Microorganisms compositions for improvement of plant traits and methods of use thereof

Abstract

Aspects of the present disclosure include compositions and methods for improving the yield of agronomically relevant crops. Aspects of the present disclosure include compositions capable of improving plant growth and performance. Aspects of the present disclosure include compositions capable of improving plant growth and performance comprising extremophile microorganisms of the species Priestia megaterium with plant growth- promoting activity.

Description

MICROORGANISMS COMPOSITIONS FOR IMPROVEMENT OF PLANT TRAITS AND METHODS OF USE THEREOF1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 564,844, filed March 13, 2024, which is hereby incorporated by reference in its entirety.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 13, 2025, is named 43003-61810_WO_seqlisting.xml and is 26 kilobytes in size.3. BACKGROUND

[0003] Worldwide population growth as well as the climate change the planet is going through have generated a need for new technologies to improve the yield of agronomically relevant crops which is greater than ever before. In this sense, the role of products such as fertilizers, stimulants, inoculants, and pesticides is key to maximizing the growth of crop plants and protecting them from potential pathogens.

[0004] In recent years, however, there has been a trend to shift from traditional chemicalbased products for agricultural use in crops to bio-based products. Bio-based products are characterized by comprising components obtained from natural sources, which exhibit much less environmental side-effects when compared to chemical-based products. Exemplary biobased products include, but are not limited to: biostimulants, biological formulations, biofertilizers, bioinoculants, soil amendments, plant amendments, and the like.

[0005] Among bio-based products, the use of microorganisms to improve the growth of agronomically relevant plants is well known in the art.

[0006] Extremophile microorganisms are those capable of growing and thriving in harsh environmental conditions, such as high or low temperatures, high soil salinity, draught, etc. There have been some reports of specific extremophile microorganisms which may be used as promoters for plant growth. For instance, patent application W02024015307A1 disclose the use of Klebsiella aerogenes, Bacillus cereus and Exiguobacterium undeae as promoters for plant growth.

[0007] However, there is a constant need for new bio-based products which exhibit high growth-promoting capabilities, as well as being resistant to different environmental conditions and other products which may be applied to crops, while being readily formulated and scalable for industrial production.4. SUMMARY

[0008] Aspects of the present disclosure include compositions for improving the yield of agronomically relevant crops. Particularly, the present invention is related to compositions capable of improving plant growth and performance. Even more particularly, the present disclosure is related to compositions capable of improving plant growth and performance comprising extremophile microorganisms with plant growth-promoting activity.

[0009] An aspect of the present disclosure includes a composition comprising: a microorganism of the species Priestia megaterium (P. megaterium).

[0010] and an agriculturally compatible carrier, wherein the microorganism is in contact with the agriculturally compatible carrier. In some embodiments, the composition comprises a culture media composition. In some embodiments, the composition comprises an agriculturally compatible carrier and a culture media composition.

[0011] In some embodiments, the composition is a synthetic composition. In some embodiments, the synthetic composition is a biological formulation. In some embodiments, the biological formulation is an inoculant, a biostimulant, a biofertilizer, soil amendment, or plant amendment.

[0012] In some embodiments, the microorganism is an endophyte. In some embodiments, the microorganism is an endophytic sporulating bacterium. In some embodiments, the microorganism is in the form of a spore, a vegetative cell, an endophyte, an endospore, or a combination thereof. In some embodiments, the microorganism comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8. In some embodiments, the microorganism comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8. In some embodiments, the microorganism comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8. In some embodiments, the microorganism comprises a nucleotide sequence having at least 97% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8. In some embodiments, the microorganism comprises a nucleotide sequence of any one of SEQ ID NOs: 1-2 and 7-8. In some embodiments, the microorganism of the species ofP. megaterium comprises a 16S rRNA gene sequence as set forth in SEQ ID NO: 1. In some embodiments, the microorganism of the species of P. megaterium comprises a nucleotide sequence as set forth in SEQ ID NO: 2. In some embodiments, the microorganism of the species of P. megaterium comprises a 16S rRNA gene sequence as set forth in SEQ ID NO: 7. In some embodiments, the microorganism of the species of P. megaterium comprises a nucleotide sequence as set forth in SEQ ID NO: 8.

[0013] In some embodiments, the microorganism of the species of P. megaterium is strain CK60 of P. megaterium having a DSMZ accession number of 35107. In some embodiments, the microorganism of the species of P. megaterium is strain CK100 of P. megaterium having a DSMZ accession number of 35108. In some embodiments, the microorganism of the species of P. megaterium is strain CK60 of P. megaterium collected at Volcan Galan, Catamarca province, Argentina, having a DSMZ accession number of 35107. In some embodiments, the microorganism of the species of P. megaterium is strain CK100 of P. megaterium collected at Volcan Galan, Catamarca province, Argentina, having a DSMZ accession number of 35108.

[0014] In some embodiments, the biological formulation is in liquid form, and wherein the microorganism of P. megaterium is present in the composition in a concentration from 1 x 102to 9 x 109CFU / mL. In some embodiments, the biological formulation is in liquid form, and wherein the microorganism of P. megaterium is present in the composition in a concentration from 1 x 103to 1 x 109CFU / mL. In some embodiments, the composition comprises strain CK60 of P. megaterium in a concentration of 2 x 107CFU / mL.

[0015] In some embodiments, the culture media composition comprises a culture medium selected from one or more of: glucose, yeast extract, meat extract, calcium chloride, magnesium sulfate, potassium chloride, iron chloride, sodium chloride, phosphate salts, ammonium sulfate, pea whey, soybean peptone, manganese chloride, chitin, sucrose, ferric chloride, dipotassium phosphate, stachyose, dihydrate, casein, soybean, hydrogen phosphate, glucose monohydrate, Calcium Chloride Dihydrate, pluripeptone, Glucose Monohydrate, Magnesium sulfate heptahydrate, Iron (III) chloride hexahydrate, Potassium hydrogen phosphate, Manganese(II) chloride tetrahydrate, and peptone.

[0016] In some embodiments, the synthetic composition further comprises one or more of: alginic acid, carrageenan, dextrin, dextran, polyethylene glycol, polyvinyl pyrrolidone,methyl cellulose, polyvinyl alcohol, gelatin, a detergent, an insecticide, a fungicide, a dressing, and combinations thereof.

[0017] In some embodiments, the culture media composition comprises a culture media composition selected from any one of the culture media compositions of Table 23 (spore composition media table; CK60). In some embodiments, the culture media composition comprises a culture media composition selected from any one of the culture media compositions of Table 24 (vegetative bacteria media table; CK60). In some embodiments, the culture media composition comprises a culture media composition selected from any one of the culture media compositions of Table 31 (spore composition media table; CK100). In some embodiments, the culture media composition comprises a culture media composition selected any one of the culture media compositions of Table 32 (vegetative bacteria media table; CK100).

[0018] In some embodiments, the microorganism of the species P. megaterium, when contacted with a plant element comprising: a plant seed, a plant or plant portion thereof, is capable of improving a plant growth-promoting trait compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when disposed via in-furrow application of soil surrounding a plant element or via foliar application of the plant element, is capable of improving a plant growth-promoting trait compared to a reference plant element contacted or inoculated or applied with the microorganism of the species P. megaterium.

[0019] In some embodiments, the plant element is a plant or portion thereof selected from: plant leaves, plant roots (e.g., primary root, root hairs, lateral root, root cap, etc.), plant stems, and plant shoots (e.g., leaf blade, stem, petiole, leaf axil, flower, terminal bud, lateral bud etc.).

[0020] In some embodiments, the microorganism of the species P. megaterium, is contacted with or disposed onto the plant element via one or more of: broadcast application, liquid or dry in-furrow application, spray application, irrigation, injection, dusting, pelleting, dressing, or coating of the plant element.

[0021] In some embodiments, the plant element is the seed, wherein the seed is a: wheat seed, potato seed, a corn seed, a barley seed, a lettuce seed, a peanut seed, a rice seed, an oat seed, a millet seed, or a tomato seed. In some embodiments, the plant element is the plantselected from: wheat, potato, corn, barley, lettuce, peanut, and tomato. In some embodiments, the microorganism is heterologously or homogeneously disposed to the plant element. In some embodiments, the microorganism is contacted with or disposed onto the plant element in an amount effective to provide one or more plant growth-promoting traits compared to a reference plant element grown without the microorganism. In some embodiments, the plant element is the seed, wherein the seed is a Gramineae seed (grasses), or a vegetable seed, or a legume seed.

[0022] In some embodiments, the one or more plant growth-promoting traits is selected from: increased germination rate, increased emergence rate, increased shoot biomass, increased seedling root length, increased seedling shoot length, increased seedling mass, increased root surface area, increased enhanced nutrient use efficiency, increased phosphate solubilization, and increased yield. In some embodiments, the plant growth-promoting trait is selected from: increased degradation of nitrogenous organic matter in the soil by which the plant element is growing and converting the nitrogenous organic matter into ammonium. In some embodiments, the plant growth-promoting trait is the microorganism’s increased production of ammonium. In some embodiments, the plant growth-promoting trait is increased hydrolysis of urea. In some embodiments, the plant growth promoting trait is the microorganism’s production and / or release of siderophores into the culture media composition. In some embodiments, the plant growth promoting trait is enhancement of the plant element’s iron nutrition. In some embodiments, the plant growth-promoting trait is an increase in flavonoids selected from one or more of: malic acid, mannitol, citric acid, tryptophan, daidzein, chrysin, apigenin, naringenin, luteolin, genistein, naringin, and naringin in its aglycone form. In some embodiments, the plant growth-promoting trait is an increase in root exudates of seeds of the plant element, wherein the plant element is the plant. In some embodiments, the plant growth-promoting trait is an increase is phosphate solubilization. In some embodiments, the microorganism comprises a phosphate solubilizing index (PSI) ranging from 3-6. In some embodiments, the microorganism comprises a phosphate solubilizing index (PSI) of about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, or about 4.9. In some embodiments, the plant growth-promoting trait is increased potassium solubilization.

[0023] In some embodiments, the plant growth promoting trait is the microorganism’s increased production of one or more phytohormones. In some embodiments, the one or more phytohormones is selected from: indole acetic acid (IAA), abscisic acid (ABA), salicylic acid(SA), gibberellic acid (GA3), jasmonic acid (JA), zeatin (ZEA), 6-benzylaminopurine (6- BAP), kinetin (KIN), and indole-3 -butyric acid (IBA).

[0024] In some embodiments, the microorganism is disposed onto or contacted with a plant seed in an amount effective to colonize the plant germinated from the microorganism formulated in the agriculturally compatible carrier as a biological formulation. In some embodiments, the colonization occurs in the root tissue of the plant. In some embodiments, the colonization results in a biofilm formation of the microorganism.

[0025] In some embodiments, the plant growth promoting trait is selected from: increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance or tolerance to drought stress, enhanced resistance to thermal stress or thermal shock, enhanced resistance to UV radiation, enhanced resistance or tolerance to different NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to stress, increased nutrient uptake, increased root hair formation, increased root branching, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof.

[0026] An aspect of the present disclosure includes methods method for improving a trait in a plant or promoting growth of the plant.

[0027] In some embodiments, the method comprises contacting a plant element comprising a seed or a plant or plant portion thereof with the synthetic composition of the present disclosure, wherein the microorganism of the species P. megaterium, and wherein the microorganism is present in the synthetic composition in an amount effective to increase colonization of the plant element and to provide a benefit to the plant compared to a reference plant not treated with the microorganisms of the species P. megaterium.

[0028] In some embodiments, the microorganism is capable of producing substances that are beneficial to plants. In some embodiments, the benefit is selected from: increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance or tolerance to drought stress, enhanced resistance or tolerance to thermal stress or thermal shock, enhanced resistance or tolerance to UV radiation, enhanced resistance or tolerance todifferent NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance or tolerance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance or tolerance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof.

[0029] In some embodiments, the method further comprises improving a plant phenotype under stress conditions as compared to reference plants not contacted or inoculated or applied with said synthetic composition. In some embodiments, the method is configured to promote growth of the plant.

[0030] In some embodiments, the contacting comprises applying the synthetic composition to the plant element via broadcast application, liquid or dry -in furrow application, spray application, irrigation, injection, dusting, pelleting, or coating of the plant element.

[0031] In some embodiments, the method comprises germinating the plant element, wherein the plant element is a seed; and growing the seed.

[0032] In some embodiments, the microorganism of the species P. megaterium, when applied via in-furrow application of soil surrounding a plant element or via foliar application of the plant element, is capable of improving a plant growth-promoting trait compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium.

[0033] In some embodiments, the plant or portion thereof is selected from: plant leaves, plant roots (e.g., primary root, root hairs, lateral root, root cap, etc.), plant stems, and plant shoots (e.g., leaf blade, stem, petiole, leaf axil, flower, terminal bud, lateral bud etc.). In some embodiments, the microorganism of the species P. megaterium, is contacted with or disposed onto the plant element via one or more of: broadcast application, liquid or dry in-furrow application, spray application, irrigation, injection, dusting, pelleting, or coating of the plant element. In some embodiments, the plant element is a seed, wherein the seed is a: wheat seed, potato seed, a corn seed, a barley seed, a lettuce seed, rice seed, oat seed, millet seed, or a tomato seed. In some embodiments, the plant element is a plant selected from: wheat, potato, corn, barley, lettuce, and tomato. In some embodiments, the microorganism is heterologouslyor homogeneously disposed to the plant element. In some embodiments, the plant element is the seed, wherein the seed is a Gramineae seed (grasses), or a vegetable seed, or a legume seed.

[0034] In some embodiments, contacting comprises homogenously or heterologously disposing the microorganism onto the plant element in an amount effective to provide one or more plant growth-promoting traits compared to a reference plant element grown without the microorganism. In some embodiments, the benefit comprises one or more of: increased germination rate, increased emergence rate, increased shoot biomass, increased seedling root length, increased seedling shoot length, increased seedling mass, increased root surface area, increased enhanced nutrient use efficiency, increased phosphate solubilization, and increased yield. In some embodiments, the benefit comprises one or more of: increased degradation of nitrogenous organic matter in the soil by which the plant element is growing and converting the nitrogenous organic matter into ammonium. In some embodiments, the benefit comprises increased production of ammonium. In some embodiments, the benefit comprises increased hydrolysis of urea. In some embodiments, the benefit comprises production and / or release of siderophores into the culture media composition. In some embodiments, the benefit comprises enhancement of the plant element’s iron nutrition. In some embodiments, the benefit comprises an increase in flavonoids selected from one or more of: malic acid, mannitol, citric acid, tryptophan, daidzein, chrysin, apigenin, naringenin, luteolin, genistein, naringin, and naringin in its aglycone form. In some embodiments, the benefit comprises an increase in root exudates of seeds of the plant element, wherein the plant element is the plant. In some embodiments, the benefit comprises an increase is phosphate solubilization. In some embodiments, the microorganism comprises a phosphate solubilizing index (PSI) ranging from 3-6. In some embodiments, the microorganism comprises a phosphate solubilizing index (PSI) of about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, or about 4.9. In some embodiments, the benefit comprises increased potassium solubilization.

[0035] In some embodiments, the benefit comprises the microorganism’s increased production of one or more phytohormones. In some embodiments, the one or more phytohormones is selected from: indole acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), jasmonic acid (JA), zeatin (ZEA), 6-benzylaminopurine (6- BAP), kinetin (KIN), and indole-3 -butyric acid (IBA).

[0036] In some embodiments, the colonization occurs in the root tissue of the plant. In some embodiments, the colonization results in a biofilm formation of the microorganism.

[0037] In some embodiments, the benefit is selected from: increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance or tolerance to drought stress, enhanced resistance to thermal stress or thermal shock, enhanced resistance to UV radiation, enhanced resistance or tolerance to different NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to stress, increased nutrient uptake, increased root hair formation, increased root branching, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof.

[0038] In some embodiments, the composition is formulated as an inoculant, and it is applied at a dose from 5 mL / kg to 10 mL / kg. In some embodiments, the plant whose growth is to be improved is wheat or barley, the composition comprises strain CK60 of P. megaterium, and it is applied at a dose of 6 mL / kg. In some embodiments, the plant whose growth is to be improved is barley, the composition comprises strain CK60 of P. megaterium, and it is applied at a dose of 6 mL / kg. In some embodiments, the plant whose growth is to be improved is wheat, the composition comprises strain CK60 of P. megaterium, and it is applied at a dose of 6 mL / kg.

[0039] In some embodiments, the method further comprises harvesting the plant.

[0040] An aspect of the present disclosure includes a method for improving soil quality , comprising applying the synthetic composition according to any the composition of the present disclosure to a plant element comprising a seed or a plant grown in the soil. In some embodiments, the method results in a reduced use of an amount of commercially available nitrogen fertilizers in the soil. An aspect of the present disclosure includes a method for improving plant quality, comprising applying the synthetic composition according to any the composition of the present disclosure to a plant element comprising a seed or a plant grown in the soil. In some embodiments, the method results in a reduced use of an amount of commercially available nitrogen fertilizers in the soil.5. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0041] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0042] FIG. 1A illustrates microscopic observation (100X) of the gram-stained smear of CK60.

[0043] FIG. IB illustrates colonies isolated in solid nutrient agar of CK60.

[0044] FIG. 2 illustrates an ANI taxonomic chart of some representative strains of the Priestia genus.

[0045] FIG. 3 illustrates a core genome phylogenetic tree of selected strains of the Priestia genus.

[0046] FIG. 4 illustrates a proposed model of EPS produced by CK60.

[0047] FIGs. 5A-5D illustrate SEM images of the upper zone of wheat roots inoculated with CK60 incubated at 15 °C. FIG. 5 A, FIG. 5B illustrate CK60 colonization on the root surface was detected. CK60 is observed alongside other bacteria, characterized by its larger diameter (> 1 pm) and length (> 5 pm), in contrast to the smaller size of the surrounding cells. FIG. 5C, FIG. 5D illustrate that in some areas, CK60 was identified immersed in a matrix, indicating the presence of a mature CK60 biofilm on the root surface after 10 days of seed inoculation.

[0048] FIGs. 6A-6D illustrate SEM images showing extensive colonization and biofilm formation of CK60 on the surface of wheat root tissue post-spore inoculation. FIG. 6A illustrates the dense aggregation of cells and the presence of a polymeric matrix, indicating active microbial interaction and establishment. FIG. 6B illustrates CK60 spores and germinating cells immersed in a polymeric matrix. The CK60 spores display dehydrated structures with an irregular surface and distinctive grooves. In contrast, the CK60 germinating cells highlight a smoother surface with less defined grooves, resulting from rehydration during the germination process. FIG. 6C, FIG. 6D illustrate CK60 spores, germinating cells, and vegetative cells colonizing the surface of wheat roots.

[0049] FIGs. 7A-7D illustrate SEM images showing extensive colonization of CK60 on the surface of wheat root tissue at 15 °C. The images illustrate the dense aggregation of cells,which can be observed forming chains, highlighting the active microbial interaction and establishment.

[0050] FIG. 8 illustrates a profile of bioactive compounds determined in the rhizodepositions of wheat seedlings (control and inoculated with CK60 and Azospirillum brasi lense).

[0051] FIG. 9 illustrates the amount of phosphate solubilized by CK60 at 3, 6, 10, and 14 days of incubation in NBRIP medium. (Analysis of variance, DGC Test, means with a common letter are not significantly different (p > 0.05)).

[0052] FIG. 10 illustrates the quantification of phosphate solubilized at 3, 6, 10, and 14 days by CK60 versus Benchmarks.

[0053] FIG. 11 illustrates the quantification of ammonium produced by CK60 in Jensen medium without nitrogen source and in Jensen medium with complex nitrogen source (Peptone), demonstrating two possible mechanisms of ammonium production and availability by the microorganism.

[0054] FIG. 12 illustrates the ability of CK60 to hydrolyze urea in a urea agar base medium is evidenced by the color change of the pH indicator from yellow to pink.

[0055] FIG. 13 illustrates the quantification of ammonium production of the strains evaluated in Jensen medium (nitrogen-free culture medium).

[0056] FIG. 14 illustrates viability of CK60 at 24 and 48 hours against different degrees of drought (PEG%).

[0057] FIG. 15 illustrates survival rate of CK60 exposed to total drought conditions at different time intervals.

[0058] FIG. 16 illustrates CK60 salinity curves.

[0059] FIG. 17 illustrates pH curves of CK60.

[0060] FIGs. 18A-18B illustrate CK60 growth curves at different temperatures. FIG.18A. Growth curves evaluated up to 24 hours. FIG. 18B. Growth curves at 30 °C, 15 °C, and 10 °C evaluated up to 168 hours.

[0061] FIG. 19 illustrates sensitivity to UV-B radiation of the strains evaluated on LB- agar plates.

[0062] FIG. 20 illustrates resistance of CK60 spores at different pH values.

[0063] FIG. 21 illustrates resistance of CK60 spores at 40 °C for 7 days.

[0064] FIG. 22 illustrates phenol ogical stages of the wheat plant. TO = Control. T1 =CK60. Dose: 6 mL of CK60 per kg of seed (6 mL / kg).

[0065] FIG. 23 illustrates the presence of CK60 formulation in the root over time.

[0066] FIG. 24 illustrates the presence of CK60 formulation in the aerial part over time.

[0067] FIG. 25 illustrates DNA concentration of CK60 in different parts of the wheat plant over time.

[0068] FIG. 26A illustrates nitrogen uptake contribution / compensation by the crop inoculated with CK60 formulation Campaign 2022.

[0069] FIG. 26B illustrates nitrogen uptake contribution / compensation by the crop inoculated with CK60 formulation Campaign 2023.

[0070] FIG. 27 illustrates average yield values (kg / ha) of the treatments.

[0071] FIG. 28 illustrates vigor analysis for CK60 formulation at 2 doses versus noninoculated control and commercial control.

[0072] FIG. 29 illustrates root dry weight in grams for CK60 formulation at 2 doses versus non-inoculated control and commercial control.

[0073] FIG. 30 illustrates root length in centimeters for CK60 formulation at 2 doses versus non-inoculated control and commercial control.

[0074] FIG. 31 illustrates number of tubers per linear meter for CK60 formulation at 2 doses versus control without inoculation and commercial control.

[0075] FIG. 32 illustrates weight of thousand tubers in kg for the different treatments evaluated.

[0076] FIG. 33 illustrates yield in tn / ha for the different treatments evaluated.

[0077] FIG. 34 illustrates average SPAD values for the different treatments.

[0078] FIG. 35 illustrates average values of dry weight per plant for the different treatments.

[0079] FIG. 36 illustrates average values of number of grains per plant for the different treatments.

[0080] FIG. 37 illustrates average values of grain weight per plant for the different treatments.

[0081] FIG. 38 illustrates weight of plants in floating system.

[0082] FIG. 39 illustrates weight of plants in NFT system.

[0083] FIG. 40 illustrates weight of plants in NFT system immersion and foliar application semi-hydroponic systems.

[0084] FIG. 41 illustrates growth of CK60 on blood agar medium, no clear halos of beta hemolysis are observed.

[0085] FIG. 42 illustrates a profile of bioactive compounds determined in the rhizodepositions of wheat seedlings (control and inoculated with CK100 and Azospirillum brasi lense).

[0086] FIG. 43 illustrates DNA concentration of CK100 in different parts of the wheat plant over time.

[0087] FIG. 44 illustrates height to flag leaf for the treatments evaluated.

[0088] FIG. 45 illustrates the average greenness index for the evaluated treatments.

[0089] FIG. 46 illustrates vegetative aerial dry weight in grams for the evaluated treatments.

[0090] FIG. 47 illustrates a number of rows of grains / cob for the treatments evaluated.

[0091] FIG. 48 illustrates a number of grains per plant for the treatments evaluated.

[0092] FIG. 49 illustrates grain weight at 13.5% in grams for the treatments evaluated.

[0093] FIG. 50 illustrates mean SPAD values for the different treatments. Equal letters indicate that there was no significant difference (p > 0.05).

[0094] FIG. 51 illustrates average values of dry weight per plant for the different treatments. Different letters indicate significant difference (p < 0.05).

[0095] FIG. 52 illustrates average values of number of grains per plant for the different treatments. Different letters indicate significant difference (p < 0.05).

[0096] FIG. 53 illustrates average values of grain weight per plant for the different treatments. Different letters indicate significant difference (p < 0.05).

[0097] FIG. 54 illustrates average values of percent confectionery per plant per plant for the different treatments. Equal letters indicate that there was no significant difference (p > 0.05).

[0098] FIG. 55 illustrates temperature record during the three days of heat stress generation.

[0099] FIGs. 56A-56B illustrate values of % electrolyte leakage measured at time Z 2.3 and Z 3.9 (FIG. 56A) and % damage coefficient (FIG. 56B) of stressed controls compared to unstressed controls for both times.

[0100] FIG. 57 illustrates values of % electrolyte leakage in the different treatments. CK100: Priestia megaterium (P. megaterium). CK100 sporulated; Z 2.3: phenological stage of main stem with three tillers; Z 3.9: phenological stage of flag leaf ligule visible.

[0101] FIGs. 58A-58B illustrate values of % electrolyte leakage (FIG. 58A) and % C.D. (FIG. 58B) measured at phenological stage Z 2.3. The arrows above the bars in the treatments indicate the decrease in the % C.D. value with respect to the stress control.

[0102] FIGs. 59A-59B illustrate electrolyte leakage % values measured at phenological stage Z 3.9 at 12 (FIG. 59A) and 20 (FIG. 59B) days after heat and water stress.

[0103] FIG. 60 illustrates % C.D. values measured at phenological stage Z 3.9 at 12 and 20 days after heat and water stress. The arrows above the bars in the treatments indicate the decrease in the % C.D. value with respect to the stress control.

[0104] FIGs. 61A-61C illustrate mean values per plant of number of spikelets (FIG. 61A), number of grains (FIG. 61B), yield in grams (FIG. 61C) of all treatments. Different letters indicate significant difference among treatments (p < 0.05).

[0105] FIGs. 62A-62C illustrate mean values per plant of number of spikelets (FIG.62A), number of grains (FIG. 62B), yield in grams (FIG. 62C) of the Z 2.3 treatments. Equal letters indicate no significant difference between treatments (p > 0.05).

[0106] FIGs. 63A-63C illustrate mean values per plant of number of spikelets (FIG. 63A), number of grains (FIG. 63B), yield in grams (FIG. 63C) of the Z 3s.9 treatments. Equal letters indicate no significant difference between treatments (p > 0.05).

[0107] FIG. 64 illustrates resistance of CK60 spores to different NaCl concentrations.

[0108] FIG. 65 illustrates percentage variation in the growth of CK60 and M. Symbioticum in response to different concentrations of PEG 600 (0%, 20%, 30%, 40%, and 50%), evaluated at 24 and 48 hours.6. DETAILED DESCRIPTION

[0109] Aspects of the present disclosure include biological formulations comprising microorganisms of the species Priestia megaterium (P. megaterium) formulated as biostimulants, bioinoculants, biofertilizers, soil amendment, and / or plant amendment, for use in promoting growth of a plant.

[0110] . The present disclosure is based on the surprising plant growth-promoting capabilities the present inventors have found for certain strains of P. megaterium. The need for improved crop yields in a worsening climate change scenario makes an invention such as this of particular relevance.[OHl] P. megaterium is an extremophile microorganism known to grow in very diverse environmental conditions. The present inventors have isolated two strains of P. megaterium from rhizosphere samples of plants collected at Volcan Galan, Catamarca Province, Argentina, with particularly good plant growth-promoting capabilities. The strains were characterized by 16s rRNA analysis, upon which their denomination as strains of P. megaterium was confirmed.

[0112] The first strain was isolated from rhizosphere samples of plants collected at Volcan Galan, Catamarca Province, Argentina, and comprises a 16S rRNA gene sequence as set forth in SEQ ID NO: 1. The strain is deposited at DSMZ (German Collection of Microorganisms and Cell Cultures GmbH), with a DSMZ accession number of 35107. Throughout this description, this strain will be referred to as “CK60”.

[0113] The second strain was isolated from rhizosphere samples of plants collected from dry, carbonic, acid and saline zones located in the salt flat at Volcan Galan, Catamarca Province, Argentina, and comprises a 16S rRNA gene sequence as set forth in SEQ ID NO: 2. The strain is deposited at DSMZ (German Collection of Microorganisms and Cell Cultures GmbH), with a DSMZ accession number of 35108. Throughout this description, this strain will be referred to as “CK100”.

[0114] CK60 and CK100 are closely related, as evidenced by their nearly identical 16S rRNA gene sequences, with 99.9% identity, as shown below.

[0115] The genome of CK60 comprises 15 copies of the 16S ribosomal gene, with two different versions differing in just two residues. The consensus sequence is provided as SEQ ID NO: 1 in Table 47, wherein R=A / G and Y=C / T (SEQ ID NO: 1).

[0116] The genome of CK100 comprises 15 copies of the 16S ribosomal gene, with two different versions differing in just two residues. The consensus sequence is provided as SEQ ID NO: 2 in Table 47, wherein R=A / G and Y=C / T (SEQ ID NO: 2).

[0117] As it will be evident from the Examples included further below in this description, CK60 and CK100 surprisingly exhibit plant growth-promoting capabilities, such as the ability to solubilize phosphorus, fix nitrogen, mineralize nitrogen compounds, hydrolyze urea, produce siderophores and synthesize auxins such as AIA. Even though applicationWO2023159168A2 discloses the potential use of P. megaterium as a nematicide in plants, the surprising capabilities of CK60 and CK100 allow them to act as plant growth promoters (PGP), regardless of whether nematode-related stress should be controlled or prevented in the target plant.6.1. Compositions

[0118] An aspect of the present disclosure comprises a composition comprising a microorganism of the species Priestia megaterium (P. megaterium),' and an agriculturally compatible carrier.

[0119] In some embodiments, the composition further comprises a culture media composition.

[0120] The compositions of the present disclosure include synthetic compositions.

[0121] In some embodiments, the composition is a biological formulation. In some embodiments, the biological formulation is in the form of an inoculant, a biostimulant, or a biofertilizer. In some embodiments, the composition is useful as a biofertilizer due to the excellent plant growth-promoting capabilities of the microorganism comprised therein, as mentioned above. The terms “biofertilizer”, “biological fertilizer”, “biostimulant” “soil amendment”, “plant amendment”, and “plant growth promoter” may be used interchangeably, and should be understood throughout this description as referring to a biobased product which may be used to improve the growth of a plant, such as the composition of the disclosure.6.1.1. Microorganism

[0122] The microorganism of the present disclosure comprises a microorganism of the species P. megaterium. In some embodiments, the microorganism is an endophyte. In some embodiments, the microorganism is an endophytic sporulating bacterium. In some embodiments, the microorganism is in the form of a spore, a vegetative cell, an endophyte, an endospore, or a combination thereof.

[0123] In some embodiments, the microorganism is of the species P. megaterium comprising a 16S rRNA gene nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the microorganism of the strain or species of P. megaterium comprises a 16S rRNA gene nucleotide sequence of SEQ ID NO: 1. In some embodiments, the composition of the present disclosure comprises a strain of P. megaterium comprising a 16S rRNA gene sequence as set forth in SEQ ID NO: 1, or a 16S rRNA gene sequence at least 99.9% identical to the nucleotide sequence set forth in SEQ ID NO: 1.

[0124] In some embodiments, the strain of P. megaterium comprised in the composition is a strain of P. megaterium comprising a 16S rRNA gene sequence as set forth in SEQ ID NO: 1. In some embodiments, the strain of P. megaterium is strain CK60 with DSMZ accession number 35107.

[0125] In some embodiments, the microorganism is of the species P. megaterium comprises a nucleotide sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 7.

[0126] In some embodiments, the strain of P. megaterium comprised in the composition is a strain of P. megaterium comprising a 16S rRNA gene sequence as set forth in SEQ ID NO: 2. In some embodiments, the strain of P. megaterium is strain CK100 with DSMZ accession number 35108. In some embodiments, the microorganism is of the species P. megaterium comprises a nucleotide sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 8.6.1.2. Concentrations of microorganisms

[0127] In some embodiments, the strain of P. megaterium is present within the composition in a concentration such that it may properly exert its plant growth-promoting activity while maintaining a proper stability and without generating any undesired effect on the plant or the soil.

[0128] In some embodiments, the concentration of the microorganism in the composition depends on the nature of said microorganism, as well as the form of the composition.

[0129] In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 101to 9 x 1012CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 101to 8 x 1012CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 101to 1 x 1012CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 102to 1 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 103to 1 x 109CFU / mL. In some embodiments, the final concentration of the composition is about 102, 103, 104, 105, 106, 107, 108, or 109spores / ml which can be reached by serial dilutions in sterile water or in an appropriate solution or buffer. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 102CFU / mL, 1 x 103CFU / mL, 1 x 104CFU / mL, 1 x 105CFU / mL, 1 x 106CFU / mL, 1 x 107CFU / mL, 1 x 108CFU / mL, 1 x 109CFU / mL, or 1 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 2 x 102CFU / mL, 2 x 103CFU / mL, 2 x 104CFU / mL, 2 x 105CFU / mL, 2 x 106CFU / mL, 2 x 107CFU / mL, 2 x 108CFU / mL, 2 x 109CFU / mL, or 2 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 3 x 102CFU / mL, 3 x 103CFU / mL, 3 x 104CFU / mL, 3 x 105CFU / mL, 3 x 106CFU / mL, 3 x 107CFU / mL, 3 x 108CFU / mL, 3 x 109CFU / mL, or 3 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 4 x 102CFU / mL, 4 x 103CFU / mL, 4 x 104CFU / mL, 4 x 105CFU / mL, 4 x 106CFU / mL, 4 x 107CFU / mL, 4 x 108CFU / mL, 4 x 109CFU / mL, or 4 x 1010CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 5 x 102CFU / mL, 5 x 103CFU / mL, 5 x 104CFU / mL, 5 x 105CFU / mL, 5 x 106CFU / mL, 5 x 107CFU / mL, 5 x 108CFU / mL, 5 x 109CFU / mL, or 5 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 6 x 102CFU / mL, 6 x 103CFU / mL, 6 x 104CFU / mL, 6 x 105CFU / mL, 6 x 106CFU / mL, 6 x 107CFU / mL, 6 x 108CFU / mL, 6 x 109CFU / mL, or 6 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 7 x 102CFU / mL, 7 x 103CFU / mL, 7 x 104CFU / mL, 7 x 105CFU / mL, 7 x 106CFU / mL, 7 x 107CFU / mL, 7 x 108CFU / mL, 7 x 109CFU / mL, or 7 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 8 x 102CFU / mL, 8 x 103CFU / mL, 8 x 104CFU / mL, 8 x 105CFU / mL, 8 x 106CFU / mL, 8 x 107CFU / mL, 8 x 108CFU / mL, 8 x 109CFU / mL, or 8 x IO10CFU / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 9 x 102CFU / mL, 9 x 103CFU / mL, 9 x 104CFU / mL, 9 x 105CFU / mL, 9 x 106CFU / mL, 9 x 107CFU / mL, 9 x 108CFU / mL, 9 x 109CFU / mL, or 9 x IO10CFU / mL.

[0130] In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 101to 9 x 1012Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 101to 8 x 1012Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 101to 1 x 1012Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 102to 1 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 103to 1 x 109Spores / mL. In some embodiments, the final concentration of the composition is about 102, 103, 104, 105, 106, 107, 108, or 109spores / ml which can be reached by serial dilutions in sterile water or in an appropriate solution or buffer. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 1 x 102Spores / mL, 1 x 103Spores / mL, 1 x 104Spores / mL, 1 x 105Spores / mL, 1 x 106Spores / mL, 1 x 107Spores / mL, 1 x 108Spores / mL, 1 x 109Spores / mL, or 1 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 2 x 102Spores / mL, 2 x 103Spores / mL, 2 x 104Spores / mL, 2 x 105Spores / mL, 2 x 106Spores / mL, 2 x 107Spores / mL, 2 x 108Spores / mL, 2 x 109Spores / mL, or2 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 3 x 102Spores / mL, 3 x 103Spores / mL, 3 x 104Spores / mL,3 x 105Spores / mL, 3 x 106Spores / mL, 3 x 107Spores / mL, 3 x 108Spores / mL, 3 x 109Spores / mL, or 3 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is presentin the composition in a concentration of 4 x 102Spores / mL, 4 x 103Spores / mL, 4 x 104Spores / mL, 4 x 105Spores / mL, 4 x 106Spores / mL, 4 x 107Spores / mL, 4 x 108Spores / mL, 4 x 109Spores / mL, or 4 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 5 x 102Spores / mL, 5 x 103Spores / mL, 5 x 104Spores / mL, 5 x 105Spores / mL, 5 x 106Spores / mL, 5 x 107Spores / mL, 5 x 108Spores / mL, 5 x 109Spores / mL, or 5 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 6 x 102Spores / mL, 6 x 103Spores / mL, 6 x 104Spores / mL, 6 x 105Spores / mL, 6 x 106Spores / mL, 6 x 107Spores / mL, 6 x 108Spores / mL, 6 x 109Spores / mL, or 6 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 7 x 102Spores / mL, 7 x 103Spores / mL, 7 x 104Spores / mL, 7 x 105Spores / mL, 7 x 106Spores / mL, 7 x 107Spores / mL, 7 x 108Spores / mL, 7 x 109Spores / mL, or 7 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 8 x 102Spores / mL, 8 x 103Spores / mL, 8 x 104Spores / mL, 8 x 105Spores / mL, 8 x 106Spores / mL, 8 x 107Spores / mL, 8 x 108Spores / mL, 8 x 109Spores / mL, or 8 x IO10Spores / mL. In an embodiment, the strain of P. megaterium is present in the composition in a concentration of 9 x 102Spores / mL, 9 x 103Spores / mL, 9 x 104Spores / mL, 9 x 105Spores / mL, 9 x 106Spores / mL, 9 x 107Spores / mL, 9 x 108Spores / mL, 9 x 109Spores / mL, or 9 x IO10Spores / mL.In some embodiments, the composition comprises vegetive cells of the microorganism of the strain P. megaterium. In some embodiments, the composition comprises spores of the microorganism of the strain P. megaterium. In some embodiments, the composition comprises a combination of vegetative cells and spores of the microorganism of the strain P. megaterium. In some embodiments, the composition comprises a ratio of vegetative cells to spores from 25:75 to 0: 100. In some embodiments, the ratio of vegetative cells to spores in the composition is 75:25 to 0: 100. In some embodiments, the ratio of vegetative cells to spores in the composition can include any known combination of vegetative cells to spores. In some embodiments, the ratio of vegetative cells to spores in the composition is 10: 10. In some embodiments, the ratio of vegetative cells to spores in the composition is 10: 20. In some embodiments, the ratio of vegetative cells to spores in the composition is 10: 25. In some embodiments, the ratio of vegetative cells to spores in the composition is 10: 30. In some embodiments, the ratio of vegetative cells to spores in the composition is 10: 35. In some embodiments, the ratio of vegetative cells to spores in the composition is 10: 40. Insome embodiments, the ratio of vegetative cells to spores in the composition is 10: 45. In some embodiments, the ratio of vegetative cells to spores in the composition is 50: 50. In some embodiments, the ratio of vegetative cells to spores in the composition is 40: 60. In some embodiments, the ratio of vegetative cells to spores in the composition is 30: 70. In some embodiments, the ratio of vegetative cells to spores in the composition is 20: 80. In some embodiments, the ratio of vegetative cells to spores in the composition is 10: 90. In some embodiments, the ratio of vegetative cells to spores in the composition is 90: 10. In some embodiments, the ratio of vegetative cells to spores in the composition is 80: 20. In some embodiments, the ratio of vegetative cells to spores in the composition is 70: 30. In some embodiments, the ratio of vegetative cells to spores in the composition is 60: 40. In some embodiments, the ratio of vegetative cells to spores in the composition is 40: 70. In some embodiments, the ratio of vegetative cells to spores in the composition is 30: 20. In some embodiments, the ratio of vegetative cells to spores in the composition is 20: 80.6.1.3. Agriculturally compatible carrier

[0131] In some embodiments, the agriculturally compatible carrier is intended to aid in the application of the composition for the microorganism comprised therein to exert its plant growth-promoting effects properly.

[0132] In some embodiments, the agriculturally compatible carrier will depend on the form adopted by the composition and the manner intended for its application to the plant. In some embodiments, the agriculturally compatible carrier comprises, among others, surfactants, emulsifiers, stabilizers, anti-foam agents, carriers, pH adjusting agents, etc.

[0133] In some embodiments, the agriculturally compatible carrier comprises one or more of: xanthan gum, polyvinylpyrrolidone (PVP), carboxymethylcellulose, sodium acetate, and potassium silicate. In some embodiments, the agriculturally compatible carrier comprises one or more polymers. In some embodiments, the one or more polymers is xanthan gum. In some embodiments, the agriculturally compatible carrier comprises polyvinylpyrrolidone. In some embodiments, the agriculturally compatible carrier comprises carboxymethylcellulose. In some embodiments, the agriculturally compatible carrier comprises sodium acetate. In some embodiments, the agriculturally compatible carrier comprises potassium silicate.

[0134] In some embodiments, the agriculturally compatible carrier comprises one or more of: polyvinylpyrrolidone (PVP), gum arabic, and xanthan gum. In some embodiments,the agriculturally compatible carrier comprises xanthan gum. In some embodiments, xanthan gum is present in the composition in a concentration of at least 0.1% w / v, at least 0.2% w / v, at least 0.3% w / v, at least 0.4% w / v, at least 0.5% w / v, at least 0.6% w / v, at least 0.7% w / v, at least 0.8% w / v, at least 0.9% w / v, or at least 1% w / v. In some embodiments, xanthan gum is present in the composition in an amount ranging from 5 to 40 g / L, 5 to 30 g / L, 5 to 20 g / L, 10 to 20 g / L, or 15 to 20 g / L. In some embodiments, polyvinylpyrrolidone is present in an amount ranging from 5 to 40 g / L, 5 to 30 g / L, 5 to 20 g / L, 10 to 20 g / L, or 15 to 20 g / L. In some embodiments, carboxymethylcellulose is present in the composition in an amount ranging from 5 to 40 g / L, 5 to 30 g / L, 5 to 20 g / L, 10 to 20 g / L, or 15 to 20 g / L. In some embodiments, sodium acetate is present in the composition in an amount ranging from 5 to 40 g / L, 5 to 30 g / L, 5 to 20 g / L, 10 to 20 g / L, or 15 to 20 g / L. In some embodiments, potassium silicate is present in the composition in an amount ranging from 5 to 40 g / L, 5 to 30 g / L, 5 to 20 g / L, 10 to 20 g / L, or 15 to 20 g / L.

[0135] In some embodiments, when formulated as a biological formulation, the composition may further comprise additional agriculturally compatible carriers, to facilitate the stability and application thereof. For instance, in some embodiments, the composition comprise stabilizers, surfactants, emulsifiers, anti-foam agents, and the like.

[0136] In some embodiments, the carrier comprises a liquid carrier. In some embodiments, the carrier comprises a solid carrier.6.1.4. Culture Media composition

[0137] The composition of the present disclosure further comprises a culture media composition.

[0138] The medium used for culturing the microorganism comprised within the composition may be any medium suitable for promoting a proper increase in the biomass thereof, and may vary according to the selected microorganism and the presentation form thereof. For instance, the strain of P. megaterium may be present mainly in sporulated or vegetative forms within the formulation. To favor the growth of each form of the strain, different media should be applied.

[0139] In some embodiments, when the strain of P. megaterium is mainly in sporulated form, the carrier comprises a culture medium composition selected from one or more of: pea serum and S23. Pea serum is a by-product of the food industry, and may be readily dissolved in water, for instance, at concentrations of 15 g / L or 30 g / L, for preparing culture media formicroorganisms. Medium S23 is a proprietary medium optimized for promoting sporulation of P. megaterium, comprising 0.5 g / L MgSO4, 0.3 g / L FeC13, and 1 g / L K2HPO4, and it is supplemented with a nutritious broth, such as Tryptic Soy Broth (TSB) or similar.

[0140] In some embodiments, when the strain of P. megaterium is mainly in vegetative form, the carrier comprises a nutritious culture medium without the addition of a specialized medium such as S23. The nutritious medium used for this scenario may be, for instance, TSB, nutrient broth or a similar medium.

[0141] In some embodiments, the composition further comprises a carrier comprising a culture medium selected from the group consisting of TSB, nutrient broth, pea serum, S23, and combinations thereof. In some embodiments, the composition is in liquid form. In some embodiments, the composition is in solid form.

[0142] In some embodiments, the culture medium composition selected from one or more of: glucose, yeast extract, meat extract, calcium chloride, magnesium sulfate, potassium chloride, iron chloride, sodium chloride, phosphate salts, ammonium sulfate, pea whey, soybean peptone, manganese chloride, chitin, sucrose, ferric chloride, dipotassium phosphate, stachyose, dihydrate, casein, soybean, hydrogen phosphate, glucose monohydrate, Calcium Chloride Dihydrate, pluripeptone, Glucose Monohydrate, Magnesium sulfate heptahydrate, Iron (III) chloride hexahydrate, Potassium hydrogen phosphate, Manganese(II) chloride tetrahydrate and peptone.

[0143] In some embodiments, the culture media composition (e.g., culture medium) comprises one or more of: glucose (dextrose), yeast extract, meat extract, calcium chloride, magnesium sulfate, potassium chloride, iron chloride, sodium chloride, phosphate salts, ammonium sulfate, pea whey, soybean peptone, manganese chloride, chitin, sucrose, ferric chloride, dipotassium phosphate, stachyose, enzymatic digest of casein, enzymatic digest of soybean, Calcium Chloride Dihydrate, pluripeptone, Magnesium sulfate heptahydrate, Iron (III) chloride hexahydrate, Dipotassium hydrogen phosphate, Monopotassium phosphate, Manganese(II) chloride tetrahydrate, and peptone, Tryptone, Magnesium sulfate anhydrous, beef peptone, starch, maltodextrin, glycerol, Disodium phosphate, sodium nitrate, zinc sulfate heptahydrate, zinc chloride, Calcium phosphate tetrahydrate, potassium nitrate, disodium hydrogen phosphate dodecahydrate, ammonium chloride, ferric ammonium citrate, potassium nitrate, Ethylenediaminetetraacetic acid, Zinc sulfate, Cobalt (II) chloride hexahydrate.

[0144] In some embodiments, the culture media comprises calcium chloride. In some embodiments, the culture media comprises magnesium sulfate. In some embodiments, the culture media comprises potassium chloride. In some embodiments, the culture media comprises Iron (III) chloride. In some embodiments, the culture media comprises sodium chloride. In some embodiments, the culture media comprises phosphate salts. In some embodiments, the culture media comprises ammonium sulfate. In some embodiments, the culture media comprises ammonium sulfate. In some embodiments, the culture media comprises one or more polymers. In some embodiments, the culture media comprises one or more of: calcium chloride, magnesium sulfate, potassium chloride, Iron (III) chloride, sodium chloride, phosphate salts, ammonium sulfate, and

[0145] In some embodiments, the culture media comprises one or more of: a carbon source, a nitrogen source, a phosphorous source, a sulfur source, magnesium, trace elements, vitamins and growth factors,

[0146] In some embodiments, the culture media comprises one or more of: glucose (dextrose), sucrose, stachyose, glycerol, and maltodextrin. In some embodiments, the culture media comprises one or more of: amino acids, nucleic acids, and proteins. In some embodiments , the culture media comprises ammonium salts and / or nitrates. In some embodiments, the culture media comprises one or more of: ammonium sulfate, ammonium nitrate, potassium nitrate, and sodium nitrate. In some embodiments, the culture media comprises monopotassium phosphate. In some embodiments, the culture media comprises dipotassium phosphate. In some embodiments, the culture media comprises magnesium sulfate. In some embodiments, the culture media comprises calcium chloride. In some embodiments, the culture media comprises one or more of: iron, zinc, and cobalt. In some embodiments, the culture media comprises thiamine and / or riboflavin.

[0147] In some embodiments, the culture media composition comprises one or more culture mediums provided in Table 1 :Table 1: list of culture media components at various concentrations

[0148] In some embodiments, the culture media composition is selected from any one of the culture media compositions of Table 23. In some embodiments, the culture mediacomposition is N°media 1 of Table 23. In some embodiments, the culture media composition is N°media 2 of Table 23. In some embodiments, the culture media composition is N°media 3 of Table 23. In some embodiments, the culture media composition is N°media 4 of Table 23. In some embodiments, the culture media composition is N°media 5 of Table 23. In some embodiments, the culture media composition is N°media 6 of Table 23. In some embodiments, the culture media composition is N°media 7 of Table 23. In some embodiments, the culture media composition is N°media 8 of Table 23. In some embodiments, the culture media composition is N°media 9 of Table 23. In some embodiments, the culture media composition is N°media 10 of Table 23. In some embodiments, the culture media composition is N°media 11 of Table 23. In some embodiments, the culture media composition is N°media 12 of Table 23. In some embodiments, the culture media composition is N°media 13 of Table 23. In some embodiments, the culture media composition is N°media 14 of Table 23. In some embodiments, the culture media composition is N°media 15 of Table 23. In some embodiments, the culture media composition is N°media 16 of Table 23. In some embodiments, the culture media composition is N°media 17 of Table 23. In some embodiments, the culture media composition is N°media 18 of Table 23. In some embodiments, the culture media composition is N°media 19 of Table 23. In some embodiments, the culture media composition is N°media 20 of Table 23.

[0149] In some embodiments, the culture media composition is selected from any one of the culture media compositions of Table 24. In some embodiments, the culture media composition is N°media 1 of Table 24. In some embodiments, the culture media composition is N°media 2 of Table 24. In some embodiments, the culture media composition is N°media 3 of Table 24. In some embodiments, the culture media composition is N°media 4 of Table 24. In some embodiments, the culture media composition is N°media 5 of Table 24. In some embodiments, the culture media composition is N°media 6 of Table 24. In some embodiments, the culture media composition is N°media 7 of Table 24. In some embodiments, the culture media composition is N°media 8 of Table 24. In some embodiments, the culture media composition is N°media 9 of Table 24. In some embodiments, the culture media composition is N°media 10 of Table 24. In some embodiments, the culture media composition is N°media 11 of Table 24. In some embodiments, the culture media composition is N°media 12 of Table 24. In some embodiments, the culture media composition is N°media 13 of Table 24. In someembodiments, the culture media composition is N°media 14 of Table 24. In some embodiments, the culture media composition is N°media 15 of Table 24. In some embodiments, the culture media composition is N°media 16 of Table 24. In some embodiments, the culture media composition is N°media 17 of Table 24. In some embodiments, the culture media composition is N°media 18 of Table 24. In some embodiments, the culture media composition is N°media 19 of Table 24. In some embodiments, the culture media composition is N°media 20 of Table 24. In some embodiments, the culture media composition is N°media 21 of Table 24. In some embodiments, the culture media composition is N°media 22 of Table 24. In some embodiments, the culture media composition is N°media 23 of Table 24. In some embodiments, the culture media composition is N°media 24 of Table 24. In some embodiments, the culture media composition is N°media 25 of Table 24. In some embodiments, the culture media composition is N°media 26 of Table 24. In some embodiments, the culture media composition is N°media 27 of Table 24. In some embodiments, the culture media composition is N°media 28 of Table 24. In some embodiments, the culture media composition is N°media 29 of Table 24. In some embodiments, the culture media composition is N°media 30 of Table 24. In some embodiments, the culture media composition is N°media 31 of Table 24. In some embodiments, the culture media composition is N°media 32 of Table 24. In some embodiments, the culture media composition is N°media 33 of Table 24. In some embodiments, the culture media composition is N°media 34 of Table 24. In some embodiments, the culture media composition is N°media 35 of Table 24. In some embodiments, the culture media composition is N°media 36 of Table 24. In some embodiments, the culture media composition is N°media 37 of Table 24. In some embodiments, the culture media composition is N°media 38 of Table 24. In some embodiments, the culture media composition is N°media 39 of Table 24. In some embodiments, the culture media composition is N°media 40 of Table 24. In some embodiments, the culture media composition is N°media 41 of Table 24. In some embodiments, the culture media composition is N°media 42 of Table 24. In some embodiments, the culture media composition is N°media 43 of Table 24. In some embodiments, the culture media composition is N°media 44 of Table 24. In some embodiments, the culture media composition is N°media 45 of Table 24. In some embodiments, the culture media composition is N°media 46 of Table 24. In someembodiments, the culture media composition is N°media 47 of Table 24. In some embodiments, the culture media composition is N°media 48 of Table 24. In some embodiments, the culture media composition is N°media 49 of Table 24. In some embodiments, the culture media composition is N°media 50 of Table 24. In some embodiments, the culture media composition is N°media 51 of Table 24. In some embodiments, the culture media composition is N°media 52 of Table 24. In some embodiments, the culture media composition is N°media 53 of Table 24. In some embodiments, the culture media composition is N°media 54 of Table 24. In some embodiments, the culture media composition is N°media 55 of Table 24. In some embodiments, the culture media composition is N°media 56 of Table 24. In some embodiments, the culture media composition is N°media 57 of Table 24. In some embodiments, the culture media composition is N°media 58 of Table 24. In some embodiments, the culture media composition is N°media 59 of Table 24. In some embodiments, the culture media composition is N°media 60 of Table 24.

[0150] In some embodiments, the culture media composition is N°media 61 of Table 24. In some embodiments, the culture media composition is N°media 62 of Table 24. In some embodiments, the culture media composition is N°media 63 of Table 24. In some embodiments, the culture media composition is N°media 64 of Table 24. In some embodiments, the culture media composition is N°media 65 of Table 24. In some embodiments, the culture media composition is N°media 66 of Table 24. In some embodiments, the culture media composition is N°media 67 of Table 24. In some embodiments, the culture media composition is N°media 68 of Table 24. In some embodiments, the culture media composition is N°media 69 of Table 24. In some embodiments, the culture media composition is N°media 70 of Table 24. In some embodiments, the culture media composition is N°media 71 of Table 24. In some embodiments, the culture media composition is N°media 72 of Table 24. In some embodiments, the culture media composition is N°media 73 of Table 24. In some embodiments, the culture media composition is N°media 74 of Table 24. In some embodiments, the culture media composition is N°media 75 of Table 24. In some embodiments, the culture media composition is N°media 76 of Table 24. In some embodiments, the culture media composition is N°media 77 of Table 24. In some embodiments, the culture media composition is N°media 78 of Table 24. In some embodiments, the culture media composition is N°media 79 of Table 24. In someembodiments, the culture media composition is N°media 80 of Table 24. In some embodiments, the culture media composition is N°media 81 of Table 24. In some embodiments, the culture media composition is N°media 82 of Table 24. In some embodiments, the culture media composition is N°media 83 of Table 24. In some embodiments, the culture media composition is N°media 84 of Table 24. In some embodiments, the culture media composition is N°media 85 of Table 24. In some embodiments, the culture media composition is N°media 86 of Table 24. In some embodiments, the culture media composition is N°media 87 of Table 24. In some embodiments, the culture media composition is N°media 88 of Table 24. In some embodiments, the culture media composition is N°media 89 of Table 24. In some embodiments, the culture media composition is N°media 90 of Table 24. In some embodiments, the culture media composition is N°media 91 of Table 24. In some embodiments, the culture media composition is N°media 92 of Table 24.

[0151] In some embodiments, the culture media composition is selected from any one of the culture media compositions of Table 31. In some embodiments, the culture media composition is N°media 1 of Table 31. In some embodiments, the culture media composition is N°media 2 of Table 31. In some embodiments, the culture media composition is N°media 3 of Table 31. In some embodiments, the culture media composition is N°media 4 of Table 31.

[0152] In some embodiments, the culture media composition is selected from any one of the culture media compositions of Table 32. In some embodiments, the culture media composition is N°media 1 of Table 32. In some embodiments, the culture media composition is N°media 2 of Table 32.

[0153] In some embodiments, the culture media composition further comprises water. In some embodiments, the culture media composition is an aqueous solution.

[0154] In some embodiments, the composition is intended to be applied by inoculating seeds of a plant with it, so that the seeds are dressed, coated, or pelleted by the composition. This kind of compositions are also known as “inoculant”, “plant inoculant”, or “bioinoculant” due to their bio-based nature. The composition may thus be referred to by using any of these terms interchangeably. To function as an inoculant, the composition comprises a component which allows for a proper coating, dressing, or pelleting of the seeds once the inoculation has taken place. The term “component for seed inoculation” is thus to be understood as referring to a component which generates a seed dressing, seed coating, or seed pelleting of thecomposition on the seeds upon which the composition has been inoculated, for the microorganism to properly exert its plant growth-promoting activity. In some embodiments, the composition is formulated as an inoculant, and the at least one agriculturally compatible carrier comprises a component for seed inoculation. For example, for seed dressing, the seed is either dressed with a dry formulation or wet treated with a slurry or liquid formulation. Another example includes seed coating, which is a special binder is used with a formulation to enhance adherence to the seed and begin to impact seed size and shape. Another example includes seed pelleting, which may result in changing the physical shape of a seed to enhance plantability and handling.

[0155] When the composition of the invention is liquid it may be prepared by culturing the selected microorganism in an appropriate culture medium such as those disclosed above for a sufficient time period to reach the desired concentration (usually measured in CFU / mL), and then adding any necessary additional agronomically acceptable component prior to packaging of the final composition. For instance, when the composition is formulated as an biological formulation, it may be prepared by culturing the selected microorganism in an appropriate culture medium for a sufficient time period to reach the desired concentration, and then adding the component for seed inoculation prior to packaging of the final composition. It is within the knowledge expected for a person of skill in the art to optimize the conditions for culturing the microorganisms according to the selected microorganism and the available resources and experimental setup.

[0156] In some embodiments, when the composition is a solid, after the culture of the microorganism described above, the microorganism may be isolated by techniques known in the art, and then formulated as needed according to the desired form for the composition.6.1.4.1 Other components of the composition

[0157] In some embodiments, the synthetic composition further comprises one or more chemicals used in an agricultural field for which the plant element is grown. In some embodiments, the synthetic composition further comprises one or more of: insecticides, fungicides, fertilizers, nematicide, herbicide, adjuvant surfactant, wetting agent, moistening and dispersing adjuvant, surfactant, or pH corrector.

[0158] In some embodiments, wherein the synthetic composition further comprises one or more of: alginic acid, carrageenan, dextrin, dextran, polyethylene glycol, polyvinyl pyrrolidone, methyl cellulose, polyvinyl alcohol, gelatin, a detergent,

[0159] In some embodiments, the composition further comprises an insecticide, a fungicide, and combinations thereof.

[0160] In some embodiments, the composition further composes one or more of: alginic acid, carrageenan, polyethylene glycol, polyvinyl pyrrolidone, methyl cellulose, sodium acetate, potassium silicate, polyvinyl alcohol, gelatin, a detergent, an insecticide, a fungicide, nematicide, and combinations thereof

[0161] In some embodiments, the synthetic composition further comprises one or more active ingredients selected from: Thiam ethoxam, Pyrethroid, Fenamiphos: (RS)-(ethyl-4- methylthio-m-m-tolyl isopropylphosphoramidate), Difenoconazole, Fludioxonil, Thiamethoxam, Difenoconazole 2.5%, Fludioxonil 2.5%, Sedaxane 5%, Metalaxyl 0.62% Difenoconazole 3.69% + Iprodione 6.25% Imidacloprid 12% w / v FS, Imidacloprid, Tebuconazole, Fluxapyroxad (Carboxamide), Fludioxonil, Metalaxyl M, Thiram, Metribuzin, l-Chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine, 2- [(phosphonomethyl) amino] acetic acid isopropylamine salt of N(-phosphonom ethyl) glycine, 2,4-Dichlorophenoxyacetic acid, Glufosinate ammonium (ammonium-(-3-amino-3 -carboxypropyl methyl phosphinate), Methylated soybean oil (MSO) and organosilicones, Monoamine ethoxylated fatty alcohol (AGE). Nonyl phenol free, Trixiloxane and copolymers. Nonyl phenol free, Nonyl phenol ethoxylate, Surfactant (Linear ethoxylated alcohol), and Polyoxymethylene nonionic surfactant.

[0162] In some embodiments, the synthetic composition further comprises one or more of: Rotam, Bifenthryn, Nemacur, Crusier plus, Compiche SX, Tregua, Philduo, Sistiva, Progus, Thiram, Sencormax, Atrazine, Glyphosate, 2,4-D 60, Liberty, Biofilm, Eco- rizospray, Silwet, tensiowett, X-trim, Tween 20, and pH corrector (3-4).

[0163] In some embodiments, the synthetic composition further comprises one or more chemicals listed in Table 22.

[0164] In some embodiments, the synthetic composition further comprises a microorganism of the species Azospirillum brasilense.

[0165] In some embodiments, the synthetic composition comprises an antibiotic. In some embodiments, the antibiotic is selected from: penicillin, amoxicillin, amoxicillin + clavulanic acid, Ampicillin, Piperacillin, Ceftriaxone, cefotaxime, Erythromycin, Azithromycin, Gentamicin, Streptomycin, Amikacin, Kanamicyn, Ciprofloxacin, Levofloxacin, Nalidixic Acid, Imipenem, Meropenem, Clindamycin, Chloramphenicol, Rifampicin, Tetracycline, andVancomycin. In some embodiments, the synthetic composition comprises an antibiotic selected from any one of the antibiotics listed in Table 21.6.1.4.2 Growth-promoting traits / properties

[0166] In some embodiments, the microorganism of the species P. megaterium, when contacted with a plant element comprising: a plant seed, a plant or plant portion thereof, is capable of improving a plant growth-promoting trait compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when disposed via in-furrow application of soil surrounding a plant element or via foliar application of the plant element, is capable of improving a plant growth-promoting trait compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium.

[0167] In some embodiments, the plant element is a plant seed, a plant or plant portion thereof. In some embodiments, the plant or portion thereof is selected from: plant leaves, plant roots (e.g., primary root, root hairs, lateral root, root cap, etc.), plant stems, and plant shoots (e.g., leaf blade, stem, petiole, leaf axil, flower, terminal bud, lateral bud etc.). In some embodiments, the plant element is a plant root. In some embodiments, the plant root comprises root exudates.

[0168] In some embodiments, the plant or plant portion thereof is a vegetable plant or plant portion thereof. In some embodiments, the plant or plant portion thereof is a fruit plant or plant portion thereof. In some embodiments, the plant or plant portion thereof is a herb plant or plant portion thereof. In some embodiments, the plant or plant portion thereof is a grain. In some embodiments, the plant or plant portion thereof is grass. In some embodiments, the plant or plant portion thereof is canola. In some embodiments, the plant or plant portion thereof is a legume plant or plant portion thereof. In some embodiments, the plant or plant portion thereof is selected from one or more of: wheat, potato, corn, barley, lettuce, peanut, and tomato.

[0169] In some embodiments, the plant element is any agronomically relevant plant. In some embodiments, the plant is com (Zea mays), wheat (Triticum spp.), soybean (Glycine max), barley (Hordeum vulgare), sugarcane (Saccharum officinarum), cotton, chickpea, rice, drybean, among others.

[0170] In some embodiments, composition comprising the microorganism of the species P. megaterium is an endophytic, sporulating bacterium capable of efficiently colonizing different plants.

[0171] In some embodiments, the microorganism of the species P. megaterium produces a biofilm, when the microorganism is contacted with or disposed onto a plant element.

[0172] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, is capable of modifying the flavonoid profile of the plant element such as root exudates. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provide an increase in flavonoids selected from one or more of: malic acid, mannitol, citric acid, tryptophan, daidzein, chrysin, apigenin, naringenin, luteolin, genistein, naringin, and naringin in its aglycone form. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in flavonoid production compared to a reference plant element that is untreated or not in contact with the synthetic composition, “untreated” as used herein, can refer to a reference plant element that has not been in contact with or treated with a composition of the present disclosure comprising the microorganism of the species P. megaterium.

[0173] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, produces siderophores into the culture media composition. In some embodiments, by producing siderophores, the microorganism enhanced the plant element’s iron (Fe) nutrition. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in siderophore production compared to a reference plant element that is untreated with the synthetic composition not contacted or inoculated or applied with the microorganism.

[0174] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, produces extracellular chitinases into the culture media composition.

[0175] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, produces auxins such as AIA. In some embodiments, AIA is produced in an amount of about 20 ug / ml, about 25 ug / ml, about 30 ug / ml, about 35 ug / ml, about 40 ug / ml, about 45 ug / ml, about 50 ug / ml, about 55 ug / ml, about 60 ug / ml, about 65 ug / ml, or about 70 ug / ml. In some embodiments, the AIA is produce in an amount of 20 ug / ml or more, about 25 ug / ml or more, about 30 ug / ml or more, about 35 ug / ml or more, about 40 ug / ml ormore, about 45 ug / ml or more, about 50 ug / ml or more, about 55 ug / ml or more, about 60 ug / ml or more, about 65 ug / ml or more, or about 70 ug / ml or more.

[0176] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, produces phytohormones. In some embodiments the phytohormones are selected from one or more of: indole acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), jasmonic acid (JA), zeatin (ZEA), 6-benzylaminopurine (6- BAP), kinetin (KIN), and indole-3 -butyric acid (IBA). In some embodiments, the phytohormone is indole acetic acid (IAA). In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in phytohormone production compared to a reference plant element that is untreated with the synthetic composition not contacted or inoculated or applied with the microorganism.

[0177] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in phosphate solubilization compared to a reference plant element that is untreated (e.g., not contacted with or inoculated with or applied with or disposed onto a reference plant element) with the synthetic composition In some embodiments, the microorganism of the composition comprises a phosphate solubilizing index (PSI) ranging from 3-6. In some embodiments, the microorganism comprises a phosphate solubilizing index (PSI) of about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, or about 4.9.

[0178] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in potassium solubilization compared to a reference plant element that is untreated with the synthetic composition not contacted or inoculated or applied with the microorganism.

[0179] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides increased degradation of nitrogenous organic matter in the soil by which the plant element is growing and converting the nitrogenous organic matter into ammonium compared to a reference plant element not treated with the composition comprising the microorganism of the species P. megaterium. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides increased production of ammonium compared to a reference plant element not treated with the composition comprising the microorganism of the species P. megaterium. In some embodiments, ammonium production is increased by at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0180] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides increased hydrolysis of urea compared to a reference plant element not treated with the composition comprising the microorganism of the species P. megaterium. Nitrogen fixation (ARA)

[0181] In some embodiments, the plant growth-promoting traits is selected from: increased germination rate, increased emergence rate, increased shoot biomass, increased seedling root length, increased seedling shoot length, increased seedling mass, increased root surface area, increased enhanced nutrient use efficiency, increased phosphate solubilization, and increased yield. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing the germination rate of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing the emergence rate of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing shoot biomass of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing seedling root length and / or seedling mass of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing root surface area of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing yield or growth of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium.

[0182] In some embodiments, the one or more growth promoting properties or traits is selected from: increasing one or more of: enhanced resistance or tolerance to drought stress, enhanced resistance to thermal stress or thermal shock, enhanced resistance to UV radiation, enhanced resistance or tolerance to different NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to stress, increased nutrient uptake, increased root hair formation, increased root branching, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof, e.g., compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium or not treated with the composition comprising the microorganism of the species P. megaterium.6.1.5. Contacted with or disposed onto plant element

[0183] The composition may be formulated in different forms able for its application to a plant whose growth is to be promoted or formulated for the surrounding environment at which the plant is grown. Correspondingly, the formulation may be either liquid or solid, and it may be formulated in any form known to be of use in the art, such as liquid suspensions, seed treatments and coatings, foliar sprays, freeze-dried reconstitutable formulations, and solid forms, among others.

[0184] In some embodiments, the composition is formulated as a biological formulation, and the microorganism is contacted with or disposed onto the plant element via one or more of: broadcast application, liquid or dry in-furrow application, spray application, irrigation, injection, dusting, dressing, pelleting, or coating of the plant element. In some embodiments, soil drench, such as in furrow, application may be performed by applying the microorganism composition to the surface of the soil and / or seed during planting. In some embodiments, the composition may comprise an microorganism suspension or an microorganism dry powder formulation. In various embodiments the microorganism may comprise vegetative cells and / or spores. In particular embodiments, the soil drench application may comprise applying the microorganism composition to the surface of the soil directly above each seed. In certainembodiments, the microorganism composition may comprise 0.01-0.1, 0.1-1, or 1-10 ml microorganism suspension, which may be a microorganism spore suspension.

[0185] In some embodiments, the composition is formulated in liquid form. In some embodiments, the formulation in liquid form is a suspension or solution. In some embodiments, the liquid form is a homogenous solution. In some embodiments, the liquid form is a heterologous suspension. In some embodiments, the composition is formulated in solid form. In some embodiments, the agriculturally compatible carrier comprises a suitable carrier, which should dissolve the component for contact with a plant element while maintaining the microorganism in a stable suspension. In some embodiments, the suitable carrier is a solid suitable carrier. In some embodiments, the liquid or solid form can comprise a medium used for culture and growth of the microorganism during the production of the composition, which may afterwards be diluted if necessary to achieve the desired concentration of the microorganism in the composition.

[0186] In some embodiments, the composition is formulated as a biological formulation such as a bioinoculant, and it is applied at a dose ranging from 1 mL / kg to 50 ml / kg. In some embodiments, the biological formulation is applied at dose of 1 ml / kg, 2 ml / kg, 3 ml / kg, 4 ml / kg, 5 ng / kg, 6 mL / kg, 7 ml / kg, 8 ml / kg, 9 ml / kg, 10 ml / kg, 11 ml / kg, 12 ml / kg, 13 ml / kg, 14 ml / kg, 15 ml / kg, 16 ml / kg, 17 ml / kg, 18 mL / kg, 19 ml / kg, or 20 ml / kg. In some embodiments, the composition is applied at a dose of at least 5 ml / kg , at least 10 ml / kg , at least 15 ml / kg , at least 20 ml / kg , at least 25 ml / kg , at least 30 ml / kg , at least 35 ml / kg , at least 40 ml / kg , at least 45 ml / kg , or at least 50 ml / kg.6.2. Methods

[0187] Aspects of the present disclosure comprise methods of improving a trait in a plant or promoting growth of a plant by contacting a plant element comprising a seed or a plant or plant portion thereof with the synthetic composition described herein.

[0188] Aspects of the present disclosure comprise methods of improving a trait in a plant or promoting growth of a plant by applying the synthetic composition described herein as a biofertilizer on the soil from which the plant element is growth.

[0189] Aspects of the present disclosure comprise methods of increasing germination rate in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing emergence rate in a plant by contacting a plant element with the synthetic composition described herein. Aspects of thepresent disclosure comprise methods of increasing shoot biomass in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing seedling root length in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing seedling mass in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing root surface area in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhancing nutrient use efficiency in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing phosphate solubilization in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing yield in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing growth in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing phytohormones produced by the microorganism by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhanced resistance or tolerance to drought stress in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhancing resistance or tolerance to thermal shock in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhancing resistance or tolerance to difference NaCl concentrations in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhancing resistance or tolerance to antibiotics by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhancing resistance or tolerance to stress (e.g., water stress, heat stress, cold stress, etc.) by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing nutrient uptake in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of increasing rot hair formation in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods ofincreasing root branching in a plant by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhancing resistance or tolerance to one or more of: salt toxicity, aluminum toxicity, fungal infection, bacterial infection, viral infection, or a combination thereof, by contacting a plant element with the synthetic composition described herein. Aspects of the present disclosure comprise methods of enhancing resistance or tolerance to UV radiation by contacting a plant element with the synthetic composition described herein.

[0190] Aspects of the present disclosure comprise a method for improving soil quality, comprising applying the composition described herein in the soil.

[0191] By such “increase”, “improvement”, or “enhancement”, the plant element shows such increased properties compared to a reference plant element not treated with the composition or microorganism within the composition described herein.6.2.1. Mode of Application

[0192] In some embodiments, contacting the plant element comprises applying the synthetic composition to the plant element via broadcast application, liquid or dry-in furrow application, spray application, irrigation, injection, dusting, pelleting, or coating of the plant element. In some embodiments, contacting the plant element comprises applying the synthetic composition to the plant element via heterologous or homogenous coating of the plant element.

[0193] In some embodiments, the microorganism, when applied via in-furrow application of soil surrounding a plant element or via foliar application of the plant element, is capable of: improving a plant growth-promoting trait compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium.

[0194] In some aspects, the methods of present disclosure further comprise germinating a plant seed and growing the plant. In some aspects, the methods of the present disclosure further comprise harvesting the plant.

[0195] In some embodiments, germinating the inoculated plant seeds and growing the plant, respectively, may be performed in an appropriate manner according to the selected plant by relying on the knowledge of the agriculturist carrying out the method.

[0196] By carrying out the method of this aspect of the methods, the treated plant experiences an improvement in its growth in comparison to an untreated plant. The growthimprovement may be ascertained by any measurement typically used in the art, such as biomass weight (either wet or dry), root development in seedlings, stem length, among others.

[0197] The improved growth of the treated plants generates an increase in the yield of a crop of said plants, wherein the increased yield of the crop is determined in comparison with an untreated crop.

[0198] As described herein, the manner in which the composition is applied depends on how it is formulated. In some embodiments, the composition is formulated as a biological formulation. In some embodiments, the biological formulation is a bioinoculant. In some embodiments, the biological formulation is a biofertilizer. In some embodiments, the biological formulation is a biostimulant. In some embodiments, the biological formulation is a soil amendment. In some embodiments, the biological formulation is a plant amendment. In some embodiments, when the composition is formulated as an inoculant, the application of the composition is carried out by inoculating seeds of the crop, and the method comprises an additional step of germinating the inoculated seeds.

[0199] In some embodiments, the biological formulation is a soil amendment. The term “soil amendment” should be understood as referring to a product which, upon application, modify soil properties to render it more suitable for plant growth. This effect may occur by different mechanisms, according to the manner in which the composition is applied. For instance, if the composition is formulated as an inoculant, the colonization of the roots of the plant upon germination of treated seed by the microorganism comprised within the composition has not only a beneficial effect on the plant itself, but also on the soil where the seeds were planted, thus generating a synergistic growth improvement in the plant, both due to the effects on the plant itself and on the soil. The improvement to the soil may be, for instance, due to the atmospheric nitrogen fixation capabilities of the microorganism comprised within the composition.

[0200] In some embodiments, the biological formulation is a plant amendment. Plant amendment is any substance applied to plants or seeds which is intended to improve germination, growth, yield, quality, reproduction, flavor or other desirable characteristics.

[0201] In an embodiment of this aspect of the invention, the composition is formulated as an inoculant, and the application of the composition to the soil is carried out by planting seeds previously inoculated with the composition, and germinating said seeds.6.2.1.1 Dosing

[0202] The composition may be formulated in different forms able for its application to a plant whose growth is to be promoted or formulated for the surrounding environment at which the plant is grown. Correspondingly, the formulation may be either liquid or solid, and it may be formulated in any form known to be of use in the art, such as liquid suspensions, seed treatments and coatings, foliar sprays, freeze-dried reconstitutable formulations, and solid forms, among others.

[0203] In some embodiments, the composition is formulated in liquid form. In some embodiments, the formulation in liquid form is a suspension or solution. In some embodiments, the liquid form is a homogenous solution. In some embodiments, the liquid form is a heterologous suspension. In some embodiments, the agriculturally compatible carrier comprises a suitable liquid carrier, which should dissolve the component for contact with a plant element while maintaining the microorganism in a stable suspension. In some embodiments, the liquid form can comprise a medium used for culture and growth of the microorganism during the production of the composition, which may afterwards be diluted if necessary to achieve the desired concentration of the microorganism in the composition.

[0204] In some embodiments, the composition is formulated as a biological formulation, and the microorganism is contacted with or disposed onto the plant element via one or more of: broadcast application, liquid or dry in-furrow application, spray application, irrigation, injection, dusting, pelleting, or coating of the plant element. In some embodiments, soil drench, such as in furrow, application may be performed by applying the microorganism composition to the surface of the soil and / or seed during planting.

[0205] In some embodiments, the composition may comprise a microorganism suspension or a microorganism dry powder formulation. In various embodiments the microorganism may comprise vegetative cells and / or spores. In particular embodiments, the soil drench application may comprise applying the microorganism composition to the surface of the soil directly above each seed. In certain embodiments, the microorganism composition may comprise 0.01-0.1, 0.1-1, or 1-10 ml microorganism suspension, which may be a microorganism spore suspension.

[0206] In some embodiments, the composition is formulated as a biological formulation such as a bioinoculant, and it is applied at a dose ranging from 1 mL / kg to 50 ml / kg. In some embodiments, the biological formulation is applied at dose of 1 ml / kg, 2 ml / kg, 3 ml / kg, 4ml / kg, 5 ng / kg, 6 mL / kg, 7 ml / kg, 8 ml / kg, 9 ml / kg, 10 ml / kg, 11 ml / kg, 12 ml / kg, 13 ml / kg, 14 ml / kg, 15 ml / kg, 16 ml / kg, 17 ml / kg, 18 mL / kg, 19 ml / kg, or 20 ml / kg. In some embodiments, the composition is applied at a dose of at least 5 ml / kg , at least 10 ml / kg , at least 15 ml / kg , at least 20 ml / kg , at least 25 ml / kg , at least 30 ml / kg , at least 35 ml / kg , at least 40 ml / kg , at least 45 ml / kg , or at least 50 ml / kg.

[0207] In some embodiments, the application of the composition is carried out in a dose such that the plant growth-promoting capabilities thereof may be fully taken advantage of, and it may depend on several factors, such as the intended application form of the composition, the plant whose growth is to be improved, the selected microorganism, environmental conditions, among others. For example, when the composition is formulated as an inoculant, the dose of the composition to be applied is usually expressed as volume of composition per weight of treated seeds.6.2.1.2 Growth promoting properties

[0208] Aspects of the present methods include contacting a plant element comprising a seed or a plant or plant portion thereof with the synthetic composition described herein. By contacting the plant element, the method results in an improvement or increase in a trait in a plant to promote growth of the plant.

[0209] In some embodiments, the microorganism is present in the synthetic composition in an amount effective to increase colonization of the plant element and to provide a benefit to the plant compared to a reference plant not treated with the microorganisms of the species P. megaterium.

[0210] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, is capable of modifying the flavonoid profile of the plant element such as root exudates. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provide an increase in flavonoids selected from one or more of: malic acid, mannitol, citric acid, tryptophan, daidzein, chrysin, apigenin, naringenin, luteolin, genistein, naringin, and naringin in its aglycone form. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in flavonoid production compared to a reference plant element that is untreated with the synthetic composition not contacted or inoculated or applied with the microorganism, “untreated” as used herein, can refer to a reference plant element that has not been in contactwith or treated with a composition of the present disclosure comprising the microorganism of the species P. megaterium.

[0211] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, produces siderophores into the culture media composition. In some embodiments, by producing siderophores, the microorganism enhanced the plant element’s iron (Fe) nutrition. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in siderophore production compared to a reference plant element that is untreated with (not contacted or inoculated or applied with) the synthetic composition comprising the microorganism.

[0212] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, produces extracellular chitinases into the culture media composition.

[0213] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, produces phytohormones. In some embodiments the phytohormones are selected from one or more of: indole acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), jasmonic acid (JA), zeatin (ZEA), 6-benzylaminopurine (6- BAP), kinetin (KIN), and indole-3 -butyric acid (IBA). In some embodiments, the phytohormone is indole acetic acid (IAA). In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in phytohormone production compared to a reference plant element that is untreated with the synthetic composition.

[0214] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in phosphate solubilization compared to a reference plant element that is untreated (e.g., not contacted with or disposed onto a reference plant element) with the synthetic composition not contacted or inoculated or applied with the microorganism. In some embodiments, the microorganism of the composition comprises a phosphate solubilizing index (PSI) ranging from 3-6. In some embodiments, the microorganism comprises a phosphate solubilizing index (PSI) of about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, or about 4.9. In some embodiments, the microorganism of the composition comprises a phosphate solubilizing index (PSI) ranging from 3-6. In some embodiments, the microorganism comprises a phosphate solubilizing index (PSI) of about 4, about 4.15, about 4.25, about 4.35, about 4.45, about 4.55, about 4.65, about 4.75, or about 4.95.

[0215] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides an increase in potassium solubilization compared to a reference plant element that is untreated with the synthetic composition not contacted or inoculated or applied with the microorganism.

[0216] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides increased degradation of nitrogenous organic matter in the soil by which the plant element is growing and converting the nitrogenous organic matter into ammonium compared to a reference plant element not treated with the composition comprising the microorganism of the species P. megaterium. In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides increased production of ammonium compared to a reference plant element not treated with the composition comprising the microorganism of the species P. megaterium. In some embodiments, ammonium production is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0217] In some embodiments, the microorganism, when contacted with or disposed onto the plant element, provides increased hydrolysis of urea compared to a reference plant element not treated with the composition comprising the microorganism of the species P. megaterium. Nitrogen fixation (ARA)

[0218] In some embodiments, the plant growth-promoting traits is selected from: increased germination rate, increased emergence rate, increased shoot biomass, increased seedling root length, increased seedling shoot length, increased seedling mass, increased root surface area, increased enhanced nutrient use efficiency, increased phosphate solubilization, and increased yield. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing the germination rate of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing the emergence rate of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capableof increasing shoot biomass of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing seedling root length and / or seedling mass of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing root surface area of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium. In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element is capable of increasing yield or growth of the plant element compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium.

[0219] In some embodiments, the one or more growth promoting properties or traits is selected from: increasing one or more of: enhanced resistance or tolerance to drought stress, enhanced resistance to thermal stress or thermal shock, enhanced resistance to UV radiation, enhanced resistance or tolerance to different NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to stress, increased nutrient uptake, increased root hair formation, increased root branching, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof, e.g., compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium or not treated with the composition comprising the microorganism of the species P. megaterium.6.2.1.3 Comparisons to control commercially available products

[0220] In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element, provides a benefit to the plant compared to a reference plant not treated with the microorganisms of the species P. megaterium.

[0221] In some embodiments, the microorganism of the species P. megaterium, when contacted with or disposed onto the plant element, provides a benefit to the plant compared to a reference plant treated with a different microorganism not of the species P. megaterium.

[0222] In some embodiments, the different microorganism not of the species P. megaterium is Azospirillum brasilense. In some embodiments, the different microorganism not of the species P. megaterium is Herbaspirillum seropedicae . In some embodiments, the different microorganism not of the species P. megaterium is Gluconacetobacter diazotrophicus. In some embodiments, the different microorganism not of the species P. megaterium is Exiguobacterium sp. SI 7. In some embodiments, the different microorganism not of the species P. megaterium is E. coli DH5a. In some embodiments, the different microorganism not of the species P. megaterium is Pseudomonas fluorescens7. EXAMPLES

[0223] The present disclosure will now be further described based on the following examples. It is to be understood that these examples are intended for illustrative purposes only, and by no means should be construed to be limiting the scope of the disclosure, which is only defined by the appended claims.7.1. Example 1: Isolation and Morphological Description of Strain CK60

[0224] CK60 was isolated from rhizosphere samples of plants collected at Volcan Galan, Catamarca Province, Argentina. The CK60 formulation, which includes the medium for collection of CK60, was TSB, whose composition in g / L is as follows: Tryptin 17.0, Soy peptone 3.0, Sodium chloride 5.0, Dipotassium phosphate 2.5, Glucose 2.5, pH 7.3 ± 0.2. Morphological Characterization

[0225] CK60, is an endophytic, sporulating bacterium, capable of efficiently colonizing different crops.

[0226] Under microscopy, CK60 appeared as a large, Gram-positive bacilli, typically arranged in clusters (FIG. 1A). Colonies on solid medium are small, white, creamy, circular, with complete and regular borders (FIG. IB).Molecular Characterization of CK60

[0227] Molecular identification of CK60 was performed. For this, from an overnight (o / n) culture of the strain, total DNA extraction was performed using a commercial kit(ZymoResearch). Conventional PCR using universal primers or primers to amplify the 16s rDNA region was performed. The primers used were 1492r (5’-TACCTTGTTACGACTT-3’ - SEQ ID NO: 9) and 8f (3’-AGAGTTTGATCCTGGCTGGCTCAG-5’ - SEQ ID NO: 10) or 27f (3’-AGAGAGTTTGATCCTGGCTCAG-5’ - SEQ ID NO: 11).

[0228] Once the 16S sequence was received, it was compared with public databases (e.g. BLAST: Basic Local Alignment Search Tool - NCBI) to find, if any, similarities with other microorganisms already registered and to elucidate the genus of the microorganism.

[0229] According to this analysis, CK60 and CK100 were identified as P. megaterium.Genome Characterization of P. megaterium Strain CK60

[0230] Methods - Genomic Sequencing and Assembly

[0231] The complete genome of the CK60 strain was obtained by short read whole genome sequencing on an Illumina Miseq platform and the assembly was performed with Unicycler.

[0232] Methods - Taxonomic Classification

[0233] Average Nucleotide Identity (ANI) was calculated with pyANI pipeline. Further, the bacterial core genome was analyzed and a phylogeny based on the concatenated sequences of the core genome genes was performed with the bcgTree pipeline.

[0234] Result - Genomic Sequencing and Assembly

[0235] The QC analysis of the reads performed with FastQC revealed that there were 4,491,863 paired end reads with 150 bp length, totalling 1,347,558,900 sequenced bases. The genome assembly obtained had a total length of 6,105,102 bp distributed in 260 contigs (N50: 436,736; N90: 50,878). Three complete plasmid sequences were retrieved from the assembly process (plasmid sizes of 70,414 bp, 9,561 bp and 6,041 bp).

[0236] Results - Average Nucleotide Identity (ANI) Analysis

[0237] ANI taxonomic assignment analysis revealed that the strain CK60 belonged to the species P. megaterium. This strain shared more than 97% of average nucleotide identity with P. megaterium strain 22-2 and P. megaterium strain CK100 as shown in FIG. 2.

[0238] Results - Core Genome Phylogeny

[0239] The CK60 strain is placed in the same cluster as P. megaterium 22-2, P. megaterium CK100 and P. aryabhattai K13. Overall, the phylogenetic analysis performed here is congruent with the ANI analysis and is shown in FIG. 3.7.2. Example 2: Biochemical Characterization of CK60 as Growth-Promoting Bacteria

[0240] The biochemical characterization of CK60 as plant growth-promoting bacteria was carried out in vitro at 30 °C. Bacterial suspensions ( 107-l 08CFU / mL) previously grown in nutrient broth medium (BRITANIA) were used unless another specific medium is indicated. These cultures were used as inocula for the various biochemical tests provided below.Colonization Abilities

[0241] The aim of this study was to determine the synergism between bacteria and plants in the context of effective colonization or colonization frequency.

[0242] CK60 colonization was determined through biofilm studies (in vitro and in vivo), molecular biology assessments, and rhizodeposition analysis.

[0243] Bacterial biofilms are clusters of bacteria attached to a surface and / or each other and embedded in a self-produced matrix. The biofilm matrix consists of substances like proteins (e.g., fibrin), polysaccharide (e.g., alginate), as well as eDNA. Biofilms create a stable microenvironment that supports sustained interaction between bacteria and plant roots, fostering long-term mutualistic relationships.

[0244] Biofilms are involved in bacterial root colonization and offer numerous benefits for the plant such as:• Improved Nutrient Uptake: bacteria in biofilms can solubilize and mobilize nutrients, such as phosphorus and nitrogen, making them more accessible to plant roots.• Growth Promotion: biofilms enhance the production of phytohormones and other growth-promoting substances.• Disease Suppression: biofilm-forming bacteria can outcompete harmful pathogens by occupying root niches and producing antimicrobial compounds.• Stress Tolerance: biofilms improve plant tolerance to abiotic stresses like drought, salinity, and heavy metals by modulating the rhizosphere environment.• Root Architecture Enhancement: biofilms stimulate root hair formation and branching, increasing the root surface area for better nutrient and water absorption.

[0245] CK60 biofilm production capacity was evaluated in vitro under normal growth conditions. Using the crystal violet staining technique, it was possible to confirm this characteristic by obtaining high DO values of 1.82 ± 0.99.

[0246] Additionally, an in-depth characterization was conducted by analyzing the exopolysaccharide present in the CK60 biofilm. The results of the study are summarized below.Determination of Monosaccharide Composition

[0247] NMR and HPAEC-PAD Analysis. As a result of this analysis, it was identified that EPS, produced by CK60, is made up of a single constituent monosaccharide: D-Fructose (homopolymer of D-Fructose). No acid sugars were identified among the EPS components.

[0248] Structural Analysis of EPS

[0249] NMR and GC-MS Analysis. In order to characterize the EPS structure and determine the sequence, anomeric configurations and branching, the following NMR experiments were performed:JH13C-HSQC, WH-TOCSY, ^-^C-HMBC and1H-1H- NOESY. With the spectra obtained from these assays it was possible to determine the sequence and branching of the EPS resulting in the following connectivity: — >6)-Fru-(2— 6)- Fru-(2— >6). The resonances obtained in the ^^H-NOESY spectra confirm that the bonding between the residues is P (2— >6).

[0250] In order to confirm the results obtained by NMR, GC-MS analysis of the monosaccharide derived from the purified EPS was performed. The mass spectrum of the identified species confirmed the presence of the monosaccharide previously assigned by NMR and HPAEC-PAD. Additionally, these results provided information on the connectivity between the EPS residues, which was in agreement with the NMR results. Based on these results, the model of the EPS structure was obtained (FIG. 4).

[0251] This model satisfied the NMR, GC-MS and HPAEC-PAD experimental data. It was determined that the structure obtained was compatible with the Levan polysaccharide.

[0252] The biofilm, particularly the production of this type of exopolysaccharide, enhanced crop growth and health by improving soil structure, increasing water retention, and enhancing nutrient availability to plants.

[0253] The colonization capacity of P. megaterium CK60 was also determined in plant trials using in all cases positive and negative controls.Microscopy

[0254] The ability of CK60 to colonize wheat roots, form biofilm, and establish itself in different parts of the plant tissue was evaluated through seed inoculation. As CK60 is a sporeforming microbe, wheat seeds were inoculated with CK60 in both its vegetative and spore forms. Biofilm formation was studied using scanning electron microscopy (SEM), and the presence of CK60 in the biofilm was confirmed by detaching the biofilm from the roots, recovering the microorganisms, and subsequently culturing them on a solid medium. The microorganism was identified based on colony morphology and its identity was confirmed through PCR using specific primers for CK60. Evaluations were conducted at two different temperatures: 30 °C and 15 °C.Seed Inoculation with Vegetative CK60

[0255] The results obtained demonstrated that CK60 was capable of effectively colonizing wheat roots and establishing itself in the stems of this crop within 10 days after seed inoculation with the microorganism. Using scanning electron microscopy (SEM), colonization of the wheat seedling roots by CK60 was observed on their surface, with both microcolonies and macro colonies detected. Additionally, CK60 cells were found embedded within a matrix in specific areas, indicating the formation of a mature biofilm on the wheat roots 10 days post-inoculation (FIG. 5). These findings highlight the ability of CK60 to colonize and establish itself in various tissues of the evaluated crop, even under suboptimal temperature conditions (15 °C) for the microorganism.Seed Inoculation with CK60 Spores

[0256] The results of this study demonstrated that when wheat seeds were inoculated with spores of CK60, they were able germinate the seeds, and the microorganism effectively colonized the wheat root tissue, forming a robust biofilm characterized by a dense aggregation of cells. The predominance of shorter vegetative cells at lower temperaturessuggested that environmental conditions significantly influenced the morphology and behavior of CK60. FIG. 6 and FIG. 7 show scanning electron micrographs of wheat roots colonized by CK60 10 days after seed inoculation with the spore form of the microorganism.

[0257] Furthermore, it was possible to recover and identify CK60 as part of the endophytic population in the stems of 10-day-old seedlings.

[0258] The CK60 spores display dehydrated structures with an irregular surface and distinctive grooves. In contrast, the CK60 germinating cells exhibit a smoother surface with less defined grooves, resulting from rehydration during the germination process. The vegetative cells show elongated morphology and a homogeneous, smooth surface without grooves, with some cells emerging from the germinating cells.

[0259] Vegetative cells are predominant with a morphology different from that observed at 30 °C, as the cells appear much shorter.Molecular Biology

[0260] Complementary to the microscopy studies, colonization of CK60 within vegetal tissues was followed using a molecular biology technique. The results demonstrated that CK60 remained present throughout the plant’s growth, ensuring its beneficial traits support plant development.Rhizodeposition Studies

[0261] The composition of the root exudates or rhizodeposition of wheat seedlings inoculated with CK60 formulation was evaluated through the determination and quantification of bioactive molecules (organic acids, flavonoids). The treatments analyzed were as follows:• Negative control: no inoculation (NC)• Positive control: Azospirillum brasilense (PC)• CK60

[0262] The results obtained are presented in Table 2.Table 2. Results of the evaluation of root exudates, or rhizodeposits, from wheat seedlings.NC (negative control); PC (positive control) = Azo spirillum brasilense', nd = not detected

[0263] The evaluation of root exudates, or rhizodeposits, from wheat seedlings treated with P. megaterium CK60 showed qualitative and quantitative differences in their composition compared to those obtained from untreated control plants (NC) as well as from the exudates of seedlings treated with Azospirillum brasilense (PC), a plant growthpromoting microorganism used as a positive control. The differences in the compositions of the rhizodeposits suggested a differential plant response to the colonization of these bacteria.

[0264] As shown in Table 2, the inoculation of wheat seeds with both CK60 and A. brasilense resulted in a modification of the flavonoid profile in the root exudates. While the composition of the other evaluated metabolites remained unchanged, a significant increase was observed in the amount of the flavonoid naringin and its aglycone form, naringenin, when wheat seeds were inoculated with CK60 (FIG. 8).

[0265] FIG. 8 illustrates a profile of bioactive compounds determined in the rhizodepositions of wheat seedlings (control and inoculated with CK60 and Azospirillum brasilense). These compounds were identified and quantified using a liquid chromatography coupled with tandem mass spectrometry (LC-MSMS, Waters Alliance 2695 and Micromass Ultima PT). Quantification was carried out using established calibration curves for each analyte with known concentrations. Monitoring was performed in Multiple Reaction Monitoring (MRM) mode, comparing the analyte in question with those of pure standards, both in terms of transitions and retention time (RT). Calculations were based on the fresh weight of the roots deposited in the nutrient solution. The concentrations for all compounds are expressed as ng / g of fresh root weight, except for luteolin, which is expressed in pg / g of fresh root weight. Statistical analysis was conducted to determine significant differences inthe amounts of each bioactive compound under the different conditions tested (Analysis of variance, DGC test: means with a common letter are not significantly different (p > 0.05)).

[0266] The flavonoid, naringenin, known to stimulate the colonization of plant growth promoting bacteria (PGPB) in the roots of wheat and other crops, such as rice and peanuts, in its glycosylated form (naringin), was found to be produced as a chemoattractant to recruit CK60 (the glycosylated form has greater mobility and reach). Subsequently, naringin was deglycosylated by CK60 into its aglycone form, naringenin, which may then facilitate the colonization of CK60 in the wheat roots by increasing infection sites.

[0267] Other flavonoids, such as genistein and daidzein, which were found in higher proportions in the root exudates of seeds treated with biological products, have also been described as compounds that stimulate root colonization of wheat by plant growth promoting bacteria (PGPB).

[0268] As shown in this study, P. megaterium CK60 was a microorganism capable of colonizing wheat plants. During this interaction, the plant modified the composition of its root exudates, releasing chemical compounds that favor the mobility, recruitment, colonization, and growth of this microorganism. The results presented in this study support the hypothesis that flavonoids, such as naringenin, play an important role in chemotaxis, mediating the colonization of wheat roots by PGPB.Nutrient Acquisition Capabilities

[0269] In terms of nutrition, this bacterium exceled in its ability to solubilize phosphate, featured an advanced and versatile nitrogen system that encompasses nitrogen fixation, mineralization of nitrogen compounds, and urea hydrolysis, enhanced iron and other minerals uptake, and synthesized enzymes responsible for solubilizing complex organic sources.

[0270] This strain demonstrated a strong ability to solubilize phosphates from insoluble inorganic sources, classifying it as an effective phosphate-solubilizing microorganism (PSM) with a solubilization index (PSI) of 4.35 recorded in a solid NBRIP (National Botanical Research Institute’s phosphate) growth medium. This capacity is beneficial for enhancing the availability of essential macronutrients for crops. The NBRIP is a defined microbiological growth medium used to screen for phosphate-solubilizing microorganisms. Typically, the NBRIP contains glucose, tricalcium phosphate (TCP), magnesium chloride hexahydrte, magnesium sulfate heptahydrate, potassium chloride, and ammonium sulfate.

[0271] In liquid medium, an increase in the amount of solubilized phosphorus was observed as a function of incubation time up to 10 days, after which a decrease in the quantified phosphate was recorded at 14 days. The accumulation of phosphate in the intracellular medium by CK60 could explain the significant reduction (p < 0.05) in the amount of phosphate quantified in the supernatant at 14 days compared to 10 days. FIG. 9 shows the results of the phosphate quantifications performed in the NBRIP medium at 3, 6, 10, and 14 days of incubation.

[0272] In addition, CK60’s performance was compared to established benchmark products, including one biological fertilizer containing Azospirillum brasilense and Pseudomonas fluorescens and another containing only Pseudomonas fluorescens. CK60 exhibited superior phosphate solubilization (345 pM phosphate solubilized), solubilizing 48% more than the Azospirillum-Pseudomonas product (236 pM phosphate solubilized) and 18% more than the Pseudomonas-only product (292 pM phosphate solubilized) after 10 days. The results are presented in FIG. 10.

[0273] Apart from phosphorus, nitrogen is recognized as a key element for crop health and development, with its availability directly influencing crop productivity. This element is found both in the earth’s atmosphere (elemental N2) and in the soil (organic nitrogen), mainly in forms that cannot be assimilated by plants. CK60 is a microorganism capable of metabolizing the different forms in which nitrogen is found in the environment, through different metabolic processes, making it available to be absorbed and used directly by plants.

[0274] Through a mineralization process, CK60 was capable of degrading nitrogenous organic matter in the soil and converting it into ammonium.

[0275] Firstly, the NH4+produced by CK60, derived from mineralization, was quantified using a commercial KIT (Ammonia Assay Kit - ab83360, ABCAM), registering a value of 659.94 pM of ammonium after 24 hours of incubation. The results obtained, compared with those obtained by other strains used as reference in the laboratory, are shown in Table 3. Importantly, this evaluation was performed on cells grown in a medium containing an organic nitrogen source.Table 3. Quantification of total NHZ derived from mineralization.

[0276] Organic nitrogen compounds’ mineralization capacity was also evaluated using the Jensen medium with peptone added (as a complex nitrogen source). Under these conditions, the ammonium ion produced by CK60 was quantified using Nessler’s reagent, and 183.76 pg / mL NHZ was registered (FIG. 11).

[0277] Ammonium was efficiently produced as a result of the hydrolysis of urea (FIG. 12) through the production of the urease enzyme. The ammonium produced was converted by this microorganism to nitrate by a nitrification process. Both ammonium (ammonia in its gaseous form) and nitrate are compounds that can be directly assimilated by plants for their growth and development. In addition, if soils are poor in nitrogenous organic matter, through a process of biological nitrogen fixation (BNF) this bacterium was also capable of utilizing gaseous nitrogen from the atmosphere and converting it into ammonia (NHZ), ensuring an effective supply of nitrogen to the plant regardless of the forms in which nitrogen is found in the environment.

[0278] The nitrogen fixation activity was also compared to microbes present in commercial products. The quantification of NHZ produced and accumulated in the extracellular medium by CK60 in a nitrogen-free medium was performed. Under these conditions, it was considered that the ammonium produced and quantified by this technique originated from the fixation of atmospheric nitrogen by the microorganism evaluated. Quantification was performed with Nessler’s reagent in Jensen’s medium, with and without nitrogen source. Methylobacterium symbiolicum. a microorganism known for its nitrogen- fixing ability and present as the active ingredient in a commercial product, was used as the positive control. Escherichia coli DH5a served as the negative control. The results obtained are shown in FIG. 13. As can be seen, under the conditions evaluated, CK60 produced approximately 20% more ammonium than the positive control.

[0279] Iron is another vital micronutrient for plants and their associated microorganisms, with their iron nutrition depending on the soil’s supply. In plants, iron is involved in the synthesis of chlorophyll, being used for the maintenance of chloroplast structure andfunction. However, its bioavailability in cultivated soils is often low. Therefore, plants and microorganisms developed active strategies for iron uptake, employing mechanisms such as acidification, chelation, and reduction processes.

[0280] The results of this study demonstrated that CK60 produced and released siderophores into the medium. The qualitative Chrome Azurol S (CAS) assay was used for their identification. This technique identifies siderophores by detecting a color change in the medium when the siderophores interact with the chromophore. Siderophore-producing bacteria (SPB) were proposed as a sustainable alternative to synthetic fertilizers. These siderophores are low-molecular-weight organic compounds with cation-chelating properties, primarily synthesized under nutrient scarcity to cope with nutritional imbalance or to alleviate the toxicity of heavy metals. By producing siderophores, CK60 enhanced the plant’s iron (Fe) nutrition. Moreover, siderophores also demonstrate an affinity for other essential elements, including Cu, Mn, Mo, and Zn, which play important roles in plant metabolism and development.

[0281] On the other hand, CK60 produced lytic enzymes involved in the degradation of complex organic compounds leaving available simpler substrates that can be taken up by plants, improving plants’ nutrition. These enzymes are also related to improvements in soil structure and biocontrol activities.

[0282] CK60 showed protease, cellulolytic, and catalase activities. Protease activity was qualitatively determined on solid media with casein, where the formation of a clear zone indicated the enzymatic hydrolysis of proteins. Cellulolytic activity was assessed on solid media containing carboxymethylcellulose (CMC), where the breakdown of CMC by bacterial cellulases resulted in the formation of a clear zone around the colony. This activity was further visualized by applying Congo Red, which stains the remaining CMC, highlighting areas of degradation. Regarding chitinolytic enzymes, CK60 successfully grew in colloidal chitin medium without forming a visible halo. Using chitin as the sole carbon source, the microorganism’s growth in the medium suggested the production of extracellular chitinases.Plant Growth-Promoting Traits

[0283] In this study, phytohormone production by CK60 was analyzed.

[0284] Phytochemicals, also known as plant hormones, play an important role in regulating plant growth and development. These organic compounds are produced in small quantities but have a profound impact on various physiological processes.

[0285] Initially, the synthesis of indole acetic acid (IAA) in CK60 was quantified using a colorimetric assay, yielding approximately 40 pg / mL after 72 h of incubation. Subsequently, the production of this phytohormone was confirmed through its identification and quantification in the culture supernatants using state-of-the-art liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). The synthesis of auxins, mainly IAA, is one of the properties linked to plant growth promotion and is associated with protection against stress factors. The inoculation of auxin-producing bacteria can cause a wide range of responses in plants, which can either promote or inhibit growth depending on the level of auxins produced. This substance (IAA), at the appropriate dose, has a positive effect on plants: it is responsible for the formation and growth of different plant organs and also participates in the improvement of secondary root proliferation. Particularly, in dicotyledons, IAA induces the formation of lateral roots, while in monocotyledons, it induces the formation of adventitious roots. This auxin not only has direct effects on the plant but also on the bacteria itself, acting as a signaling molecule that triggers the expression of genes involved in adhesion and adaptation to plant tissues, thereby favoring colonization in the plant. The application of IAA leads to an increase in crop yields and quality.

[0286] Additionally, the identification and quantification of other phytohormones in the culture supernatant were performed using LC-MS / MS. The phytohormones analyzed by this technique, in addition to IAA, included: abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), jasmonic acid (J A), zeatin (ZEA), 6-benzylaminopurine (6-BAP), kinetin (KIN), and indole-3 -butyric acid (IB A).

[0287] The results obtained are available in Table 4.Table 4. Phytohormones identified and quantified in the culture supernatants of CK60 and other microorganisms of interest.

[0288] All the phytohormones under study were detected in the culture supernatants of CK60. However, CK60 particularly stood out as a producer of GA-3.

[0289] Gibberellins (GAs), including GA-3, are involved in a range of developmental and physiological processes, such as seed germination, seedling emergence, stem and leaf growth, floral induction, and flower and / or fruit growth, regulation of vegetative and reproductive dormancy (bud dormancy), and delaying senescence. Gibberellin-producing bacteria regulate plant hormone levels in various ways: through a direct synthesis of GAs, deconjugation of glucosylated gibberellins, and conversion of inactive gibberellin states into active GAs, which promotes improved crop performance.

[0290] Gibberellin-producing bacteria play an important role in response to abiotic stress, particularly in conditions of drought and salinity, explaining CK60’s ability to tolerate extreme water stress conditions, its drought resistance, and its capacity to grow in up to 5% NaCl concentrations.Conclusions

[0291] The growth-promoting capacity resulted from the additive effect of more than one mode of action, determined by analyzing various properties, such as colonization abilities, nutrient acquisition capabilities, plant growth-promoting traits, yield, and tolerance to abiotic stress. Thus, of CK60 resulted from a synergistic effect between CK60 and the plant as reflected by the yield increase.7.3. Example 3: Genomic Analysis of CK60 for Phosphate Solubilization Genes

[0292] Phosphate solubilization is facilitated by a spectrum of genes governing phosphatase activity, metabolism, and the production of both organic and inorganic acids. The interplay between these genetic components orchestrates the liberation of phosphate from insoluble forms, thereby enhancing its availability to plants for uptake and utilization.

[0293] The analysis of the number of genes associated with phosphate solubilization in the genome of CK60 was performed.

[0294] The genomic analysis was conducted through a multi-faceted approach. Firstly, the genome was annotated using Prokka, facilitating the identification of protein-coding genes and functional elements. Subsequently, the EggNOG database was utilized to classify gene functions and assign orthologous groups. Additionally, a custom database specific toplant growth-promoting traits was employed to pinpoint genes associated with phosphate solubilization.

[0295] Specifically focusing on phosphate solubilization, genes were categorized into four main groups: phosphatase activity, phosphatase metabolism, organic acids, and other acids. The search for genes within these categories was conducted for CK60.

[0296] Following gene identification, visualization of the genomic data was achieved through the construction of bubble graphics. These graphics illustrated the distribution and abundance of phosphate solubilization genes across the genomes, providing valuable insights into the genetic landscape of each strain. Additionally, bar plots were generated to display the total number of genes within each category, facilitating comparative analysis among the strains.7.4. Example 4: Abiotic tolerance of CK60

[0297] Abiotic Stress Tolerance

[0298] Due to CK60’s isolated origin, its tolerance to various stress conditions was evaluated, whether in the vegetative or spore stage, yielding the following results.CK60 Vegetative Form Characterization

[0299] Tolerance to Partial and Total Drought

[0300] For the partial drought test, CK60 cultures were grown in culture media with different concentrations of PEG (0, 10, 20, 30, 40, and 50%) to simulate different degrees of drought. Viability was evaluated at 24 and 48 hours by counting CFU / mL. The results obtained are shown in FIG. 14.

[0301] Considering that the PEG concentrations used simulate high to extreme drought conditions (4% PEG = mild; 8% PEG = moderate; greater than 10% = high), it can be concluded that CK60 had an adequate tolerance to extreme levels of drought.

[0302] As for the evaluation of total drought tolerance, a microorganism is considered to have a good tolerance to total drought when its survival rate is greater than 5%. In this case, a survival rates of 35%, 25% and 15% were recorded for CK60 at 16, 24, and 48 hours of evaluation, respectively, standing out positively relatively to the other strains evaluated (Table 5, FIG. 15).Table 5. Survival rate (%) of CK60 and other Laboratory Reference Strains under total drought at different time intervals.

[0303] Tolerance to Salinity

[0304] It was found that CK60 was capable of growing in NaCl concentrations up to 5%. A longer lag phase was observed as the NaCl concentration increases from 0.8% to 5%.

[0305] These findings suggest that CK60 is a halophilic microorganism, as it can tolerate moderate salinity (up to 5% NaCl). CK60 salinity curves are presented on FIG. 16.

[0306] pH Tolerance

[0307] Growth curves were performed at pH 5, 5.5, and 7. As shown in FIG. 17, CK60 tolerates all the pH levels tested, demonstrating its versatility in adapting to diverse conditions. In addition, in all the conditions tested, in which CK60 growth was recorded, the medium pH rose gradually with culture time, with pH values above 7.5 being recorded at 24 hours (data not shown).

[0308] Temperature Tolerance

[0309] Thermal Shock. As can be seen in Table 6, CK60 exhibited high stability at elevated temperatures, remaining viable at 50 °C for 1 hour.Table 6. Thermal stability of CK60.

[0310] Growth at Different Temperatures. The growth of CK60 was evaluated at different temperatures: 10 °C, 15 °C, 20 °C, 30 °C, and 40 °C. The results are shown in FIG. 18 (A and B). CK60 demonstrated the ability to grow across the entire temperature range tested reaching high biomass ranges.

[0311] UV Resistance

[0312] CK60 was exposed to UV-B radiation (280-320 nm) for different times: 0, 5, 10, 20, and 30 minutes. As a negative control, the UV-sensitive strain E. coli DH5a was employed. Subsequently, their recovery capacity was evaluated in darkness. Microbial growth was recorded relative to the control culture of unexposed cells (Table 7, FIG. 19).Table 7. UV resistance profile of the evaluated strains.++: Growth is slightly reduced compared to the control without UV. +: Growth is significantly lower than the non-UV control. No growth observed in any dilution tested (sensitive strain).

[0313] To investigate UV-B radiation tolerance, 10 pL of serial dilutions of a CK60 culture were loaded onto LB agar plates and were further exposed to different irradiation times (0, 5, 10, 10, 20 and 30 min). Finally, the plates were incubated at 30 °C wrapped in aluminum foil to study their recovery in the dark.

[0314] Strain CK60 exhibited moderate growth at the tested doses, demonstrating a higher tolerance to UV radiation compared to the UV-sensitive E. coli DH5a control.

[0315] CK60 Spores Characterization

[0316] The resistance of CK60 spores to different pH ranges, temperatures and NaCl concentrations was evaluated.

[0317] Resistance to Different pH

[0318] CK60 spores were exposed to a wide range of pH (2, 5, 7, 9 and 12) for 30, 60 and 90 minutes. Spore survival was evaluated by counting CFU / mL after heating the samples at 80 °C for 20 minutes. The spore count remained stable in all conditions between 30 and 90 minutes evaluated (FIG. 20).

[0319] Temperature Resistance

[0320] CK60 spores were exposed for 7 days at 40 °C maintaining constant viability throughout the evaluation. Survival was evaluated by CFU / mL count after heating the samples at 80 °C for 20 minutes (FIG. 21).

[0321] Resistance to Different NaCl Concentrations

[0322] CK60 spores were exposed to a wide range of NaCl concentrations (0.8, 5, 10, 15 and 25%) for 30, 60 and 90 minutes. Spore survival was assessed by CFU / mL count after heating the samples at 80 °C for 20 minutes. Spore counts remained stable at all conditions and times evaluated (FIG. 64).

[0323] Compared to other bacteria present in currently marketed benchmark products for wheat and other crops, CK60 showed significantly superior tolerance to water stress. FIG. 65 presents the percentage variation in growth of CK60 and M. symbioticum relative to time zero, in response to different concentrations of PEG 600 (0%, 20%, 30%, 40%, and 50%), evaluated at 24 and 48 hours. It was observed that CK60 grows normally up to a 50% PEG 600 concentration, while M. symbioticum can only grow at concentrations up to 20% PEG, with a decrease in viability at higher concentrations.

[0324] CK60 showed growth even at concentrations up to 50% PEG, whereas M. symbioticum only exhibits growth at concentrations up to 20% PEG, with a decrease in viability at higher concentrations.7.5. Example 5: Evaluation of the Potential of P. megaterium CK60 to Promote the Growth of Wheat (Triticum Aestivum L.) Through its Application as a Field Seed Treatment

[0325] The performance of P. megaterium CK60 (CK60 formulation) applied as a seed treatment in wheat was evaluated through trials in two seasons. The results indicate that the interaction of CK60 formulation with wheat, evaluated under diverse agroecological and edaphic conditions, generated stimuli in crop growth, which led to an increase in yields (kg / ha) achieved in various wheat-growing regions of Argentina.

[0326] The increase in global food demand, in the face of a limited productive surface, requires maximizing agricultural productivity through the rational use of resources and sustainable practices, especially in areas with adverse conditions. In this context, bioinputs, such as biofertilizers, soil amendments, plant amendments, and biostimulants, play a key role in improving nutrient availability and promoting plant growth. The application of bacteria on seeds establishes a beneficial interaction between roots and microorganisms, such as synergistic effects, that favor nitrogen fixation, phosphorus solubilization and the production of compounds that increase crop productivity and quality.

[0327] In this study, extremophilic microorganisms that evolved in extreme conditions and develops innovative bioinputs for seed treatments were researched. These microorganisms have proven to be plant growth promoters. They perform functions such as nitrogen fixation, auxin production and phosphorus solubilization. After promising results in the laboratory, field trials were proposed in various productive regions of Argentina.

[0328] The objective of these studies was to evaluate the impact on productivity (Kg / ha) generated by the use of CK60 formulation (commercial name), a product formulated with P. megaterium CK60 in wheat, during the 2022 and 2023 seasons.

[0329] Materials and Methods

[0330] Seed inoculation

[0331] For seed treatment, the following protocol was carried out:

[0332] Before starting the seeds must be dry. The temperature of the place chosen to work should not be higher than 30°C, with avoiding environments with high relative humidity (environments saturated with steam). The amount of seed to be treated was placed in a dry and clean container that exceeded the volume occupied by the seeds, so that they were adequately mixed, ensuring that the inoculating liquid was distributed among all the seeds. After putting the seeds in the selected container, the inoculating liquid was applied on the seeds, maintaining the ratio indicated by the dosage. The inoculation seed mixture is mixed by moving the container without causing physical damage to the seed to ensure a homogeneous distribution of the inoculant. The duration of this procedure depends on the amount of seed to be inoculated and the capacity of the equipment used to do so. After mixing, the seeds were left to dry in the open air for a few minutes being used for sowing.

[0333] Trials were conducted throughout the agroecological production zones defined for wheat cultivation in Argentina by the local regulatory entities. Each selected region is characterized by contrasting agroecological and edaphic conditions.

[0334] In the years 2022 and 2023, 22 field trials were conducted. In all cases (22 trials), studies were carried out in the same way, with the same agronomic practices, formulation and dosage of CK60 formulation as an aqueous solution for seed treatment.

[0335] The varieties planted (Table 8) were selected by the trialists, according to the recommendation of use for each zone. Due to the diversity of zones, it was possible to evaluate the CK60 formulation symbiosis in varieties with different cycle lengths andqualities (Table 8). Seed therapeutics used in the trials included: Fluxapiroxad (33.3%, at 77 mL / 100 kg seed); Prothioconazole 3.5g / L + Fluoxastrobin 3.75g / L + Tebuconazole 0.5g / L at 150 mL / 100 kg seed; Thiram 30% + Carbendazim 30% at 100 mL / 100 kg seed with Imidacloprid 60% at 100 mL / 100 kg seed.Table 8. Wheat varieties used during the 2022 and 2023 season-Argentina.

[0336] In Argentina, soils vary significantly according to their physicochemical behavior and intrinsic properties, as a consequence of the climatic characteristics that influence their development, the presence of specific horizons, compositional characteristics and morphological features (amount and type of clay, structure, color, etc.). In the trials conducted, the work was performed on soils with different productivity indexes, such as Argiudols and Hapludols, with organic matter from 1.6 to 6.7%, electrical conductivity from 0.16 to 0.60 dS / m, silt loam and sandy loam textures, and pH between 5.6 and 6.6.

[0337] Environmental conditions also varied, with maximum temperatures from 25 to 36 °C, minimum temperatures from -2 to 8 °C, rainfall from 308 to 586 mm, photoperiods from 10.3 to 14.5 hours, and average monthly radiation between 6.2 and 23.2 Mj / m2 / day.

[0338] Results

[0339] All the sites tested showed a positive difference with respect to the control (Table 9), with the range of differences varying according to the area, variety, environment, and conditions of growth and development of the wheat crop.

[0340] The average of the evaluations in the areas of highest productivity presented a value of +588 kg / ha above the control (Table 9), with positive values in all sites, indicating a positive response rate of CK60 formulation.Table 9. Yields of CK60 formulation versus uninoculated control in trials for the 2023-Argentina season.

[0341] Including the northern zone of Argentina in the previous analysis, the values were equally favorable, with a total average value of +532 kg / ha (+13.3%) above the control (Table 10).

[0342] Yield data obtained (kg / ha) in the trials showed that, in almost all sites, CK60 formulation generated a positive difference concerning the control.

[0343] In these sites, the trials were conducted in microplots with homogeneous soil fractions. In some cases, this did not cover the heterogeneity found in a real field production situation, but in spite of this, a positive response rate of CK60 formulation was observed, with an average value of differences with respect to the control of +11% (Table 10).Table 10. Yields of CK60 formulation versus uninoculated control trials 2022 and 2023 in Argentina.

[0344] Conclusions

[0345] The results obtained confirmed the efficacy and versatility of CK60 formulation as an innovative solution to improve wheat (Triticum aestivum L.) crop productivity, highlighting the following:• Positive effect on wheat yield: Under conditions similar to those observed in productive establishments, the use of CK60 formulation generated an average increase of 14% in wheat yield, demonstrating its efficacy as an agronomic tool.• High plasticity in response: CK60 formulation showed an outstanding capacity for adaptation, reaching a positive response rate of over 95%. This performance was maintained when evaluated under a wide range of soil and climatic conditions, wheat varieties and planting dates, demonstrating its flexibility and robustness in different production scenarios.• Applicability in diverse areas: The data presented indicate that CK60 formulation is a versatile and effective tool in areas with high, medium and low productive capacity, achieving consistent benefits regardless of the soil productivity index.7.6. Example 6: Recovery of P. megaterium CK60 From Wheat Plants by qPCR: Seed Treatment

[0346] The aim of the study was the recovery of CK60 formulated in different phenological stages of the wheat plant by a molecular biology technique: qPCR.

[0347] Methods and Results

[0348] The wheat seeds were treated with CK60 formulation, and to understand how it colonizes the wheat plant over time, the qPCR technique was used with specific primers designed for CK60. In qPCR, fluorescent dyes that bind to double-stranded DNA are incorporated into the PCR reaction, and the formation of the product is monitored in real-time after each PCR cycle. The dye is added to the reaction, and the fluorescence is measured in each PCR cycle. Since the fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored by the increase in fluorescent signal. The main result of a real-time PCR experiment is the amplification curve, which shows the accumulation of the amplified product in the form of a fluorescent signal relative to the number of cycles.

[0349] The accumulation of CK60 genetic material was evaluated at the phenological stages of the wheat plant depicted in FIG. 22. Two treatments, TO and Tl, were applied to the wheat plants.

[0350] To examine the presence of CK60 on wheat plants, DNA from different parts (aerial part, and root) of the plants was purified using a commercial DNA Extraction Kit. After that, the purified DNA was used to carry out qPCR using specific primers for CK60. The results are shown in FIGs. 23 and 24.

[0351] CK60 was found in the root of the wheat plant during all the studied phenological stages. No presence of CK60 was found in the control.

[0352] CK60 was found in the aerial part during the Z5.9 and Z9.4 stages. No presence of CK60 was found in the control.

[0353] The results demonstrated that CK60 remains present throughout the plant’s growth, ensuring its beneficial traits support plant development.

[0354] Specific Primer Design for P. megaterium Strain CK60

[0355] In this study, the method used to design specific primers for P. megaterium strain CK60 is presented. The process involved identifying genomic regions with low homology to other sequences within the Priestia genus and verifying these regions in the NCBI non- redundant database and several DNA samples for specificity.

[0356] Methodology

[0357] Horizontal gene transfer events within the CK60 genome were predicted using the Alien Hunter software. Specific regions adjacent to predicted horizontal gene transfer sites were prioritized for primer design, since these regions tend to show a low degree of conservation. This characteristic enhances the likelihood of identifying unique genomic sequences suitable for specific primer targeting.

[0358] The final selected specific regions were inputted into PeriPrimer to identify sequences from which primers were designed.

[0359] Primer Testing and Validation

[0360] The designed primers were synthesized and tested for specificity by performing PCR on complex metagenomic samples, several strains from our microbial collection, and DNA extracted from P. megaterium strain CK60.

[0361] The specificity of the PCR amplification was confirmed by analyzing gel electrophoresis results and sequencing the PCR products.

[0362] PCR Cycling Conditions

[0363] The PCR amplification was performed under the following cycling conditions: an initial denaturation step at 95 °C for 3 minutes, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 59 °C for 30 seconds, and extension at 72 °C for 1 minute. A final extension was conducted at 72 °C for 5 minutes. This specific annealing temperature of 59 °C was chosen based on the melting temperatures of the primers designed to ensure high specificity under the defined experimental conditions.

[0364] Specific CK60 Selected Sequence

[0365] >CK60 specific sequence

[0366] TTTGGACATGGAGTTATGCCTGAAGGGTTTAGTCCAAGGTTGAGTAGT TATTTTAATTTAGTACCAATTGATATATATCAAACACGAGACTTTACAGGTGGCA (SEQ ID NO: 7)

[0367] Specific CK60 Primers

[0368] >CK60 ss Fw

[0369] TTTGGACATGGAGTTATGCCT (SEQ ID NO: 12)

[0370] >CK60 ss Rv

[0371] TGCCACCTGTAAAGTCTCGT (SEQ ID NO: 13)

[0372] The described methodology successfully identified and verified specific primer sequences for targeting unique genomic regions of interest in P. megaterium strain CK60. This approach ensures the primers specificity and applicability in diverse biological samples.Example 7: Recovery of P. megaterium CK60 from Wheat Plants by qPCR: Foliar Treatment

[0373] The aim of the study was the recovery of CK60 formulated at different times post- foliar treatments from the wheat plants using a molecular biology technique: qPCR.

[0374] Methods and Results

[0375] The wheat plants were independently treated with CK60 using foliar treatments. To understand how CK60 colonizes the wheat plant over time the qPCR technique with specific primers designed for CK60 was used. In qPCR, fluorescent dyes that bind to doublestrand DNA are incorporated into the PCR reaction, and the formation of the product ismonitored in real-time after each PCR cycle. The dye is added to the reaction, and the fluorescence is measured in each PCR cycle. Since the fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored by the increase in fluorescent signal. The main result of a real-time PCR experiment is the amplification curve, which shows the accumulation of the amplified product in the form of a fluorescent signal relative to the number of cycles.

[0376] The accumulation of CK60 genetic material was evaluated at three different days post-infection (5, 12, and 19 dpi). The roots and root exudate of the wheat plants treated with 2750 cm3 / h of these microorganisms were studied.

[0377] To examine the presence of CK60 in wheat plants, DNA from different parts (root and root exudate) of the plants was purified using a commercial Soil DNA Extraction Kit. After that, purified DNA was used to carry out qPCR using specific primers for CK60.

[0378] The Presence of CK60 in Wheat Plants Over Time

[0379] As can be seen in FIG. 25, CK60 formulated, 5 dpi is in the roots and root exudate, but the DNA concentration in the latter is less. DNA concentration increases over time in the roots part. Meanwhile, the DNA concentration in root exudates increases after 5 dpi and is stable with time.

[0380] CK60 was found in the root and root exudates of the wheat plant at least 19 dpi. No presence of CK60 was found in the control.

[0381] CK60 was found to be translocated to roots and root exudates when applied as a foliar treatment being capable of colonizing the vegetal tissues.7.7. Example 8: Analysis of the Use of P. megaterium CK60 as a Seed Treatment for the Reduction of Nitrogen Fertilization in Wheat - Campaign 2022-2023, Argentina.

[0382] The use of P. megaterium CK60 in wheat as a seed treatment was evaluated as a biological alternative to reduce the use of synthetic nitrogen fertilizers. The results of field trials in two seasons showed that the use of this extremophilic bacterium CK60 has a positive effect on crop yield, with reductions in UREA input of 20-25%. It also reduces greenhouse gas emissions (N2O) by significant amounts.

[0383] Nitrogen fertilizers applied in excess are highly susceptible to losses, either by leaching (due to their high mobility in the soil in the form of nitrate) or by volatilization (lossas a gas into the atmosphere in the form of nitrous oxide, nitrogen or ammonium). Both cases represent a detriment to the environment and the health of the population.

[0384] Because of a direct relationship between excess application rate and N2O emission flux, a higher nitrogen use efficiency combined with a reduction in application rate adjusted to the maximum rate of return of nitrogen is considered a suitable agricultural strategy.

[0385] With the purpose of proposing a biological tool to accompany the development of a more sustainable agriculture, trials were conducted with P. megaterium CK60 (formulated strain “CK60 formulation”), a microorganism found to be capable of generating a more efficient use of nitrogen by plants, stimulating their growth and productivity with lower inputs of synthetic nitrogen fertilizers.

[0386] Materials and Methods

[0387] Table 11 presents general data of the sites where the trials were conducted. Data on soil characteristics, fertilization, varieties used and planting dates in the trials are presented.Table 11. General data of the sites where the trials were conducted.

[0388] During the 2022 and 2023 seasons, trials on wheat (Trilicum aestivum L.) were conducted at the locations indicated in Table 11 with variable nitrogen inputs in the form of UREA (46:0:0) at the phenol ogical stage corresponding to tillering.

[0389] Similarly, in each location (Table 11), base fertilizations were made at planting with different nitrogen sources and with other essential mineral elements (monoammonium phosphate, diammonium phosphate, triple superphosphate; calcium sulfate). Among the seed therapeutants used in the trials were: Fluxapiroxad (33.3%, at 77 mL / 100 kg of seed);Prothioconazole 3.5g / L + Fluoxastrobin 3.75g / L + Tebuconazole 0.5g / L at 150 mL / 100 kg of seed.

[0390] In all cases, the crop was cultivated with the necessary cultural practices to ensure normal growth (chemical control of weeds, insects and diseases).

[0391] All trials were set up as microplot trials of 8 rows by 15 meters long with 4 replications, arranged in a randomized block design.

[0392] Results

[0393] The yield results obtained in the field trials are presented below (Table 12), as well as the corresponding nitrogen reduction value for each location and year of the trial.Table 12. Yield results obtained in the field trials.

[0394] The following procedure was used to calculate the contribution / compensation of nitrogen uptake by the crop inoculated with CK60 formulation (Table 13, FIGs. 54A-B):• 1- The difference in yield between control 100% and control 75% (or control 80%) was calculated.• 2- The difference in N achieved by reducing 75% (or 80%) of the total requirement (100%) was calculated.• 3- An Equivalent index was calculated by making the difference of the previous items (Kg of grains / Kg of Nitrogen).• 4- The difference in yield between CK60 formulation 75% (or 80%) and 100% control was calculated.• 5- The Equivalent index was applied inversely using the difference in yield from the previous point.• 6- The value of the previous point was added to the difference of Nitrogen (point 2).Table 13. Calculation of Nitrogen uptake / compensation of the inoculated crop with CK60 formulation.

[0395] Yield values in kg / ha in the different treatments did not differ significantly in the years considered, since the value of the difference between the means found among the treatments was less than the PCALT. As can be seen in the graphs in FIG. 26, when seeds were treated with CK60 formulation with a 75% (or 80%) reduction of fertilizer, the yield exceeded the control with 100% fertilizer.

[0396] In other words, the yield compensation measured in kg / ha obtained with the use of CK60 formulation exceeds the yield reduction obtained by reducing nitrogen fertilization by up to 25% (or 20%).Table 14. Analysis of variance for the mean yields obtained in the evaluated treatments.

[0397] By expressing the compensation obtained by the use of CK60 formulation (36 kg N / ha average 2022-2023) in the different types of nitrogen fertilizers available in the market, the following values were obtained:• - in kg of UREA (46-0-0): 78.2• - in liters of UAN (32-0-0): 112.3• - in kg PMA (11-23-0): 327.2• - in kg of PDA (18-20-0): 200

[0398] According to the equivalences of Kg of Urea to CO2 equivalent and its respective equivalence to N2O presented below:• 0.051 kg C per kg Urea - Equivalent value of CO2 released to the atmosphere per kg Urea applied to crop according to Won Kim et al., 2017.• 0.2 kg of C per kg of Urea - Equivalent value of CO2 released to the atmosphere per kg of Urea applied to crop according to Intergovernmental Panel of Climate Change (IPCC) 2021.• 273 kg of CO2 is equivalent to 1 kg of N2O according to the Intergovernmental Panel of Climate Change (IPCC) 2021.

[0399] The amounts of Kg CCheq corresponding to the Kg of UREA not applied to the field would be 15.64 to 3.99, according to the estimation methods, which is equivalent to not emitting 0.057 to 0.015 kg of N2O per hectare of wheat produced.7.8. Example 9: Evaluation of the Potential of P. megaterium CK60 to Promote the Growth of Malting Barley (Hordeum Vulgare L.) Through its Application as a Seed Treatment in the Field

[0400] An exploratory study was carried out under the seed treatment modality in the cultivation of malting barley (Hordeum vulgare L) in the localities of Miramar, Tandil, and Balcarce in the province of Buenos Aires. It was carried out on microplots. Among the treatments were P. megaterium CK60 at 2 doses and a control without inoculation. Yield, protein percentage, and percentage of grains with a caliber of 2.5 mm were evaluated according to local regulations in force. A differential increase in yield was observed depending on the dose of P. megaterium CK60 used and no modification in protein or size was observed, thus maintaining the quality standard for commercialization and industrialization.

[0401] P. megaterium CK60 strain was selected for the development and formulation of a biological fertilizer called CK60 formulation for barley.

[0402] Materials and Methods

[0403] Seed inoculation was conducted using the protocol as described in Example 5 on wheat seeds.

[0404] Trials were conducted in the localities of Tandil, Miramar and Balcarce in barley under seed treatment. Table 15 shows the locations where the trials were conducted, cultivars, type of fertilization, and soil data, among other characteristics. The agricultural cultural practices used were specific for barley cultivation and according to the edaphoclimatic conditions of each locality.Table 15. Locations of malting barley cultivation trials 2023 crop year.

[0405] Several treatments were evaluated, among which the following were developed:• Tl: Absolute control + Fungicide (Fluxapyroxad 33.3% - (75 mL / kg) + Tri ti conazole 10% (25 mL / kg)).• T2: CK60 formulation at a dose of 8 mL / kg seed + Fungicide (Fluxapyroxad 33.3% - (75 mL / kg) + Triticonazole 10% (25 mL / kg)).• T3: CK60 formulation at a dose of 6 mL / kg of seed + Fungicide (Fluxapyroxad 33.3% - (75 mL / kg) + Triticonazole 10% (25 mL / kg)).

[0406] The methodology used was a microplot trial in a completely randomized block experimental arrangement, consisting of experimental units of 7 furrows spaced 0.21 meters apart for a length of 6 meters. Each treatment consisted of 6 experimental units for yield and 3 experimental units for the rest of the variables. For methodological reasons, 3 replicates were used for the corresponding analysis in order to have the same sample number for all the variables analyzed.

[0407] The effect of the application of the treatments and their agronomic efficacy was measured by a comparative analysis of the yields obtained in kg / ha of barley in each of the detailed locations. Protein and caliber values were also analyzed on a 2.5 sieve.

[0408] The statistical technique of analysis of variance was used, applying the DGC test with a significance level of 5% for the comparison of yield means.

[0409] Results

[0410] Performance

[0411] It was observed that the application of CK60 formulation treatment generated an increase in yield compared to the control without inoculation in two of the three locations evaluated (Table 16, FIG. 27). In the location where no yield improvement was observed, yields were close to those obtained by the control without inoculation (Table 16). Theproduct CK60 formulation showed efficacy in its use on malting barley with average increases in kg / ha close to 300 kg / ha for the 6 mL / kg dose, and approximately 500 kg / ha for the 8 mL / kg dose concerning the control treatment without inoculation.Table 16. Yield increase of CK60 formulation treatment versus control without inoculation.

[0412] Protein Percentage and Caliper Percentage on 2.5mm Sieve

[0413] As for the barley quality standards established by the local regulatory entity, the values of protein and caliber on a 2.5 mm sieve were within adequate values in the three locations (Table 17), which indicated that the seed treatment did not alter the quality parameters. This demonstrated efficacy in its use in barley cultivation since it increased yield, as detailed in the previous point, and did not compromise quality standards.Table 17. Protein and caliber values of CK60 formulation treatments vs. control without inoculation.

[0414] The application of CK60 formulation based on P. megaterium CK60 as seed treatment at the tested doses was efficient and effective in obtaining increases in yield and quality of the malting barley crop when compared to the control without inoculation.

[0415] Yield increases with the application of CK60 formulation were observed in two of the three areas evaluated for malting barley, even though these areas had different soil conditions and environmental conditions.

[0416] The treatment with a dose of 6 mL / kg showed a greater difference in the percentage increase values compared to the control without inoculation. For this reason, the application of the CK60 formulation treatment on malt barley at a dose of 6 mL / kg of seed is recommended.7.9. Example 10: Evaluation of the Potential of P. megaterium CK60 to Promote the Growth of Potato (Solatium Tuberosum L.) Through its Application as a Furrow Inoculation

[0417] In the 2023 season, an exploratory study was conducted on potato cultivation under in-furrow inoculation, evaluating P. megaterium CK60 at two doses (500mL / ha and 1000 mL / ha), a commercial control and an absolute control. Vigor, aerial dry weight, and root length at V5 were measured, as well as tuber number, tuber weight, and harvest yield. Inoculation showed significant positive effects, increasing vigor, tuber number, and tuber weight, which resulted in higher yields. P. megaterium CK60 stood out in all variables compared to the non-inoculated and commercial controls.

[0418] The aim of the study was the promoting effect of extremophilic microorganisms in field conditions.

[0419] To understand when and how these microorganisms benefited crop growth and yield, a series of evaluations were carried out at different specific phenological moments and were aimed at interpreting the symbiotic and synergistic relationship between the two. The evaluations were oriented to quantify the effect on plant vigor, and on root development by dry weight and length. During the reproductive stage, they were oriented on the numerical components of yield and the final yield obtained.

[0420] Since yield (tn / ha) is a variable generated as a result of the stimulus to growth and normal development experienced by plants throughout their life cycle, the assessment of the positive effect of extremophilic microorganisms on the potato crop was defined through theincrease in yield achieved. The following summarizes the evaluations and results obtained for the product CK60 formulation based on the P. megaterium CK60 strain.

[0421] Materials and Methods

[0422] The experimental design used was “Blocks Completely Randomized” (BCA) with 6 treatments and 4 replications. The experimental unit (plot) consisted of 4 furrows of 10 m long with a distance between furrows of 0.80 m and aisles between replicates of 1 m long. The applications for weed, pest and disease control respected the agronomic management carried out in the establishment according to need, in order to avoid variations in the trial site.

[0423] The field was prepared for planting the crop. It was corroborated that the pressure of the equipment used was constant and that the flow rate was uniform. In the field, planting lines were opened and seed potatoes were placed at the bottom according to the established planting density. Inoculation was then carried out by applying the liquid on the seed potatoes in the planting line, at the indicated dose. After the application, the planting line was closed with the same soil.

[0424] As for the system used in the treatments, the same planter was used at the time of planting with the in-furrow spraying system for the incorporation of the products. Table 18 shows the data pertinent to the trial and Table 19 shows the treatments / doses evaluated. The commercial treatment used for the comparison is registered for potato and consists of a mixture of Azospirilum brasilense and Pseudomonas fluorescens.Table 18. Planting density, fertilization, Evaluation of extremophiles in the potato crop.Table 19. Doses and treatments evaluated. Evaluation of extremophiles in potato crop, season 2023-2024 Tucuman-Argentina.

[0425] Results

[0426] InfoStat statistical software was used for data analysis. Given the low variability among treatments (low overall CV%) within the tested locality, it was not considered necessary to block additional cases. An analysis of variance (ANOVA) with DGC test at 5% significance level was performed to evaluate the effect of inoculation.

[0427] Vigor

[0428] This was carried out at phenological stage V5 using a score from 1 to 5, always assigning the mean value (3) to the control and comparing the rest of the plots with the same score, where 5 was the best value and 1 the worst. FIG. 28 shows that the treatments with CK60 formulation showed significant differences for this variable, presenting better vigor compared to the control without inoculation and equaling the commercial control used at a dose of lOOOcc / ha.

[0429] Root Dry Weight and Root Length

[0430] Both variables were evaluated at the phenological stage of V5, as shown in FIGs. 29 and 30, and there were no significant differences between the treatments evaluated.

[0431] Number of Tubers per Linear Meter

[0432] This is one of the most important numerical components in this crop, since a greater number of tubers per linear meter can reflect a higher productivity of the crop, provided that these tubers reach an adequate commercial size. An increase was observed in the number of tubers per linear meter for CK60 formulation of +5.8% for the 500 cc / ha dose and +18.6% for the 1000 cc / ha dose compared to the absolute control, while the commercial control presented a decrease of -4.5% compared to the absolute control (FIG. 31).

[0433] Weight of Thousand Tubers in kg

[0434] For this variable, significant differences were observed between the inoculated treatments and the control without inoculation, with an increase of approximately +10 to +20% depending on the treatment (FIG. 32), with the CK60 formulation treatments showing the greatest increase. Quantitative differences of +4 to +10% for CK60 formulation were observed with respect to the commercial control, depending on the dose.

[0435] Performance

[0436] For this variable, although no statistically significant differences were observed, there is a marked quantitative difference between CK60 formulation at both doses, showing differences of +1.6 to +2.8 tn / ha with respect to the control without inoculation, depending on the dose (FIG. 33). This would be equivalent to an increase of 80 to 140 bags of 20 kg of potato. Quantitative differences of +0.8 to 2 tn / ha of potato were also observed compared to the commercial control.

[0437] In-furrow inoculation of potato showed a significant positive effect, promoting greater vigor, an increase in the number of tubers per linear meter and an increase in tuber weight. These combined effects generated an increase in crop yield.

[0438] The adequate performance of CK60 formulation at both doses for the different variables in comparison with the control without inoculation and the commercial control was highlighted.

[0439] The results obtained reaffirmed the efficacy of biological treatments on potato crops, contributing to the sustainable and productive management of horticultural systems.7.10. Example 11: Evaluation of P. megaterium CK60 as Seed Treatment in Peanut (Arachis Hypogaea L.) Under Greenhouse Conditions. Campaign 2023-2024, Tucuman-Argentina

[0440] During the summer season of 2023-24, the promoter effect of P. megaterium CK60 (CK60 formulation) applied as a treatment on the sowing line in the peanut crop was evaluated. The results of the trial show a positive effect of the use of P. megaterium CK60 on the variables analyzed. A positive effect of the use of CK60 formulation was observed directly impacting on the numerical components of yield.

[0441] P. megaterium CK60 (commercially CK60 formulation) was selected to evaluate its effect on peanut crops.

[0442] Materials and Methods

[0443] The crop was planted on December 15, 2023, in 20-liter pots filled with MultiPro substrate from the company Terrafertil. Before planting, the pots were leveled to achieve a flat planting bed. It was verified that the pressure of the equipment used was constant and that the flow rate was uniform. In each pot, 6 cm deep planting lines were opened; the seeds were placed at the bottom of the planting line in each pot at the established planting density.Inoculation was then carried out by applying the liquid on the seeds in the sowing line, at a dose of 10 ml / kg of seed with an application rate of 20 liters of water per hectare. After the application, the sowing line was closed with the same substrate as the pot and pressure was applied to ensure that the seeds remained in contact with the substrate.

[0444] The trial was carried out under irrigation, using a planting density of ± 150,000 pl / ha (density used in the productive establishments of this latitude), with a separation of 0.70 m between plant rows and 0.10 m between plants. Peanut runner type granoleic variety was used, whose seeds were treated with fungicide (Fludioxonil 2.5% + Metalaxyl-M 3.75%) at 100 mL / 100 kg of seeds. The application of the biological products was carried out on the sowing line, on the seed (simulating the inoculation carried out in the field).

[0445] The trial was conducted up to phenological stage R8 (harvest maturity).

[0446] The treatments were:• Tl: Control (without biological products)• T2: CK60 formulation 10 cc / kg

[0447] The variables analyzed were selected with the objective of comparing crop growth and yield among the selected treatments.

[0448] In relation to growth (defined as the irreversible increase of dry matter in different organs), the variable considered was the dry weight of the aerial part of the plant.

[0449] The SPAD variable, or leaf greenness index, expresses the amount of chlorophyll in the leaf and is an indicator of the nutritional status of the plant. SPAD was measured on the leaf corresponding to the penultimate node developed. Four SPAD readings were taken on each tetrafoliate leaf, one for each leaflet, from which the average value of the leaf was obtained for each plant. The measurement was taken at phenological stage R2.

[0450] Regarding yield, the variable taken as a variable was the weight of seeds / pl. for each treatment. In addition, the variables number of grains / pl. were included, considered as a direct component of yield.

[0451] Results

[0452] Table 20 shows the average values of the variables.Table 20. Average values of the variables analyzed for the different treatments

[0453] Greenness Index-SPAD

[0454] As shown in FIG. 34, no significant differences were observed in this variable for the treatments evaluated.

[0455] Dry Weight per Plant

[0456] When evaluating plant dry weight, CK60 formulation showed an increase in dry matter of +38.6% with respect to the uninoculated control (FIG. 35).

[0457] Number of Grains per Plant

[0458] Regarding the variable number of grains, CK60 formulation showed an increase of +76% with respect to the control without inoculation (FIG. 36).

[0459] Grain Weight per Plant

[0460] When analyzing grain weight per plant or yield per plant, as with the previous variable, CK60 formulation showed an increase of more than 80% (FIG. 37).

[0461] The treatment representing CK60 formulation presented an excellent performance for the variables aerial dry weight, grain weight and grain number, which are the constituents of crop yield.7.11. Example 12: Evaluation of P. megaterium CK60 as Plant Growth Promoter (PGPM) in Lettuce Hydroponic System

[0462] Objective

[0463] The objective of the study was to evaluate the efficacy of CK60 treatment as a PGPR in two hydroponic systems: Floating and NFT, using a foliar application.

[0464] Materials and Methods

[0465] Seeds were sown in a moistened substrate, covered with vermiculite, and irrigated regularly to maintain adequate humidity. After seven days, when the seedlings had developed1-2 true leaves, they were fertilized with 15 g / 100 L of a mixture with NPK and micronutrients composition (15% nitrogen, 5% phosphorus, and 30% potassium; it also includes micronutrients such as iron (0.1%), manganese (0.1%), zinc (0.05%), copper (0.005%), boron (0.02%), and molybdenum (0.001%)).

[0466] Once the plants had developed 3-4 true leaves, they were transferred to a hydroponic system: Nutrient film technique (NFT) and floating. After 24 hours of acclimatization, the fertilization process was repeated under the same conditions.

[0467] CK60 was activated by culturing in nutrient broth and then prepared for application.

[0468] Two foliar applications were made every 10 days, ensuring complete coverage of the plants to the point of runoff.

[0469] pH (5.5-6.5) and electrical conductivity (1.6 - 1.9 dS / cm) were monitored and adjusted weekly to maintain optimal conditions.

[0470] The trial was completed 50 days after the transplant.

[0471] Treatments• Control without biological treatment• P. megaterium CK60

[0472] Measured parameters• Root fresh weight• Aerial fresh weight• Total fresh weight• Number of sheets

[0473] Results

[0474] In both hydroponic systems, Floating and NFT, greater growth was observed with the CK60 treatment compared to the control (FIGs. 38 and 39). The plants treated with CK60 showed improved development, indicating its potential as a plant growth-promoter (PGPR). In the floating system, the treated plants weighed 173 g, while in the NFT system, it was 281 g. These results suggest that the CK60 treatment enhances plant growth in both systems, and it is a promising alternative for improving crop yields in hydroponic cultivation.7.12. Example 13: Evaluation of CK60 as Plant Growth Promoter (PGPM) in Tomatoes Semi Hydroponic System

[0475] Objective

[0476] The objective of the study was to evaluate the efficacy of CK60 treatment as a plant growth promoter (PGPR) in tomatoes in semi-hydroponics systems in immersion and foliar application.

[0477] Materials and Methods

[0478] Plant Material

[0479] Solanum lycopersicum L. (common name: tomato), cv. ‘Chalchalero’

[0480] Bioassay 1: Immersion Inoculation

[0481] A completely randomized design with five replicates per treatment, was conducted in pots. Treatments including CK60 and control without a biological treatment, were applied by immersion to tomato plants of the cv. ‘Chalchalero’ at 15 days postgermination under controlled phytotron conditions, alongside their respective control plants. After 7 days, plants were transferred to final pots under greenhouse conditions. Total plants for Bioassay 1 = 25.

[0482] Bioassay 2: Foliar Application

[0483] A completely randomized design with five replicates per treatment was conducted in pots. Treatments, including CK60, were applied as a monthly foliar spray at a concentration of IxlO8CFU / ml on tomato plants of the cv. ‘Chalchalero’ throughout the growing season, alongside their respective control plants. The total number of plants for Bioassay 2 = 25.

[0484] Results

[0485] In both semi-hydroponic systems, immerse and foliar application, greater fruit production was observed with the CK60 treatment compared to the control. The plants treated with CK60 showed an increase in fruits number and weight, indicating the potential of CK60 as a plant growth-promoter (PGPR). Immersion was more efficient than foliar application (FIG. 40). These results suggest that the CK60 treatment enhances tomatos production in both systems, and it is a promising alternative for improving crop yields in semi-hydroponic cultivation.7.13. Example 14: Antibiotic susceptibility CK60 Biosafety

[0486] Strain CK60 was identified as P. megaterium. It is a genus of Gram-positive, aerobic, rod-shaped bacteria, has a low G+C genome (-38%) and forms endospores. Prestia members are species that formerly belonged to Bacillus. P. megaterium is a ubiquitous organism in the environment around us. Besides being a common soil bacterium and an endophyte, it can be found in honey, wine, raw meat , fish, seawater , and the oral cavity of humans. P. megaterium offers a set of advantages that have made them an ideal organism for industry for more than 50 years (. Several authors postulate it as one of the first biotechnological producers of vitamin B12, there are reports of strains capable of producing polyhydroxybutyrate (biodegradable plastics), and, among other properties, it is used as an inoculant for agriculture.

[0487] In the current market, Prestia megaterium is a component of several commercial products, among which are:

[0488] Tango R (Mexican product). Inoculant formulated with five beneficial bacteria; Bacillus sublilis. Bacillus licheniformis, Bacillus megaterium, Pseudomonas fluorescens and Azotobacter sp.

[0489] Gaia R. Megaterium, product manufactured in Cordoba, Argentina.

[0490] Bactrium, produced by Atens, a Spanish company.

[0491] Although P. megaterium is considered a non-pathogenic microorganism, the biosafety of strain CK60 was evaluated by means of an antibiotic resistance assay in addition to the evaluation of hemolytic activity.

[0492] Antibiotic Resistance

[0493] The antibiotic resistance of the CK60 strain was evaluated using the guidelines established by the Clinical and Laboratory Standards Institute (CLSI).

[0494] Antibiotic susceptibility testing was carried out using the agar diffusion method, which follows the guidelines set by CLSI. This method involves assessing the ability of antibiotics to inhibit bacterial growth on an agar plate. The results obtained were classified according to the cutoff points defined by CLSI. These values are updated periodically and are specific to each type of microorganism and antibiotic. Therefore, the results were interpreted based on these cutoff points, and each antibiotic tested was classified as Sensitive, Intermediate, or Resistant.• Sensitive (S): The microorganism is susceptible to the action of the antibiotic at achievable concentrations in the body.• Intermediate (I): The microorganism shows a partial response to the antibiotic. In some cases, the antibiotic may be effective if higher concentrations are reached.• Resistant (R): The microorganism is not inhibited by the antibiotic at achievable concentrations in the body, suggesting that it will not respond to treatment with that antibiotic.

[0495] All results were recorded following standard laboratory documentation procedures.

[0496] Table 21 presents the antibiotic susceptibility results for CK60.Table 21. Antibiotic susceptibility test results for the CK60 strain.

[0497] Keys: S = Susceptible or Sensitive > 15 mm diameter; I = Intermediately Susceptible / Sensitive 10-15 mm diameter; and R = Resistant < 10 mm diameter.

[0498] Hemolysis in Blood

[0499] Following the methodology of hemolytic activity of Delftia lacustris PPO-1, the blood-agar test for CK60 was performed. FIG. 41 demonstrates the strain’s inability to produce beta hemolysis on blood (no hemolytic halos are observed), which may indicate a non-pathogenic characteristic of the strain.

[0500] Resistance to Chemical Products

[0501] The tolerance of CK60 to different chemicals used in the field was evaluated. Table 22 summarizes the results obtained.Table 22. The tolerance of CK60 to different chemicals.R = resistant, therefore compatible with the product analyzed7.14. Example 15: Evaluation of culture media for CK60 spores

[0502] The objective of a culture medium for spore formulation is to cultivate the microorganism (P. megaterium CK60) under specific conditions (such as nutrient depletion, temperature fluctuations, pH changes) that trigger the sporulation process. This enables the cells to form spores, which are more resilient to harsh environments, ensuring that the microorganisms can survive until the conditions become more favorable for growth. The use of spores in the formulation has advantages related to durability and stability. Spores are highly resistant to environmental stressors such as heat, desiccation, and UV radiation.Additionally, spores can germinate and grow rapidly when the conditions are favorable. This can be beneficial for applications in agriculture, where quick colonization or microbial activity is needed.

[0503] The presence of specific salts in the culture media can help to mimic stress conditions (such as nutrient limitation, changes in osmotic pressure, or metal ion depletion) that naturally trigger the sporulation process in microorganisms. Calcium chloride, magnesium sulfate, potassium chloride, and iron (III) chloride are some of the most common salts used in culture media to induce sporulation.

[0504] Calcium chloride - Role in Sporulation: Calcium ions are involved in the formation of the spore cortex and the development of spore resistance.

[0505] Magnesium sulfate - Role in Sporulation: Magnesium is another important ion that influences sporulation. It is involved in enzyme activation and other metabolic processes that can trigger sporulation under nutrient limitation or stress conditions.

[0506] Potassium chloride - Role in Sporulation: Potassium ions are important for maintaining cellular osmotic balance and enzyme function. Potassium chloride in the culture medium can help induce sporulation, particularly when the cells are exposed to nutrient depletion or other stress factors.

[0507] Iron (III) chloride - Role in Sporulation: Iron deficiency or the presence of specific iron salts (like iron chloride) in the medium can sometimes trigger sporulation in bacteria, especially in response to nutrient scarcity or environmental stress.

[0508] Sodium chloride - Role in Sporulation: Sodium chloride can influence the osmotic pressure in the medium. While it is typically used to maintain osmotic balance, in high concentrations, sodium chloride may induce stress that could lead to sporulation in some microorganisms.

[0509] Phosphate salts - Role in Sporulation: Phosphate is essential for energy metabolism and cellular signaling. In some species, phosphate depletion in the medium can induce sporulation. Additionally, high concentrations of phosphate salts can be used to trigger sporulation under certain conditions.

[0510] Ammonium sulfate - Role in Sporulation: Ammonium salts, particularly ammonium sulfate, can sometimes stimulate sporulation in certain bacterial species when nitrogen becomes limited or when other nutrients are in low supply. Ammonium ions can trigger the metabolic pathways that lead to sporulation

[0511] Polymers - Functions: In the preparation of culture media, polymers serve various functions that can enhance the physical and chemical properties of the medium, as well as facilitate the growth and development of microorganisms or cells in culture. Some polymers can encapsulate nutrients releasing them in a controlled and prolonged manner. Other polymers can retain large amounts of water, maintaining moisture in the culture medium and promoting cell growth. The incorporation of polymers can modify the viscosity, elasticity, and other physical properties of the medium, adapting it to the specific needs of the culture.

[0512] Polymers - Examples: Xanthan gum

[0513] The media and components tested for the sporulation of CK60 are described in Table 23. The spores are expressed in CFU / mL.

[0514] Table 23. CK60 spores (CFU / mL) in different culture media tested for sporulation.7.15. Example 16: Evaluation of culture media for CK60 (vegetative)

[0515] A culture medium is a nutrient-rich solution designed for the proliferation of microorganisms under controlled conditions. These media provide essential nutrients and conditions for the survival and reproduction of cultured organisms.

[0516] The key components of culture media and their functions are described below.

[0517] Carbon Source - Functions: Serves as the primary energy source and a fundamental building block for cellular components.

[0518] Carbon Source - Examples: Some simple carbon sources used are glucose (dextrose), sucrose, stachyose, glycerol, and maltodextrin. These organic compounds are easily metabolized by microorganisms. Other complex carbon sources used in culture media are yeast extract, meat extract, peptones, caseins, and strach, among others.

[0519] Nitrogen Source - Functions: Essential for the synthesis of amino acids, nucleic acids, and proteins, which are vital for cell growth and function.

[0520] Nitrogen Source - Examples: Ammonium salts or nitrates are typical nitrogen sources incorporated into culture media. Some inorganic nitrogen sources used in culture media are ammonium sulfate, ammonium nitrate, potassium nitrate, and sodium nitrate.

[0521] Phosphorus Source - Functions: Crucial for the formation of nucleic acids (DNA and RNA) and ATP, the energy currency of the cell.

[0522] Phosphorus Source - Examples: Phosphates, such as monopotassium phosphate and dipotassium phosphate are commonly included in culture media for formulations.

[0523] Sulfur Source - Functions: Integral for synthesizing certain amino acids (like cysteine and methionine) and vitamins, contributing to protein structure and function.

[0524] Sulfur Source - Examples: Sulfates, such as magnesium sulfate, provide sulfur in culture media.

[0525] Magnesium and other minerals - Functions: Act as cofactors for various enzymatic reactions and are important for maintaining cell membrane stability and function.

[0526] Magnesium and other minerals - Examples: Magnesium sulfate supplies magnesium ions, while calcium chloride provides calcium ions.

[0527] Trace elements - Functions: Required in minute amounts, these elements are essential for the activity of specific enzymes and overall cellular metabolism.

[0528] Trace elements - Examples: Elements like iron, zinc, and cobalt are often included in trace element solutions added to culture media.

[0529] Vitamins and growth factors - Functions: Facilitate various metabolic pathways and support growth, especially for fastidious organisms that cannot synthesize these compounds.

[0530] Vitamins and growth factors - Examples: B-vitamins, such as thiamine and riboflavin, are commonly supplemented in culture media.

[0531] pH Buffers - Functions: Maintain the optimal pH range for organism growth by neutralizing acids or bases produced during metabolism.

[0532] pH Buffers - Examples: Phosphate buffers are frequently used to stabilize pH levels in culture media.

[0533] Polymers - Functions: In the preparation of culture media, polymers serve various functions that can enhance the physical and chemical properties of the medium, as well as facilitate the growth and development of microorganisms or cells in culture. Some polymers can encapsulate nutrients releasing them in a controlled and prolonged manner. Other polymers, can retain large amounts of water, maintaining moisture in the culture medium and promoting cell growth. The incorporation of polymers can modify the viscosity, elasticity, and other physical properties of the medium, adapting it to the specific needs of the culture.

[0534] Polymers - Example: Xanthan gum, polyvinylpyrrolidone, and carboxymethylcellulose

[0535] The objective of a culture media formulation was to grow the microorganism (P. megaterium CK60) in optimal conditions testing different sources such as carbon, nitrogen, phosphorus to maintain its plant growth-promoting properties. In the present case, at least 1E+3 colony-forming units per milliliter (CFU / mL) of vegetative cells needed to be reached. The media and components tested for the best growth of CK60 are described in Table 24. The vegetative growth is expressed in CFU / mL.Table 24. CK60 growth (CFU / mL) in different culture media tested.7.16. Example 17: Molecular Characterization of CK100

[0536] The molecular identification of CK100 was performed. First, conventional PCR using universal primers or primers to amplify the 16S rDNA region was performed. The primers used were 1492r (5'-TACCTTGTTACGACTT-3') (SEQ ID NO: 14) and 8f (3'- AGAGTTTGATCCTGGCTGGCTCAG- 5) (SEQ ID NO: 15) or 27f (31- AGAGAGTTTGATCCTGGCTCAG-51) (SEQ ID NO: 16). Once the 16S sequence was received, it was compared with public databases (e.g. BLAST: Basic Local Alignment Search Tool - NCBI) to find, if any, similarities with other microorganisms already registered and to elucidate the genus of the microorganism.

[0537] Second, the complete genome sequencing of this strain allowed performance of the ANI analysis, and CK100 was identified as P. megaterium.

[0538] Specific primers that amplified a specific sequence of DNA, belonging to this microbe, were designed and identified following the same steps described for the CK60 strain.

[0539] P. megaterium CK100 Specific Strain PCR Sequence and Primers

[0540] >CK100_specific_sequence

[0541] CCCACCAGCATTAACTACCCATAGGTCCACACATAAAGACACTCTAGA ATCAACCTTCCTTTTTTTGTTTTTACTTATTATAGAAAATAAAAAAAGAATGTGGG CAAAGCATTTCGGAATAGTCAATAGCGGTAA (SEQ ID NO: 8)

[0542] >CK100_ss_Fw

[0543] CCCACCAGCATTAACTACCC (SEQ ID NO: 17)

[0544] >CK100_ss_Rv

[0545] TTACCGCTATTGACTATTCCGA (SEQ ID NO: 18)

[0546] Furthermore The biochemical characterization of CK100 as plant growthpromoting bacteria was carried out in vitro at 30 °C. Bacterial suspensions (107-108 CFU / mL) previously grown in nutrient broth medium (BRITANIA) were used unless another specific medium is indicated. These cultures were used as inocula in the examples below.7.17. Example 18: Resistance of CK100 to Different Chemicals

[0547] The tolerance of CK100 to different chemicals used in the field was evaluated.Table 25 summarizes the results obtained.

[0548] It can be seen that CK100 is resistant to a variety of chemical compounds used in crops, which shows that they can be used in combination with them.7.18. Example 19: Physiological Characterization of CK100 (Mode of Action)

[0549] The growth-promoting capacity of CK100 results from a synergistic effect of several mechanisms. This microorganism stands out for its ability to fix atmospheric nitrogen, produce ammonium, solubilize phosphorus, and synthesize phytohormones, such as indole-3 -acetic acid (IAA), among other plant hormones. Additionally, CK100 produces siderophores and exhibits enzymatic activity, including proteases, cellulases, and chitinases, all contributing to its growth-promoting ability and improving crop conditions.

[0550] One of the key mechanisms of CK100 is its nitrogen availability, an essential nutrient for plants. CK100 has been shown to fix atmospheric nitrogen, a crucial process in conditions where traditional nitrogen sources are limited. Additionally, the strain can produce ammonium from complex nitrogen compounds, allowing it to mineralize nitrogen and release it in forms easily assimilated by plants. CK100 also has the ability to perform nitrification, transforming ammonium into nitrate, a nitrogen source quickly absorbed by plants. Its urease activity facilitates the breakdown of urea, releasing ammonia that can be utilized both by theplant and the microorganism itself, contributing to the nitrogen cycle and enhancing soil fertility.

[0551] Furthermore, CK100 improved the availability of essential macronutrients for plants, such as phosphorus and potassium. This strain solubilizes phosphates from insoluble inorganic sources by producing organic acids and excreting siderophores, which act as metalchelating agents, releasing phosphates that become available for plant uptake. Additionally, CK100 can solubilize potassium from insoluble inorganic sources, improving the availability of this essential nutrient.

[0552] CK100 is also notable for its ability to produce phytohormones, which play a crucial role in promoting plant growth. Among the phytohormones synthesized by CK100 are salicylic acid (SA), indole-3 -acetic acid (IAA), indole-3 -butyric acid (IB A), jasmonic acid (JA), cytokinins such as kinetin (KIN), zeatin (ZEA), and 6-benzylaminopurine (6 BAP), as well as abscisic acid (ABA) and gibberellic acid (GA3). Among these, IAA (indole-3 -acetic acid) stands out for its critical role in promoting root development, cell elongation, and overall plant growth. IAA is a key auxin that plays a central role in the regulation of plant growth processes, and CKlOO’s ability to produce this phytohormone enhances its potential to positively influence plant root architecture and overall vigor.

[0553] CK100 also excels in the production of jasmonic acid (JA), abscisic acid (ABA), and gibberellic acid (GA3). JA is associated with the plant's defense response against pathogens. ABA regulates plant water status, enhances drought tolerance, and plays a key role in stress response by promoting stomatai closure. GA3 plays a key role in seed germination, stem elongation, and root development. This phytohormone enhances plant height and biomass in different crops. The production of these phytohormones by CK100 contributes not only to direct plant growth but also to plant adaptation to stress conditions, with IAA further promoting root branching and growth under stress conditions.

[0554] In addition to its phytohormone production, CK100 is a microorganism that produces lytic enzymes, such as proteases, cellulases, and chitinases, which facilitate the degradation of the cell wall and contribute to its biocontrol against phytopathogens. The production of siderophores is also linked to this mechanism, as they compete for essential nutrients with pathogens. To confirm its potential as a biocontrol agent, antagonism tests against specific pathogens are needed.

[0555] To gain a deeper understanding of the effect of CK100 as a plant growthpromoting rhizobacteria (PGPR), the composition of the root exudates (or rhizodeposits) from wheat seedlings inoculated with CK100 was analyzed by determining and quantifying bioactive molecules, including organic acids and flavonoids. The treatments analyzed were as follows:

[0556] Negative control: no inoculation (NC)

[0557] Positive control: Azospirillum brasilense (PC)

[0558] CK100

[0559] The results obtained are presented in FIG. 42, which illustrates a profile of bioactive compounds determined in the rhizodepositions of wheat seedlings (control and inoculated with CK100 and Azospirillum brasilense'). These compounds were identified and quantified using a liquid chromatograph coupled with tandem mass spectrometry (LC- MSMS, Waters Alliance 2695 and Micromass Ultima PT). Quantification was carried out using established calibration curves for each analyte with known concentrations. Monitoring was performed in Multiple Reaction Monitoring (MRM) mode, comparing the analyte in question with those of pure standards, both in terms of transitions and retention time (RT). Calculations were based on the fresh weight of the roots deposited in the nutrient solution; therefore, the concentrations for all compounds are expressed as ng / g of fresh root weight, *except for luteolin, which is expressed in pg / g of fresh root weight. Statistical analysis was conducted to determine significant differences in the amounts of each bioactive compound under the different conditions tested (Analysis of variance, DGC test: means with a common letter are not significantly different (p > 0.05)).

[0560] According to these results, CK100 positively affected the production of certain secondary metabolites, such as apigenin, daidzein, genistein, and luteolin, while no citric acid, chryseine, naringin, or malic acid was detected in the root exudates. Apigenin, daidzein, and genistein are known for their antioxidant and antimicrobial properties, while luteolin contributes to stress tolerance and defense mechanisms in plants. These results suggested that CK100 may influence the synthesis of metabolites with antioxidant and defensive properties, potentially contributing to plant adaptation to stress and promoting plant growth.Analysis of Results for Biochemical Characterization of CK100

[0561] Table 26 below summarizes the results obtained for the biochemical characterization of CK100 as plant growth promoters of Examples 17-19.Table 26. Summary of plant growth-promoting capabilities of CK100.

[0562] CK100 exhibited protease and cellulolytic activity. It grew in colloidal chitin medium, without visible halo formation. It did not exhibit catalase activity, and it did not produce HCN. It was characterized by its ability to solubilize phosphorus, produce siderophores and synthesize auxins such as AIA. A production of 35 pg / mL of AIA was quantified after 48 h of incubation (below CK1 contro 1= 60 pg / mL. CK100 did not exhibit ACC deaminase activity.7.19. Example 20: Recovery of P. megaterium CK100 from Wheat Plants by qPCR: Foliar Treatment

[0563] The aim of the study was the recovery of CK100 formulation at different times post-foliar treatments from the wheat plants using a molecular biology technique: qPCR.

[0564] Methods and Results

[0565] The wheat plants were independently treated with CK100 using foliar treatments. The temperature of the place chosen to work was no higher than 30°C, and environments with high relative humidity (steam saturated environments) was avoided. It was verified that the pressure of the equipment used was constant and that the flow rate was uniform. A Jacto backpack model DJB-20 was used, with a JSF 110 01 flat fan sprayer. The application rate was 80 liters of water / hectare. The Jacto backpack was filled to half volume with water, then the inoculant was poured into the backpack and the backpack was brought to fullvolume. The mechanical agitators of the backpack were turned on for 5 minutes to achieve a correct mixing of the inoculant in the application vehicle (water). The application proceeded at a constant speed through the use of a step indicator provided by the Jacto backpack. The application was carried out with total coverage. The application was made in the late afternoon, ensuring that the applied plants were not exposed to direct sunlight or high temperatures.

[0566] To understand how CK100 colonizes the wheat plant over time the qPCR technique with specific primers designed for CK100 was used. In qPCR, fluorescent dyes that bind to double-strand DNA are incorporated into the PCR reaction, and the formation of the product is monitored in real-time after each PCR cycle. The dye is added to the reaction, and the fluorescence is measured in each PCR cycle. Since the fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored by the increase in fluorescent signal. The main result of a real-time PCR experiment is the amplification curve, which shows the accumulation of the amplified product in the form of a fluorescent signal relative to the number of cycles.

[0567] The accumulation of CK100 genetic material was evaluated at three different days post-infection (5, 12, and 19 dpi). The roots and root exudate of the wheat plants treated with 2750 cm3 / h of these microorganisms were studied.

[0568] To examine the presence of CK100 in wheat plants, DNA from different parts (root and root exudate) of the plants was purified using a commercial Soil DNA Extraction Kit. After that, purified DNA was used to carry out qPCR using specific primers for CK100.

[0569] The Presence of CK100 in Wheat Plants Over Time

[0570] As can be seen in FIG. 43, CK100 formulated, 5 dpi is in the roots and root exudate, but the DNA concentration in the last one is less. The DNA concentration decreases over time in the roots part. Meanwhile, the DNA concentration in root exudates increases after 5 dpi and is stable with time.

[0571] Conclusions

[0572] CK100 was found in the root and root exudate of the wheat plant at least 19 dpi.No CK100 was found in the control.

[0573] The DNA concentration of CK60 was larger than CK100 in roots and root exudates in wheat plants, suggesting that CK60 has better translocation than CK100.

[0574] It can be concluded, that CK100 is translocated to roots and root exudates when applied as a foliar treatment being capable of colonizing the vegetal tissues.7.20. Example 21: Growth promotion Trial in Maize (Zea mays Linn) by Seed Treatment with P. megaterium CK100 Under Greenhouse Conditions

[0575] Tests were conducted on the com crop under controlled conditions, simulating the planting framework and management characteristic of the area. Throughout the crop cycle, height, aerial dry weight and greenness index were measured at different phenological stages. Also, the number of grain rows, number of grains and weight of grains per plant were recorded. Three treatments were evaluated: P. megaterium CK100, a control without inoculation and a commercial control composed of Azospirillum brasilense and Pseudomonas fluorescens, applied by seed treatment prior to planting. The results showed positive quantitative differences in greenness index, number of kernels and kernel weight per plant in favor of the P. megaterium CK100 strain compared to the commercial control and the absolute control.

[0576] The maize (Zea mays') crop is one of the main pillars of agriculture worldwide, playing a fundamental role in food security and various industries. However, adverse environmental conditions, such as drought, high temperatures or saline soils, can significantly limit its productivity. In this context, extremophilic microorganisms have attracted increasing interest due to their ability to survive and thrive in extreme environments, offering potential biotechnological applications to improve resilience and productivity in maize cultivation. The main objective was to evaluate under controlled conditions the impact of different strains of extremophilic microorganisms on maize growth and development, with a view to identify sustainable strategies to increase agricultural productivity in challenging scenarios. The comparison of the Priesta megaterium CK100 strain with a non-inoculated control and a commercial control under the preplant seed treatment modality was developed.

[0577] Materials and Methods

[0578] The crop was planted on December 14, 2023, in 20-liter pots, with MultiPro substrate from Terrafertil. The trial was carried out under irrigation, using a planting density of ± 60,000 plants / ha (density used in the productive establishments of this latitude), with a separation between plant rows of 0.60 m and 0.30 m between plants. A DUO 225 PWU corn variety was used. The seeds were treated with a mixture of fungicides (Thiabendazole 30% + Fludioxonil 3.75% + metalaxyl-M 3% + Azoxystrobin 1.5%) at 100 mL / 100 kg of seeds, andinsecticide (Clotianidin 60%) at 0.5 mL / 1000 seeds. The trial was conducted until phenol ogical stage R6 (physiological maturity). Table 27 shows the treatments and doses used.Table 27. Treatments evaluated in the corn crop under controlled conditions.

[0579] The variables analyzed were selected with the objective of comparing growth, vegetative development and crop yield. In relation to growth (defined as the irreversible increase of dry matter in different organs), the variables considered were height and aerial dry weight. Height measurements were made in correspondence to the advance of the phenological stage, being the point of comparison developed in VT (anthesis), since this is the phenological stage in which the height growth of the plant culminates. Height was taken from the ground to the visible collar of the flag leaf.

[0580] Aerial dry weight was determined at the end of the cycle. The plant was considered without reproductive structures in this variable. The material was dried in a forced ventilation oven at 60 °C until a constant weight value was reached.

[0581] The SPAD variable, or leaf greenness index, expresses the amount of chlorophyll in the leaf and is an indicator of the nutritional status of the plant. It was measured with SPAD in three positions on the plant: leaf +1, -1 and on the spike leaf. The measurement was made between R1-R2.

[0582] Regarding yield, the seed weight was taken as a variable for each treatment with a moisture content of 13.5%, which determines the time of harvest. In addition, the variables number of grains / pl and number of rows / cob, which are considered yield components, were included.

[0583] Results

[0584] Height to Flag Leaf

[0585] As shown in FIG. 44, this variable does not show significant differences among the treatments evaluated.

[0586] Average SPAD Index

[0587] FIG. 45 shows that the use of CK100 in the treatment of corn seeds increased the SPAD by an average of 2 points with respect to the control without inoculation, despite not presenting significant differences. The increase in chlorophyll observed ranges approximately 5% with respect to the control without inoculation and is superior to the commercial control.

[0588] Vegetative Air Dry Weight

[0589] FIG. 46 shows that there were no significant differences between the treatments evaluated for this variable. However, there was a notorious difference in the aerial dry weight achieved with the use of CK100 as opposed to the commercial product used in corn.

[0590] Number of Rows of Grains per Cob

[0591] FIG. 47 illustrates that this variable did not show significant differences among the treatments evaluated. CK 100 has a lower number of rows with respect to the control treatment, but a higher number with respect to the commercial treatment.

[0592] Number of Grains per Plant

[0593] Number of Grains per Plant was one of the most relevant numerical components in the construction of yield. FIG. 48 shows that the treatment corresponding to CK100 presented a quantitative increase of +11.78% with respect to the control without inoculation and +7.8% compared to the commercial control, despite not showing significant differences.

[0594] Grain Weight at 13.5% in Grams

[0595] FIG. 49 illustrates the behavior of CK100, which showed increases with respect to both controls. Compared to the control without inoculation, CK100 showed an increase of +6.78%. Compared to the commercial control, CK showed an increase of +18%.

[0596] Conclusions

[0597] The use of Priesta megaterium strain CK100 as seed treatment showed an increase in yield, kernel number and greenness index in maize. No significant differences were observed with respect to plant height, aerial dry weight and number of rows of grain per ear, but their response was positive in most of the variables analyzed.7.21. Example 22: Evaluation of Extremophilic Bacteria as Seed Treatments in Peanut (Arachis Hypogaea L.) Cultivation Under Greenhouse Conditions

[0598] The peanut (Arachis hypogaea L.) is one of the most important oilseed crops in tropical and subtropical regions of the world. The Argentine peanut industry stands out due to its high-quality standards, which allows it to position itself as one of the main players in international trade. Being the 9thworld producer, the low domestic consumption allows it to export about 95% of what it produces, making Argentina the first world exporter always with added value, making it the main exporter of quality peanuts and peanut oil.

[0599] During the summer season of 2023-24, the promoter effect of P. megaterium CK100 applied as a treatment on the sowing line in the peanut crop was evaluated. The results of the trial showed a positive effect of the use of P. megaterium CK100 on the quality and quantity of peanuts produced per plant.

[0600] Materials and Methods

[0601] The crop was planted on December 15, 2023, in 20-liter pots filled with MultiPro substrate from the company Terrafertil. Before planting, the pots were leveled to achieve a flat planting bed. It was verified that the pressure of the equipment used was constant and that the flow rate was uniform. In each pot, 6 cm deep planting lines were opened; the seeds were placed at the bottom of the planting line in each pot at the established planting density. Inoculation was then carried out by applying the liquid on the seeds in the sowing line, at a dose of 10 ml / kg of seed with an application rate of 20 liters of water per hectare. After the application, the sowing line was closed with the same substrate as the pot and pressure was applied to ensure that the seeds remained in contact with the substrate

[0602] The crop was planted in 20-liter pots, under irrigation, using a planting density of ± 150,000 pl / ha (density used in the productive establishments of this latitude), with a separation between plant rows of 0.70 m and 0.10 m between plants. Peanut runner type granoleic variety was used, whose seeds were treated with fungicide (Fludioxonil 2.5% + Metalaxyl-M 3.75%) at 100 mL / 100 kg of seeds. The application of the biological products was carried out on the sowing line, on the seed (simulating the inoculation in furrow that is carried out in the field).

[0603] The trial was conducted up to phenological stage R8 (harvest maturity).

[0604] The treatments were:

[0605] 1 - P. megaterium CK100 10 cc / kg

[0606] 2- Control (without biological products)

[0607] The variables analyzed were selected with the objective of comparing the growth, commercial quality and yield of the crop among the selected treatments.

[0608] In relation to growth (defined as the irreversible increase of dry matter in different organs), the variable considered was the dry weight of the aerial part of the plant.

[0609] The SPAD variable, or leaf greenness index, expresses the amount of chlorophyll in the leaf and is an indicator of the nutritional status of the plant. SPAD was measured on the leaf corresponding to the penultimate node developed. Four SPAD readings were taken on each tetrafoliate leaf, one for each leaflet, from which the average value of the leaf was obtained for each plant. The measurement was taken at phenological stage R2.

[0610] Regarding yield, the variable taken as a variable was the weight of seeds / plant for each treatment. In addition, the variables number of grains / plant, considered as direct components of yield, were included.

[0611] The variable referring to commercial quality was the % for confectionery, which expresses the % of grains with a grain size greater than 7.5 mm. This classification distinguishes the grains suitable for direct consumption, which represent a higher value, from those grains with a smaller grain size that are destined for the oil industry.

[0612] Results

[0613] Table 28 shows the average values of the variables evaluated.Table 28. Average values of the variables analyzed for the different treatments.

[0614] Greenness Index-SPAD

[0615] As shown in FIG. 50, CK100 presented the best performance with respect to the commercial control, but with no significant difference. The CK100 treatment outperformed the control by approximately 4 points in chlorophyll index.

[0616] Dry Weight per Plant

[0617] When evaluating plant dry weight, CK100 generated increases in plant dry weight with significant differences with respect to the control (FIG. 51). CK100 generated plants with a dry matter value double that of the control plants.

[0618] Number of Grains per Plant

[0619] As for the variable number of grains, CK100 differed significantly from the control (FIG. 52). CK100 generated more than twice the number of grains per plant compared to the control treatment.

[0620] Grain Weight per Plant

[0621] CK100 differed significantly from the control treatment, doubling the yield obtained in each plant (FIG. 53).

[0622] Percentage Confectionery

[0623] When evaluating the variable percent confectionery, CK100 outperformed the control treatment, but without significant differences (FIG. 54).

[0624] From the results shown, it was concluded that:• The use of P. megaterium CK100 applied as an in-furrow treatment on peanut seeds generated significant increases in the productivity of the peanut crop. In addition, it was possible to improve the quality standards of this crop.• P. megaterium CK100 is a new biological alternative with high potential to improve peanut quality and quantity.7.22. Example 23: Antibiotic Resistance of CK100

[0625] The antibiotic resistance of the CK100 strain was evaluated using the guidelines established by the Clinical and Laboratory Standards Institute (CLSI).

[0626] Antibiotic susceptibility testing was carried out using the agar diffusion method. This method involves assessing the ability of antibiotics to inhibit bacterial growth on an agarplate. The results obtained were classified according to the cutoff points. These values are updated periodically and are specific to each type of microorganism and antibiotic. Therefore, the results were interpreted based on these cutoff points, and each antibiotic tested was classified as Sensitive, Intermediate, or Resistant.• Sensitive (S): The microorganism is susceptible to the action of the antibiotic at achievable concentrations in the body.• Intermediate (I): The microorganism shows a partial response to the antibiotic. In some cases, the antibiotic may be effective if higher concentrations are reached.• Resistant (R): The microorganism is not inhibited by the antibiotic at achievable concentrations in the body, suggesting that it will not respond to treatment with that antibiotic.

[0627] Table 29 presents the antibiotic susceptibility results for P. megaterium - CK100.Table 29. Antibiotic susceptibility test results for the CK100 strain.S = Susceptible or Sensitive > 15 mm diameter; I = Intermediately Susceptible / Sensitive 10-15 mm diameter; and R = Resistant < 10 mm diameter. The result was similar to other Bacillus sp.

[0628] The results obtained show that strain CK100 exhibits sensitivity to all the antibiotics tested.

[0629] Resistance of CK100 to Different Chemical Products Used in the Field

[0630] The tolerance of CK100 to different chemicals used in the field was evaluated.Table 30 summarizes the results obtained.Table 30. The tolerance of CK100 to different chemicals.R = resistant, and therefore compatible with the product analyzed7.23. Example 24: Bioformulation of culture media for CK100 spores

[0631] The objective of a culture medium for spore formulation is to cultivate the microorganism (P. megaterium CK100) under specific conditions (such as nutrient depletion, temperature fluctuations, pH changes) that trigger the sporulation process. This enables the cells to form spores, which are more resilient to harsh environments, ensuring that the microorganisms can survive until the conditions become more favorable for growth. The use of spores in the formulation has advantages related to durability and stability. Spores are highly resistant to environmental stressors such as heat, desiccation, and UV radiation. This makes them ideal for products that need to be stored for long periods without losing potency or effectiveness. Additionally, spores can germinate and grow rapidly when the conditions are favorable. This can be beneficial for applications in agriculture, where quick colonization or microbial activity is needed.

[0632] The presence of specific salts in the culture media can help to mimic stress conditions (such as nutrient limitation, changes in osmotic pressure, or metal ion depletion)that naturally trigger the sporulation process in microorganisms. Calcium chloride, magnesium sulfate, potassium chloride, and iron (III) chloride are some of the most common salts used in culture media to induce sporulation. The role of each salt in the sporulation process is described below:

[0633] Calcium chloride - Role in Sporulation: Calcium ions are involved in the formation of the spore cortex and the development of spore resistance.

[0634] Magnesium sulfate - Role in Sporulation: Magnesium is another important ion that influences sporulation. It is involved in enzyme activation and other metabolic processes that can trigger sporulation under nutrient limitation or stress conditions.

[0635] Potassium chloride - Role in Sporulation: Potassium ions are important for maintaining cellular osmotic balance and enzyme function. Potassium chloride in the culture medium can help induce sporulation, particularly when the cells are exposed to nutrient depletion or other stress factors.

[0636] Iron (III) chloride - Role in Sporulation: Iron deficiency or the presence of specific iron salts (like iron chloride) in the medium can sometimes trigger sporulation in bacteria, especially in response to nutrient scarcity or environmental stress.

[0637] Sodium chloride - Role in Sporulation: Sodium chloride can influence the osmotic pressure in the medium. While it is typically used to maintain osmotic balance, in high concentrations, sodium chloride may induce stress that could lead to sporulation in some microorganisms.

[0638] Phosphate salts - Role in Sporulation: Phosphate is essential for energy metabolism and cellular signaling. In some species, phosphate depletion in the medium can induce sporulation. Additionally, high concentrations of phosphate salts can be used to trigger sporulation under certain conditions.

[0639] Ammonium sulfate - Role in Sporulation: Ammonium salts, particularly ammonium sulfate, can sometimes stimulate sporulation in certain bacterial species when nitrogen becomes limited or when other nutrients are in low supply. Ammonium ions can trigger the metabolic pathways that lead to sporulation.

[0640] Polymers - Functions: In the preparation of culture media, polymers serve various functions that can enhance the physical and chemical properties of the medium, as well as facilitate the growth and development of microorganisms or cells in culture. Some polymerscan encapsulate nutrients releasing them in a controlled and prolonged manner. Other polymers can retain large amounts of water, maintaining moisture in the culture medium and promoting cell growth. The incorporation of polymers can modify the viscosity, elasticity, and other physical properties of the medium, adapting it to the specific needs of the culture.

[0641] Polymers - Examples: Xanthan gum.

[0642] In the present case, at least 1E+3 spores per milliliter (CFU / mL) needed to be reached. All the media and components tested for the sporulation of CK100 are described in Table 31. The spores are expressed in CFU / mL.Table 31. CK100 spores (CFU / mL) in different culture media tested for sporulation.7.24. Example 25: Bioformulation of culture media for CK100 vegetative

[0643] A culture medium is a nutrient-rich solution designed for the proliferation of microorganisms under controlled conditions. These media provide essential nutrients and conditions for the survival and reproduction of cultured organisms.

[0644] The key components of culture media and their functions are described below.

[0645] Carbon source - Functions: Serves as the primary energy source and a fundamental building block for cellular components.

[0646] Carbon source - Examples: Some simple carbon sources used are Glucose (dextrose), Sucrose, Stachyose, Glycerol, and Maltodextrin. These organic compounds are easily metabolized by microorganisms. Other complex carbon sources used in culture media are Yeast extract, Meat extract, Peptones, Caseins, and Strach, among others.

[0647] Nitrogen source - Functions: Essential for the synthesis of amino acids, nucleic acids, and proteins, which are vital for cell growth and function.

[0648] Nitrogen source - Examples: Ammonium salts or nitrates are typical nitrogen sources incorporated into culture media. Some inorganic nitrogen sources used in culture media are Ammonium Sulfate, Ammonium Nitrate, Potassium Nitrate, and Sodium Nitrate.

[0649] Polymers - Functions: In the preparation of culture media, polymers serve various functions that can enhance the physical and chemical properties of the medium, as well as facilitate the growth and development of microorganisms or cells in culture. Some polymers can encapsulate nutrients releasing them in a controlled and prolonged manner. Other polymers, can retain large amounts of water, maintaining moisture in the culture medium and promoting cell growth. The incorporation of polymers can modify the viscosity, elasticity, and other physical properties of the medium, adapting it to the specific needs of the culture.

[0650] Polymers - Example: Xanthan gum, Polyvinylpyrrolidone, and Carboxymethylcellulose

[0651] The objective of a culture media formulation was to grow the microorganism (P. megaterium CK100) in optimal conditions testing different sources such as Carbon, Nitrogen, Phosphorus (already described above) to maintain its plant growth-promoting properties. In the present case, at least 1E+3 colony-forming units per milliliter (CFU / mL) of vegetative cells needed to be reached. All the media and components tested for the best growth of CK100 are described in Table 32. The vegetative growth is expressed in CFU / mL.Table 32. CK100 growth (CFU / mL) in different culture media tested.7.25. Example 26: Performance Evaluation of Foliar-Applied Extremophilic Bacteria on Wheat Grown Under Abiotic Stress Conditions

[0652] In the assay, the growth promoting and abiotic stress mitigating effect of P. megaterium CK100 formulated “CK100 formulation”, foliar applied on wheat, was evaluated. The results of the trial showed a positive effect on the use of the bacteria on thecrop, which was reflected in productivity and in the stability of the cell membranes of its foliar tissues.

[0653] Most plant species are sensitive to temperature stress and suffer when temperatures are low or very high with respect to the thresholds defined for each one. Small temperature increases (30 to 35 °C) can damage the reproductive organs of many crops, including wheat (Triticum aestivum (L.) Thell), maize (Zea mays L.), rice (Oryza sativa L.), peanut (Arachis hypogaea L.) and tomato (Solarium lycopersicum L.).

[0654] Exposure of plants to very high temperatures (> 50 °C) results in severe damage at the cellular level within minutes and rapid collapse of cellular organization. However, when moderately high temperatures are present, damage occurs after longer exposures.

[0655] On the other hand, drought can have devastating effects on agriculture, especially in regions where water is scarce. Crops that face a lack of moisture show limited growth, lower yields and inferior quality of agricultural products. Soil properties are also affected, as it loses water and loses structure, causing greater difficulty for plants to absorb nutrients and water.

[0656] Materials and Methods

[0657] The crop was planted in 10-liter pots.

[0658] The trial design was a completely randomized 3x2x2 (dose x timing x condition) factorial arrangement, with 5 replicates per treatment, plus an absolute control (Table 48). The perimeter of the trial was planted with pots as a border. In this way, all the plants evaluated were grown in competition.

[0659] Fourteen seeds of wheat variety Minerva (germination power 96% - Intermediate Cycle) were planted at ± 2 cm depth per pot. The sowing density was ± 62 kg of seed / ha (density used in productive establishments at this latitude). Between each planting row the spacing was ± 0.52 with the intention of generating the commercial spacing between rows.

[0660] The trial was conducted under irrigation so that the crop does not grow under water stress conditions, except in the treatments and at the times when it was generated.

[0661] Insecticide and fungicide applications were made according to the corresponding pest in order to ensure the sanitary status of the crop. Weed control was manual.

[0662] The trial was conducted up to phenological stage Z 9.2 (harvest maturity - cariopse duro) according to the phenological scale of Zadoks.

[0663] Stress Generation

[0664] The generation of water stress was achieved by restricting irrigation to the treatments. To determine the water content at which the plant reaches a stress situation, the weight of the pot was used as an indicator of water content. First, the weight of a pot at Wcc (water content at field capacity) was determined by taking the amount of substrate necessary to fill a pot, watering it to saturation point, allowing it to drain freely for three days and finally weighing it to determine its weight at Wcc. It was determined that, with a pot weight of 2.5 kg, the substrate had a water content corresponding to a decrease of 60% of the water content at field capacity, which is equivalent to water hardly usable for cultivation.

[0665] For the generation of thermal stress, an iron tent lined with polyethylene for long thermal duration and a heat generator were used to raise the temperature and reach values higher than the normal greenhouse temperature. The duration of the stress was 3 hours with a temperature above 40 °C, the maximums reached were 43 °C to 45 °C (FIG. 55). The temperature was recorded with a digital thermometer placed inside the tent. This situation was replicated for 3 consecutive days.

[0666] After the generation of heat and water stress, irrigation was restored to the treatments (Table 33).Table 33. Treatments applied with their doses, times of application and condition with respect to stress. CK100: Priestia m. CK100 sporulated; Z 2.3: phenological stage of main stem with three tillers; Z 3.9: phenological stage of visible flag leaf ligule.

[0667] Foliar applications were made at phenological stages Z2.3 (main stem with three tillers) and Z3.9 (flag leaf ligule visible). A Jacto knapsack model DJB-20 was used, with JSF 110 01 flat fan tablet. An adjuvant formulated with ethoxylated monoramidated fatty alcohol (AGE) was used at a dose of 40 cc for 100 liters of water.

[0668] Variables Analyzed

[0669] The effect of abiotic stress on the plant was quantified through the % electrolyte leakage and the membrane damage coefficient expressed in % (C.D.%). Both variables refer to cell membrane damage caused by the appearance and presence of reactive oxygen species (ROS) caused by the occurrence of abiotic stress (heat and water) to which the plants were subjected. A Metrohm model 644 conductivity meter was used for these measurements. The evaluation of damage and electrolyte leakage was performed on the penultimate developed leaf (visible ligule) present at the time of evaluation in both phenological stages.

[0670] ROS damage the cell membrane by altering its selective permeability quality and, as a consequence, the membrane fails to retain electrolytes inside the cell. This type of damage correlates with electrolyte leakage.

[0671] On the other hand, the C.D.% allows a comparison to be made between the membrane damage of a cell subjected to one stress condition with respect to the membrane damage of a cell subjected to another condition.

[0672] As for crop performance, the variable seed weight / plant was taken as a variable to evaluate the yield achieved in each treatment. In addition, the variables number of grains / plant and number of ears / plant were included.

[0673] Results

[0674] FIG. 56 shows the effect of stress on cell membranes in plants grown under nonstressed and stressed conditions. The damage caused in the cell membrane when comparing the value of % electrolyte leakage and C.D.% between the Control without stress and the Control with stress for both phenological stages, Z 2.3 and Z 3.9 measured 20 days after the generation of heat and water stress indicates that the stress effect generated damage at the tissue level in the wheat crop (FIG. 56).

[0675] It was observed in both phenological stages that the unstressed control showed lower values of % electrolyte leakage than the stressed control, thus showing the damage caused on the cell membrane of the stressed control. In the same way, the % of D.C. showedthat the stressed control presents higher values compared to the unstressed control for both phenological stages (FIG. 57).

[0676] FIG. 57 shows that the % of electrolyte leakage increased in the stressed treatments at the Z 3.9 stage. In addition, it can be observed that treatments including CK100 generated a decrease in electrolyte leakage in the stressed treatments compared to their respective controls at Z 2.3 and Z3.9.

[0677] FIG. 58A presents how the analyzed treatments presented lower % of electrolyte leakage than the stressed control, thus showing the attenuation of membrane damage.Similarly, in In FIG. 58B, the treatments decreased the % of D.C. with respect to the stressed control.

[0678] FIGs. 59A, 59B show how the treatments presented values of % electrolyte leakage lower than those of the stressed control and in some cases, values very close to those of the unstressed control.

[0679] FIG. 60 illustrates % C.D. values measured at phenological stage Z 3.9 at 12 and 20 days after heat and water stress. The arrows above the bars in the treatments indicate the decrease in the % C.D. value with respect to the stress control. In the graph corresponding to the % of C.D., it can be seen how all treatments showed a decrease in membrane damage with respect to the stressed control. Similarly, it can be observed that with time, the damage values decreased, which could be due to the fact that the plants were irrigated to optimum values after being subjected to stress.

[0680] FIG. 61 shows the average values of the variables related to crop yield. It is observed that CK100 increased the value of the variables that influence crop yield in both stress and non-stress conditions.

[0681] Among the variables presented, significant differences were observed in grain number and yield in the CK100 treatment with respect to the control. The number of ears per plant was not significantly affected by the doses, application times and the presence or absence of stress.

[0682] Similarly, the stress generated at Z3.9 generated the most detrimental effects on yield components. This phenological moment is close to the critical yield period in wheat, and any unfavorable effect at this phenological stage directly affects yield.

[0683] The results of the control treatments indicated that the occurrence or nonoccurrence of stress and the time at which it occurred generated a decrease in plant yield, which affirmed that the stress condition was successful.

[0684] The yield per plant achieved indicated that the two doses with and without stress generated significantly higher yields than the stressed control treatment (Z 2.3; Stress; Control) when the application was made at Z 2.3. Stressed and non-stressed plants at the Z 2.3 stage outperformed the unstressed control treatment, indicating the positive effect of CK100 as a growth stimulant, in addition to the stress mitigation effect.

[0685] When the plants were subjected to stress and applied with CK100 at Z 3.9 stage, it was observed that the two doses evaluated generated increases in yield and number of grains per plant with respect to the stressed control treatment at the same phenological stage (FIG. 62).

[0686] Grouping the treatments by phenological stages (Z 2.3 and Z3.9), it can be observed that CK100 generated positive effects on stressed and non-stressed plants.

[0687] When plants were subjected to water and heat stress at Z 2.3, the application of CK100 generated superior results to both control treatments (stressed and non-stressed plants). In the same sense, the application of CK100 at 2250 cc / ha on non-stressed plants outperformed both controls, and the highest dose (2750 cc / ha) outperformed the stressed control (FIG. 63).

[0688] When plants were subjected to stress at Z 3.9, a significant difference was observed in the number of grains and yield per plant with respect to the non-stressed treatments.

[0689] The foliar application of CK100 in its two doses (1750 cc / ha and 2250 cc / ha) generated better results compared to the control when plants were stressed. Similarly, the application of CK100 at dose 1 outperformed the control in non-stressed plants.

[0690] By means of the methodology employed, the plants were subjected to abiotic stress. This condition affected membrane permeability in leaf tissues and negatively influenced crop productivity to different degrees depending on the phenological stage.

[0691] The extremophilic bacterium P. megaterium CK100 increased the productive capacity of the wheat crop when applied foliarly at two phenological moments on stressed and non-stressed plants.

[0692] When plants were stressed near the critical period of the crop, foliar application with P. megaterium CK100 improved plant productivity.8. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0693] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0694] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.TABLE 34: LIST OF SEQUENCESSEQ DESCRIPTION SEQUENCETCGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGCONSENSUSGCGGCGTGCCTAATACATGCAAGTCGAGCGAACTGSEQUENCE FORATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACCK60 16S RRNAGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACGENETGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACRAGAGTAACTGCTYGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT - (R: A / G and Y:C / T)TTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT - (R: A / G ANDY:C / T)TCGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACTGCOEGMCEA 00007ATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGAC16S RIBOSOMALGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACRNATGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTTCGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGCK60GCGGCGTGCCTAATACATGCAAGTCGAGCGAACTGCOEGMCEA 00043ATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGAC16S RIBOSOMALGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACRNATGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTTCGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGCK100GCGGCGTGCCTAATACATGCAAGTCGAGCGAACTGNGKBNCCO 00007ATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGAC16S RIBOSOMALGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACRNATGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATCGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGCK100GCGGCGTGCCTAATACATGCAAGTCGAGCGAACTGNGKBNCCO 00252ATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGAC16S RIBOSOMALGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACRNATGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATT

Claims

What is Claimed is:

1. A synthetic composition comprising: a microorganism of the species Priestia megaterium (P. megalerium and an agriculturally compatible carrier, wherein the microorganism is in contact with the agriculturally compatible carrier.

2. The synthetic composition of claim 1, wherein the synthetic composition is a biological formulation and further comprises a culture media composition.

3. The synthetic composition of claim 2, wherein the biological formulation is an inoculant, a biostimulant, a biofertilizer, a soil amendment, or a plant amendment.

4. The synthetic composition of any one of claims 1-6, wherein the microorganism is an endophyte.

5. The synthetic composition of any one of claims 1-3, wherein the microorganism is an endophytic sporulating bacterium.

6. The synthetic composition of any one of claims 1-5, wherein the microorganism is in the form of a spore, a vegetative cell, an endophyte, an endospore, or a combination thereof.

7. The synthetic composition of any one of claims 1-6, wherein the microorganism comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8.

8. The synthetic composition of any one of claims 1-6, wherein the microorganism comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8.

9. The synthetic composition of any one of claims 1-6, wherein the microorganism comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8.

10. The synthetic composition of any one of claims 1-6, wherein the microorganism comprises a nucleotide sequence having at least 97% sequence identity to any one of SEQ ID NOs: 1-2 and 7-8.

11. The synthetic composition of any one of claims 1-6, wherein the microorganism comprises a nucleotide sequence of any one of SEQ ID NOs: 1-2 and 7-8.

12. The composition of any one of claims 1-6, wherein the microorganism of the species of P. megaterium comprises a 16S rRNA gene sequence as set forth in SEQ ID NO: 1.

13. The composition of any one of claims 1-6, wherein the microorganism of the species of P. megaterium comprises a nucleotide sequence as set forth in SEQ ID NO: 2.

14. The composition of any one of claims 1-6, wherein the microorganism of the species of P. megaterium comprises a 16S rRNA gene sequence as set forth in SEQ ID NO: 7.

15. The composition of any one of claims 1-6, wherein the microorganism of the species of P. megaterium comprises a nucleotide sequence as set forth in SEQ ID NO: 8.

16. The synthetic composition of any one of claims 1-6, wherein the microorganism of the species of P. megaterium is strain CK60 of P. megaterium having a DSMZ accession number of 35107.

17. The synthetic composition of any one of claims 1-6, wherein the microorganism of the species of P. megaterium is strain CK100 of P. megaterium having a DSMZ accession number of 35108.

18. The synthetic composition of any one of claims 2-17, wherein the biological formulation is in liquid form, and wherein the microorganism of P. megaterium is present in the composition in a concentration from 1 x 103to 1 x 109CFU / mL.

19. The synthetic composition of claim 18, wherein the composition comprises strain CK60 of P. megaterium in a concentration of 2 x 107CFU / mL.

20. The synthetic composition of any of claims 2 to 19, wherein the culture media composition comprises a culture medium selected from one or more of: glucose, yeast extract, meat extract, calcium chloride, magnesium sulfate, potassium chloride, iron chloride, sodium chloride, phosphate salts, ammonium sulfate, pea whey, soybean peptone, manganese chloride, chitin, sucrose, ferric chloride, dipotassium phosphate, stachyose, dihydrate, casein, soybean, hydrogen phosphate, glucose monohydrate, Calcium Chloride Dihydrate, pluripeptone, Glucose Monohydrate, Magnesium sulfate heptahydrate, Iron (III) chloride hexahydrate, Potassium hydrogen phosphate, Manganese(II) chloride tetrahydrate, and peptone.

21. The synthetic composition of claim 20, wherein the synthetic composition further comprises one or more of: alginic acid, carrageenan, dextrin, dextran, polyethyleneglycol, polyvinyl pyrrolidone, methyl cellulose, polyvinyl alcohol, gelatin, a detergent, an insecticide, a nematicide, a herbicide, a fungicide, and combinations thereof.

22. The synthetic composition of any one of claims 2-21, wherein the culture media composition comprises a culture media composition selected from any one of the culture media compositions of Table 23 (spore composition media table; CK60).

23. The synthetic composition of any one of claims 2-21, wherein the culture media composition comprises a culture media composition selected from any one of the culture media compositions of Table 24 (vegetative bacteria media table; CK60).

24. The synthetic composition of any one of claims 2-21, wherein the culture media composition comprises a culture media composition selected from any one of the culture media compositions of Table 31 (spore composition media table; CK100).

25. The synthetic composition of any one of claims 2-21, wherein the culture media composition comprises a culture media composition selected any one of the culture media compositions of Table 32 (vegetative bacteria media table; CK100).

26. The synthetic composition of any one of claims 1-25, wherein the microorganism of the species P. megaterium, when contacted with a plant element comprising: a plant seed, a plant or plant portion thereof, is capable of improving a plant growth-promoting trait compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium.

27. The synthetic composition of any one of claims 1-26, wherein the microorganism of the species P. megaterium, when disposed via in-furrow application of soil surrounding a plant element or via foliar application of the plant element, is capable of improving a plant growth-promoting trait compared to a reference plant element not contacted or inoculated or applied with the microorganism of the species P. megaterium.

28. The synthetic composition of any one of claims 27-28, wherein the plant element is a plant or portion thereof selected from: plant leaves, plant roots (e.g., primary root, root hairs, lateral root, root cap, etc.), plant stems, and plant shoots (e.g., leaf blade, stem, petiole, leaf axil, flower, terminal bud, lateral bud etc.).

29. The synthetic composition of any one of claims 26-28, wherein the microorganism of the species P. megaterium, is contacted with or disposed onto the plant element via one ormore of: broadcast application, liquid or dry in-furrow application, spray application, irrigation, injection, dusting, dressing, pelleting, or coating of the plant element.

30. The synthetic composition of any one of claims 26-28, wherein the plant element is the seed, wherein the seed is a: wheat seed, potato seed, a com seed, a barley seed, a lettuce seed, a peanut seed, or a tomato seed.

31. The synthetic composition of any one of claims 26-28, wherein the plant element is the plant selected from: wheat, potato, corn, barley, lettuce, peanut, and tomato.

32. The synthetic composition of any one of claims 26-31, wherein the microorganism is heterologously or homogeneously disposed to the plant element.

33. The synthetic composition of any one of claims 26-32, wherein the microorganism is contacted with or disposed onto the plant element in an amount effective to provide one or more plant growth-promoting traits compared to a reference plant element grown without the microorganism.

34. The synthetic composition of claim 33, wherein the one or more plant growth-promoting traits is selected from: increased germination rate, increased emergence rate, increased shoot biomass, increased seedling root length, increased seedling shoot length, increased seedling mass, increased root surface area, increased enhanced nutrient use efficiency, increased phosphate solubilization, and increased yield.

35. The synthetic composition of any one of claims 26-32, wherein the plant growthpromoting trait is selected from: increased degradation of nitrogenous organic matter in the soil by which the plant element is growing and converting the nitrogenous organic matter into ammonium.

36. The synthetic composition of claim 35, wherein the plant growth-promoting trait is the microorganism’s increased production of ammonium.

37. The synthetic composition of any one of claims 26-36, wherein the plant growthpromoting trait is increased hydrolysis of urea.

38. The synthetic composition of any one of claims 26-37, wherein the plant growth promoting trait is the microorganism’s production and / or release of siderophores into the culture media composition.

39. The synthetic composition of any one of claims 26-38, wherein the plant growth promoting trait is enhancement of the plant element’s iron nutrition.

40. The synthetic composition of any one of claims 26-39, wherein the plant growthpromoting trait is an increase in flavonoids selected from one or more of: malic acid, mannitol, citric acid, tryptophan, daidzein, chrysin, apigenin, naringenin, luteolin, geni stein, naringin, and naringin in its aglycone form.

41. The synthetic composition of any one of claims 26-40, wherein the plant growthpromoting trait is an increase in root exudates of seeds of the plant element, wherein the plant element is the plant.

42. The synthetic composition of any one of claims 26-41, wherein the plant growthpromoting trait is an increase is phosphate solubilization.

43. The synthetic composition of claim 42, wherein the microorganism comprises a phosphate solubilizing index (PSI) ranging from 3-6.

44. The synthetic composition of claim 43, wherein the microorganism comprises a phosphate solubilizing index (PSI) of about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, or about 4.9.

45. The synthetic composition of any one of claims 26-44, wherein the plant growthpromoting trait is increased potassium solubilization.

46. The synthetic composition of any one of claims 26-45, wherein the plant growth promoting trait is the microorganism’s increased production of one or more phytohormones.

47. The synthetic composition of claim 46, wherein the one or more phytohormones is selected from: indole acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), jasmonic acid (JA), zeatin (ZEA), 6-benzylaminopurine (6-BAP), kinetin (KIN), and indole-3 -butyric acid (IBA).

48. The synthetic composition of any one of claims 1-47, wherein the microorganism is disposed onto or contacted with a plant seed in an amount effective to colonize the plant germinated from the microorganism formulated in the agriculturally compatible carrier as a biological formulation.

49. The synthetic composition of claim 48, wherein the colonization occurs in the root tissue of the plant.

50. The synthetic composition of any one of claims 48-49, wherein the colonization results in a biofilm formation of the microorganism.

51. The synthetic composition of any one of claims 26-50, wherein the plant growth promoting trait is selected from: increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance or tolerance to drought stress, enhanced resistance to thermal stress or thermal shock, enhanced resistance to UV radiation, enhanced resistance or tolerance to different NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to stress, increased nutrient uptake, increased root hair formation, increased root branching, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof.

52. A method for improving a trait in a plant or promoting growth of the plant, the method comprising: contacting a plant element comprising a seed or a plant or plant portion thereof with the synthetic composition of any one of claims 1-25, wherein the microorganism of the species P. megaterium, and wherein the microorganism is present in the synthetic composition in an amount effective to increase colonization of the plant element and to provide a benefit to the plant compared to a reference plant not treated with the microorganisms of the species P. megaterium.

53. The method of claim 52, wherein the microorganism is capable of producing substances that are beneficial to plants.

54. The method of any one of claims 52-53, wherein the benefit is selected from: increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance or tolerance to drought stress, enhanced resistance or tolerance to thermal stressor thermal shock, enhanced resistance or tolerance to UV radiation, enhanced resistance or tolerance to different NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance or tolerance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance or tolerance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof.

55. The method of any one of claims 52-54, wherein the method further comprises improving a plant phenotype under stress conditions as compared to reference plants not contacted with or not derived from plant elements inoculated with said synthetic composition.

56. The method of any one of claims 52-55, wherein the method is configured to promote growth of the plant.

57. The method of claim 55, wherein the contacting comprises applying the synthetic composition to the plant element via broadcast application, liquid or dry -in furrow application, spray application, irrigation, injection, dusting, pelleting, or coating of the plant element.

58. The method of claim 57, wherein the method comprises germinating the plant element, wherein the plant element is a seed; and growing the seed.

59. The synthetic composition of any one of claims 57-58 wherein the microorganism of the species P. megaterium, when applied via in-furrow application of soil surrounding a plant element or via foliar application of the plant element, is capable of improving a plant growth-promoting trait compared to a reference plant element not applied with the microorganism of the species P. megaterium.

60. The synthetic composition of any one of claims 52-59, wherein the plant or portion thereof is selected from: plant leaves, plant roots (e.g., primary root, root hairs, lateral root, root cap, etc.), plant stems, and plant shoots (e.g., leaf blade, stem, petiole, leaf axil, flower, terminal bud, lateral bud etc.).

61. The synthetic composition of any one of claims 26-28, wherein the microorganism of the species P. megaterium, is contacted with or disposed onto the plant element via one ormore of: broadcast application, liquid or dry in-furrow application, spray application, irrigation, injection, dusting, pelleting, or coating of the plant element.

62. The method of any one of claims 52-57, wherein the plant element is a seed, wherein the seed is a: wheat seed, potato seed, a com seed, a barley seed, a lettuce seed, or a tomato seed.

63. The method of any one of claims 52-57, wherein the plant element is a plant selected from: wheat, potato, corn, barley, lettuce, tomato, legume, and grass.

64. The method of any one of claims 52-63, wherein the microorganism is heterologously or homogeneously disposed to the plant element.

65. The method of any one of claims 52-64, wherein said contacting comprises homogenously or heterologously disposing the microorganism onto the plant element in an amount effective to provide one or more plant growth-promoting traits compared to a reference plant element grown without the microorganism.

66. The method of any one of claims 52-64, wherein the benefit comprises one or more of: increased germination rate, increased emergence rate, increased shoot biomass, increased seedling root length, increased seedling shoot length, increased seedling mass, increased root surface area, increased enhanced nutrient use efficiency, increased phosphate solubilization, and increased yield.

67. The method of any one of claims 52-64, wherein the benefit comprises one or more of: increased degradation of nitrogenous organic matter in the soil by which the plant element is growing and converting the nitrogenous organic matter into ammonium.

68. The method of any one of claims 52-67, wherein the benefit comprises increased production of ammonium.

69. The method of any one of claims 52-68, wherein the benefit comprises increased hydrolysis of urea.

70. The method of any one of claims 52-69, wherein the benefit comprises production and / or release of siderophores into the culture media composition.

71. The method of any one of claims 52-70, wherein the benefit comprises enhancement of the plant element’s iron nutrition.

72. The method of any one of claims 52-71, wherein the benefit comprises an increase in flavonoids selected from one or more of: malic acid, mannitol, citric acid, tryptophan, daidzein, chrysin, apigenin, naringenin, luteolin, genistein, naringin, and naringin in its aglycone form.

73. The method of any one of claims 52-72, wherein the benefit comprises an increase in root exudates of seeds of the plant element, wherein the plant element is the plant.

74. The method of any one of claims 52-73, wherein the benefit comprises an increase is phosphate solubilization.

75. The synthetic composition of claim 74, wherein the microorganism comprises a phosphate solubilizing index (PSI) ranging from 3-6.

76. The synthetic composition of claim 75, wherein the microorganism comprises a phosphate solubilizing index (PSI) of about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, or about 4.9.

77. The method of any one of claims 52-76, wherein the benefit comprises increased potassium solubilization.

78. The method of any one of claims 52-77, wherein the benefit comprises the microorganism’s increased production of one or more phytohormones.

79. The synthetic composition of claim 78, wherein the one or more phytohormones is selected from: indole acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), jasmonic acid (JA), zeatin (ZEA), 6-benzylaminopurine (6-BAP), kinetin (KIN), and indole-3 -butyric acid (IBA).

80. The method of claim 52, wherein the colonization occurs in the root tissue of the plant.

81. The method of claim 52, wherein the colonization results in a biofilm formation of the microorganism.

82. The method of any one of claims 52-81, wherein the benefit is selected from: increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance or tolerance to drought stress, enhanced resistance to thermal stress or thermal shock, enhanced resistance to UV radiation, enhanced resistance or tolerance to different NaCl concentrations, enhanced resistance or tolerance to antibiotics, enhanced tolerance to stress, increased nutrient uptake, increased root hair formation, increased rootbranching, enhanced tolerance to water stress, increased drought tolerance, enhanced resistance to cold stress, enhanced resistance or tolerance to heat stress, enhanced resistance or tolerance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance or tolerance to aluminum toxicity, enhanced resistance or tolerance to fungal infection, enhanced resistance or tolerance to bacterial infection, enhanced resistance or tolerance to viral infection, and a combination thereof.

83. The method of any one of claims 52-82, wherein the composition is formulated as an inoculant, and it is applied at a dose from 5 mL / kg to 10 mL / kg.

84. The method of any one of claims 52-53, wherein the plant whose growth is to be improved is wheat or barley, the composition comprises strain CK60 of P. megalerium. and it is applied at a dose of 6 mL / kg.

85. The method of any one of claims 52-85, wherein the method further comprises harvesting the plant.

86. A method for improving soil qualityand / or plant quality, comprising applying the synthetic composition according to any one of claims 1-25 to a plant element comprising a seed or a plant grown in the soil.

87. The method of claim 86, wherein the method results in a reduced use of an amount of commercially available nitrogen fertilizers in the soil.