WO2017089641A1 - Compositions biostimulantes de plantes qui comprennent des souches de microorganismes - Google Patents

Compositions biostimulantes de plantes qui comprennent des souches de microorganismes Download PDF

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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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English (en)
Spanish (es)
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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/fr

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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

La présente invention concerne des souches microbiennes qui sont des promoteurs de la croissance végétale pouvant être combinées avec des co-formulants, et destinées à être utilisées en tant que biostimulants applicables à divers substrats dans des cultures agricoles. Les compositions biostimulantes selon la présente invention se caractérisent en ce qu'elles comprennent la bactérie Pseudomonas fluorescens CECT 9015, et peuvent également comprendre d'autres microorganismes sélectionnés parmi les genres Pseudomonas, Bacillus, Arthrobacter, Trichoderma ou une combinaison de ces derniers. L'effet sur la culture où on applique la composition est caractérisé en ce que certains des indicateurs suivants ou combinaisons d'indicateurs produisent: une augmentation de la production sans réduire la taille ou calibre du fruit, une augmentation du nombre de fruits tout en maintenant la qualité desdits fruits, une augmentation de la masse végétale et radiculaire de la plante, une augmentation de l'activité de photosynthèse des végétaux dans des conditions difficiles et l'augmentation de la disponibilité, de l'absorption et de la teneur en nutriments de la plante.
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CN110896966A (zh) * 2019-12-19 2020-03-24 河南农贝得农业科技有限公司 一种针对农作物全蚀病的微生物菌剂及其制备方法
CN111826319A (zh) * 2020-07-31 2020-10-27 西南林业大学 一种微生物促生剂及其应用
WO2021091365A1 (fr) * 2019-11-06 2021-05-14 Becerra Carranza Luis Rodrigo Mélange et produit organique à usage agricole, et leurs procédés de préparation et d'application
ES2902976A1 (es) * 2021-09-21 2022-03-30 Biobab R&D S L Pseudomonas atacamensis BBB003 mejorada de la producción vegetal y estimulante del metabolismo secundario de compuestos fenólicos y de la capacidad de los extractos de fresa y frambuesa para inhibir enzimas relacionadas con el síndrome metabólico
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Cited By (9)

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Publication number Priority date Publication date Assignee Title
ES2706099A1 (es) * 2017-09-27 2019-03-27 Univ Almeria Nueva cepa de Trichoderma aggressivum fsp europaeum, composiciones y aplicaciones de la misma
WO2019063865A1 (fr) * 2017-09-27 2019-04-04 Universidad De Almería Nouvelle souche de trichoderma aggresivum fsp europaeum, , compositions et applications de celle-ci
EP3863408A4 (fr) * 2018-10-09 2022-10-05 Locus IP Company, LLC Matériaux et procédés pour une utilisation et/ou une séquestration améliorée du carbone ainsi que pour la réduction des gaz atmosphériques délétères
WO2021091365A1 (fr) * 2019-11-06 2021-05-14 Becerra Carranza Luis Rodrigo Mélange et produit organique à usage agricole, et leurs procédés de préparation et d'application
CN110896966A (zh) * 2019-12-19 2020-03-24 河南农贝得农业科技有限公司 一种针对农作物全蚀病的微生物菌剂及其制备方法
CN111826319A (zh) * 2020-07-31 2020-10-27 西南林业大学 一种微生物促生剂及其应用
CN111826319B (zh) * 2020-07-31 2023-02-24 西南林业大学 一种微生物促生剂及其应用
ES2902976A1 (es) * 2021-09-21 2022-03-30 Biobab R&D S L Pseudomonas atacamensis BBB003 mejorada de la producción vegetal y estimulante del metabolismo secundario de compuestos fenólicos y de la capacidad de los extractos de fresa y frambuesa para inhibir enzimas relacionadas con el síndrome metabólico
WO2023047005A1 (fr) * 2021-09-21 2023-03-30 Biobab R&D, S.L. Pseudomonas atacamensis cect 30419 améliorant la production végétale et stimulant le métabolisme secondaire de plantes

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