injertada sobre patrón de la variedad Arnold, mediante la técnica de entutorado con descuelgue de tipo holandés. El ensayo se llevó a cabo de forma paralela sobre suelo y sobre sustrato inerte con fertirrigación. En suelo se aplicaron dos composiciones (IM1 y IM2) a dos sectores de una parcela (3157 plantas), y se compararon con un sector Testigo de las mismas dimensiones. Para el ensayo en sustrato con fertirrigación se utilizaron 2 sectores, uno en lana de roca y otro en fibra de coco, a los que se aplicó la composición IM3, y se utilizó un sector de lana de roca como Testigo en el que se aplicó el tratamiento habitual de la finca sin aplicación de la composición bioestimulante de la presente invención. El producto IM1 contiene P. fluorescens CECT 9015 y los coformulantes S. cerevisiae, ácidos húmicos y aminoácidos. El producto IM2 contiene la formulación IM 1 y además P. putida CECT 9011. El producto IM3 contiene P. fluorescens CECT 9015 y S. cerevisiae formuladas sobre un hidrolizado proteico de origen vegetal. El producto IM3 se aplica a una dosis de 200 g/ha, mientras que los productos IM1 y IM2 se aplican a una dosis de 2 kg/ha. A lo largo del ciclo de cultivo se hicieron 6 aplicaciones de cada uno de los productos, aproximadamente una vez al mes con el siguiente protocolo de aplicación: The Mariana hybrid tomato variety grafted onto the Arnold variety pattern was cultivated using the Dutch-type pick-up technique. The test was carried out in parallel on soil and on inert substrate with fertirrigation. In soil two compositions (IM1 and IM2) were applied to two sectors of a plot (3157 plants), and compared with a Witness sector of the same dimensions. For the fertirrigation substrate test, 2 sectors were used, one in rock wool and the other in coconut fiber, to which the IM3 composition was applied, and a rock wool sector was used as a Witness in which the usual treatment of the farm without application of the biostimulant composition of the present invention. The product IM1 contains P. fluorescens CECT 9015 and the coformulants S. cerevisiae, humic acids and amino acids. The product IM2 contains the formulation IM 1 and also P. putida CECT 9011. The product IM3 contains P. fluorescens CECT 9015 and S. cerevisiae formulated on a protein hydrolyzate of vegetable origin. The IM3 product is applied at a dose of 200 g / ha, while the IM1 and IM2 products are applied at a dose of 2 kg / ha. Throughout the crop cycle, 6 applications of each of the products were made, approximately once a month with the following application protocol:
Se diluye el producto en polvo en 100-200 litros de agua no clorada  The powder product is diluted in 100-200 liters of unchlorinated water
- Se deja el producto reposando en el agua durante al menos 30 minutos para que los microorganismos liofilizados se rehidraten correctamente y recuperen su actividad. Se aplica el producto inyectándolo al sistema de riego por goteo, con el objetivo de que llegue al suelo.  - The product is left standing in the water for at least 30 minutes so that the lyophilized microorganisms rehydrate correctly and recover their activity. The product is applied by injecting it to the drip irrigation system, in order to reach the ground.
La Figura 10A muestra la producción acumulada. Los datos indican claramente que el tratamiento con el inoculo microbiano IM2 (representado por triángulos en la gráfica) aumenta la producción acumulada al final del ensayo en un 42,41 % respecto al testigo, mientras que la parcela tratada con el inoculo IM1 (representado por cuadrados en la gráfica) presenta inicialmente menor producción que el testigo, y posteriormente lo supera con un 6,02% más de producción acumulada al final del ensayo. Los resultados indican que la producción acumulada de las plantas tratadas con la combinación de cepas P. fluorescens CECT 9015 y P. putida CECT 9011 es mayor que la producción con plantas tratadas con la composición que comprende P. fluorescens CECT 9015, que a su vez es superior que la producción en las plantas sin tratar. Figure 10A shows the cumulative production. The data clearly indicate that the treatment with the microbial inoculum IM2 (represented by triangles in the graph) increases the cumulative production at the end of the test by 42.41% with respect to the control, while the plot treated with the inoculum IM1 (represented by squares in the graph) initially shows lower production than the control, and subsequently exceeds it with 6.02% more accumulated production at the end of the test. The results indicate that the cumulative production of the plants treated with the combination of P. fluorescens CECT 9015 and P. putida CECT 9011 strains is greater than the production with plants treated with the composition comprising P. fluorescens CECT 9015, which in turn It is higher than the production in untreated plants.
Las plantas se clasifican de acuerdo a su categoría y calibre de acuerdo con lo descrito en el apartado 2.4 del presente documento. La Figura 10B muestra clasificación por calibres y categorías de la producción acumulada en cada parcela del ensayo de suelo. La aplicación de la composición IM2 aumenta la producción respecto al Testigo y el tratamiento con IM1 , favoreciendo de forma equilibrada un incremento de la producción de tomate en todos los calibres. También se aprecia que la mayor parte (80,1 1 %) de la producción de la parcela tratada con IM2 se clasifica como Categoría Extra, la de mayor valor económico. Plants are classified according to their category and size according to what is described in section 2.4 of this document. Figure 10B shows classification by caliber and categories of cumulative production in each plot of the soil test. The application of the IM2 composition increases the production with respect to the Witness and the treatment with IM1, favoring in a balanced way an increase in tomato production in all sizes. It is also appreciated that the majority (80.1%) of the production of the plot treated with IM2 is classified as Extra Category, the one with the highest economic value.
Los datos de producción acumulada del cultivo hidropónico se representan en la Figura 11 A. En lana de roca se observa que el tratamiento con la composición bioestimulante IM3 (representado por cruces en la gráfica) aumenta la producción acumulada en un 20,03% respecto al testigo (representado por rombos en la gráfica). La distribución por categorías de la producción acumulada hasta el final del ensayo muestra que en lana de roca el porcentaje de tomates de categoría Extra en el sector tratado con IM3 es similar al Testigo. Sin embargo, si se tiene en cuenta que hay una diferencia de la producción acumulada de 1 ,76 kg/planta entre la parcela tratada con IM3 y la parcela Testigo, la distribución por categorías indica que con la composición IM3 se han obtenido 1 ,47 kg de tomates de Categoría Extra más por planta que en la parcela Testigo, lo que revertirá en mayores beneficios para el agricultor. The cumulative production data of the hydroponic culture are represented in Figure 11 A. In rock wool it is observed that the treatment with the biostimulant composition IM3 (represented by crosses in the graph) increases the accumulated production by 20.03% with respect to the witness (represented by rhombuses in the graph). The distribution by categories of the accumulated production until the end of the test shows that in rock wool the percentage of tomatoes of Extra category in the sector treated with IM3 is similar to the Witness. However, if one takes into account that there is a cumulative production difference of 1.76 kg / plant between the plot treated with IM3 and the Witness plot, the distribution by categories indicates that with the IM3 composition 1, 47 have been obtained kg of Extra Category tomatoes more per plant than in the Witness plot, which will revert to greater benefits for the farmer.
La clasificación final por calibres y categorías de la producción acumulada en los sectores del ensayo en hidroponico se muestra en la Figura 1 1 B. Se observa que la aplicación de la composición IM3 aumenta la producción respecto al Testigo en lana de roca, favoreciendo sobre todo un incremento de los tomates de calibre Mp, y en menor medida Mg y MMg. También se aprecia que la mayor parte (81 , 15%) de la producción de la parcela tratada con IM3 se clasifica como Categoría Extra, la de mayor valor económico. The final classification by caliber and categories of cumulative production in the hydroponic test sectors is shown in Figure 1 1 B. It is observed that the application of the IM3 composition increases the production with respect to the Witness in rock wool, favoring above all an increase in tomatoes of caliber Mp, and to a lesser extent Mg and MMg. It is also appreciated that most (81, 15%) of the production of the plot treated with IM3 is classified as Extra Category, the one with the highest economic value.
Como conclusión a este ensayo, se puede afirmar que las composiciones comprenden P fluorescens CECT 9015 tienen un efecto bioestimulante y biofertilizante sobre un cultivo comercial de tomate. La aplicación de la composición bioestimulante IM2 (P. fluorescens CECT 9015 y P. putida CECT 901 1) aumenta significativamente la producción acumulada respecto al Testigo (42,41 %), predominando los tomates de categoría Extra (82,69%) y calibre Mp. Los resultados del ensayo sobre lana de roca indican que el inoculo microbiano IM3 (P fluorescens CECT 9015) aumenta significativamente (20%) la producción acumulada respecto al testigo, predominando también los tomates de categoría Extra (84%) y calibre Mp. As a conclusion to this test, it can be stated that the compositions comprise P fluorescens CECT 9015 have a biostimulant and biofertilizing effect on a commercial tomato crop. The application of the IM2 biostimulant composition (P. fluorescens CECT 9015 and P. putida CECT 901 1) significantly increases the accumulated production compared to the Witness (42.41%), predominantly tomatoes of Extra category (82.69%) and caliber Mp. The results of the rock wool test indicate that the IM3 microbial inoculum (P fluorescens CECT 9015) significantly increases (20%) the accumulated production with respect to the control, also predominantly tomatoes of Extra category (84%) and Mp caliber.
3.3.5 Ensayo de estimulación radicular y foliar sobre el cultivo de uva 3.3.5 Root and foliar stimulation test on grape cultivation
Se cultivó uva de mesa de 6 años de edad variedad Red Globe. El ensayo se llevó a cabo de aplicando dos tratamientos (To: testigo y T1 : composición bioestimulante) con tres repeticiones, donde cada una corresponde a lotes completos en los que el lote tratado es de 8.16 ha y el lote Testigo de 1.14 ha.  Red Grape 6-year-old table grape was grown. The test was carried out by applying two treatments (To: control and T1: biostimulant composition) with three repetitions, where each corresponds to complete batches in which the treated batch is 8.16 ha and the Witness batch of 1.14 ha.
T1 es una composición compuesta por los siguientes microorganismos y coformulantes: Inoculo microbiano (108 UFC/g) que consiste en Bacillus subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015, y los coformulantes S. cerevisiae, 40% p/p de carbono orgánico total, 41 % ácidos húmicos, 19% ácidos fúlvicos, 6.5% p/p nitrógeno total, 4.5% p/p potasio (K2O) total y 1 1 % p/p aminoácidos libres. Se realizaron tres aplicaciones de 2 kg/ha. La primera aplicación fue en el momento del flash radicular de finales de verano, la segunda unos 40-60 días después de la poda de producción (inicio del crecimiento radicular de primavera, y la tercera antes de la "pinta" o envero de la uva, momento en el que el fruto se ablanda, aumentando los azúcares y disminuyendo su acidez, cambia de color y aumenta de tamaño. T1 is a composition composed of the following microorganisms and coformulants: Microbial inoculum (10 8 CFU / g) consisting of Bacillus subtilis CECT 9016, B. amyloliquefaciens CECT 9017, Pseudomonas fluorescens CECT 9015, and coformulants S. cerevisiae, 40% p / p total organic carbon, 41% humic acids, 19% fulvic acids, 6.5% w / w total nitrogen, 4.5% w / w total potassium (K2O) and 1 1% w / w free amino acids. Three applications of 2 kg / ha were made. The first application was at the time of the root flash of late summer, the second about 40-60 days after the pruning of production (beginning of spring root growth, and the third before the "pint" or envero of the Grape, at which time the fruit softens, increasing sugars and decreasing acidity, changes color and increases in size.
Se evaluaron las raíces de las plantas mediante calicatas de 1 m2 de superficie en dos líneas de la parcela tratada y dos de la parcela testigo. La superficie de cada calicata se dividió en 100 cuadrantes de 10 m2, y se contó el número de cuadrantes que tienen raíces, y entre éstos, aquellos que presentan raíces nuevas de color claro, no suberizadas. Como se indica en la Tabla 22, se observa que en la parcela tratada con la composición T1 haya mayor porcentaje de cuadrantes con raíces, y más raíces nuevas, lo que demuestra que el producto estimula el desarrollo radicular. El porcentaje representa el número de cuadrantes en la superficie de 1 m2 en los que se detectó la presencia de raíces (raíces totales) y la presencia de raíces nuevas. The roots of the plants were evaluated by calicatas of 1 m 2 of surface in two lines of the treated plot and two of the control plot. The surface of each calicata was divided into 100 quadrants of 10 m 2 , and the number of quadrants that have roots was counted, and among these, those that have new, light, non-sub-rooted new roots. As indicated in Table 22, it is observed that in the plot treated with the composition T1 there is a higher percentage of quadrants with roots, and more new roots, which shows that the product stimulates root development. The percentage represents the number of quadrants on the surface of 1 m 2 in which the presence of roots (total roots) and the presence of new roots were detected.
Tabla 22: Resultados de las calicatas realizadas en dos líneas de la parcela tratada y la parcela testigo Table 22: Results of the calicatas made in two lines of the treated plot and the control plot
% Raíces % Raíces  % Roots% Roots
totales nuevas  new totals
LINEA 8 - ■T1 78 30 LINE 8 - ■ T1 78 30
LÍNEA 21 - T1 67 36  LINE 21 - S1 67 36
LÍNEA 31 - To 40 7  LINE 31 - To 40 7
LÍNEA 41 - To 54 19 Para evaluar el peso seco de las raíces, instaló una jaula en los sectores tratado y testigo. Las jaulas se instalan en el medio de cada parrón del cultivo y entre plantas con las mismas características fenológicas. Se dividió el área ocupada por cada planta en 4 cuadrantes, y en uno de ellos (el mismo en todas las plantas) se instaló la jaula antes de la aplicación de la composición. Estas jaulas se retiraron al final del ensayo (post cosecha) para comparar el desarrollo y peso radicular fresco. La composición T1 incrementa significativamente el desarrollo de raíces respecto del testigo, aumentando el peso de raíces dentro de la jaula colocada en más del doble. Los datos de la parte tratada son la media de dos jaulas instaladas (Figura 12).  LINE 41 - To 54 19 To assess the dry weight of the roots, a cage was installed in the treated and control sectors. The cages are installed in the middle of each crop stop and between plants with the same phenological characteristics. The area occupied by each plant was divided into 4 quadrants, and in one of them (the same in all the plants) the cage was installed before the application of the composition. These cages were removed at the end of the trial (post harvest) to compare the development and fresh root weight. The T1 composition significantly increases the development of roots with respect to the control, increasing the weight of roots within the cage placed more than double. The data of the treated part is the average of two cages installed (Figure 12).
Como conclusión, indicar que la composición que comprende la cepa P. fluorescens CECT 9015 promueve el desarrollo radicular, produciendo un incremento en la superficie radicular del 50% respecto al testigo y estimula el desarrollo de raíces nuevas hasta valores de más del doble en la parte tratada respecto al cultivo sin tratar. In conclusion, indicate that the composition comprising the strain P. fluorescens CECT 9015 promotes root development, producing a 50% increase in root surface area compared to the control and stimulates the development of new roots to values more than double in the part treated with respect to untreated culture.
3.3.6 Ensayo de estimulación radicular, absorción de nutrientes y mejora de la cantidad y calidad de la cosecha de un cultivo de aguacate Se cultivó aguacate variedad Hass en árboles de 10 años. El ensayo se llevó a cabo de aplicando dos tratamientos (To: testigo y T1 : composición descrita en el ejemplo 3.3.5). Para su aplicación en el cultivo, T1 se disuelve en agua y se inyecta en el sistema de riego. El tratamiento consistió en tres aplicaciones de T1 a una dosis de 2 kg/ha al inicio de la floración, a los 30 días de la primera aplicación y 30 días después de la segunda aplicación (1 , 2 y 3). 3.3.6 Test of root stimulation, nutrient absorption and improvement of the quantity and quality of the harvest of an avocado crop Hass avocado variety was grown on 10-year-old trees. The test was carried out by applying two treatments (To: control and T1: composition described in example 3.3.5). For application in cultivation, T1 is dissolved in water and injected into the irrigation system. The treatment consisted of three applications of T1 at a dose of 2 kg / ha at the beginning of flowering, 30 days after the first application and 30 days after the second application (1, 2 and 3).
En primer lugar, se analizó el suelo tomando una muestra lo más cercana posible a las raíces, en la estrata de mayor concentración de raíces en diferentes momentos del ensayo (antes de la aplicación de la composición T1 , 15 días después de la aplicación, 30 días después de la aplicación y al término del ensayo). Se evaluaron parámetros como la conductividad eléctrica, pH, fósforo disponible, caliza activa, nitrógeno Dumas, materia orgánica oxidable, capacidad de intercambio iónico efectiva, propiedades físicas (contenido en arena, arcilla, limo y granulometría), relación C/N, calcio, magnesio, potasio, sodio y zinc disponibles, la distribución de estos compuestos y la relación entre ellos, calcio, magnesio, potasio, sodio y bases de cambio, boro, cobre (DTPA) y hierro (DTPA). Los resultados de la analítica presentados en la Figura 13 indican que al final del ciclo de cultivo, tras tres aplicaciones de T1 , se incrementa la disponibilidad de macroelementos primarios (nitrógeno, fósforo, potasio), macroelementos secundarios (calcio, magnesio) y microelementos como el hierro, que suele presentar problemas de disponibilidad. También se ha demostrado que estos microorganismos aumentan la eficiencia en el uso del nitrógeno, y pueden favorecer la absorción de cationes como el potasio. Por otro lado, se observa que el producto incrementa el contenido en materia orgánica del suelo y la capacidad de intercambio catiónico (Figura 13 G-H). First, the soil was analyzed by taking a sample as close as possible to the roots, in the layer with the highest concentration of roots at different times of the test (before the application of the T1 composition, 15 days after the application, 30 days after application and at the end of the test). Parameters such as electrical conductivity, pH, available phosphorus, active limestone, Dumas nitrogen, oxidizable organic matter, effective ion exchange capacity, physical properties (content in sand, clay, silt and grain size), C / N ratio, calcium, were evaluated. magnesium, potassium, sodium and zinc available, the distribution of these compounds and the relationship between them, calcium, magnesium, potassium, sodium and exchange bases, boron, copper (DTPA) and iron (DTPA). The analytical results presented in Figure 13 indicate that at the end of the culture cycle, after three applications of T1, the availability of primary macroelements (nitrogen, phosphorus, potassium), secondary macroelements (calcium, magnesium) and microelements such as iron, which usually presents problems of availability. It has also been shown that these microorganisms increase the efficiency in the use of nitrogen, and may favor the absorption of cations such as potassium. On the other hand, it is observed that the product increases the organic matter content of the soil and the cation exchange capacity (Figure 13 G-H).
Para evaluar las raíces de las plantas se llevó a cabo un conteo de presencia de raíces en cada uno de los cuadrantes de la calicata siguiendo el procedimiento explicado en el ensayo 3.3.5. To assess the roots of the plants, a root presence count was carried out in each of the quadrants of the calicate following the procedure explained in the test 3.3.5.
Como se observa en la Tabla 23, en la parcela tratada con la composición T1 hay mayor porcentaje de cuadrantes con raíces, y más raíces nuevas, lo que demuestra que el producto estimula el desarrollo radicular, más del doble que la composición testigo To. El porcentaje representa el número de cuadrantes en la superficie de 1 m2 en los que se detectó la presencia de raíces (raíces totales) y la presencia de raíces nuevas. As can be seen in Table 23, in the plot treated with the composition T1 there is a higher percentage of quadrants with roots, and more new roots, which shows that the product stimulates root development, more than double the control composition To. The percentage represents the number of quadrants on the surface of 1 m 2 in which the presence of roots (total roots) and the presence of new roots were detected.
Tabla 23: Resultados de las calicatas realizadas en dos líneas de la parcela tratada y la parcela testigo Table 23: Results of the calicatas made in two lines of the treated plot and the control plot
Tratamiento % Raíces totales % Raíces nuevas  Treatment% Total roots% New roots
T1 74 31 Por último, se evaluó la producción de aguacate al final del ciclo de cultivo. Para ello se muestrearon 10 árboles escogidos al azar en la parcela tratada con T1 y en la parcela testigo, y se contó el número de frutos por árbol (Figura 14). Se observa que la composición bioestimulante T1 favorece un incremento del número de frutos por árbol
Figure imgf000044_0001
,811 ; P=0, 195) que podría suponer un aumento de ingresos para el agricultor. S1 74 31 Finally, avocado production was evaluated at the end of the growing cycle. For this, 10 randomly selected trees were sampled in the plot treated with T1 and in the control plot, and the number of fruits per tree was counted (Figure 14). It is observed that the biostimulant composition T1 favors an increase in the number of fruits per tree
Figure imgf000044_0001
, 811; P = 0, 195) which could mean an increase in income for the farmer.
Como conclusiones, indicar que la composición T1 que comprende P. fluorescens CECT 9015 aumenta la disponibilidad de nutrientes en el suelo, en particular de nitrógeno y potasio, aumenta el desarrollo radicular, tanto en superficie como en peso y favorece un aumento del número de frutos por árbol. 3.3.7 Ensayo de evaluación de la efectividad biológica de un inoculante microbiano en el cultivo de tomate. As conclusions, indicate that the composition T1 comprising P. fluorescens CECT 9015 increases the availability of nutrients in the soil, in particular nitrogen and potassium, increases root development, both in surface and weight and favors an increase in the number of fruits per tree 3.3.7 Test of evaluation of the biological effectiveness of a microbial inoculant in tomato cultivation.
El ensayo se llevó a cabo en un cultivo de tomate (Solanum lycopersicum var. Toro) al aire libre en Yechar (Muía). Se utilizaron 6 composiciones diferentes (M1 , M2, M3, M4, M5 y M6) y se realizaron 4 aplicaciones de cada tratamiento vía riego durante el ciclo de cultivo, con 20 plantas por repetición, en la zona radicular de las plantas. Las aplicaciones tuvieron lugar en intervalos de aproximadamente 15 días. La densidad de la siembra fue de 0.5 x 2 m, 10.000 plantas por ha al aire libre y con riego por goteo (4 l/h). Se aplicó a una dosis de 250 litros por hectárea, por lo que la dosis por planta y aplicación fue 25 ml/planta.  The test was carried out in a tomato crop (Solanum lycopersicum var. Toro) outdoors in Yechar (Muía). 6 different compositions (M1, M2, M3, M4, M5 and M6) were used and 4 applications of each treatment were made via irrigation during the crop cycle, with 20 plants per repetition, in the root zone of the plants. Applications took place at intervals of approximately 15 days. The sowing density was 0.5 x 2 m, 10,000 plants per ha outdoors and drip irrigation (4 l / h). It was applied at a dose of 250 liters per hectare, so the dose per plant and application was 25 ml / plant.
Se realizaron tres recolecciones en las que se evaluaron 40 plantas por cada uno de los tratamientos. Three collections were made in which 40 plants were evaluated for each of the treatments.
M1 : Control. Solución de aminoácidos 0.2%.  M1: Control. 0.2% amino acid solution.
M2: P. fluorescens CECT 9015 108 UFC/g + aminoácidos 0.2%. M2: P. fluorescens CECT 9015 10 8 CFU / g + amino acids 0.2%.
M3: P fluorescens CECT 9015 + P putida CECT 9011 , ambas a 108 UFC/g+ aminoácidos 0.2%. M3: P fluorescens CECT 9015 + P putida CECT 9011, both at 10 8 CFU / g + amino acids 0.2%.
M4: P fluorescens CECT 9015 + P putida CECT 9011 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, todas a 108 UFC/g + aminoácidos 0.2%. M4: P fluorescens CECT 9015 + P putida CECT 9011 + B. subtilis CECT 9016 + B. licheniformis CECT 9018 + B. amyloliquefaciens CECT 9017 + A. oxydans CECT 7170 + T. harzanium CECT 20946, all at 10 8 CFU / g + 0.2% amino acids.
M5: P fluorescens CECT 378 + aminoácidos 0.2%.  M5: P fluorescens CECT 378 + amino acids 0.2%.
M6: P fluorescens CECT 378 + P. putida MTCC 5670 + aminoácidos a 0.2%. Las cepas P. fluorescens CECT 378 y P putida MTCC 5670 son cepas comerciales, disponibles en el estado de la técnica, utilizadas en el presente documento como controles positivos para valorar la capacidad bioestimulante de las composiciones de la invención.  M6: P fluorescens CECT 378 + P. putida MTCC 5670 + amino acids at 0.2%. The P. fluorescens CECT 378 and P putida MTCC 5670 strains are commercial strains, available in the state of the art, used herein as positive controls to assess the biostimulant capacity of the compositions of the invention.
Los parámetros fotosintéticos se midieron durante el cultivo, antes de la cuarta aplicación, mientras que el resto de parámetros se evaluaron al final de cada cosecha. Se realizó un ANOVA simple para analizar la significación de las diferencias debidas al tratamiento. Las medias de los tratamientos fueron separadas con la prueba de la mínima diferencia significativa (LSD) (P<0,05). Las distintas letras en las gráficas indican diferencias significativas entre los tratamientos ensayados. La fotosíntesis de las plantas de tomate del cultivo se midió de acuerdo a la metodología indicada en el apartado 2.4 de esta memoria. La Figura 15 recoge el resultado de los parámetros fotosintéticos medidos. En la Figura 15A se ve que las composiciones M3 y M5 producen mayor estrés a la planta (incremento significativo de Fo) y aumentan el quenching fotoquímico del fotosistema II (OPSII) también es significativamente mayor en las combinaciones M3 y M5. Es interesante destacar que la disipación de calor durante la fotosíntesis es menor en las plantas tratadas con M3, lo que indica una mayor eficiencia fotosintética. A la luz de todos los datos descritos, se concluye que la combinación de las cepas P. fluorescens CECT 9015 y P. putida CECT 901 1 son mejores para favorecer la fotosíntesis. La producción del cultivo se midió como el peso en toneladas de los frutos totales por hectárea de cultivo. Se realizó una medida después de cada una de las cosechas, y el resultado se indica en la Tabla 24 junto con la producción total del cultivo para cada composición. Photosynthetic parameters were measured during cultivation, before the fourth application, while the rest of the parameters were evaluated at the end of each harvest. A Simple ANOVA to analyze the significance of the differences due to treatment. The means of the treatments were separated with the test of the least significant difference (LSD) (P <0.05). The different letters in the graphs indicate significant differences between the treatments tested. The photosynthesis of the tomato plants of the crop was measured according to the methodology indicated in section 2.4 of this report. Figure 15 shows the result of the measured photosynthetic parameters. Figure 15A shows that the M3 and M5 compositions produce greater stress to the plant (significant increase in Fo) and increase the photochemical quenching of photosystem II (OPSII) is also significantly greater in the M3 and M5 combinations. It is interesting to note that heat dissipation during photosynthesis is lower in plants treated with M3, which indicates greater photosynthetic efficiency. In light of all the data described, it is concluded that the combination of the P. fluorescens CECT 9015 and P. putida CECT 901 1 strains are better for promoting photosynthesis. The crop production was measured as the weight in tons of total fruits per hectare of crop. A measurement was made after each of the crops, and the result is indicated in Table 24 together with the total crop production for each composition.
Tabla 24: Producción en cada una de las cosechas (Tn/ha) Table 24: Production in each of the crops (Tn / ha)
Figure imgf000045_0001
A partir de los datos presentes en la Tabla 25 se observa que el tratamiento M2 es el más productivo de forma total y el más precoz, pero la composición que comprende las cepas P. fluorescens CECT 9015 y P. putida CECT 9011 (M3) y la composición que comprende todas las cepas son las más productivas en la segunda cosecha. La cepa de P. fluorescens CECT 9015 es más productiva que la cepa comercial de referencia (CECT 378). De los frutos del cultivo se analizaron el número de frutos por planta, el contenido en azúcares, su grado de madurez y la acidez.
Figure imgf000045_0001
From the data present in Table 25, it is observed that the M2 treatment is the most productive in total and the earliest, but the composition comprising the P. fluorescens CECT 9015 and P. putida CECT 9011 (M3) and The composition that comprises all the strains are the most productive in the second harvest. The strain of P. fluorescens CECT 9015 is more productive than the commercial reference strain (CECT 378). From the fruits of the crop, the number of fruits per plant, the sugar content, its maturity level and acidity were analyzed.
Se analizó el número de frutos por planta, comprobándose que para cada cosecha y tratamiento presenta el mismo patrón que en el caso de la producción. Tabla 25: Número de frutos por planta. The number of fruits per plant was analyzed, checking that for each harvest and treatment it has the same pattern as in the case of production. Table 25: Number of fruits per plant.
Figure imgf000046_0001
Figure imgf000046_0001
El tratamiento que produjo mayor número de frutos fue M2 (44,8 frutos/planta), mientras que M6 fue el tratamiento menos eficaz (36,4 frutos/planta).  The treatment that produced the greatest number of fruits was M2 (44.8 fruits / plant), while M6 was the least effective treatment (36.4 fruits / plant).
Para medir la calidad interna del fruto se realizó un triturado con cinco tomates por repetición y se midió la acidez y el índice de madurez después de las tres cosechas. La Tablas 26 y 27 muestran los resultados de la medida de estos parámetros en cada tratamiento. To measure the internal quality of the fruit, crushing with five tomatoes per repetition was performed and the acidity and maturity index were measured after the three harvests. Tables 26 and 27 show the results of the measurement of these parameters in each treatment.
El índice de madurez se midió como el cociente °Brix/% de acidez titulable. Este valor se obtuvo midiendo el volumen desplazado mediante la titulación con bureta (25 mi) y NaOH 0, 1 N. The maturity index was measured as the quotient Brix /% of titratable acidity. This value was obtained by measuring the volume displaced by titration with burette (25 ml) and 0.1 N NaOH.
Tabla 26: índice de madurez (E/A) Table 26: Maturity index (I / O)
Figure imgf000046_0002
Figure imgf000046_0002
Se observa que los frutos de las plantas a las que se administró composición M2 tienen mayor grado de madurez que los de las plantas a las que se les administró M5, lo que demuestra que la cepa CECT 9015 de P. fluorescens de la invención es mejor que otras cepas de la misma especie. It is observed that the fruits of the plants to which M2 composition was administered have a higher degree of maturity than those of the plants to which M5 was administered, which demonstrates that the CECT 9015 strain of P. fluorescens of the invention is better than other strains of the same species.
La evaluación de la acidez en el zumo de tomate se cuantificó mediante una valoración ácido- base, en donde los ácidos presentes en el zumo fueron neutralizados con una solución básica de hidróxido de sodio. Se produce una reacción de neutralización del ácido con el hidróxido de sodio. Tabla 27: Acidez del fruto. The acidity evaluation in tomato juice was quantified by an acid-base titration, where the acids present in the juice were neutralized with a basic solution of sodium hydroxide. A neutralization reaction of the acid with sodium hydroxide occurs. Table 27: Fruit acidity.
Figure imgf000047_0001
Figure imgf000047_0001
Los frutos del cultivo tratado con las composiciones que contienen la cepa cepas P fluorescens CECT 9015 (M2 y M3) son menos ácidos que el resto de los frutos. Las composiciones que comprenden la cepa comercial de P. fluorescens (M5 y M6) son más ácidos, lo que demuestra el mejor efecto de la cepa reivindicada frente a otras de la misma especie. The fruits of the culture treated with the compositions containing the strain P fluorescens CECT 9015 strains (M2 and M3) are less acidic than the rest of the fruits. The compositions comprising the commercial strain of P. fluorescens (M5 and M6) are more acidic, demonstrating the best effect of the claimed strain against others of the same species.
Se analizaron los componentes bioactivos en hojas y frutos tales como los fenoles y flavonoles de alto potencial antioxidante. La Figura 16A muestra el contenido de fenoles en las hojas de tomate medidos como equivalentes mg/ácido gálico por cada 100 gramos de peso fresco. La Figura 16B muestra el contenido en flavonoles de hojas, medido como equivalentes mg de catequina por 100 gramos de peso fresco. En la Figura 16A se observa que las composiciones M2 y M5 incrementan la concentración de fenoles en las hojas de tomate, mientras que la composición M2 es la que más cantidad de flavonoles contiene, lo que corrobora una vez más que la cepa P. fluorescens CECT 9015 tiene un efecto en las plantas mejor que otras cepas de la misma especie. Los flavonoles, además de antioxidantes, tienen un efecto muy importante en la defensa de la planta, por lo que se concluye que la cepa P. fluorescens CECT 9015 es la que tiene un mayor potencial defensivo. Además, los efectos de la combinación de las cepas P. fluorescens CECT 9015 y P. putida CECT 9011 son también mejores que la combinación de las cepas respectivas de las mismas especies descritas en el estado del arte (P. fluorescens CECT 378 y P. putida MTCC). Bioactive components in leaves and fruits such as phenols and flavonols with high antioxidant potential were analyzed. Figure 16A shows the phenol content in tomato leaves measured as mg / gallic acid equivalents per 100 grams of fresh weight. Figure 16B shows the flavonial content of leaves, measured as mg equivalent of catechin per 100 grams of fresh weight. Figure 16A shows that compositions M2 and M5 increase the concentration of phenols in tomato leaves, while composition M2 is the one that contains the most flavonols, which confirms once more than the strain P. fluorescens CECT 9015 has a better effect on plants than other strains of the same species. Flavonols, in addition to antioxidants, have a very important effect on the defense of the plant, so it is concluded that the strain P. fluorescens CECT 9015 is the one with the greatest defensive potential. In addition, the effects of the combination of the P. fluorescens CECT 9015 and P. putida CECT 9011 strains are also better than the combination of the respective strains of the same species described in the state of the art (P. fluorescens CECT 378 and P. MTCC putida).
La Figura 17 muestra los fenoles (Figura 17) y los flavonoles (Figura 18) en los frutos de tomate en cada una de las tres cosechas. Mientras que no se aprecian diferencias en los contenidos de fenoles en las composiciones empleadas en este ensayo, el contenido en flavonoles después de la cosecha 3 es mucho mayor en las composiciones M3 y M4. En los 0.15 mg de equivalentes de catequinas observados después de la tercera cosecha supone 5 veces más flavonoles en estos frutos, lo que representa un aumento de su calidad. De estos resultados se concluye que las plantas a las que se les aplican composiciones que comprenden la cepa P. fluorescens CECT 9015, sola o en combinación con otros microorganismos tienen mejor potencial defensivo, mejor producción, sus frutos maduran antes y son menos ácidos, y contienen bioactivos beneficiosos para la salud del consumidor, lo que dota de gran valor a esta cepa, frente a otras cepas de la misma especie, que no producen estos efectos estimulantes en las plantas. Figure 17 shows the phenols (Figure 17) and flavonols (Figure 18) in tomato fruits in each of the three crops. While there are no differences in the phenol contents in the compositions used in this test, the content of flavonols after harvest 3 is much greater in the compositions M3 and M4. In the 0.15 mg of catechins equivalent observed after the third harvest, it represents 5 times more flavonols in these fruits, which represents an increase in their quality. From these results it is concluded that the plants to which compositions comprising the strain P. fluorescens CECT 9015 are applied, alone or in combination with other microorganisms have better defensive potential, better production, their fruits ripen before and are less acidic, and They contain bioactive beneficial to the health of the consumer, which gives this strain great value, compared to other strains of the same species, which do not produce these stimulating effects on plants.
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Claims

REIVINDICACIONES
1. Composición bioestimulante de plantas que comprende la cepa Pseudomonas fluorescens CECT 9015. 1. Biostimulant composition of plants comprising the strain Pseudomonas fluorescens CECT 9015.
2. Composición de acuerdo con la reivindicación 1 , que además comprende al menos un segundo microorganismo seleccionado entre los géneros Pseudomonas, Bacillus, Arthrobacter, Trichoderma, o una combinación de los mismos. 2. Composition according to claim 1, further comprising at least a second microorganism selected from the genera Pseudomonas, Bacillus, Arthrobacter, Trichoderma, or a combination thereof.
3. Composición de acuerdo con la reivindicación 2, en que el segundo microorganismo del género Pseudomonas es Pseudomonas putida.  3. Composition according to claim 2, wherein the second microorganism of the genus Pseudomonas is Pseudomonas putida.
4. Composición de acuerdo con cualquiera de las reivindicaciones 1 a 3, caracterizada porque además comprende un coformulante. 4. Composition according to any one of claims 1 to 3, characterized in that it further comprises a coformulant.
5. Composición de acuerdo con la reivindicación 4, en la que el coformulante se selecciona del grupo que consiste en: abonos, abonos compuestos por los elementos nitrógeno, fósforo y/o potasio y sus combinaciones dobles o triples, productos fertilizantes, polisorbatos asociados a ácidos grasos, asparagina, manitol, ácidos orgánicos, medio CAS, medio nutritivo para el cultivo de bacterias, sustancias quelante de macro y microelementos nutricionales, algas o sus extractos y levaduras.  5. Composition according to claim 4, wherein the coformulant is selected from the group consisting of: fertilizers, fertilizers composed of the elements nitrogen, phosphorus and / or potassium and their double or triple combinations, fertilizer products, polysorbates associated with fatty acids, asparagine, mannitol, organic acids, CAS medium, nutritive medium for the cultivation of bacteria, macro chelating substances and nutritional microelements, algae or their extracts and yeasts.
6. Composición de acuerdo con cualquiera de las reivindicaciones 1 a 5, que se encuentra en forma sólida o líquida.  6. Composition according to any of claims 1 to 5, which is in solid or liquid form.
7. Composición de acuerdo con cualquiera de las reivindicaciones 2 a 6, en la que el segundo microorganismo se selecciona de las cepas Bacillus subtilis CECT 9016, Pseudomonas putida CECT 9011 , Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Trichoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, o combinaciones de las mismas. 7. Composition according to any of claims 2 to 6, wherein the second microorganism is selected from the strains Bacillus subtilis CECT 9016, Pseudomonas putida CECT 9011, Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Trichoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, or combinations thereof.
8. Método para bioestimular de plantas en cultivos que consiste en aplicar en los mismos la composición de las reivindicaciones 1 a 7. 8. Method for biostimulating plants in crops which consists in applying the composition of claims 1 to 7 therein.
9. Método según la reivindicación 8, en que el efecto bioestimulante sobre las plantas en el cultivo al que se aplica la composición de las reivindicaciones 1 a 7 se mide por cualquiera de los parámetros, o por una combinación de los mismos, seleccionados entre los que consisten en: incremento de la producción en un 10-45% sin reducir el tamaño o calibre del fruto, incremento del número de frutos en un 11-40%, incremento de la masa vegetal y radicular en un 5-35%, incremento de la actividad fotosintética de los vegetales en condiciones adversas en un 4-30%, incremento de la absorción y contenido de potasio (K) en un 2-21 %, y/o de hierro (Fe) en un 11-100%, y/o de fósforo (P) en un 6-40%, y/o de nitrógeno (N) en un 5-25%, y/o de otros nutrientes como el magnesio (Mg), el zinc (Zn) o el boro (B) por mecanismos que incrementan la solubilidad del elemento, y/o su disponibilidad, y/o su absorción a través de la membrana. 9. Method according to claim 8, wherein the biostimulant effect on plants in the crop to which the composition of claims 1 to 7 is applied is measured by any of the parameters, or by a combination thereof, selected from among the which consist of: increase in production by 10-45% without reducing the size or size of the fruit, increase in the number of fruits by 11-40%, increase in plant and root mass by 5-35%, increase of the photosynthetic activity of vegetables in adverse conditions by 4-30%, increased absorption and content of potassium (K) by 2-21%, and / or iron (Fe) by 11-100%, and / or phosphorus (P) in 6-40%, and / or of nitrogen (N) by 5-25%, and / or other nutrients such as magnesium (Mg), zinc (Zn) or boron (B) by mechanisms that increase the element's solubility, and / or its availability , and / or its absorption through the membrane.
10. Método según cualquiera de las reivindicaciones 8 y 9 en que la composición bioestimulante de las reivindicaciones 1 a 7, se aplica en forma líquida y en cultivo hidropónico o en cultivo sobre suelo.  10. Method according to any of claims 8 and 9 wherein the biostimulant composition of claims 1 to 7 is applied in liquid form and in hydroponic culture or in soil culture.
1 1. Uso de Pseudomonas fluorescens CECT 9015 para la fabricación de composiciones bioestimulantes de plantas.  1 1. Use of Pseudomonas fluorescens CECT 9015 for the manufacture of plant biostimulant compositions.
12. Uso de Pseudomonas fluorescens CECT 9015 de acuerdo con la reivindicación 11 , en combinación con al menos un segundo microorganismo seleccionado entre los géneros 12. Use of Pseudomonas fluorescens CECT 9015 according to claim 11, in combination with at least a second microorganism selected from the genera
Pseudomonas, Bacillus, Arthrobacter, Trichoderma, o una combinación de los mismos.Pseudomonas, Bacillus, Arthrobacter, Trichoderma, or a combination thereof.
13. Uso de Pseudomonas fluorescens de acuerdo con la reivindicación 12, en el que el segundo microorganismo del género Pseudomonas es la especie Pseudomonas putida.13. Use of Pseudomonas fluorescens according to claim 12, wherein the second microorganism of the genus Pseudomonas is the species Pseudomonas putida.
14. Uso de acuerdo con cualquiera de las reivindicaciones 12 a 13 en que la composición bioestimulante comprende al menos una cepa seleccionada entre: Bacillus subtilis CECT14. Use according to any of claims 12 to 13 wherein the biostimulant composition comprises at least one strain selected from: Bacillus subtilis CECT
9016, Pseudomonas putida CECT 901 1 , Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Trichoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, o combinaciones de las mismas. 9016, Pseudomonas putida CECT 901 1, Bacillus amyloliquefaciens CECT 9017, Bacillus licheniformis CECT 9018, Trichoderma harzanium CECT 20946, Arthrobacter oxydans CECT 7170, or combinations thereof.
15. Uso de acuerdo con cualquiera de las reivindicaciones anteriores, en el que la composición bioestimulante además comprende un coformulante seleccionado del grupo que consiste en: abonos, abonos compuestos por los elementos nitrógeno, fósforo y/o potasio y sus combinaciones dobles o triples, productos fertilizantes, polisorbatos asociados a distintos ácidos grasos, asparagina, manitol, ácidos orgánicos, medio CAS, medio nutritivo para el cultivo de bacterias, sustancias quelante de macro y microelementos nutricionales, algas o sus extractos, y levaduras.  15. Use according to any of the preceding claims, wherein the biostimulant composition further comprises a coformulant selected from the group consisting of: fertilizers, fertilizers composed of the elements nitrogen, phosphorus and / or potassium and their double or triple combinations, fertilizer products, polysorbates associated with different fatty acids, asparagine, mannitol, organic acids, CAS medium, nutritive medium for the cultivation of bacteria, macro chelating substances and nutritional microelements, algae or their extracts, and yeasts.
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