Bacterial strains, probiotic mixture, formulation and method that stimulate plant growth under abiotic stress

Abstract

The invention relates to isolated strains of Bacillus subtilis identified as H72, Pseudomonas koreensis identified as H121, Enterobacter cancerogenus identified as H104 and Bacillus licheniformis identified as H169, which comprise the characteristics of the strains deposited with deposit numbers CM-CNRG TB227, CM-CNRG TB228, CM-CNRG TB229 and CM-CNRG TB226, respectively. The invention also relates to a probiotic mixture for preparing a formulation that stimulates plant development under abiotic stress, which comprises at least two isolated bacterial strains, selected from the following group of deposited strains: CM-CNRG TB227, CM-CNRG TB229, CM-CNRG TB228 and CM-CNRG TB226. The invention additionally relates to a formulation for stimulating plant development under abiotic stress, which comprises: a probiotic mixture according to the present invention; at least one prebiotic substance; and at least one excipient. The invention further relates to a method for stimulating plant development under abiotic stress, which comprises applying an effective amount of the formulation of the invention.

Description

[0001] BACTERIAL STRAINS, PROBIOTIC MIXTURE, FORMULATION AND METHOD, WHICH STIMULATE PLANT DEVELOPMENT UNDER ABIOTIC STRESS

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the technical fields of biotechnology and agriculture, as it relates to endophytic bacterial strains from various plants; a probiotic mixture comprising said bacterial strains; and a formulation and method for protecting and stimulating plant growth under stress conditions caused by abiotic factors.

[0004] BACKGROUND OF THE INVENTION

[0005] Dehydration is a common threat to plants experiencing osmotic stress caused by salinity, drought, cold conditions, and high temperatures. The elevated accumulation of osmoprotectants in plants under water stress can increase cellular osmotic pressure to lower cell water potential and, consequently, water loss, and can also stabilize protein and membrane structures (Lui and Zhang, 2015). The abiotic stress tolerance of soil microorganisms has been studied to provide a biological understanding of the adaptation and survival of living microorganisms in extreme environments (Marulanda et al., 2009). Drought stress induces microorganisms to produce a variety of compounds that affect community stability.For example, treated dry soils contain more antibiotics, leading to the hypothesis that they are produced by drought-tolerant bacteria as a physiological response to outcompete other bacteria due to nutrient limitation, or possibly as a signal to induce drought response pathways such as biofilm formation. Some of these compounds include amino acids, such as proline, glutamine, and glycine betaine, and carbohydrates, such as trehalose and ectoine (Naylos and Coleman-Derr, 2018). Endophytic microorganisms are beneficial microorganisms that can stimulate plant growth or increase stress tolerance. Some of these microorganisms have been applied in agriculture, resulting in increased shoot emergence, plant weight, increased crop yield, and disease resistance (Lui and Zhang, 2015).

[0006] Stress situations in plants can be classified into two large groups according to the factor that causes them:

[0007] • Biotic factors. These are produced by other living beings, animals, insects, plants, fungi, bacteria, pathogens, etc.

[0008] • Abiotic factors. These include physical and chemical factors such as temperature, irradiation, drought, flooding, salinity, agricultural management, the use of machinery or other farming tools, etc.

[0009] The use of microorganisms as biofertilizers has emerged as an alternative not only environmentally friendly but also a way to increase crop yield under stressful conditions caused by abiotic factors. It also provides various nutrients, including nitrogen, for proper plant development.

[0010] US10645934B2 relates to an isolated culture of Enterobacter sp. 638, which is a non-phytopathogenic bacterial strain isolated under aerobic conditions from surface-sterilized root and stem samples of hybrid poplar H11-11 grown in silt loam soil with groundwater contaminated with carbon tetrachloride or trichloroethylene. A biological deposit of Enterobacter sp. 638 was made on March 4, 2011 with ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110. The Enterobacter sp. 638 includes a single circular chromosome of 4,518,712 bp with an overall G+C content of 52.98% and stably includes a plasmid pENT638-1 of 157,749 bp, with an overall G+C content of 50.57%. Plasmid pENT638-1 exhibits, based on its GC content, at least four distinct regions. Plasmid pENT638-1 is related to F plasmids found in other Enterobacteriaceae.Plasmids in this family are involved in host interaction and virulence, such as the pFra plasmid from the pest microbe Yersinia pestis. However, in pENT638-1, the pFra pathogenicity island is replaced by a single putative 23 kb genomic island (flanked by an integrase gene and having a GC content that is significantly different from the rest of the plasmid). In another aspect, the invention relates to a method of increasing drought tolerance and / or resistance in a plant. The method includes treating the plant with a composition that includes an isolated culture of Enterobacter sp. 638; although not limited to a particular strain, Enterobacter sp. 638 may increase drought tolerance and / or resistance in a plant due to the production of acetoin and 2,3-butanediol by Enterobacter sp. 638. While it is true that this document proposes the use of an isolated strain of Enterobacter spp.638 and a method for increasing a plant's tolerance to drought, but it is a single strain completely different from the Enterobacter cancerogenus H104 strain, while the present invention proposes a consortium of endophytic strains isolated from different plants in Mexico, with the purpose of protecting plants under conditions of water stress. Therefore, it is very likely that the characteristics of said Enterobacter spp 638 strain will not manifest optimally in the regions of our country; hence the need to isolate and validate the possible antagonistic and promoting characteristics in the protection of plant plants against conditions of water stress, of endophytic bacterial strains in the geographical region where they will be used, to guarantee a high effectiveness of said isolated strains.

[0011] Patent document IN201637040339A discloses a novel strain that has a fertilizing effect and can be used as a new type of biopesticide. Multifunctional plant endophytic bacteria were isolated that can broadly suppress the growth of plant pathogenic bacteria and fungi and specifically target rice as a host plant, thereby retaining both a disease resistance-inducing effect and a plant growth-promoting effect, followed by mass cultivation and formulation. The present invention provides a novel microorganism Bacillus oryzicola that has a DNA-to-DNA ratio of 70% or more with the Bacillus oryzicola species. It can be seen that this is a single isolated strain of B. oryzicola, and its usefulness is in suppressing the growth of pathogenic fungi and bacteria, diseases, and as a plant growth promoter, exclusively in rice; but not for protecting or stimulating plants under water stress.

[0012] Regarding patent document CN201710868805A it describes a bioproduct for plant plantations, comprising an inoculum having 20-30% solid preparation of rhizobia, 20-30% solid preparation of ACC deamination enzyme bacteria from the rhizosphere, 20-30% solid preparation of IAA producing bacteria from the rhizosphere. Among its beneficial effects of this invention, is that it can rapidly promote the growth and reproduction of plant roots, promote nodulation of legumes, increase plant resistance to stress and improve soil fertility with high slope and rocky. While it is true that document CN201710868805A proposes the treatment of plantations in conditions of sloping soils that are poor or eroded, but does not propose the isolation of endophytic microorganisms to induce the growth of plant crops under stress conditions.

[0013] For its part, patent document US2018368420A1 relates to methods and products based on microorganisms, to provide a benefit to seeds or agricultural seedlings, such as an agricultural plant, particularly a cereal, or an agricultural plant such as an agricultural herb derived from the seed or seedling. For example, this document provides purified bacterial populations that include novel bacterial endophytes of seed origin and synthetic combinations of seeds and / or seedlings with heterologous bacterial endophytes derived from seeds. Such bacterial endophytes of seed origin can provide beneficial properties to the seed, seedling or agricultural plant derived from the seed or seedling, including metabolism, transcription, proteome alterations, morphology and resistance to a variety of environmental stresses and the combination of such properties.From what is understood, this document provides combinations of isolated microorganisms that are already in the state of the art. One of the drawbacks is that the geographic origin of the isolation of these strains is not well known, nor from which plants they are isolated. Therefore, it is very likely that their growth and microbial invasion characteristics are not optimally expressed under the conditions of our country. This is why, once again, there is great interest in isolating and validating the potential antagonistic and development-promoting characteristics of endophytic bacterial strains in the region where they will be applied, to ensure high effectiveness of these isolated strains.

[0014] Therefore, in order to contribute to the solution of the problems encountered in the state of the art, endophytic bacterial strains were isolated from various plants, which demonstrated growth-stimulating activity in plants under stress conditions caused by abiotic factors, such as high temperature and drought. Therefore, a probiotic mixture with the isolated bacterial strains, a formulation, and a method were also developed to stimulate plant growth under stress conditions caused by abiotic factors.

[0015] The characteristic details of the present invention are clearly shown in the following detailed description of the invention, examples and accompanying figures, where:

[0016] Figure 1 is a graph showing the proline concentration in different treatments of selected bacterial strains and their mixtures, in germinated corn seeds, subjected to water stress.

[0017] Figure 2 is a graph illustrating the concentration of peroxidase (POD) in different treatments of selected bacterial strains and their mixtures, in germinated corn seeds, subjected to water stress.

[0018] Figure 3 is a graph illustrating the proline concentration in different treatments of selected bacterial strains and their mixtures in corn seedlings, subjected to water stress.

[0019] Figure 4 is a graph of the behavior of the absorbances obtained from the production of superoxide ion (Ü2') of the different treatments of selected bacterial strains and their mixtures, applied to corn seedlings.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to an isolated strain of Bacillus subtilis identified as H72, which has the characteristics of the strain deposited under deposit number CM-CNRG TB227; which is an endophyte of Larrea tridentata.

[0022] This invention also relates to an isolated strain of Enterobacter cancerogenus identified as H104, which comprises the characteristics of the strain deposited under No. CM-CNRG TB229; which is an endophyte of Ferocactus viridescens.

[0023] The present invention provides an isolated strain of Pseudomonas koreensis identified as H121, which comprises the characteristics of the strain deposited under accession number CM-CNRG TB228; which is an endophyte of Yucca treculeana.

[0024] This invention relates to an isolated strain of Bacillus licheniformis identified as H169, which has the characteristics of the strain deposited under accession number CM-CNRG TB226; said strain is also an endophyte of Ferocactus viridescens.

[0025] The strains CM-CNRG TB227, CM-CNRG TB229, CM-CNRG TB228 and CM-CNRG TB226, in accordance with the present invention, were found to exhibit plant growth-stimulating activity under abiotic stress conditions; preferably, water stress conditions, high temperature stress, among others, and their combination.

[0026] The invention in question also relates to a probiotic mixture for preparing a formulation that stimulates plant growth under abiotic stress conditions; said mixture contains at least two isolated bacterial strains selected from the following group of deposited strains: CM-CNRG TB227, CM-CNRG TB229, CM-CNRG TB228 and CM-CNRG TB226, and mixtures thereof.

[0027] An embodiment of the probiotic mixture of the present invention is when the CM-CNRG TB227 strain is an endophyte of Larrea tridentata.

[0028] Another embodiment of the probiotic mixture in question is when the CM-CNRG TB228 strain is an endophyte of Yucca treculeana.

[0029] One embodiment of the probiotic mixture of the present invention is when the strains CM-CNRG TB226 and CM-CNRG TB229 are endophytes of Ferocactus viridescens.

[0030] In a further embodiment of the probiotic mixture according to the present invention, the aforementioned bacterial strains exhibit activity that stimulates plant growth under abiotic stress conditions; water stress, high temperature stress, and their combination are more preferred.

[0031] In another embodiment of the mixture according to the present invention, said mixture is made up of the strains: CM-CNRG TB228 and CM-CNRG TB229; a preferred embodiment being when the mixture, in addition to the previous strains, comprises the strain CM-CNRG TB226; a more preferred embodiment being when said mixture also comprises the strain CM-CNRG TB227. This invention also provides a formulation to stimulate the development of plants under abiotic stress conditions, which comprises: a probiotic mixture, which in turn comprises at least two strains selected from the following group: CM-CNRG TB227, CM-CNRG TB229, CM-CNRG TB228 and CM-CNRG TB226; at least one prebiotic substance; and at least one excipient.

[0032] Where an embodiment of the formulation to stimulate the development of plants under abiotic stress conditions, of the present invention, is when the abiotic stress is water stress, high temperature stress, and a combination thereof.

[0033] Therefore, this invention also relates to a method for stimulating the development of plants under abiotic stress conditions, wherein said method comprises applying an efficient amount of the formulation that stimulates the development of plants under abiotic stress conditions, in accordance with what is proposed by the present invention.

[0034] Being a preferred embodiment of the method for stimulating plant development under abiotic stress conditions, the invention in question is when the abiotic stress is water stress, high temperature stress, and their combination.

[0035] EXAMPLES

[0036] The following examples are included for the sole purpose of illustrating the conception of the present invention and some preferred embodiments thereof, and should therefore not be considered as limiting the scope of protection of said invention.

[0037] Example 1. Select and identify microorganisms that promote plant growth under abiotic stress conditions.

[0038] Materials and methods

[0039] With the aim of selecting microorganisms that promote or stimulate the growth or development of plants under abiotic stress conditions, microorganisms were isolated from wild tomatillo plants (Solatium elaeagnifolium), rock tea (Jasonia glutinosa), Gauda (Reseda luteola), Agave (Agave chrysantha), Nopal (Opuntia ficus-indica), Yucca (Yuca treculeana), Cactus (Ferocactus viridescens), Creosote bush (Larrea tridentata), Biznaga llavina (Mammillaria dioica), Dry grass (Cynodon dactylon) and a species of dry shrub from the San Luis Potosí desert, which were collected near the borders between the states of Jalisco and San Luis Potosí, Mexico; during the months of May 2021 to May 2022. The plants were selected according to their appearance and the degree of drought of the environment where they were.

[0040] The plants were transported in paper envelopes and plastic bags to minimize root damage. Once they arrived at the Microendo Laboratory in Atotonilco el Alto, Jalisco, Mexico, the plants were carefully removed from their plastic bags and rinsed with running water to remove excess soil.

[0041] Once the plants were washed, the roots were sectioned into sections to subsequently follow the endophyte extraction protocol. The roots were washed with 75% alcohol for 30 s, then with 3% sodium hypochlorite for 10 min, and finally, washed three times with sterile, double-distilled water for 5 min each. One mL of water was taken from the last wash and placed in a Petri dish of trypticase soy agar to ensure proper washing.

[0042] The washed root (approximately 100 g per plant) was then ground using a mortar and pestle with 30 mL of isotonic saline solution to form a suspension. A plate count of 1 mL was taken from the suspension using the serial dilution technique. 8 dilutions were made for each plant, and the final dilutions were plated onto conventional Nutrient Agar.

[0043] When the suspensions were diluted and plated, increasingly less concentrated colonies were formed. From these dilutions, colonies with different morphologies (color, size, texture) were taken and reseeded several times on trypticase soy agar until they were purified, that is, until they only had a single morphology. From this extraction of endophytes from all the plants, a total of 192 bacterial colonies with different characteristics and properties were isolated. These were identified from H1 to H192, and are illustrated in Table 1. Agronomic tests

[0044] The 192 colonies isolated from the different collected plants were subjected to the following agronomic tests: nitrogen fixation test (NFT), phosphate solubilization test (NBRIP Ca, and NBRIP Fe), iron chelators (siderophores) test, phytohormone (auxin) production test, and acc deaminase (ACC) enzyme production test. In this case, since our main objective was to select microorganisms that induced plant growth, we emphasized the production of auxins, since this is a phytohormone related to growth, as well as nitrogen fixation, an essential element for the plant and, especially, the production of the ACC deaminase enzyme, an enzyme whose function is to inhibit the synthesis of ethylene, a molecule related to fruit ripening.In addition, we also measured exopolysaccharide (Exo) production, since according to the literature, exopolysaccharide production improves soil texture and creates micro- and macroaggregates by binding to soil particles, retaining water and slowing drying. However, we also considered the results of other tests to generate a product that not only achieves adequate growth but also provides other properties that may be beneficial to the plant. Table 1 summarizes the results of the agronomic tests for all the isolated strains, with the goal of selecting those that yield the best results in the agronomic tests.

[0045] When analyzing all the isolates, we selected 4 strains of the genera Bacillus (2 strains), Enterobacter (1 strain) and Pseudomonas (1 strain) from the total of 192 isolated strains, because these strains showed greater production of metabolites such as ACC deaminase, siderophores and auxins, metabolites that stimulate plant growth, as well as nitrogen fixation and phosphate solubilization, in addition some showed to be capable of producing exopolysaccharide and poly hydroxybutyrate, molecules that help the formation of biofilms and water retention. Also these strains showed the ability to produce various enzymes such as oxidases and catalases (enzymes that help combat oxidative stress), in addition to chitinases, proteases and lipases, enzymes that can help combat pathogenic fungi. The selected bacterial strains were the following:

[0046] - Bacillus subtilis H72 (Firm cutes), was selected because it solubilises organic and inorganic phosphates, produces siderophores, auxins and ACC deaminase, generates exopolysaccharide, produces the enzymes oxidases, catalases, chitinases, proteases and lipases, in addition to producing PHB and ammonium.

[0047] - Enterobacter cancerogenus. H104 (Proteobacteria), was selected because it was able to fix nitrogen, solubilize organic phosphates, produce siderophores, ACC deaminase, HCN, PHB and ammonium, produce the enzymes catalase, cellulase, chitinase and lipase, and be the largest producer of auxins and the only one capable of solubilizing potassium.

[0048] - Pseudomonas koreensis H121 (Proteobacteria), was selected because it fixes nitrogen, solubilises organic phosphates, produces siderophores, ACC deaminase, PHB and ammonium, produces the enzymes oxidases amylases, cellulases, proteases and lipases, is capable of solubilising potassium and is the largest producer of HCN.

[0049] - Bacillus licheniformis H169 (Firmicutes), was selected because it solubilises organic and inorganic phosphates, produces siderophores, aminase ACC, exopolysaccharide, HCN, PHB and ammonium, in addition to producing the enzymes catalase, cellulase, chitinase, protease and lipase, and is the largest producer of amylases.

[0050] Also in Table 1 we observe that 2 of the 4 strains are positive to the nitrogen fixation test, with abundant fixation, due to the color change they showed. For the solubilization of inorganic phosphates, the halo generated around the colony inoculated in the medium is measured, the only strains that presented minimal solubilization of inorganic phosphates were strains H72 and H169. However, for the solubilization of organic phosphates, the 4 strains showed the ability to solubilize organic phosphates, with strain H104 presenting the largest halo at 0.5 cm. All four strains were shown to be effective for the production of siderophores. Table 1. Agronomic tests of all isolated strains. Table 1. Continued... Table 1. Continued... Table 1. Continued... Table 1. Continued... Table 1. Continued... Table 1. Continued...

[0051] Where, lig + is minimal production. + is low production. ++ is moderate production and +++ is abundant production. In the production of auxins, the strain that produced the greatest amount of this phytohormone was strain H104, followed by strain H72, with poor production of this molecule by having a cross of three, while strains H121 and H169 were not able to produce this molecule. For the production of the ACC deaminase enzyme, as we observe in Table 1 , the strain with the highest production was H169 with three crosses of three, followed by strains H72 and H104 with moderate production and strain H121 with poor production of this enzyme. Finally, in the production of exopolysaccharide, only strains H72 and H169 produced this molecule.

[0052] Biochemical tests

[0053] Subsequently, the 4 selected strains underwent biochemical tests: production of antioxidant enzymes such as catalases and oxidases, production of hydrocyanic acid (HCN), polyhydroxybutyrate (PHB), ammonium and production of enzymes such as amylases, cellulases, proteases, pectinases, lipases and chitinases, in addition to measuring the capacity of bacterial strains to solubilize potassium. This was done to ensure that the selected strains are the most complete in the production of molecules that can be beneficial to the plant. The results obtained from the aforementioned tests are reported in Table 2.

[0054] The strains that showed the highest production of oxidases were strains H72 and H121, which present an abundant production of this enzyme, while H104 and H169 did not produce this enzyme. On the other hand, for catalases, the strains that showed abundant production of this enzyme were H72, H104 and H169, while H121 did not produce this molecule. Hydrocyanic acid production was abundant for strain H121, while for H72 it was low production and for strains H104 and H169 the production was minimal. All strains produced PHB abundantly. Strains H121 and 169 showed little production of cellulases, while strain H104 had a minimal production of this enzyme, strain H72 did not present any cellulase production. For amylases, only strain H169 produced this enzyme abundantly and H121 had poor production. Strains H72 and H169 showed abundant production of proteases, while strain H121 produced minimally.For pectinases, no strain produced this enzyme. Regarding ammonium (NH4) production, strains H104 and H121 produced this molecule abundantly, while strains H72 and H169 produced it moderately. Three of the four strains that produced the highest amount of chitinases are H72, H104 and H169 with a halo formed of 0.1 cm. In the case of lipases, strains H72 and H169 had moderate production, while strains H104 and H121 had minimal production. Finally, for potassium solubilization, only strain H104 presented a halo of 0.4 cm of solubilization and strain H121 slightly solubilized potassium, the rest of the strains did not have the capacity to solubilize potassium.

[0055] Table 2. Biochemical tests of the 4 selected bacterial strains.

[0056] Where: lig+ is minimal production, + is low production, ++ is moderate production, and +++ is abundant production.

[0057] Salinity resistance

[0058] Once the agronomic and biochemical tests were completed, the selected strains were tested for salinity resistance. This test demonstrated that the selected bacterial strains were able to withstand high salt concentrations, as occurs in soils when they are dry. The bacteria were cultured in Ashby broth supplemented with sodium chloride. Three tests were performed per bacterium according to the following parameters: Ashby broth with a sodium chloride concentration of 0.2 g / L; Ashby broth with 15 times more sodium chloride; and Ashby broth with 30 times more sodium chloride. The sterile broths were inoculated with the bacteria, and the optical density at 600 nm was measured at 24, 48, and 72 h using a visible spectrophotometer. Table 3 shows the optical density values ​​obtained.

[0059] Table 3. Optical densities (OD) obtained post-inoculation in Ashby broth with increasing salt concentration, of the four selected bacterial strains.

[0060] As can be seen in Table 3, the strain that showed the least tolerance to salts is strain H72, since it only showed optical density at 24 h, but after 48 h there was no reading, which could mean a decrease in colony forming units. On the other hand, strains H104, H121 and H169 showed high tolerance to high salinity by gradually increasing the optical density over time, reaching 0.598, 0.783 and 0.747 respectively, at 72 h after inoculation with Ashby broth that contained 30 times more salt concentration than the initial broth. This indicates that the strains will be able to withstand high salt concentrations.

[0061] Trehalose production

[0062] The selected strains were also tested for trehalose production, which is important because it protects against various abiotic stress conditions such as drought, salinity, and high temperatures. To measure trehalose production, the bacteria were grown in minimal medium (M9) containing glucose, magnesium sulfate, and calcium chloride. After 24 h, they were centrifuged to obtain the pellet. The pellet was washed and resuspended in 1 mL of the same medium. The cells were lysed and then centrifuged. 100 pL of the supernatant was taken, and 90 pL of a sodium acetate buffer solution and 0.02 U of trehalase were added. They were incubated overnight at 37°C. Subsequently, 150 pL of the reaction mixture was taken and 150 pL of a mixture containing Aspergillus niger glucose oxidase, horseradish peroxidase in a phosphate buffer and 50 pL of o-toluidine was added. After 30 min of incubation at 37°C, 1 .5 mL of water were added and read at 420 nm. The results are shown in Table 4.

[0063] Table 4. Trehalose production of the four selected bacterial strains.

[0064] Table 4 shows the concentrations of trehalose obtained, the strain with the highest trehalose production is strain H72 with 1.5x10 -5 M, followed by strain H169 with 1.1x10' 5 M, this could be due to the ability of these strains to produce exopolysaccharide. While strains H104 and H121 produced this molecule in smaller quantities, managing to produce 1.7x10 -6 and 6.2x10 -6 M, respectively. In summary, the four selected strains proved to be effective in producing not only trehalose, but also other molecules that can help the plant withstand water stress and high temperatures, in addition to providing other molecules for healthy growth.

[0065] ID

[0066] The four selected strains were identified. Bacterial identification was performed using the MALDI-TOF technique, which involves obtaining a protein profile of the microorganism being analyzed and comparing it with a database. The result is a measure of the accuracy of the identification. The score ranges from 0 to 3, with a score of 0-1,599 indicating an unreliable identification; a score of 1,600-1,999 indicating a high probability that the identified genus is correct; a score of 2,000-2,299 indicating a 100% match for the identified genus and a high probability for the species; and finally, a score of 2,300-3,000 indicating a 100% match for the genus and species. The identification results are shown in Table 5.

[0067] Table 5. MALDI-TOF identification of the selected strains.

[0068] Example 2. Compatibility between the four selected bacterial strains.

[0069] Once the four selected strains were identified, a compatibility test was conducted between them to determine the feasibility of mixing them and measure their effect on plant growth, as well as their protection against water stress and high temperatures.

[0070] The compatibility test was performed as reported by James and Mathew in 2017. In a Petri dish containing thypticasein soy agar, one of the bacterial strains was inoculated horizontally and the other bacterial strain was inoculated vertically, forming a cross, and they were incubated at 37°C for 24 h. Growth was observed at the intersection where the two strains met.

[0071] When testing the four selected strains, strains H104 and H121 were compatible with each other, and strain H169 was compatible with both strains, but a decrease in the biomass of this strain was observed. On the other hand, H72 was only compatible with strain H169.

[0072] Example 3. Determination of the protective effect of bacterial strains against water stress in corn seeds.

[0073] To determine the effectiveness of the selected bacterial strains against water stress, an experiment was carried out with corn seeds, in which the following treatments (T) shown in Table 6 were tested.

[0074] Fifteen corn seeds were selected per treatment. The seeds were immersed in their respective treatment for 30 minutes. After this time, the seeds were placed in a Petri dish containing a filter impregnated with 15% polyethylene glycol 6000, to generate an osmotic pressure effect that resembles the water stress experienced by seeds in the field. Five seeds were placed per Petri dish. The seeds were incubated at 25°C, with a 12 / 12 h light / dark period for 3 weeks.

[0075] After three weeks, the seeds were removed, and the following parameters were quantified: number of germinated seeds (NSG), average weight (PP) in g of the seedlings, average root length (LPR) in cm, and average first leaf length (LPH) generated; extractions were subsequently performed to measure the proline, peroxidase, and superoxide contents. The results obtained are summarized in Table 7. Table 6. Treatments to determine the efficacy of the selected bacterial strains against water stress in corn seeds.

[0076] Proline Determination For the determination of proline in the tissues of germinated seeds, 0.5 g of tissue was first macerated with 10 mL of 3% sulfosalicylic acid and filtered. 2 mL of the filtrate was then reacted with 2 mL of ninhydrine acid and 2 mL of glacial acetic acid in a tube at 100°C for 1 h. After this time, the reaction was stopped in an ice bath. 4 mL of toluene was added to the mixture and mixed for 20 s. The aqueous phase was extracted from the mixture and read in a visible spectrophotometer at 520 nm. A proline curve was prepared to determine the concentration of proline present in the plant tissue. The proline concentrations obtained are shown in Table 7. Figure 1 shows the behavior of the proline concentration produced by the sprouts under osmotic stress conditions. The treatment that produces the highest proline concentration is the treatment

[0077] 13 with a production of 560.98 mg / L of proline, followed by treatments 8 and 14 with a production of 547.92 mg / L and 462.78 mg / L, respectively. The induction of proline production in the plant is important since proline maintains cellular water, helps preserve membranes and proteins when under stress, as it acts as an osmolyte. In addition, it helps eliminate hydroxyl radicals and moderates the NAD / NADH ratio.

[0078] Determination of peroxidase (POP)

[0079] For tissue peroxidase determination, the first leaf to sprout from seeds germinated under osmotic stress conditions was taken and macerated with a pH 7 buffer solution containing EDTA, polyvinylpyrrolidone, and triton X-100. The resulting liquid was taken and centrifuged at 5000 rpm for 30 min. The supernatant was recovered and stored at 4°C until analysis.

[0080] From the supernatant, 20 pL were taken and mixed with 1 mL of a pH 7 phosphate buffer containing hydrogen peroxide and guaiacol. The resulting mixture was analyzed in a visible spectrophotometer at 470 nm, measuring the absorbance every minute for 5 min. The concentration of peroxidase present in the tissue is shown in Table 7.

[0081] In Figure 2, the production of peroxidase can be better observed, where the treatment that presented the highest concentration of peroxidase was the treatment

[0082] 14 with a concentration of 0.0068 pM of peroxidase, followed by treatments 13 and 16 with a concentration of 0.0064 and 0.0062 pM, respectively. To reduce the damage caused by reactive oxygen species (ROS), plants usually adjust the activity of their antioxidant system to improve their resistance to water stress, in this case, the levels of antioxidant enzymes such as peroxidase (POD) should increase, since this is indicative of a good antioxidant capacity. Of all the treatments tested for peroxidase, treatment 14 is the treatment with the best antioxidant capacity since it is the treatment that induces a greater production of said enzyme. Table 7. Parameters of germinated corn seeds under osmotic pressure conditions.

[0083] T = treatments, NSG = number of germinated seeds, LPR = average root length, LPH = average length of the first leaf, and PP = average seedling weight. Example 4. Determination of the protective effect of bacterial strains against water stress in corn seedlings.

[0084] To determine the efficacy of the selected strains against water stress, an experiment was conducted with corn seedlings. Corn seeds were germinated in a germination tray and approximately 7 days were allowed before transferring the seedlings to pots containing 100 g of a 1:1 sand-perlite mixture. The plants were kept outdoors and watered daily for a week. The plants were subsequently inoculated with various selected bacteria and their mixtures. The following treatments were tested, as shown in Table 8. Treatments were administered in triplicate. After inoculation, the plants were watered for a period of one week, and after the end of the irrigation period, irrigation was suspended for 7 days.Once the time had passed, the leaves were taken from the treatments to subsequently perform extractions and measure proline and superoxide.

[0085] Determination of proline

[0086] To determine proline, dried leaves were macerated from a plant with 10 mL of 3% sulfosalicylic acid and filtered. Two mL of the filtrate were then reacted with 2 mL of acid ninhydrin and 2 mL of glacial acetic acid in a tube at 100°C for one hour. After this time, the reaction was stopped in an ice bath. 4 mL of toluene was added to the mixture and the mixture was mixed for 20 s. The aqueous phase was extracted from the mixture and read in a visible spectrophotometer at 520 nm. A proline curve was prepared to determine the proline concentration present in the plant tissue. The proline concentrations obtained are shown in Table 9.

[0087] Figure 3 shows the concentration of proline produced by plants subjected to water stress, where the treatment that produced the highest concentration of proline was treatment 14 with a production of 30.31 mg / L of proline, followed by treatments 2 and 12 with a production of 25.59 and 25.33 mg / L respectively. The induction of proline production in the plant is important since proline maintains cell water, helps preserve membranes and proteins when they are under stress, since it acts as an osmolyte. In addition, it helps eliminate hydroxyl radicals and moderates the NAD / NADH ratio. Table 8. Treatments to determine the efficacy of the selected bacterial strains against water stress in corn seedlings.

[0088] Determination of superoxide in corn seedlings. For the determination of superoxide in the tissue, the leaves were taken and macerated with the help of a pH 7.8 buffer solution. The resulting liquid was taken and centrifuged at 5000 rpm for 5 min. The supernatant was recovered and stored at 0°C until further use. 1 mL of the supernatant was mixed with 0.9 mL of a pH 7.8 phosphate buffer solution and 0.1 mL of hydroxylamine hydrochloride. The samples were placed in a water bath at 25°C for 20 min, then 1 mL of sulfanilamide and 1 mL of α-naphthylamine were added, mixed and placed in a water bath at 25°C for 20 min. The resulting mixture was analyzed in a visible spectrophotometer at 530 nm. The absorbances obtained are shown in Table 9. In Figure 4, we can see that the treatment with the highest absorbance is treatment 8, which had an absorbance of 0.353, followed by treatments 14 and 2 with absorbance values ​​of 0.3143 and 0.3123 respectively.Treatment 14 was the second-best treatment, and compared to treatment 8, there was no significant difference in the values ​​obtained. In this case, the absorbance values ​​are directly proportional to the superoxide ion concentration; that is, the higher the absorbance, the higher the concentration of this molecule. This ion indicates a healthy oxidative system in the plant, since its presence indicates the induction of an enzyme called superoxide dismutase, which has antioxidant activity.

[0089] Table 9. Parameters of the different treatments in corn seedlings under osmotic pressure conditions. STORAGE OF ISOLATED BACTERIAL STRAINS

[0090] Since the isolated bacterial strains H72, H104, H121 and H169 showed agronomic potential, they were deposited on January 13, 2023, in the Microorganism Collection of the National Center for Genetic Resources, which belongs to the National Institute of Forestry, Agriculture and Livestock Research, with registration number 1006 before the World Federation of Culture Collection (CM-CNRG) and International Deposited Authority with notification 308 for the purposes of patent proceedings in accordance with the Budapest Treaty; and which has its address at Boulevard de la Biodiversidad 400, CP 47600, Tepatitlán de Morelos, Jalisco, Mexico. The bacterial strain H72 Bacillus subtilis was assigned deposit number CM-CNRG TB227, the strain H104 Enterobacter cancerogenus was assigned accession No. CM-CNRG TB229, the strain H121 Pseudomonas koreensis was given deposit number No. CM-CNRG TB228, and the strain H169 Bacillus licheniformis was assigned accession No. CM-CNRG TB226.

Claims

CLAIMS 1. An isolated strain of Bacillus subtilis identified as H72, characterized in that it comprises the characteristics of the strain deposited under deposit number CM-CNRG TB227.

2. The strain of the preceding claim, wherein said strain is an endophyte of Larrea tridentata.

3. An isolated strain of Pseudomonas koreensis identified as H121, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB228.

4. The strain of the preceding claim, wherein said strain is an endophyte of Yucca treculeana.

5. An isolated strain of Enterobacter cancerogenus identified as H104, characterized in that it comprises the characteristics of the strain deposited with deposit No. CM-CNRG TB229.

6. An isolated strain of Bacillus licheniformis identified as H169, characterized in that it comprises the characteristics of the strain deposited under accession No. CM-CNRG TB226.

7. The strains of claims 5 and 6, wherein such strains are endophytes of Ferocactus viridescens.

8. Strains according to any of the preceding claims, wherein said strains exhibit plant development stimulating activity under abiotic stress conditions.

9. The strains of the preceding claim, wherein the abiotic stress is water stress, high temperature stress, salinity stress, and a combination thereof.

10. A probiotic mixture for preparing a formulation that stimulates plant growth under abiotic stress conditions; characterized in that it comprises at least two isolated bacterial strains selected from the following group of deposited strains: CM-CNRG TB227, CM-CNRG TB229, CM-CNRG TB228, and CM-CNRG TB226.

11. The mixture of the preceding claim, wherein the strain CM-CNRG TB227 is an endophyte of Larrea tridentata.

12. The mixture of claim 10, wherein the strain CM-CNRG TB228 is an endophyte of Yucca treculeana.

13. The mixture of claim 10, wherein the strains CM-CNRG TB226 and CM-CNRG TB229 are endophytes of Ferocactus viridescens.

14. The mixture according to claim 10, wherein said strains exhibit plant development stimulating activity under abiotic stress conditions.

15. The mixture of the preceding claim, wherein the abiotic stress is water stress, high temperature stress, salinity stress, and a combination thereof.

16. The mixture according to claim 10, wherein the selected strains are: CM-CNRG TB228 and CM-CNRG TB229.

17. The mixture of the preceding claim, characterized in that it also comprises the CM-CNRG TB226 strain.

18. The mixture according to the preceding claim, characterized in that it also comprises the CM-CNRG TB227 strain.

19. A formulation for stimulating plant growth under abiotic stress conditions, characterized in that it comprises: a probiotic mixture according to claims 10 to 18; at least one prebiotic substance; and at least one excipient.

20. The formulation of the preceding claim, wherein the abiotic stress is water stress, high temperature stress, salinity stress, and a combination thereof.

21. A method for stimulating the development of plants under abiotic stress conditions, characterized in that it comprises applying an efficient amount of the formulation that stimulates the development of plants under abiotic stress conditions, in accordance with claims 19 and 20.

22. The method of the preceding claim, wherein the abiotic stress is water stress, high temperature stress, salinity stress, and a combination thereof.

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