Modified and isolated strains of pseudomonas stutzeri RDC-01 and bacillus velezensis ca1, compositions used as inoculum for improving plant growth and abiotic stress resistance, methods and uses thereof

Abstract

Modified bacterial strain of the Pseudomonas sp. genus, the strain of Pseudomonas stutzeri deposited under Deposit Accession No. RGM3505 and a modified bacterial strain of the Bacillus sp. genus, a strain of Bacillus velezensis deposited under Accession No. RGM3504. The strains may be combined at a Pseudomonas stutzeri to Bacillus velezensis ratio from 1 :10 to 10:1 CFU / mL. The strains were modified to have greater chemotactic attraction towards plant roots and to improve the beneficial features of crops, for example, of legumes.

Description

[0001]Docket No.1063.034 MODIFIED AND ISOLATED STRAINS OF PSEUDOMONAS STUTZERI RDC-01 AND BACILLUS VELEZENSIS CA1, COMPOSITIONS USED AS INOCULUM FOR IMPROVING PLANT GROWTH AND ABIOTIC STRESS RESISTANCE, METHODS AND USES THEREOF The present invention refers to modified bacterial strains of the Pseudomonas sp. genus, to the strain of Pseudomonas stutzeri deposited under Deposit Accession No. RGM3505 and to a modified bacterial strain of the Bacillus sp. genus, a strain of Bacillus velezensis deposited under Deposit Accession No. RGM3504, to compositions containing them, and to methods and uses thereof. The strains may be combined at a ratio of Pseudomonas stutzeri : Bacillus velezensis from 1:10 to 10:1 CFU / mL. The strains were modified to improve their chemotactic attraction towards plant roots, e.g., of legumes, and to enhance their beneficial effects on plants. BACKGROUND OF THE INVENTION According to the United Nations, the current world population of 7.6 billon people is expected to increase to more than 9.8 billon by the year 2050. An equivalent of almost three planets would be needed to provide the natural resources required to sustain current lifestyles. The negative global impact of anthropogenic climatic change on food yield and safety is a great concern, with a possibility that 200 million more people may be at risk of suffering from hunger by the year 2100. This dramatic expansion of human population comes with environmentally destructive activities such as deforestation and excessive use of chemical fertilizers and pesticides in agriculture. Furthermore, global heating, caused by greenhouse gas emissions, worsen the abiotic stress and leads to a reduction in the extension of agricultural land, and agricultural productivity. Abiotic stresses, such as drought, soil salinity, extreme temperatures, ultraviolet radiation and nutrient deficiency and / or unavailability, are the cause of more than 50% of yield losses in major crops. As a result of incorrect land management and of environmental factors related to climatic change, 1-2% of productive agricultural areas are lost each year. Soil salinization affects about 7% of land worldwide, with a deteriorating trend in Eurasia Docket No.1063.034 and South America. In addition, water shortage in soils is responsible for a significant reduction in crop yields. Surface and groundwater supplies are also reduced during periods of drought, which affects water availability for crop irrigation. Consequently, the need to find environmentally friendly, cost-effective and sustainable approaches to guarantee food availability for the growing population has become the subject of extensive research worldwide. There are very huge and urgent issues for crop production. Studies show that 70% of the yield shortfall between potential yield and actual yield may be attributed to abiotic factors, mainly adverse soil and climate conditions. To address these problems, the inventors have developed microorganisms which improve plant growth and resistance to biotic and abiotic stress. So far, the development of inocula has been mainly based on the beneficial properties exerted on plants by the microorganisms. Some products include a combination of microbes. However, the selection of each component does not take into account possible interactions between the members of the multispecific inoculum and the natural microbiota. The aim of the invention is to develop a product based on a microbial consortium having an optimal synergy among its components and those of the natural microbiota, thus mimicking the natural mechanisms that occur in microbiota associated to plants. SUMMARY OF THE INVENTION There is provided a modified bacterial strain of the Pseudomonas sp. genus, a strain of Pseudomonas stutzeri deposited under Deposit Accession No. RGM3505 and a modified bacterial strain of the Bacillus sp. genus, and a strain of Bacillus velezensis deposited under Accession No. RGM3504. Further provided is a combination of modified bacterial strains comprising: the strain of Pseudomonas stutzeri deposited under Deposit Accession No. RGM3505 and the strain of Bacillus velezensis deposited under Accession No. RGM3504, wherein the combination may comprise a Pseudomonas stutzeri to Bacillus velezensis ratio from 1:10 to 10:1 CFU / mL. The strains were modified to Docket No.1063.034 improve their chemotactic attraction towards plant roots, preferably of legumes and more preferably of soybean, and thus achieve more potent beneficial effects. Also provided is a composition for increasing growth and / or tolerance to abiotic stress in a plant, comprising the strain of Pseudomonas stutzeri deposited under Deposit Accession No. RGM3505 or the strain of Bacillus velezensis deposited under Deposit Accession No. RGM3504, or both strains (combinations thereof); and an agricultural inoculum medium. The agricultural inoculum medium may comprise one or more of the following compounds: salts, organic acids, polymers, polysaccharides, surfactants, or the culture medium of the corresponding strain or the culture medium of both strains. Each composition comprises from about 108to about 109CFU / mL. There is provided a process for preparing a composition comprising the following steps: a. culturing strains of Pseudomonas stutzeri bacteria deposited under Accession No. RGM3505, or the strain of Bacillus velezensis deposited under Accession No. RGM3504 in a production medium until stationary phase is reached; b. preparing a liquid agricultural inoculum c. adding an amount of the culture of step a. to an amount of the inoculum of step b. at a ratio of 1:5 v / v. In case the composition comprises both strains, before step c, a combination of both strains is prepared at a Pseudomonas stutzeri to Bacillus velezensis ratio from 1:10 to 10:1 CFU / mL. The method comprises contacting a plant or a plant part with the composition described in the previous paragraph, where said contact may be selected from various methods, for example, spraying, dipping, soil inoculation. The abiotic stress suffered by the plant may be, for example, high salinity soils, drought, soils having an electrical conductivity equal to or greater than dS / m, very high temperatures, and other conditions. Just as the compositions are of use for a plant subjected to abiotic stress, said compositions may also be used for enhancing Docket No.1063.034 non-stressed plant growth under normal conditions. The use of the compositions for any plant under different growth conditions falls within the scope of the invention. The plant part may be a seed, root, stem, leaf, or fruit. In one preferred embodiment the part of plant is a seed. The use of any of the strains and combinations thereof for preparing a composition improving growth and / or tolerance to abiotic stress of a plant is also provided. In addition, the invention provides the use of the culture supernatant of any of the strains for preparing a cell-free composition for increasing growth and / or tolerance to abiotic stress of a plant. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the adaptation cycles. Figure 2 is a schematic representation of interspecies interactions. The numbers indicate taxonomically different isolates. The Figure should be interpreted as follows: the color of each square corresponds to the effect on the isolate indicated by the number on the left when interacting with the isolate indicated on the top. Dark gray denotes antagonism, gray squares of medium intensity indicate beneficial interactions, white squares indicate neutral interactions and light gray squares indicate interactions with particular properties, such as biofilm improvement. Figure 3 shows representative images of plants grown in soil having an electrical conductivity in a range of 4-7 dS / m and inoculated with the different compositions. Figure 4 shows nodulation in control plants and in plants treated with RDC- 01-A and CA1-A) strains. B. Enlarged view of roots (control versus RDC-01-A and versus CA1-A). Results obtained in field trials. DETAILED DESCRIPTION OF THE INVENTION Docket No.1063.034 Definitions: RDC-01 refers to a strain of Pseudomonas stutzeri bacteria isolated from soil. CA1 refers to a strain of Bacillus velezensis bacteria isolated from soil. RDC-01-A refers to a strain of modified RDC-01 bacteria adapted to the rhizosphere and high salinity. CA1-A refers to a strain of modified CA1 bacteria adapted to the rhizosphere and high salinity. Composition 1 is RDC-01-A formulated with chemical substances to improve seed shelf life and survival (described below). Composition 2 is CA1-A formulated with chemical substances to improve seed shelf life and survival (described below). Composition 3 is RDC-01-A and CA1-A taken as a consortium formulated with chemical substances to improve seed shelf life and survival (described below). On September 22, 2023, Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A were deposited, under the Budapest Treaty, with Colección Chilena de Recursos Genéticos Microbianos [Chilean Collection of Microbial Genetic Resources] under Accession Nos. RGM3505 and RGM3504, respectively. Isolation of bacterial strains: two isolates were selected from all the resulting isolates, based on their biochemical properties as described below. These isolates were designated RDC-01 and CA1. Strain RDC-01 was isolated from a rhizosphere in Tucumán (Argentina). Species assignment was performed by sequencing 16S rDNA and subsequent sequence analysis using BLAST. The RDC-01 isolate showed 100% sequence identity with ATCC 17588 of Pseudomonas stutzeri. Genomic sequencing of the RDC-01 isolate allowed for a phylogenetic analysis with reference and representative genomes and an in silico DNA-DNA hybridization analysis. Both Docket No.1063.034 analyses showed that RDC-01 is closely related to Pseudomonas stutzeri A1501, which confirms the genus and species designation. After species designation, the RDC-01 isolate was designated Pseudomonas stutzeri RDC-01. The CA1 strain was isolated from marginal agricultural soil (Tucumán, Argentina). Genus assignment was performed by sequencing 16S rDNA and subsequent sequence analysis using BLAST. The CA1 isolate showed 98.79% sequence identity with Bacillus velezensis FZB42. The CA1 strain was designated Bacillus velezensis CA1. Genomic sequencing was also performed for Bacillus velezensis CA1. Biochemical characterization of the isolated strains: Tolerance of Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 to sodium chloride and their biochemical properties associated to plant-growth promoting characteristics (production of siderophores, phosphate solubilization and phytohormone production) are detailed in Table 1. Both strains were able to tolerate NaCl, showing positive biochemical features associated with plant-growth promoting rhizobacteria (indole and siderophore production, and phosphate solubilization). Table 1 Tolerance to NaCl and biochemical properties of the isolated strains Strain Tolerance to Production of Production Solubilization Tolerance to NaCl: The strains were classified based on their growth after 24 h on LB plates supplemented with 500 mmol / L: highly resistant (+++), growth observed at dilutions of 10-3or higher: moderately resistant (++), growth observed Docket No.1063.034 at dilutions of 10-2: low resistance (+), growth observed at dilutions of 10-1Production of indole acetic acid: (+) IAA ≤10 μg mL, (++) IAA 10–20 μg mL and (+++) IAA ≥20 μg mL. Production of siderophores: SP (production of siderophores) = Halo diameter / Colony diameter SP = 1 (negative), SP from 1.0 to 1.2 (+), SP from 1.2 to 1.5 (++) and SP ≥ 1.5 (+++). Phosphate solubilization index: PSI (phosphate solubilization index) = Halo diameter / Colony diameter PSI = 1 (negative), PSI from 1.0 to 1.2 (+), PSI from 1.2 to 1.5 (++) and PSI ≥ 1.5 (+++). Adaptation cycles of Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 to soybean plant rhizospheres growing under saline conditions: The Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 strains were adapted to the rhizosphere of soybean plants growing under saline conditions by submitting each bacterial strain separately to successive cycles of inoculation of seeds, sowing seeds in sterile sand, growing of seedlings under controlled conditions of light, humidity and watering with saline solution, and collection of bacteria from the rhizosphere. This cycle is shown in Figure 1. Five cycles were performed and then the bacteria were isolated. All selected and isolated strains showed a similar or higher positive chemotaxis towards soybean root exudates compared to Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 parental strains that had not been adapted by the method shown in Figure 1. Isolate 3 resulting from the adaptation cycles of Pseudomonas stutzeri RDC-01, showed a stronger chemotaxis effect (Table 2) and further improved soybean plant yield (Tables 3 and 4). This isolate was designated Pseudomonas stutzeri RDC-01-A. Isolate 7, resulting from the adaptation cycles of Bacillus velezensis CA1, showed the strongest chemotaxis effect (Table 2), improved soybean plant yield (Tables 3 and 4) and was designated Bacillus velezensis CA1- A. Several isolations of both strains (10 isolates per strain) were performed, and one of them was selected. In all cases, the isolates showed that they exceeded the chemotactic values of their strain of origin. Docket No.1063.034 Table 2 Chemotaxis Strain CFU / mL e compos ons were prepared usng modified bacterial Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A strain, which were grown in a production medium (MP), comprising 12 g / L Tryptone, 3 g / L yeast extract, and 15 g / L NaCl, until the stationary phase was reached. The Pseudomonas stutzeri RDC- 01-A and Bacillus velezensis CA1-A strains were resuspended, for example, in inoculum medium comprising 0.90 g / L KH2PO4, 0.1 g / L citric acid, 10 g / L polyvinyl acetate (PVA), 1.4 g / L Guar gum, and 1.4 g / L xanthan gum. Accordingly, final compositions 1 and 2 comprised bacterial cells, metabolites present in the culture medium and chemicals of the inoculum medium. The results shown in the tables for compositions 1 and 2 correspond to a dose of 1.5 mL / Kg seed. Each composition comprised from about 108to about 109CFU / mL. For composition 3, compositions 1 and 2 were used in a combination resulting in a Pseudomonas stutzeri RDC-01-A to Bacillus velezensis CA1-Ain ratio of 1:1, 1:2, 1:5, 1:10, 2:1, 5:1, and 10:1 CFU / mL, respectively. The results obtained with composition 3 shown in the tables correspond to a Pseudomonas stutzeri RDC- 01-A to Bacillus velezensis CA1-A ratio of 1 : 1 , respectively and a dose of 1.5 mL Docket No.1063.034 of composition 3 per Kg seed. All ratios evaluated in the range from 1:10 to 10:1 showed a positive effect on the plants, where the preferred ratio corresponds to equal parts of each strain (1:1). Four seed inoculum media (F1, F2, F3, and F4) were also evaluated for bacterial cell viability over time. F1 contained 0.90 g / L KH2PO4, 0.1 g / L citric acid, 10 g / L polyvinyl acetate (PVA), 1.4 g / L Guar gum, 1.4 g / L xanthan gum. F2 contained 3 g / L xanthan gum, 20 g / L polyvinylpyrrolidone, 5 g / L Tween 20, 2 g / L potassium sorbate. F3 contained 1 g / L carboxymethyl cellulose, 20 g / L polyvinylpyrrolidone, 5 g / L Tween 20, 2 g / L potassium sorbate. F4 contained 3 g / L xanthan gum, 5 g / L Tween 20, 2 g / L potassium sorbate, 10 g / L polyethylene glycol. In order to select the preferred medium, seeds were evaluated for shelf life and cell viability over time. F1 was selected and showed a viability and shelf life of at least 200 days with a CFU / mL value of 109and viable cells detected in seeds for up to 7 days. Seeds treated with the compositions improved plant growth features under non-saline and saline conditions (Tables 3 to 5). Table 3 Growth of soybean on commercial substrate watered with a solution without added NaCl Impact on plant growth Docket No.1063.034 Bacillus velezensis CA1-A 17.6 38.1 96 5347 a e Growth of soybean on commercial substrate watered with a solution containing 100 mM NaCl Impact on plant growth Docket No.1063.034 Composition 1 comprising 16.4 37.2 98 5252 Pseudomonas stutzeri RDC- Growth of soybean on commercial substrate watered with a solution containing 200 mM NaCl Impact on plant growth Docket No.1063.034 Composition 2 comprising 11.5 22.1 77 2587 Bacillus velezensis CA1-A Growth of soybean in soil with different electrical conductivities. Root length (cm) Shoot length Germination Vigor index 1 2 6 8 8 9 1 6 Docket No.1063.034 Compositio 18. 23 16. 14. 42 44 24. 14 9 9 9 7 58 65 37 22 n 2 1 2 8 9 2 7 8 7 8 0 6 50 85 35 57 4 7 Adapted modified strains (Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A) were evolved strains obtained in the laboratory from Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 exposed to selective pressure in order to increase their agronomic properties and affinity for crops. The treated seeds improved plant growth characteristics under both non- saline and saline conditions (Tables 3 to 5). Although no viable cells were detected in the seeds after 7-10 days of inoculation, the sown seeds showed a positive impact on plant growth features for up to 21-30 days after inoculation. Positive effects were detected even for longer periods of time after seed inoculation. The effect on soybean growth and saline stress attenuation of compositions comprising Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A was evaluated. The possibility of combining different strains originated in an embodiment of a previous study of intraspecific interactions where taxonomically different microorganisms were co-cultivated in pairwise combinations on a solid medium. Docket No.1063.034 Traits such as bacterial growth promotion, inhibition and changes in colony morphology were monitored over time in order to establish ecological relationships between different microorganisms classified as neutral (no obvious interaction was observed), beneficial (commensals or mutualists), antagonic or special (Figure 2). It was determined that Bacillus velezensis CA1 favors production of extracellular matrix of Pseudomonas stutzeri RDC-01, and this is critical for the impact of Pseudomonas stutzeri RDC-01 on plant growth. A composition combining both strains for inoculation of soybean seeds was developed. Soybean seeds were inoculated with the composition and its impact on plants was evaluated. For example, plants growing in commercial substrate and watered with saline solution (Tables 3, 4, and 5) and plants growing in soil having different electrical conductivities (Table 6). Figure 3 shows a representative image of the synergic impact of inoculating soybean seeds with both strains separately and with a combination of both in an inoculum (composition 3). Treatment of seeds with the compositions increased the number of germinated seeds and also both root and aerial development, where the best result was observed with composition 3. Inoculation of soybean seeds with Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 resulted in increased soybean growth under controlled conditions involving a plant growth substrate without sodium chloride supplements or with the addition of sodium chloride as an irrigation solution at a concentration of up to 200 mM (Tables 3 to 5). Both Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 showed a plant growth promoting effect and the ability of improving saline stress resistance under controlled conditions. Adaptation of Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 to soybean roots (see adaptation cycles) generated the Pseudomonas stutzeri RDC- 01-A and Bacillus velezensis CA1-A strains, which enhanced the positive impact on plants (Tables 3 to 5). Compositions 1 and 2 improved the impact on soybean growth even more (Tables 3 to 5). Composition 3, resulting from using Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A formulated at a Docket No.1063.034 ratio of 1:1, had an additional beneficial impact on plant growth (Tables 3 a5). Although a certain range of NaCl concentrations was used (0 to 200 mM) it was observed that inoculation of seeds with composition 3 had less impact when the NaCl content was greater than 200 mM. A positive impact on plant growth was also observed when soybean seeds were inoculated with Pseudomonas stutzeri RDC-01, Bacillus velezensis CA1, Pseudomonas stutzeri RDC-01-A, or Bacillus velezensis CA1-A and then seeded in soils with an electrical conductivity ranging from <4 to >10 dS / m (Table 6). The soybean plants got stressed when irrigation was discontinued. Seeds inoculated with compositions 1, 2, or 3 showed a significantly improved ability to withstand stress by drought (Table 7), defined as the time until onset of wilt is observed, which was delayed in plants inoculated with the strains. Survival rate also improved with inoculation of the compositions. As observed with plant growth under saline conditions, composition 3 showed the best results in terms of tolerance to drought. Table 7 Impact of drought stress on soybean plants Condition Wilting occurrence (days Survival Rate Docket No.1063.034 RDC-01-A and Bacillus velezensis CA1-A to enhance plant growth and also to alleviate plant stress caused by salinity or water deficiency. The strains modified by adaptation, Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1–A, used as inocula of seeds positively impacted plant growth, outperforming unmodified isolated strains. Composition 3 showed synergism, increasing plant growth under non-saline and saline conditions. Field trials were conducted on soybean, corn, sunflower, chickpea, pea, and wheat plants. The trials were carried out in 3 different agroecological regions in Argentina. Trial design involved randomly distributed blocks with at least 5 replicates for each condition. Seeds of each crop type were inoculated with compositions 1 or 2 using standard farming practices (i.e., use of fungicides, insecticides, and other biological products). Crops were analyzed according to different parameters such as plant density, nodulation (legumes), fresh weight of stems, fresh weight of roots, plant height, and yield. The results are shown below in Table 8. Table 8 Results of the field trials: numbers are percentages of change with respect to control treatment (normal farming practices) plants compositio density nodulatio Fresh Fresh hei Yield Docket No.1063.034 Soybea composition 8*** 66*** 23*** 55*** 18** 18** n in low 1 * NS, non-significant result (p<0.1); *, significant result (p<0.1); **, significant result (p<0.05); ***, significant result (p<0.001).N / A, not applicable; ND, not Docket No.1063.034 determined The field trials showed a positive impact of compositions 1 and 2 comprising Pseudomonas stutzeri RDC-01-A or Bacillus velezensis CA1-A on various crops. Furthermore, this indicated that the present technology worked well on a broad range of plant species comprising monocotyledonean and dicotyledonean crops. Table 9 Sterile sand Commercial soil Germination Germination Stem len th Root len th Germination percentages (%) of soybean seeds after 7 days in sterile sand under saline conditions (watered with 100 mM NaCl). Germination percentages (%) of soybean seeds after 7 days, stem and root lengths of each treatment after 14 days in commercial soil under saline conditions (watered with 100 mM NaCl). SN means supernatant. Saline control, corresponds to irrigation with salt but with no supernatant applied to the seeds. Field trials results: field trials were conducted at Saladillo, province of Buenos Aires, in medium salinity soils. This trial was carried out on soybean using compositions comprising the adapted strains (Pseudomonas stutzeri RDC-01-A or Bacillus velezensis CA1-A). Positive results were observed one month after seeding. A significant increase of plant density was observed (Table 10) with both strains and also a remarkable increase of nodulation (Figures 4 A and B). Table 10 Treatment pl / m Docket No.1063.034 RDC-01-A 28 tutzeri RDC-01-A or Bacillus velezensis CA1-A strains have the ability of promoting plant growth and also of ameliorating plant stress caused by salinity or water deficiency. Adaptation of wild type isolates to soybean roots and formulation of the compositions increased even more the ability of these strains to positively affect plant growth. In addition, a combination of both enhanced effect of individual strains, thus demonstrating a synergic effect evidenced by plant growth under non-saline or saline growth conditions. Experiments were carried out using a cell-free culture supernatant of the Pseudomonas stutzeri RDC-01-A strain. Supernatants were obtained by centrifuging the cultures at 8000 rpm and subsequently sterilizing them using 0.22 µm pore filters. Table 9 shows that a supernatant of the strain (RDC-01-A) improves germination, stem and root lengths of soybean plants. The following examples should not be construed as a limitation of the scope of the claims, but instead as exemplary embodiments among other possible examples. This invention is better illustrated in the following examples, which should not be construed as a limitation of the scope thereof. On the contrary, it should be clearly understood that other embodiments, modifications and equivalents thereof may be possible after reading the present description, which may be suggested to a person skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims. Examples Example 1: Bacterial sampling and isolation: Docket No.1063.034 The bacteria were isolated with a collection permit (Document 228-2022) granted by the local Flora, Wildlife, and Soils Authority of the province of Tucumán, Argentina. In order to isolate bacteria from the rhizosphere of halophyte plants, loosely adhering soil was removed from the roots by gentle shaking. Roots having firmly adhering soil particles were cut off and placed in sterile 50 mL screw-capped tubes containing 5 mL of M9 minimal salts medium (Sigma Aldrich, Munich, Germany) (Sambrook and Russell 2001) supplemented with 0.2% casamino acids and 0.2% glucose. The tubes were shaken with 4 g of sterile glass beads (3 mm diameter) for 2 minutes and then the beads were left to settle. Serial dilutions of the supernatant were made in M9 medium supplemented with 0.2 % casamino acids and 0.2 % glucose. The tubes were shaken during 2 minutes and then the beads were left to settle. Serial dilutions of the supernatant were made in M9 minimal salts medium (Sigma Aldrich, Munich, Germany) (Sambrook and Russell 2001) supplemented with 0.2% casamino acids and 0.2% glucose. Designation of the bacterial genus: In order to identify CA1 isolates and RdC-01 strains, 16S rDNA of each isolate was amplified using the universal oligonucleotide primers described by Lane (1991). The resulting sequences were analyzed using the Targeted Loci Nucleotide tool BLAST available at the National Center for Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / ) Biochemical characterization of the isolated strains: Total indole production: Total indole production was detected using the method described by Glickmann and Dessaux (1995). Bacterial cultures were grown in M9 medium supplemented with 0.2% casamino acids, 0.2% glucose, 1 mmol / L MgSO4, and 1 mg / mL vitamin B1 and 0.1 mg / mL L-tryptophan for 48 h at 30°C. Cultures were centrifuged at 5000 rev / min for 10 min. The supernatant was mixed with Salkowski reagent (12.5 g FeCl3, 7.5 mol / L H2SO4 (1:1) and left in the dark during 30 min. Docket No.1063.034 The development of pink color indicates production of indole. Optical density was measured with a spectrophotometer at 540 nm. The concentration of indoles was measured using IAA (Sigma-Aldrich, Saint Louis, MO, USA.) as a standard, in a range from 3 to 30 μg / mL. Production of siderophores: The production of siderophores by PGPR was detected using the chromium azurol S assay (Schwyn and Neilands 1987). The isolates were detected on chromium azurol S agar plates (10 μL 106CFU per mL) and development of an orange-yellow halo surrounding the colony, indicative of production of siderophores, was analyzed after 48 h (Pérez-Miranda et al., 2007). The experiments were repeated at least three times. The production of siderophores was normalized with respect to colony growth and expressed as a halo diameter to colony diameter ratio (HD / CD), as previously described (Edi-Premono et al., 1996). Phosphate solubilization: The ability to solubilize inorganic phosphate was tested in triplicate by growing the bacterial isolates on NBRIP agar plates (Nautiyal 1999) for 6 days at 30 °C, as described by Gaur (1990). Formation of a clear halo surrounding the colonies was indicative of inorganic phosphate solubilization. Halo diameters were normalized as a function of colony growth and expressed as a halo diameter to colony diameter ratio (HD / CD). Example 2: Plant growth under saline and non-saline conditions by application of bacterial strains: In order to evaluate plant growth promotion and abiotic stress attenuation by isolated bacterial strains (Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1), bacteria adapted (A) to the rhizosphere of soybean (see below) (Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A) and compositions comprising adapted bacteria (RDC-01-A, CA1-A and a combination containing both RDC-01-A and CA1-A), seeds were inoculated with 0.15 to 4 mL / kg Docket No.1063.034 seed. The inoculated seeds were seeded on plastic trays containing commercial plant substrate (Growmix). The trays were incubated in a plant incubator (Panasonic Versatile Environmental Test Chamber MLR-352H) and watered regularly with a NaCl solution ranging from 0 a 200 mM. The plant growth parameters recorded after 18 days of incubation were shoot length, root length, germination rate, and vigor index. For the drought tolerance study, the seeds were inoculated as described hereinabove. Fourteen days after germination, drought stress was induced by stopping irrigation and recording the time to occurrence of wilt and the survival rate upon 28 days of germination. Example 3: Adaptation cycles: The seeds were surface disinfected with ethanol and sodium hypochlorite and then they were inoculated with Pseudomonas stutzeri RDC-01 or Bacillus velezensis CA1 at a dose of 1.50 mL (109CFU / mL) / 1 kg seed. The inoculated soybean seeds were sown in sterile sand contained in Falcon tubes and then placed in a plant incubator (Panasonic Versatile Environmental Test Chamber MLR-352H) for 14 days at a temperature, humidity, and light cycles optimized for soybean (De Weert et al; 2014). The plants were watered periodically with 150 mM NaCl. Two weeks after inoculation, the plants were carefully removed from the sand, roots were cut off and then placed in sterile Falcon tubes containing glass beads and sterile saline solution. The tubes were vortexed to separate bacteria from the root surface. The bacterial suspensions (obtained from the 1stcycle) were collected to inoculate a new set of seeds for the 2ndcycle. This process was subsequently repeated for 5 cycles, where each cycle lasted 2 weeks (Figure 1). At the end of cycle 5, isolates (at least 10 isolates) of the bacterial suspension were obtained and the phenotypic variance was evaluated by analyzing the chemotaxis of each isolate towards soybean root exudates and the impact of each isolate on soybean plants. The strains obtained by this process were: Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A were deposited under the Budapest Treaty on September 22, 2023, at the Colección Chilena de Recursos Genéticos Microbianos [Chilean Collection of Microbial Genetic Resources] under Accession Nos. Docket No.1063.034 RGM3505 and RGM3504, respectively. Example 4: Analysis of the modified bacterial strains Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A: Chemotaxis towards soybean root exudates: Chemotactic response towards soybean root exudates of Pseudomonas stutzeri RDC-01 and Bacillus velezensis CA1 strains and of strains modified by the adaptation cycles (Pseudomonas stutzeri RDC-01-A or Bacillus velezensis CA1-A) was evaluated. The soybean root exudates were obtained from plants hydroponically grown for 6 days in 5 mL of MS medium with 150 mM NaCl, under sterile conditions. The exudates were collected and filtered through a 0.22 µm membrane and used for quantitative chemotaxis tests based on the microcapillary method of Martín-Mora et al., (2016) with some modifications. The cultures were grown in a production medium (PM) containing 15 g / L NaCl, 12 g / L Tryptone, and 3 g / L yeast extract diluted 1 / 10 to an OD600 = 0.4, the cells were washed and resuspended in chemotaxis buffer (30 mM K2HPO4, 19 mM KH2PO4, 20 μM EDTA and 0.05% glycerol (v / v), pH 7.0) to a final OD600 = 0.1. Two hundred thirty μl of bacterial suspension were placed in each well of a polystyrene multiwell plate. The capillaries (Microcaps, Drummond Scientific, USA) were heat sealed at one end, then filled with the resulting root exudates and dipped into the bacterial suspension by the open end. After 30 min of incubation, the capillary was removed, washed on the outside with sterile water and emptied into an Eppendorf tube which contained 1 mL of M9 medium. Serial dilutions in PM were made to determine the number of bacteria which had migrated into the capillary (CFU / mL). Positive and negative controls were included (0,1% casamino acids and chemotaxis buffer, respectively). The data are the mean values of three experiments carried out independently and by triplicate. Example 5: Preparation of the Compositions: Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A bacterial strains were grown in production medium (MP), comprising 12 g / L Tryptone, 3 g / L yeast extract, and 15 g / L NaCl, until stationary phase was reached. The Pseudomonas stutzeri RDC-01-A and Bacillus velezensis CA1-A strains were Docket No.1063.034 resuspended at a 1:5 ratio in inoculum medium comprising 0.90 g / L KH2PO4, 0.1 g / L citric acid, 10 g / L polyvinyl acetate (PVA), 1.4 g / L Guar gum, and 1.4 g / L xanthan gum. Accordingly, final compositions 1 and 2 were comprised by bacterial cells, metabolites present in the culture medium and chemicals of the inoculum medium. The results shown in the tables for compositions 1 and 2 correspond to a dose of 1.5 mL / Kg seed, both with 108to 109CFU / mL. For composition 3, compositions 1 and 2 were used in a combination resulting in a Pseudomonas stutzeri RDC-01-A to Bacillus velezensis CA1-Ain ratio of 1:1, 1:2, 1:5, 1:10, 2:1, 5:1, and 10:1 CFU / mL, respectively. The results obtained with composition 3 shown in the tables correspond to a Pseudomonas stutzeri RDC- 01-A to Bacillus velezensis CA1-A ratio of 1: 1, respectively and a dose of 1.5 mL of composition 3 per Kg seed. Shelf Life: The compositions were kept at room temperature and viability of the bacteria was evaluated over time by determination of Colony Forming Units per mL (CFU / mL). Viability on seed surfaces (CFU / g): 1.5 mL of the compositions was used for the inoculation by kg seeds. Viability of the bacteria was measured over time by washing 25 seeds with 5 mL of sterile saline solution and vortexing for 2 min. The cells detached from the seeds and present in the supernatant were seeded in solid medium for the CFU / mL count. Seed inoculation dosage To test the ability of the inoculated bacteria to affect plant growth, inoculation doses ranging from 0.15 to 4 mL / kg seed were evaluated Example 6: Field tests Field trials were carried out using a dosage of 1.5 mL of compositions 1 and 2 per kg of seeds. Prior to seeding of each crop, said dosage was applied directly Docket No.1063.034 on the seeds and immediately mixed to achieve a homogeneous distribution. Further, depending on each crop, the seeds received, or not, on their surface, other biological products or chemicals according to usual farming practices for each of these crops. Each treatment was compared to a control consisting of said normal farming practice without addition of compositions 1 or 2. "The biological material described herein has been deposited with the “Colección Chilena de Recursos Genéticos Microbianos” [Chilean Collection of Microbial Genetic Resources] (CChRGM), Avenida Vicente Méndez 515, Chillán, CHILE, under accession number RGM 3504 on August 1st2023. The deposited material is available to the public upon request in accordance with the terms and conditions of the CChRGM." "The biological material described herein has been deposited with the “Colección Chilena de Recursos Genéticos Microbianos” [Chilean Collection of Microbial Genetic Resources] (CChRGM), Avenida Vicente Méndez 515, Chillán, CHILE, under accession number RGM 3505 on August 1st2023. The deposited material is available to the public upon request in accordance with the terms and conditions of the CChRGM." REFERENCES de Weert, S.; Dekkers, L.C.; Kuiper, I.; Bloemberg, G. V; Lugtenberg, B.J.J. Role of Chemotaxis Toward Fusaric Acid in Colonization of Hyphae of Generation of Enhanced Competitive Root-Tip-Colonizing Pseudomonas Bacteria through Accelerated Evolution. J. Bacteriol. 2014, Vol. 186, 3153–3159, doi: 10.1128 / JB.186.10.3153 Edi-Premono, M., Moawad, A.M. and Vlek, P.L.G. (1996) Effect of phosphate- solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizosphere. Indonesian J Crop Sci 11, 13–23 Gaur, A.C. (1990) Phosphate Solubilizing Microorganisms as Biofertilizers; New Delhi: Omega Scientific Publishers no.198. Docket No.1063.034 Glickmann, E. and Dessaux, Y. (1995) A critical examination of the specificity of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria; Appl Environ Microbiol 61, 793–796. Lane, D.J. (1991) rRNA sequencing; In Nucleic Acid Techniques in Bacterial Systematics ed. Stackebrandt, E. and Goodfellow, M. pp.115–175. UK: John Wiley and Sons. Martín-Mora D, Ortega A, Reyes-Darias JA, García V, López-Farfán D, Matilla MA, Krell T; Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward α-Ketoglutarate; Front Microbiol., 2016 Nov 29; 7: 1937. doi: 10.3389 / fmicb.2016.01937. eCollection 2016. Nautiyal, C.S. (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms; FEMS Microbiol Lett 170, 265–270. Pérez-Miranda, S., Cabirol, N., George-Tellez, R., Zamudio-Rivera, L.S. and Fernandez, F.J. (2007) O-CAS, a fast and universal method for siderophore detection; J Microbiol Methods 70, 127–131. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual (3rd edn) New York: Cold Spring Harbor Laboratory Press. Schwyn, B. and Neilands, J.B. (1987) Universal chemical assay for the detection and determination of siderophores; Anal Biochem 160, 47–56.

Claims

Docket No.: 1063.034 CLAIMS: Having thus specifically described and determined the nature and the best mode for carrying out the present invention, we claim exclusive property and right on:

1. A modified bacterial strain of the Pseudomonas sp. genus, characterized by comprising the strain of Pseudomonas stutzeri deposited under Accession No. RGM3505.

2. A modified bacterial strain of the genus Bacillus sp., characterized by comprising the strain of Bacillus velezensis deposited under Accession No. RGM3504.

3. A combination of modified bacterial strains, characterized by comprising the Pseudomonas stutzeri strain deposited under Accession No. RGM3505 and the Bacillus velezensis strain deposited under Accession No. RGM3504.

4. The combination according to claim 3, characterized in that the combination comprises a Pseudomonas stutzeri to Bacillus velezensis rario from 1:10 to 10:1 CFU / mL.

5. A composition for increasing growth and abiotic stress tolerance in a plant, characterized by comprising a bacterial strain selected from the group consisting of Pseudomonas stutzeri deposited under Accession No. RGM3505, Bacillus velezensis deposited under Accession No. RGM3504 and combinations thereof, and an agricultural inoculum medium.

6. The composition according to claim 5, characterized in that the agricultural inoculum medium comprises at least salts, organic acids, polymers, and polysaccharides.

7. The composition according to claim 6, characterized in that the agriculturalDocket No.: 1063.034 inoculum medium further comprises a culture medium selected from the group consisting of a culture medium of the Pseudomonas stutzeri strain deposited under Accession No. RGM3505, the Bacillus velezensis strain deposited under Accession No. RGM3504 and mixtures of both culture media.

8. The composition according to claim 5, characterized in that the plant is selected from the group consisting of soybean, corn, sunflower, chickpea, wheat, beans, safflower, and turf.

9. The composition according to claim 8, characterized in that the plant is soybean.

10. The composition according to claim 5, characterized by comprising from 108to 109CFU / mL.

11. A process for preparing the composition of any of claims 5 to 8, characterized by comprising the steps of: a) culturing bacterial strains selected from Pseudomonas stutzeri deposited under Accession No. RGM3505, and Bacillus velezensis deposited under Accession No. RGM3504 in a production medium until stationary phase is reached; b) preparing a liquid agricultural inoculum; and c) adding an amount of the culture of step a. to an amount of the inoculum of step b. at a ratio of 1:5 v / v.

12. The process according to claim 11, characterized in that when the composition comprises both strains, step c. comprises combining both strains at a ratio of Pseudomonas stutzeri : Bacillus velezensis from 1:10 to 10:1 CFU / mL.

13. A method for increasing growth and / or abiotic stress tolerance of a plant or plant part, characterized by comprising contacting a plant or plant part with the composition of claim 5.

14. The method according to claim 13, characterized in that the abiotic stress is selected from the group consisting of saline soils and drought.Docket No.: 1063.034 15. The method according to claim 13, characterized in that the plant or plant part is selected from the group consisting of soybean, corn, sunflower, chickpea, wheat, beans, safflower, and turf.

16. The method according to claim 13, characterized in that the plant or plant part is soybean.

17. The method according to claim 13, characterized in that the plant part is selected from the group consisting of seed, root, stem, leaf, and fruit.

18. The method according to claim 17, characterized in that the plant part is a seed.

19. The method according to claim 13, characterized in that the step of contacting the plant or plant part with the composition is carried out by a method selected from the group consisting of spraying, dipping and inoculation in the soil.

20. The method according to claims 1 and 18, characterized by comprising contacting an amount from 0.15 to 4 mL / kg seeds.

21. The use of the strain of claim 1, characterized in that it is for preparing a composition to increase growth and / or abiotic stress tolerance of a plant.

22. The use of the strain of claim 2, characterized in that it is for preparing a composition to increase growth and / or abiotic stress tolerance of a plant.

23. The use of the combination of strains of claim 3, characterized in that it is for preparing a composition to increase growth and / or abiotic stress tolerance of a plant.

24. The use of the culture supernatant of the strain of la claim 1, characterized in that it is for preparing a cell free composition to increase growth and / or abiotic stress tolerance of a plant.Docket No.: 1063.034 25. The use of the culture supernatant of the strain of claim 2, characterized in that it is for preparing a cell free composition to increase growth and / or abiotic stress tolerance of a plant.

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