Biostimulant composition

Abstract

The present disclosure refers to a microbial consortium containing microorganisms from at least two species selected from the group consisting of Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans, Priestia megaterium, and Pichia kluyveri. In addition, also disclosed are compositions comprising said consortia, in particular, together with extract of algae, and use of the consortia and compositions thereof for stimulating plant growth.

Description

[0001] Biostimulant composition

[0002] This application claims the benefit of European Patent Application EP23383091.8 filed on 25.10.2023.

[0003] Technical Field

[0004] The present disclosure relates to the field of agriculture, more particularly to a new environmentally-friendly biostimulant composition.

[0005] Background Art

[0006] The consistent use of synthetic fertilizers and chemicals in traditional agriculture has not only compromised the fragile agroecosystems but has also adversely affected human, aquatic, and terrestrial life. A promising and environmental-friendly innovation is the use of natural plant biostimulants that enhance flowering, plant growth, fruit set, crop productivity, and nutrient use efficiency, and are able also to improve the tolerance against a wide range of abiotic stressors.

[0007] Biostimulants play a substantial role in agro-environmental sustainability as their use in crop production model seeks to protect the environment by reducing detrimental amounts of chemical inputs to crop fields, while maintaining crop yield and quality. As a consequence, the demand of such biostimulants has steadily grown over the years.

[0008] However, available biostimulants are not always compatible with existing commercially available crop management products, or do not possess good properties regarding shelllife, colonizing ability, and biostimulant activity, all of which limit their ultimate performance when administered to the plant.

[0009] Therefore, there is a continued need for developing new biostimulants to improve the availability of nutrients in the soil for the plant, to increase the efficiency of their utilization by the plant, and to improve stress-tolerance.

[0010] Summary of Invention

[0011] The inventors have developed a new biostimulant composition that overcomes the drawbacks mentioned above. The new composition comprises a microbial consortium that has been judiciously selected to act synergically and provide improved biostimulant activity. Thus, a first aspect of the disclosure provides a microbial consortium containing microorganisms from at least two species selected from the group consisting of Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans, Priestia megaterium, and Pichia kluyveri.

[0012] As shown in the examples below, a combination of microbial species according to the first aspect provides several advantages: promotes nitrogen fixation, increases Phosphorous and Potassium solubilization, thus increasing their availability to the plant, increases iron absorption, and increases stress resistance, in particular, to drought conditions, through increasing root water storage. Ultimately, the consortium of the first aspect has been shown to improve crop yield. Moreover, the microorganisms integrating the consortium are considered safe in agriculture, are compatible with each other, and also possess good properties regarding shell-life and colonizing ability.

[0013] A second aspect refers to a composition comprising the consortium of the first aspect.

[0014] The combination of multiple biostimulating activities and commercial fitness mentioned above render the microbial consortium of the first aspect, or composition comprising it, useful for agricultural purposes. Thus, a third aspect refers to use of the of the microbial consortium of the first aspect or the composition of the second aspect in agriculture.

[0015] A fourth aspect relates to the use of the microbial consortium of the first aspect or composition comprising it for improving nutrient uptake by plants, for improving nutrient utilization by plants, for improving resistance to abiotic stress in plants, or for improving plant growth and / or crop productivity. This aspect may also be formulated as a method for improving nutrient uptake by plants, for improving nutrient utilization by plants, for improving resistance to abiotic stress in plants, or for improving plant growth and / or crop productivity the method comprising administering to the plant the microbial consortium of the first aspect or the composition of the second aspect.

[0016] A fifth aspect refers to the use of the microbial consortium of the first aspect for improving plant growth and / or crop productivity. Otherwise stated, this aspect provides a method for improving plant growth and / or crop productivity by administering the microbial consortium of the first aspect.

[0017] The inventors have also identified new strains of interest for use in the consortium of the present invention. Therefore, a sixth aspect of the disclosure refers to a strain selected from the group consisting of Pseudomonas canadensis strain deposited at the "Coleccion Espanola de Cultivos Tipo” (CECT) with identification reference Pseudomonas canadensis 30930, an Azotobacter chroococcum strain deposited at the CECT with identification reference Azotobacter chroococcum 30924, a Peribacillus frigoritolerans strain deposited at the CECT with identification reference Peribacillus frigoritolerans 30923, a Pichia kluyveri strain deposited at the CECT with identification reference Pichia kluyveri 13224, and a Priestia megaterium strain is deposited at the CECT with identification reference Priestia megaterium 30922.

[0018] Further aspects of the invention refer to compositions comprising any of said strains and use of any of the strains or compositions comprising them in agriculture, as well for improving nutrient uptake by plants, for improving nutrient utilization by plants, for improving resistance to abiotic stress in plants, or for improving plant growth and / or crop productivity.

[0019] Brief description of the drawings

[0020] Figure 1. Effect of the biostimulant according to the invention (Composition A) on Potato var. Vivaldi during the autumn period in Sa Pobla, Mallorca (Spain). CONTROL 100%: 100% of water and fertilizer representing optimal conditions for the growth of the potato crop; CONTROL 70%: 70% of water and fertilizer (i.e. 30% reduction water and fertilizer with respect to the optimal conditions); COMPOUND A 70%: compound A together with 70% of water a fertilizer.

[0021] Detailed description of the invention

[0022] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition. The definitions given herein are included for the purpose of understanding and expected to be applied throughout description, claims and drawings.

[0023] As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun.

[0024] "Biostimulant" or "plant biostimulant" is understood as a substance that stimulates the nutrition processes of plants independently of the nutrients it contains, for the purpose of improving nutrient use efficiency, tolerance to abiotic stress, qualitative characteristics and / or availability of nutrients confined to soil or rhizosphere.

[0025] In the sense of the present disclosure “biostimulants” encompass “phytostimulants”. The term “phytostimulant” is understood as substances and materials that, when applied to plants, seeds or growing substrates in specific formulations, have the capacity to modify physiological processes of plants in a way that provides potential benefits to growth, development, and / or stress response.

[0026] “Fertilizer” is any material of natural or synthetic origin that is applied to soil or to plant tissues to supply plant nutrients.

[0027] “Macrocystis” is a genus of kelp (large brown algae) . It is commonly known as giant kelp or bladder kelp. Macrocystis is found widely in subtropical, temperate, and sub-Antarctic oceans of the Southern Hemisphere (e.g. Chile, New Zealand, Australia, Falkland Islands, Auckland Islands, etc.) and in the northeast Pacific from Baja California to Sitka, Alaska. Macrocystis is often a major component of temperate kelp forests. Appropriate species of Macrocystis in the sense of the present disclosure is Macrocystis pyrifera.

[0028] “Agriculturally acceptable compounds” refer to those compounds and I or materials which are suitable and generally accepted for use in agriculture. In general such compounds should be non-toxic to humans and should preferably be environmentally friendly.

[0029] The term "mutants" refer to strains obtained using the bacterial strains of the invention as starting material and characterized by maintaining the herein described blood glucose controlling effects. A "mutant" of the strain is also understood according to the invention as a "variant" or “derivative”.

[0030] As used herein, "culture supernatant" or “conditioned culture media” refer to culture media in which microbial cells have been cultured for a suitable period of time and into which the cells have secreted active agent(s) to sufficient levels to provide a desired biological activity, in particular, biostimulating activity. A culture supernatant may contain one or more soluble metabolites of the bacterium useful in the methods described herein. The skilled in the art will know which media is suitable for culturing the microorganism, and for how long it should be cultured in order to obtain a supernatant with enough active metabolites using its common general knowledge. The culture supernatant may be obtained, for example, by settling, precipitating or centrifuging the cells in culture. The supernatant may be filtered. A culture supernatant may contain one or more soluble metabolites. As used herein "lysate" refers microbial cells which has been subject to lysis. A lysate may contain one or more soluble metabolites of the microorganism useful in the methods described herein. The skilled person knows several methods to carry out the lysis which belong to the common general knowledge. For example, lysis may occur by chemical or physical disruption, such as by addition of an osmotic agent or enzyme to the bacteria, or by the application of physical pressure, for example through sonic disruption. The microorganisms may be washed prior to lysis. The lysate may be filtered and / or sterilized after lysis to remove any whole bacteria remaining. The lysate may be suspended, for example in aqueous medium.

[0031] The present disclosure refers to a microbial consortium containing microorganisms from at least two species selected from the group consisting of Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans, Priestia megaterium, and Pichia kluyveri. This combination of microorganisms has unexpected advantages in that they act synergistically to provide several biostimulating activities to the plant.

[0032] In one embodiment, the consortium comprises at least Azotobacter chroococcum. In another embodiment, the consortium comprises at least Pseudomonas canadensis. In one particular embodiment, the consortium comprises Pseudomonas canadensis, Azotobacter chroococcum, and Peribacillus frigoritolerans. In a more particular embodiment, the consortium comprises Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans, and Pichia kluyveri. In another more particular embodiment, the consortium comprises Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans, and Priestia megaterium. In an even more particular embodiment, the consortium comprises Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans, Priestia megaterium and Pichia kluyveri.

[0033] In other embodiments, the consortium comprises:

[0034] - Azotobacter chroococcum and Peribacillus frigoritolerans, or

[0035] - Azotobacter chroococcum and Pichia kluyveri, or

[0036] - Azotobacter chroococcum and Priestia megaterium, or

[0037] - Azotobacter chroococcum and Pseudomonas canadensis, or

[0038] - Azotobacter chroococcum, Priestia megaterium, and Pseudomonas canadensis, or

[0039] - Azotobacter chroococcum, Priestia megaterium, and Pichia kluyveri, or

[0040] - Azotobacter chroococcum, Peribacillus frigoritolerans, and Pichia kluyveri.

[0041] In a particular embodiment, the Pseudomonas canadensis is a strain deposited at the CECT with identification reference 30930. In another particular embodiment, the Azotobacter chroococcum is a strain deposited at the CECT with identification reference 30924. In another particular embodiment, the Peribacillus frigoritolerans is a strain deposited at the CECT with identification reference 30923. In another particular embodiment, the Pichia kluyveri is a strain deposited at the CECT with identification reference 13224. In another particular embodiment, the Priestia megaterium is a strain deposited at the CECT with identification reference 30922.

[0042] The strain of Pseudomonas canadensis 30930, isolated from soil samples of irrigated common bean crop in the Leon (Spain), was deposited by the applicant, according to the Budapest Treaty, on 20.09.2023 in the Spanish Type Culture Collection (Coleccion Espanola de Cultivos Tipo, CECT), located at Universidad de Valencia C.P 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain). The strain was identified by the depositor with the reference CD_FICOS_03 and was declared viable.

[0043] The strain of Azotobacter chroococcum 30924, isolated from soil samples of irrigated maize crop in Leon (Spain), was deposited by the applicant, according to the Budapest Treaty, on 14.09.2023 in the Spanish Type Culture Collection (Coleccion Espanola de Cultivos Tipo, CECT), located at Universidad de Valencia C.P 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain). The strain was identified by the depositor with the reference CD_FICOS_04 and was declared viable.

[0044] The strain of Peribacillus frigoritolerans 30923, isolated from soil samples of irrigate common bean crop in Leon (Spain), was deposited by the applicant, according to the Budapest Treaty, on 14.09.2023 in the Spanish Type Culture Collection (Coleccion Espanola de Cultivos Tipo, CECT), located at Universidad de Valencia C.P 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain). The strain was identified by the depositor with the reference CD_FICOS_02 and was declared viable.

[0045] The strain of Priestia megaterium 30922, isolated from soil samples of irrigate pepper crop in Leon (Spain), was deposited by the applicant, according to the Budapest Treaty, on 14.09.2023 in the Spanish Type Culture Collection (Coleccion Espanola de Cultivos Tipo, CECT), located at Universidad de Valencia C.P 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain). The strain was identified by the depositor with the reference CD_FICOS_01 and was declared viable.

[0046] The strain of Pichia kluyveri 13224, isolated from the surface of grapes grown in the province of Leon (Spain), was deposited by the applicant, according to the Budapest Treaty, on 14.09.2023 in the Spanish Type Culture Collection (Coleccion Espanola de Cultivos Tipo, CECT), located at Universidad de Valencia C.P 46980 Catedratico Agustin Escardino N° 9 Paterna, Valencia (Spain). The strain was identified by the depositor with the reference CD_FICOS_05 and was declared viable.

[0047] The microbial consortium of the first aspect may be obtained by mixing the microorganisms integrating the consortium at appropriate proportions. In particular embodiments, the microbial strains are provided as dry microorganisms, for example, as lyophilised microorganisms. In particular embodiments, the microorganisms integrating the consortium are mixed at equal proportions. In other particular embodiments, the microorganisms integrating the consortium are mixed at the following proportions (Pseudomonas canadensis / Azotobacter chroococcum / Peribacillus frigoritolerans): 1-3 / 1- 3 / 1-3.

[0048] The microorganisms integrating the consortium may be obtained from a process comprising culturing each of the strains under conditions appropriate for growth of the strain, separating the cultured cells from the medium, and drying the cells, for example by freeze-drying. Appropriate conditions for growing the strains include appropriate culture medium (preferably liquid) and temperature. Appropriate culture media for growth of the strains will be apparent to the skilled person and may be selected, for example, from TSB (Tryptic Soy Broth) and Ashby-mannitol, the latter being particularly suitable for Azotobacter species. Appropriate temperature for growth is from 25 to 30 °C. Other conditions appropriate for growth of the microorganisms of the present disclosure are aerobic or semi-aerobic conditions, 24 to 72 hours incubation, etc.

[0049] The microbial consortium of the first aspect may be provided to a plant alone, or as a component of a composition including other agriculturally acceptable components. Moreover, the composition may contain the microbial cells or, alternatively, a lysate thereof or culture supernatant thereof. It is also contemplated that the composition contains the microbial cells and, additionally, a lysate or culture supernatant thereof.

[0050] A second aspect of the disclosure, as mentioned above, refers to a composition comprising the consortium of the first aspect. All embodiments mentioned above for the consortium of the first aspect also apply to the composition of the second aspect.

[0051] As outlined above, the composition may alternatively or additionally contain a cell lysate or culture supernatant of the consortium of the first aspect. In one particular embodiment, the composition of the second aspect further comprises extract of algae, in particular selected from Macrocystis, Laminaria, and combinations thereof. More in particular, the extract is selected from Macrocystis pyrifera, Laminaria ochroleuca, and combinations thereof. The extracts of Macrocystis and Laminaria have been found to be an excellent support for the microorganisms, as well as providing nutrients to the plant and having biostimulating properties of themselves. In some embodiments, the algae extract is a dry extract. In other embodiments, the algae extract is liquid extract. More in particular, the algae extract is a dry, powdered extract, which is water soluble or water suspendable. For example, the algae extract is a dry, powdered extract, which is water soluble (+97%). By “water suspendable” it is understood that the particles of the dry extract may form a suspension or colloid suspension in water.

[0052] Extract of Macrocystis is commercially available from different sources. For example, a suitable dry Macrocystis pyrifera extract is Cystium-d, marketed by Ficosterra (Spain). Moreover, extract of Macrocystis may be obtained following methods known in the art, for example, as described in US9854810. A suitable method to obtain dry extract of Macrocystis is alkaline extraction plus subsequent dehydration. Briefly, the process comprises:

[0053] - Harvesting of seaweed and treatment with acid to stabilise the seaweed and paralyse possible hydrolysis or degradation.

[0054] - Cooking at 50-60°C with the addition of K. The purpose of potash is to facilitate the breaking of the algae cell wall during the cooking process.

[0055] - Addition of a quantity of leonardite to increase solids in suspension and improve the efficiency of the filtering process.

[0056] - Filtration

[0057] - Drying in heat exchanger, the resulting product is a 'sheet' with moisture <10%.

[0058] - Chopping and sieving.

[0059] The present disclosure also refers to a composition comprising extract of Macrocystis, for example, one obtained as disclosed above, and Azotobacter chroococcum. This particular composition provides unexpected biostimulant activity because of the synergies stablished between the extract and the microorganism. The Azotobacter chroococcum strain in the composition may be the strain deposited at the CECT with identification reference 30924. This composition may preferably include other microorganisms mentioned above, in particular Pseudomonas canadensis and / or Peribacillus frigoritolerans.

[0060] Also disclosed is a composition comprising extract of Macrocystis, for example, one obtained as disclosed above, and Pseudomonas canadensis. This composition may preferably include other microorganisms mentioned above, in particular Azotobacter chroococcum and / or Peribacillus frigoritolerans. In further embodiments of the second aspect, the composition further comprises one or more agriculturally acceptable compounds. In a particular embodiment, the composition of the invention may contain compounds for improving the adherence of the strains in the plants to be treated, phytofortifying compounds, nutrients, humectants, stabilizers, osmoprotectants, antioxidants, sunscreens, buffering compounds or combinations thereof. The composition of the invention may also contain water. Some adhesion enhancing compounds are gelatins, starches, pectins, alginates and various types of gums such as xanthans. Many of these compounds are also humectants. Sunscreens include dyes such as Congo red. In particular the compositions of the invention contain at least one osmoprotector. Non-limiting examples of osmoprotective compounds are betaines, amino acids and trehalose.

[0061] The composition of the second aspect may be in solid form, aqueous liquid form, oily liquid form, emulsion form, semi-solid form or in the form of gel. In particular embodiments, the composition is in solid form, more preferably, the composition is in powder form. In one embodiment, the composition is a water soluble solid, or a water suspendible solid, or is in the form of water dispersible granules. The amount of the microbial consortium in the composition may be from 1% to 10% (mass percentage), in particular from 2 to 7%, for example, around 5%. The amount of microbial cells in the composition may be from 106to 1O10cfu per g composition, in particular from 107to 109, for example, around 109cfu / g composition.

[0062] For field use, the composition is preferably diluted in water to form an aqueous solution or suspension. In certain embodiments the composition is an aqueous solution. In other embodiments the composition is an aqueous suspension. In particular embodiments, the amount of dry composition comprising extract from algae (Macrocystis and / or Laminaria) and the microbial consortium in the aqueous solution or suspension is from 0.005 % to 1 % (w / v %, i.e. 0.005-1 g / 100 mL), in particular from 0.01% to 0.1 % (w / v %), more particularly from 0.01% to 0.05% (w / v %), for example around 0.025% (w / v %).

[0063] The composition of the second aspect may be considered a biostimulant composition. It can also be considered a phytostimulant composition. Moreover, the composition including extract of macrocystis may also be considered a fertilizing composition.

[0064] The present disclosure also contemplates a method for preparing a composition according to the first aspect, said method comprising the following steps:

[0065] - Providing a microbial consortium as defined in the first aspect;

[0066] - Mixing the consortium with extract from Macrocystis and / or Laminaria;

[0067] - Optionally adding further agriculturally acceptable compounds; and

[0068] - Optionally, dissolving or suspending the mixture in water. The third to fifth aspects of the disclosure refer to use of the of the microbial consortium of the first aspect or the composition of the second aspect in agriculture, in particular for improving nutrient uptake by plants, for improving nutrient utilization by plants, for improving resistance to abiotic stress in plants, or, altogether, for improving plant growth and / or crop productivity. Relevant biostimulant properties of the microbial consortium and the composition include Nitrogen fixation, Phosphorous mobilization, and Potassium solubilization. Thus, in more particular embodiments the use of the microbial consortium or composition comprising it is for increasing Nitrogen fixation, Phosphorous mobilization, and Potassium solubilization by plants. Other biostimulating properties include iron absorption, and root water storage. Thus, additional particular uses of the consortium or composition comprising it are for increasing iron absorption and drought resistance. In some embodiments, the composition, preferably diluted in water, is applied as seed coating, by soil application, for example, by drip irrigation system or drench, or by foliar application. In a particular embodiment, the composition is applied to the soil, more in particular, by drip irrigation.

[0069] The plants or crops to be treated may be selected from any food crop, feed crop, fiber crop, oil crop, ornamental crop, and industrial crop. Non-limiting crops to be treated are selected from the group consisting of orchards, berries, vines, olive trees, and cereal.

[0070] The dosage will depend on the crop that is to be treated. For example, for intensive crops, such as orchards, the dosage of the composition comprising macrocystis extract and microbial consortium can be from 200 to 600 g, per hectare per month. In one embodiment, the of the composition comprising macrocystis extract and microbial consortium is from 250 to 400 g per hectare per month. This dosage is appropriate, for example, for berries. In another embodiment, the of the composition comprising macrocystis extract and microbial consortium is from 400 to 600 g per hectare per month. This dosage is appropriate, for example, for orchards. The dosage of the composition comprising macrocystis extract and microbial consortium for extensive crops, such as cereal, vines and olive trees, can be from 100 to 400 g per hectare once, two or three times per year. For example, in one embodiment, the dosage for vines is from 200 to 300 g per hectare twice a year, which is convenient for olive trees and vines. In another embodiment, the dosage is from 200 to 300 g per hectare once a year, which is convenient for cereal.

[0071] In general, the dosage of the composition administered to the plants is calculated such that it provides around 10E+8 / hectare, or around 10E+7 / plant. The present disclosure also contemplates particular strains of the microbial species integrating the consortium of the first aspect which have been found to be particularly useful in the sense of the invention. The strains are, in particular, selected from the group consisting of Pseudomonas canadensis 30930, Azotobacter chroococcum 30924, Peribacillus frigoritolerans 30923, Pichia kluyveri 13224, and Priestia megaterium 30922. The disclosure also contemplates mutants of said strains which maintain the biostimulating features of the original strain. An expert in the art will understand that using the strains described herein for the first time as starting material it is routinely possible to obtain, for example by spontaneous mutation or directed mutagenesis, mutants that retain the characteristics and relevant advantages described herein. Methods for obtaining mutants of a given bacterial strain are known in the art. Examples can be found in (Sambrook, J. and Russell, DW "Molecular Cloning: A Laboratory Manual", Chapter 13, "Mutagenesis", Cold Spring Harbor, 3rd Ed., 2001). The mutants usually retain above 99% DNA sequence identity, for example above 99.9% sequence identity, (along the full length of the genome) with the original strain from which they derive.

[0072] Thus, also contemplated in the present disclosure is method to obtain a mutant of a strains elected from the groups consisting of Pseudomonas canadensis 30930, Azotobacter chroococcum 30924, Peribacillus frigoritolerans 30923, Pichia kluyveri 13224, and Priestia megaterium 30922, the method comprising the step of subjecting the original strain Pseudomonas canadensis 30930, Azotobacter chroococcum 30924, Peribacillus frigoritolerans 30923, Pichia kluyveri 13224, or Priestia megaterium 30922 to a DNA recombinant technique, for example mutagenesis, or to conditions to generate spontaneous mutations, for example UV light.

[0073] In a further aspect the disclosure also refers to the use of Pseudomonas canadensis or a composition comprising the same for improving nutrient uptake by plants, for improving nutrient utilization by plants, for improving resistance to abiotic stress in plants, or, altogether, for improving plant growth and / or crop productivity. Relevant biostimulant properties of Pseudomonas canadensis and the composition comprising the same include Nitrogen fixation, Phosphorous mobilization, and Potassium solubilization. Thus, in more particular embodiments the use of Pseudomonas canadensis or composition comprising the same is for increasing Nitrogen fixation, Phosphorous mobilization, and Potassium solubilization by plants. Other biostimulating properties include iron absorption, and root water storage. Thus, additional particular uses of Pseudomonas canadensis or composition comprising it are for increasing iron absorption and drought resistance. Embodiments disclosed above for the compositions, uses and strains also apply to this aspect. Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0074] Examples

[0075] 1. Selection of microorganisms

[0076] The microorganisms were selected for showing superior plant growth promoting (PGP) properties based on in vitro assays. For this selection, the following criteria were used: 1) Plant growth promoting properties that can contribute to the better development of the crop; 2) compatibility between strains with a view to the possible formulation of biofertilizers that combine several of them, and 3) compatibility between strains and cultivation model plants.

[0077] Microorganisms were screened considering the above criteria, from which the following were finally selected for their superior and equilibrated plant growth promoting (PGP) properties: Pseudomonas canadensis 30930 (from now on, simply 30930), Azotobacter chroococcum 30924 (from now on, simply 30924), Peribacillus frigoritolerans 30923 (from now on, simply 30923), Pichia kluyveri 13224 (from now on, simply 13224), and Priestia megaterium 30922 (from now on, simply 30922).

[0078] The sections below illustrate the methods and results of the screening.

[0079] 1.1. Plant growth promoting properties

[0080] 1.1.1. Quantitative analysis of siderophores production

[0081] The solubilization ability of inorganic phosphate was evaluated in NBRIP medium. The isolates were cultured in Petri dishes with NBRIP medium (Table 1) and incubated at 28 ° C for 5-7 days. The formation of a transparent halo in the culture medium around each colony was evaluated which indicated the solubilization of the phosphate present in the medium. The phosphate solubilization index (PSI) was calculated by measuring the diameter of the colony and the diameter of the halo using the formula:

[0082] Phosphate solubilization index (PSI) = (colony + halo zone diameter) / colony diameter

[0083] Table 2 shows the values of the siderophore production index (SPI): (colony + halo zone diameter of) / colony diameter.

[0084] 1.1.2. Quantitative analysis the phosphates solubilization ability

[0085] Forty ml of liquid NBRIP medium (no agar) pH 7 was prepared, following autoclaving in 250 ml flasks. They were inoculated with 160 pl of inoculum of each strain (1.8 x 108cfu I ml-1) and incubated at 30 °C at 120 rpm I min for 7 days.

[0086] One and a half ml of each culture was taken every 24 hours. They were centrifuged at 8,000 g for 15 minutes. One ml of supernatant was taken to which 9 ml distilled water was added to estimate the soluble phosphate content by the Molybdenum Blue Colorimetric method. Molybdenum blue binds to soluble phosphorus forming Blue- phosphomolybdenum causing a measurable color change at a wavelength of 882nm.

[0087] To 10 ml of sample (1 ml sample + 9 ml water; 1 :10) was added 2 ml of reagent, allowed to stand for 10 minutes in the dark and measured by spectrophotometry at 882 nm. Blanks were made with 10 ml distilled water + 2 ml reagent.

[0088] A pattern curve (Table 3) was prepared as follows from a stock solution of KH2PO4 (0.1757 g / 1) (dilute 1:10; 17.5 mg in 100 ml), to make a concentration of 40 mg phosphorus per liter. Dilutions were made in distilled water to obtain concentrations of: 0- 1- 2- 4- 8- 16 pg / ml.

[0089] The reagents needed for the blue molybdenum method were the following

[0090] • 5 N sulfuric acid: 70 ml in 500ml water (125 ml)

[0091] • Ammonium molybdate: 20g in 500ml keep cold (37.5 ml)

[0092] • Ascorbic Acid: 1.32 g / 75 ml (75ml) (Prepare at the moment)

[0093] • Potassium antimonyltartate: 0.2743 g / 100 ml (12.5 ml)

[0094] Qualitative analysis the phosphates solubilization capacity

[0095] The phosphate solubilization index (PSI) was calculated from the halos produced by the colonies growing in NBRIP medium (Table 4). This measurement is an indicator of the capacity of each strain to solubilize phosphate.

[0096] Quantitative analysis the phosphates solubilization capacity

[0097] The results, expressed in pg phosphate solubilized per ml of extract inoculated with 108cfu / ml of the corresponding strain strain, are shown in Table 5.

[0098] 1.1.3. Analysis of the potassium solubilization ability

[0099] The inorganic potassium solubilization capacity was qualitatively evaluated in modified Aleksandrov medium (Table 6). This method is based on the ability of some microorganisms to solubilize insoluble forms of potassium adhering to minerals by secreting organic acids that can directly release potassium or chelate the silicon of these minerals, leaving potassium free in solution. Therefore, this method consists of the direct visualization on the agar plates of a halo around the solubilizing potassium colonies, by means of the inclusion of bromitol blue, which will act as an acid-base indicator. The organic acids on the plate diffuse radially and form halo zones in response to reaction with the acid-base indicator dye BTB. Potassium plaque solubilization with this method can be observed in 48-72 h.

[0100] From pure cultures, the bacterial strains were plated onto TSA medium dishes for 48 hours. Subsequently, the strains were resuspended from TSA plates in 4 ml of 0.9% saline solution (1x108cfu I ml approx.). From this liquid culture, a 10 pl drop of each strain was applied to the center of its corresponding plate of modified Aleksandrov medium.

[0101] Plates were incubated at 28 ° C for 48-72 hours. After this time, it was possible to observe the formation of a yellow halo around each colony. This halo was qualitatively evaluated. Another essential nutrient for the optimal development of any crop is potassium. A qualitative analysis based on the intensity of color change around the inoculum was carried out with the collection strains. The different intensities of each strain made it possible to assess the ability to solubilize inorganic potassium of the bacterial strain using a qualitative scale. The data are shown in Table 7. 1. 1.4. Indoleacetic acid (IAA) production ability

[0102] Auxin production in the presence of L-tryptophan was determined calorimetrically. The bacterial strains were resuspended from TSA plates in 4 ml of 0.9% saline solution (1x108cfu I ml approx.) and 500 pl were incubated in 4 ml of sterile TSB for 3 days in the dark at

[0103] 28 °C. Subsequently, 500 pl were transferred to vials with 4 ml of modified JMM minimal medium (Table 8). In addition, 1 ml was taken to measure optical density (OD) at 600 nm for the determination of bacterial growth. The vials were incubated for 4 days in the dark at 28 °C. After this incubation phase, 1.5 ml were taken from each vial and centrifuged at 5,000 g for 10'. One ml of supernatant was taken and 1 ml of Salkowski agent (0.01 M

[0104] FeCI3 and 34.3% perchloric acid) was added. The mixture was allowed to stand 30 'and the OD was measured at 535 nm. Stock of L-tryptophan and vitamins were filtered by Millipore filters and stored at -20 ° C. They were added to the medium after autoclaving when the temperature was below 50 ° C. The determination of auxin production was made by quantifying the equivalent pg I ml of IAA, by comparison with a standard curve made with IAA (0-50 pg / ml). Indoleacetic acid (IAA) production capacity results, expressed as pg IAA per ml of bacterial suspension, normalized to a density of 1 x 108cfu / ml, are shown in Table 9. 1.1.5. Analysis of 1 -aminocyclopropane-1 -carboxylic acid (ACC) deaminase activity

[0105] 1.1.5.1. Bacterial biomass production.

[0106] The bacterial isolates were first plated onto commercial TSA medium (Sigma-Aldrich) for 2 days and then transferred to Petri dishes with DF salts medium (Table 10), with 0.5 M ACC (sterilized by filtration, 300 pl per plate) as the sole nitrogen source and incubated for 2 days at 28 °C, until obtaining sufficient bacterial growth. Strains that did not grow on this medium lacked ACC deaminase activity.

[0107] * Adjust to pH 7.2 and sterilize in autoclave. If the medium is solid, use 1.5% agar (nitrogen free).

[0108] The bacterial mass of each isolate was resuspended in 2.5 ml of sterile distilled water, to a concentration of 6x108cfu / ml. Twenty microlitres of each bacterial solution were taken and inoculated in 15 ml of TSB medium. It was incubated overnight at 28 °C and 200 rpm. To rescue the bacterial mass produced, it was centrifuged at 8,000 g at 4 ° C, the supernatant was discarded and the bacterial mass was resuspended in 7.5 ml of liquid DF salts minimal medium; 45 pl of 0.5 M ACC were added and incubated overnight at 28 ° C and 200 rpm. After this time, it was centrifuged at 8,000 g at 4 °C, the supernatant was removed and the cells were washed and resuspended in 5 ml of 0.1 M Tris-HCI pH 7.6, centrifuged again and the supernatant was discarded. The washing procedure was repeated twice in order to remove traces of the culture medium. The bacterial mass was stored at 4 ° C.

[0109] 1.1.5.2. Measurement ACC deaminase activity.

[0110] The ACC deaminase enzyme catalyzes the hydrolysis of ACC, the products of which are a-ketobutyrate and ammonium; this procedure indirectly measures the action of the enzyme, since it is based on the measurement of a-ketobutyrate production.

[0111] The bacterial biomass obtained by the procedure described above was resuspended in 1 ml of 0.1 M Tris-HCI pH 7.6, transferred to a 1.5 ml eppendorf tube and centrifuged at 16,000 g for 5 minutes. The supernatant was discarded and the pellet was resuspended in 600 pl of 0.1 M Tris-HCI pH 8.5; 30 pl of toluene was added and vortexed for 30 seconds. The blank was made from this point with 600 pl of 0.1 M Tris-HCI pH 8.5 and 30 pl of toluene. A 100 pl aliquot of the "toluenized cells" was stored at 4 °C for subsequent determination of protein activity.

[0112] Two hundred microliters of the "toluenized cells" were transferred to a fresh Eppendorf tube, 20 pl of 0.5 M ACC was added, the tube was shaken and incubated at 30 °C for 15 minutes. Then 1 ml of 0.56 M HCI was added, stirred and centrifuged for 5 minutes at 16,000 g at room temperature. One millilitre of the supernatant was taken and transferred to a test tube, 800 pl of 0.56 M HCI were added, it was stirred and 300 pl of 2,4- dinitrophenylhydrazine (0.2% in 2 M HCI) were added, once more the whole mixture was shaken and incubated at 30 ° C for 30 minutes. After this time, 2 ml of 2 N NaOH were added and the OD was measured at 540 nm.

[0113] The amount of pmol of a-ketobutyrate produced was determined by comparing the absorbance at 540 nm of the sample with the standard curve of a-ketobutyrate (Sigma- Aldrich), with concentrations from 200-1000 pM. A 100 mM a-ketobutyrate stock solution diluted in 0.1 M Tris-HCI pH 8.5 was prepared and stored at 4 °C. From the stock solution, a 10 mM solution was made for immediate use in the preparation of the solutions for the generation of the standard curve; 1.8 ml of each solution (of each concentration) were placed with 300 pl of 2,4-dinitrophenylhydrazine (0.2% in 2M HCI) and the steps detailed in the previous paragraph were continued, and finally the OD was measured at 540 nm.

[0114] The ACC deaminase activity is shown in Table 11. Different studies have demonstrated the agrobiotechnological importance of this enzyme since it is a mechanism used by some PGPRs to promote plant growth, especially in plants subjected to different types of stress. This enzyme provides two important advantages: on the one hand, it reduces the concentrations of ethylene in the plant, since ethylene is a key molecule to the detriment of plant growth and development under stress conditions, and it increases the availability of ammonia in the rhizosphere. With this, the activity of the ACC deaminase enzyme improves plant nutrition and resistance to stress factors.

[0115] Table 11

[0116] 7.2. Assessment of the compatibility of the different fertilizer’s components

[0117] 1.2.1. Strain-strain compatibility (bacteria - bacteria and bacteria - yeast)

[0118] 1.2.1.1. Bacteria-bacteria compatibility

[0119] For bacteria, the compatibility of the strains was assessed by means of dual co-growth assay in Petri dishes. All the possible combinations in the fertilizer were tested.

[0120] In Petri dishes with the growth medium suitable for the corresponding strain (TSA from Sigma-Aldrich, except for Azotobacter that was Ashby-mannitol-agar), each microorganisms was inoculated, from 100 microliters of a suspension with 108cfu I ml of inoculum in liquid form that was distributed by means of a Digralsky loop onto the Petri dish. Circular slits with a diameter of 0.5 cm were made perpendicular to the base of the plate, into which 50 microliters of a suspension with 108cfu I ml of the bacteria with which it was intended to check compatibility were introduced. The plate was incubated at 28 °C for 3-5 days and the possible presence of an inhibition halo was observed and if it effectively appeared, it was measured.

[0121] None of the selected bacteria showed incompatibility with the others.

[0122] 1.2.1.2. Bacteria-yeast compatibility

[0123] In this case, a simplified methodology was used. The test consisted on putting the inoculum of the yeast strain in just one stripe, in the middle of a Petri dish with the adequate growth medium (TSA). Following, each one of the bacteria strains were put on a single stripe, perpendicular to the yeast stripe, with one contact point between both of them. The presence of a discontinuity in the yeast’s stripe appearing in the contact point between a yeast and a bacteria strip would indicate growth inhibition and thus incompatibility.

[0124] 13224 showed no incompatibility with the selected bacteria. 7.2.2. Strain-plant compatibility

[0125] The objective of this test was to analyze the compatibility between the previously selected microorganisms and the plant. Even if the strains had been selected based on their in vitro PGP properties, due to the high number of factors that participates in the microbes-plant interaction and that some of them cannot be analysed in vitro, it is necessary to assess the microbial - plant interaction.

[0126] For such an in vivo test, barley variety Scarlett was used as a control plant. The parameters used to evaluate the compatibility between the microbes and the plant were the dry weight of the root and the aerial part and the chlorophyll content.

[0127] The barley seeds were disinfected with 70% ethanol for 1 minute, followed by 2% sodium hypochlorite for 5 minutes and finally 3 washes with sterile distilled water. They were then placed in agar-water medium (20 g 1-1 ) in the dark at room temperature for 48 hours. After this time the germinated seeds were sown in 1,000 ml pots filled with sterile washed vermiculite, at a rate of 1 germinated seed per pot.

[0128] The selected microbes were grown in TSA medium. Bacterial / yeast solutions were prepared from these cultures using 0.9% saline solution (6x108cfu ml-1). Each seed was inoculated with 1 ml of said bacterial / yest solution at the time of sowing.

[0129] The plants were watered weekly with 150 ml of distilled water and every 15 days with sterile Rigaud and Puppo solution (Table 12). After 4 weeks it was harvested and the fresh and dry weight of the aerial part of the plant was obtained, in addition to the chlorophyll content.

[0130] None of the selected microorganisms showed incompatibility with the plant.

[0131] 1.2.3. Strain- Macrocystis pyrifera seaweed (in these examples termed as “MDE”) compatibility

[0132] Bacteria / yeast strains were grown at 30 °C in agitation at 80 rpm on TSB (Ashby-mannitol broth for Azotobacter) until they reached a concentration of approximately 108cfu I ml. It took between 24 and 48 h for most of the strains. Following the bacterial suspension was thoroughly homogenized and it was split in three sterile test tubes, each one with 5 ml. One was used as control with no MDE addition, whilst the other two received MDE (Cystium-d, Ficosterra) at a final concentration of 5g / l and 10g / I respectively. The mother solution of MDE was sterilised by filtration through sterile 0.45 pm cellulose acetate syringe filters. The test tubes were incubated at 30 °C in continuous agitation (80 rpm) for 48 hours. After this time, the final microbial concentration was estimated by two different procedures: i) count of the number of colony former units (cfu) after serial dilutions and plating onto TSA (Ashby-mannitol-agar for Azotobacter); ii) measurement of the optical density at 600 nm of each condition, versus controls consisting of clean medium, medium with 5 g / l MDE and medium with 10 g / l MDE.

[0133] All the selected strains produced higher growth with MDE compared to the control, although the optimal dose depends on the strain.

[0134] 2. Selection under hydroponic conditions of various combinations of MDE- microorganism strains

[0135] 2.1. Methodology

[0136] For the microcosm test, barley was grown in 1L containers under hydroponic and axenic conditions, with previously autoclaved vermiculite as substrate. Two plants per container and 8 replicates (8 containers) per treatment were planted. The treatments consisted of plants inoculated with the product Cystium-d, provided by Ficosterra, and each of the combinations of microorganisms indicated in Table 13, consisting of the strains 30924 of Azotobacter chroococcum, 30930 of Pseudomonas canadensis, 30922 of Priestia megaterium, 30923 of Peribacillus frigoritolerans and / or 13224 of Pichia kluyveri. The plant growth promoting properties of the strains are as follows. 30924: N fixation and P solubilization. 30922: IAA producer and ACC producer. 30923: IAA producer and ACC producer. 30930: P solubilization and K solubilization. 13224: K solubilization. Two controls, a blank (no biostimulant added) and a positive control, consisting of the product Cystium-d and 3 strains of commercial microorganisms, were used. The commercial strains in the positive control were Priestia megaterium CB (P solubilization), Bacillus tequilensis (K mobilizer) and Bacillus pseudomycoides (N fixator), all of the microorganisms obtained from Ceres Biotech (Madrid, Spain, provider’s references: CB36, CB38 and CB39).

[0137] Table 13. Combinations of microorganisms and concentrations used in the microcosm test treatments.

[0138] The product was applied using the following methodology:

[0139] 1. Preparation of the final bacterial inoculum: To prepare the bacterial inoculum, 10 g of cystium-d was dissolved in 1 litre of water and 7 ml of the liquid culture medium of each of the strains was added (total bacterial inoculum 21 ml). The value of 7 millilitres of bacterial inoculum is calculated using the cfu present in the culture medium, ensuring a cfu similar to that obtained in the POSITIVE CONTROL product (approximately 10E+8 cfu).

[0140] 2. Preparation of the positive control: For the preparation of the positive control, 10 g of cystium-d was dissolved in 1 litre of water and 7 g of each of the commercial strains were added (final microbial biomass 21 g) the cfu of the positive control were 10E+8 cfu.

[0141] 3. Application of the microbial inoculum: 0.1 mL of the final solution was inoculated per plant after germination. This dose contained a microbial and cystium-d concentration that simulates the dose received per plant in the field (plant calculation: 10,000 plants / ha, dose per ha: 1 litre of product).

[0142] Table 14. Summary table of doses and concentrations

[0143] 4. Vegetative growth period: Plants were kept at a constant temperature of 21 °C and watered alternately with distilled water and Rigaud-Puppo solution with a limited source of nitrogen (Table 15).

[0144] Table 15. Rigaud-Puppo Nutrient Solution.

[0145] 2.2. Results

[0146] 2.2.1. Biomass

[0147] With the data obtained for the fresh and dry weight of the aerial part of the plants, it can be seen that all the combinations of strains tested together with cystium-d caused an increase in the production of the aerial biomass of the plant with respect to the control, and this increase was also significant in the CD-H treatment (Tables 16 and 17). As for the root fresh weight data, all combinations except CD-E and CD-F produced an increase in the root biomass of inoculated plants, although none of the differences between treatments was statistically significant (Tables 16 and 17). However, when measuring root biomass in dry weight, we observed that only three combinations of microorganisms caused an increase with respect to the control, although in the other combinations the percentage decrease was very low (Tables 16 and 17). With these results it was found that any of the combinations of microorganisms evaluated would be capable of replacing the microbial consortium that makes up the Cystium-d.

[0148] The best performing combination in terms of aerial biomass was CD-H, followed by CD-C and CD-E. The CD-H consortium contains Azotobacter chroococcum strain 30924, Pseudomonas canadensis strain 30930 and Peribacillus frigoritolerans strain 30923. This combination covers all the PGPB properties taken into account for this study, which could explain the large increase in aboveground biomass compared to the control of 41.3 and 34.4% in fresh and dry weight, respectively (Table 17). In terms of root biomass, the CD-H combination also showed the best result when fresh weight was taken into account (Table 17). However, when root biomass is measured in dry weight, the treatment inoculated with the CD-H combination not only does not increase, but also shows a slight decrease compared to the positive control inoculated with Cystium-d + commercial microorganisms (Table 17). This large variation between root fresh weight and root dry weight could be explained by the fact that plants inoculated with the CD-H combination store a large amount of water in their root system, which could also explain the observed increase in aerial biomass. If this is the case, the CD-H combination could provide the plant with greater tolerance to drought stress as the plant would have greater water reserves to withstand dry periods.

[0149] On the other hand, the inoculum that provided the greatest increase in root biomass, except for the value for fresh weight of the CD-H consortium, was CD-A, which only includes Azotobacter chroococcum strain 30924, but with a microbial concentration equal to the sum of the other treatments (Table 17). Therefore, this strain is a strong promoter of plant root growth, which is also translated into an increase in the biomass of the aerial part of the plant, although in this variable it is outperformed by the CD-C, CD-E and CD-H consortia (Table 17).

[0150] Table 16. Analysis of Variance (ANO A) for the dependent variables fresh and dry aboveground and root biomass and N content measured in barley plants grown in microcosms.

[0151] Table 17. Mean values, standard error and percentage increase with respect to the control of the fresh and dry biomass of the aerial and root part of the barley plants in the microcosm trial. Asterisks indicate the significance level of the differences between each treatment and the control by Dunnet's test. The absence of differences compared to the control is expressed as ns. Significance level: *p< 0.05.

[0152] 2.2.2. N content

[0153] In the results of plant N content it can be seen how, as with aboveground biomass, all the treatments inoculated with the different microbial consortia managed to increase the N content with respect to the control inoculated with Cystium-d (Tables 16 and 18).

[0154] Moreover, in five of the treatments the differences were statistically significant (Table 18).

[0155] The consortium that produced the greatest increase in N content was CD-G, consisting of Azotobacter chroococcum strain 30924, Priestia megaterium strain 30922 and Pichia kluyveri strain 13224 (Table 18). Among the other consortia that showed significant differences were CD-A, CD-C and CD-H, which were mentioned in the previous section as being good promoters of root and / or aerial growth, as well as CD-B (Table 18).

[0156] These results indicate that all the consortia evaluated improve the nutritional status of the plant, at least in terms of N. Thus, the high capacity of Azotobacter chroococcum strain 30924, which is present in all the consortia inoculated in the microcosm test, is further confirmed. Furthermore, the CD-H, CD-A and CD-C consortia with good results in biomass production also coincide in improving the nutritional status of the plant, making them the best potential substitutes for the strains that make up Cystium-d.

[0157] Table 18. Mean values and standard error of N content of barley plants in the microcosm trial. Asterisks indicate the significance level of differences between each treatment and the Control by Dunnet's test. The absence of differences compared to the control is expressed as ns. Significance level: *p< 0.05.

[0158] 2.3. Conclusions

[0159] The best combination of biostimulants would be the one formed by cystium-d and the strains 30924 of Azotobacter chroococcum, 30930 of Pseudomonas canadensis and 30923 of Peribacillus frigoritolerans due to the results obtained in aerial biomass and root fresh weight, as well as being among the consortia that improve N content. Also noteworthy are the results obtained with the inoculation of the consortia CD-C (C- D+30924+30923) and CD-E (C-D+30924+ 13224) for their increase in the biomass of the aerial part of the plant and CD-G (C-D+30924+30922+ 13224) for its higher N content. Therefore, the consortia CD-C, CD-E, CD-G and CD-H have been selected to pass to the next phase of testing in microcosms with real soil.

[0160] 3. Evaluation of biostimulant activity of the selected consortia under microcosm conditions

[0161] The effect of selected biostimulant combinations on the biomass production and nutrient content of the crop was further analysed under microcosm conditions with barley and broccoli in neutral and acidic soil and compared to the positive control.

[0162] The selected consortia were those that provided the best results in the previous study disclosed in section 2, namely: CD-H, CD-C, CD-E and CD-G. The results confirmed the improved biostimulant effect found in section 2 for the selected combinations when compared to the control. The biostimulant effect was outstanding for CD-H, outperforming, not only the positive control, but the other consortia, under all conditions and for all variables.

[0163] 4. Determination of quality traits resulting from the use of a plant biostimulant: according to the invention on potato against water and fertilizer reduction

[0164] SUMMARY

[0165] The action of a biostimulan composition according to the invention (herein called Composition A) on Potato var. Vivaldi was tested during the autumn period in Sa Pobla, Mallorca (Spain) against water and fertilizer reduction. The trial was started on October 25th with the first application, 2023, in outdoor experimental plots.

[0166] A randomized complete block design with 3 thesis: Control with 100% of water and fertilizer representing optimal conditions for the growth of the potato crop, a second control with 70% of water and fertilizer (i.e. 30% reduction water and fertilizer with respect to the optimal conditions) and compound A together with 70% of water a fertilizer. 4 replicates per thesis were used in this trial. The average yield per plot was recorded adding the weight of potatoes per plot.

[0167] EXPERIMENTAL CONDITIONS

[0168] 1.1 OBJECTIVE

[0169] The present example reports a trial to test the increase of Quality Traits on Potato var. Vivaldi in adverse conditions (adverse conditions = 30% reduction water and fertilizer with respect to optimal conditions) when supplementing with a composition A consisting of extract of Macrocystis (Cystium-d) 98% w / w and the microbial consortium CD-H 2% w / w (Peribacillus frigoritolerans, Pseudomonas canadensis and Azotobacter chroococcum).

[0170] Methodologies used:

[0171] CEN / TS17700-1 , CEN / TS17700-2, CEN / TS17700-3, CEN / TS 17700-4, CEN / TS17700-5 PP1 / 181 Conduct and reporting of efficacy evaluation trials, including good experimental practices.

[0172] PP1 / 152 Design and analysis of efficacy evaluation trials.

[0173] PP1 / 135 Phytotoxicity assessment.

[0174] Treatment list:

[0175] A: Cover fertilizer - 25 / 10 / 2023

[0176] B: 20 DAA- 14 / 11 / 2023

[0177] No pH control

[0178] Composition: Target: Biostimulant effect

[0179] Trial environment: Outdoor

[0180] Type of intended action Increase of the yield

[0181] 1.2 SITE DESCRIPTION

[0182] Location Name: Poligono 12 Parcela 287

[0183] City: Sa Pobla

[0184] Country: ES SPAIN

[0185] State: Mallorca

[0186] Postal / Zip Code: 07420

[0187] Facility: FIELD SITE

[0188] GPS Coordinates in Decimal Degrees

[0189] Latitude of LL Corner °: 39°46' 48.5" N

[0190] Longitude of LL Corner °: 3° 01 ' 57.3" E

[0191] 1.3 TRIAL CONDITIONS

[0192] Crop Description: Solanum tuberosum (Potato) var. Vivaldi

[0193] Experimental plot

[0194] N9plants / plot 100

[0195] N® replicates 4

[0196] Distance between lines (m) 0,75

[0197] Distance between plants (m) 0,3 Plot width (m) 7,5

[0198] Plot length (m) 3

[0199] Plot surface (m2) 22,5

[0200] The plot had 10 rows with 10 plants

[0201] Planting date: 15 / 09 / 2023

[0202] Irrigation system: Sprinkler irrigation

[0203] Frequency:

[0204] - September and October 3 times per week for 2 hours

[0205] - November and December 2 times per week for 2 hours

[0206] Control plots with 100% of water were supplied with a 30% more water than other plots.

[0207] Soil information

[0208] Texture Group: Clay Soil Drainage: Good

[0209] Tillage Type: Conventional % Sand: 20,9 % Silt: 35,6 % Clay: 43,4 pH: 8,32 %OM: 2,47

[0210] Maintenance:

[0211] Fertilization for plots with 100% was:

[0212] Entec 20 / 10 / 10 - 714 Kg / Ha - 15 / 09 / 2023 - Ubesol 21 / 0 / 0 - 714 Kg / Ha - 23 / 10 / 2023

[0213] Fertilization for plots with 70% was:

[0214] Entec 20 / 10 / 10 - 500 Kg / Ha - 15 / 09 / 2023

[0215] Ubesol 21 / 0 / 0 - 500 Kg / Ha - 23 / 10 / 2023 No phytosanitary product was applied during the trial period.

[0216] 1.4 DESIGN AND LAY OUT

[0217] 2.1. APPLICATION CONDITIONS

[0218] No problems were observed during product handling and application. Easy to dissolve in water.

[0219] 2.2. WEATHER CONDITIONS (STATION SA POBLA < 10 KM)

[0220] 3.1. EFFICACY

[0221] The assessments were performed according to the claim Quality traits:

[0222] • Enhancing of yield (Kg / plot; Kg / Ha)

[0223] 3.2. STATISTICAL ANALYSIS

[0224] The Statistical analysis of raw data was made according to EPPO guideline number PP 1 / 152(4). The data were subjected to analysis of variance (ANOVA), at 95% significance. When significant differences were obtained, a Student-Newman-Keuls test was applied to separate the means. The values followed with the same letter did not show significant differences. Bartlett’s test was applied to study the homogeneity of variances.

[0225] RESULTS AND CONCLUSIONS

[0226] The results are shown in figure 1. It may be observed that the use of the biostimulant composition A on Potato var. Vivaldi during the study was tested applying the product at 500 g / Ha two times at cover fertilizer and 20 days after first application to know if there were significant differences with the control.

[0227] The trial was set up in Sa Pobla (Mallorca) in open field and the maintenance of the plots were similar. The average yield per plot was recorded adding the weight of potatoes per plot.

[0228] All the applied products were completely safe and no phytotoxicity effects were observed. Effects on non-target individuals were neither observed.

[0229] Yield assessment. The results under claim - Increase of yield due to the action of composition A against water and fertilizer reduction were positive. The results showed significant differences at LSD 0,05 between the Control 70% and the strategy with composition A, increasing Kg / Ha by 4266,67 Kg. There were not significant differences between Control 100% and composition A.

[0230] Citation List

[0231] • Sambrook, J. and Russell, DW "Molecular Cloning: A Laboratory Manual", Chapter 13, "Mutagenesis", Cold Spring Harbor, 3rd Ed., 2001.

[0232] • PP1 / 181 Conduct and reporting of efficacy evaluation trials, including good experimental practices. EPPO Bulletin. 2021 ;00:1-13. DOI: 10.1111 / epp.12788

[0233] • PP1 / 152 Design and analysis of efficacy evaluation trials. Bulletin OEPP / EPPO Bulletin (2012) 42 (3), 367-381. DOI: 10.1111 / epp.2610

[0234] • PP1 / 135 Phytotoxicity assessment. Bulletin OEPP / EPPO Bulletin (2014) 44 (3), 265- 273. DOI: 10.1111 / epp.12134

[0235] • CEN / TS17700-1

[0236] • CEN / TS 17700-2

[0237] • CEN / TS17700-3

[0238] • CEN / TS 17700-4

[0239] • CEN / TS17700-5

[0240] Clauses

[0241] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0242] 1. A microbial consortium containing microorganisms from at least two species selected from the group consisting of Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans, Priestia megaterium, and Pichia kluyveri.

[0243] 2. The microbial consortium according to clause 1 , comprising Pseudomonas canadensis, Azotobacter chroococcum, Peribacillus frigoritolerans. 3. The microbial consortium according to clause 2, further comprising Pichia kluyveri, Priestia megaterium, or combinations thereof.

[0244] 4. The microbial consortium according to any one of clauses 1-3, wherein the Pseudomonas canadensis is a strain deposited at the "Coleccion Espanola de Cultivos Tipo” (CECT) with identification reference 30930, the Azotobacter chroococcum is a strain deposited at the CECT with identification reference 30924, the Peribacillus frigoritolerans is a strain deposited at the CECT with identification reference 30923, the Pichia kluyveri is a strain deposited at the CECT with identification reference 13224, and the Priestia megaterium is a strain deposited at the CECT with identification reference 30922.

[0245] 5. A composition comprising the microbial consortium as defined in any one of clauses 1- 4.

[0246] 6. The composition according to any one of clauses 1-5, further comprising extract from algae selected from extract from Macrocystis algae, in particular in particular Macrocystis pyrifera, extract from Laminaria algae, in particular Laminaria ochroleuca, and a combination thereof.

[0247] 7. The composition according to clause 6, wherein the extract from algae is a dry extract.

[0248] 8. The composition according to any one of clauses 5-7, further comprising one or more agriculturally acceptable compounds.

[0249] 9. The composition according to any one of clauses 5-8, which is a biostimulant composition.

[0250] 10. The composition according to any one of clauses 5-8, which is a fertilizer.

[0251] 11. Use of microbial consortium as defined in any one of clauses 1-4, or of a composition as defined in any one of clauses 5-9, for improving nutrient uptake by plants, for improving nutrient utilization by plants, and / or for improving the resistance of plants to abiotic stress, in particular, to drought.

[0252] 12. Use of microbial consortium as defined in any one of clauses 1-4, or of a composition as defined in any one of clauses 5-9, for improving plant growth and / or crop productivity.

[0253] 13. Use according to any one of clauses 11-12, for increasing Nitrogen fixation, Phosphorous mobilization, and Potassium solubilization by plants. 14. Use according to any one of clauses 11-13, comprising applying the composition by a drip irrigation system. 15. A strain selected from a Pseudomonas canadensis strain deposited at the "Coleccion

[0254] Espanola de Cultivos Tipo” (CECT) with identification reference 30930, a Azotobacter chroococcum strain deposited at the CECT with identification reference 30924, a Peribacillus frigoritolerans strain deposited at the CECT with identification reference 30923, a Pichia kluyveri a strain deposited at the CECT with identification reference 13224, and a Priestia megaterium strain deposited at the CECT with identification reference 30922.

Claims

Claims1. A microbial consortium containing Pseudomonas canadensis and one or more further microorganisms from a species selected from the group consisting of Azotobacter chroococcum, Peribacillus frigoritolerans, Priestia megaterium, and Pichia kluyveri.

2. The microbial consortium according to claim 1 , comprising Pseudomonas canadensis, Azotobacter chroococcum, and Peribacillus frigoritolerans.

3. The microbial consortium according to claim 2, further comprising Pichia kluyveri, Priestia megaterium, or Pichia kluyveri and Priestia megaterium.

4. The microbial consortium according to any one of claims 1-3, wherein the Pseudomonas canadensis is a strain deposited at the "Coleccion Espanola de Cultivos Tipo” (CECT) with identification reference 30930, the Azotobacter chroococcum is a strain deposited at the CECT with identification reference 30924, the Peribacillus frigoritolerans is a strain deposited at the CECT with identification reference 30923, the Pichia kluyveri is a strain deposited at the CECT with identification reference 13224, and the Priestia megaterium is a strain deposited at the CECT with identification reference 30922.

5. A composition comprising the microbial consortium as defined in any one of claims 1-4.

6. The composition according to any one of claims 1-5, further comprising extract from algae selected from extract from Macrocystis algae, in particular Macrocystis pyrifera.

7. The composition according to claim 6, wherein the extract from algae is a dry extract.

8. The composition according to any one of claims 5-7, further comprising one or more agriculturally acceptable compounds selected from the group consisting of phytofortifiers, nutrients, humectants, stabilizers, osmoprotectants, antioxidants, sunscreens, buffering compounds, adherence improving compounds such as gelatins, starches, pectins, alginates, and gums, or combinations thereof.

9. The composition according to any one of claims 5-8, which is a plant biostimulant composition.

10. The composition according to any one of claims 5-8, which is a fertilizer.

11. Use of microbial consortium as defined in any one of claims 1-4, or of a compositionas defined in any one of claims 5-9, for improving nutrient uptake by plants, for improving nutrient utilization by plants, and / or for improving the resistance of plants to abiotic stress, in particular, to drought.

12. Use of microbial consortium as defined in any one of claims 1-4, or of a composition as defined in any one of claims 5-9, for improving plant growth and / or crop productivity.

13. Use according to any one of claims 11-12, for increasing Nitrogen fixation, Phosphorous mobilization, and Potassium solubilization by plants.

14. Use according to any one of claims 11-13, comprising applying the composition by a drip irrigation system.

15. A strain selected from a Pseudomonas canadensis strain deposited at the "Coleccion Espanola de Cultivos Tipo” (CECT) with identification reference 30930.

16. Use of Pseudomonas canadensis for improving nutrient uptake by plants, for improving nutrient utilization by plants, and / or for improving the resistance of plants to abiotic stress, in particular, to drought.

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