Bacillus nakamurai CECT 30581 and plant growth-promoting metabolic products thereof under conditions of water stress

Abstract

The present invention relates to bacterial strain Bacillus nakamurai CECT 30581, a microorganism of the gram-positive group of bacteria, genus Bacillus, and the metabolic products resulting from fermentation in the culture medium, total metabolic liquid (TML), hexane fraction (HF) and hexane subfraction (HS), nitrogen, phosphorous, potassium and manganese absorption stimulants in plants, and atmospheric CO2 fixation and transpiration, even under conditions of water stress. Once this strain has been isolated from the rhizosphere of Pinus pinaster and characterised from a physiological and genetic standpoint, the TML is separated / extracted and the metabolites responsible for the effects thereof, such as a characterisation profile of the bacterial products for different uses, are identified. Both the bacterial strain and its compositions can be used as the basis for producing different preparations intended to optimise the photosynthetic process of plants, improving plant growth and production in adverse situations of drought or reduced irrigation water. They can also be used within the field of organic biofertilisers to improve mineral nutrition in the indicated nutrients.

Description

[0001] DESCRIPTION

[0002] Bacillus nakamurai CECT 30581 and its metabolic products that promote plant growth under water stress conditions.

[0003] The present invention relates to a new strain of Bacillus nakamurai and to the metabolites produced during its fermentation process, both components being used in the treatment of plants subjected to water stress conditions, with the aim of improving mineral nutrition in nitrogen, phosphorus, potassium and manganese, and improving the uptake and fixation of CO2, increasing biomass production (dry weight), the efficiency in the use of water, and plant production in drought environments or in the absence of water at risk.

[0004] This strain has been deposited for patent purposes with the Spanish Type Culture Collection (CECT) on March 29, 2022, where it has been assigned deposit number 30581. The CECT is headquartered in the research building of the University of Valencia, located on the Burjassot campus (DP 46100 - Valencia, Spain).

[0005] FIELD OF APPLICATION. -

[0006] This invention is situated in the field of agro-food biotechnology, specifically within plant growth promoting bacteria (PGPB), since the aforementioned bacterial strain and its metabolites can serve as a basis for the preparation of various products intended to increase CO2 uptake and optimize the photosynthetic process of plants, which translates into an increase in dry weight and production.

[0007] And also in the field of organic biofertilizers, since these bacterial products can be used to improve the mineral nutrition of plants with the indicated nutrients.

[0008] STATE OF THE ART. -

[0009] Nitrogen (N) and carbon (C) are essential elements for plant development and crop production. Improving the utilization of these elements can increase agricultural productivity and reduce the need for fertilizers. While N is mainly supplied through the roots, CO2 is obtained through the process of photosynthesis. Photosynthesis is a vital process in plant physiology, where light energy is converted into chemical energy: H2O + CO2 + E (light energy) produces C6H12O6 + O2. This process involves the absorption of water through the roots and the uptake of carbon dioxide in the leaves, and the capture of energy from solar radiation.Photosynthesis consists primarily of two stages: first, the capture of light energy and its conversion into chemical energy (in the form of ATP and NADPH), and second, the use of this energy to fix CO2 and build organic compounds that will provide energy for vital processes.

[0010] During photosynthesis, photosynthetic pigments play an essential role in capturing light energy, but this process also generates oxidative stress in the form of free radicals, primarily reactive oxygen species (ROS). The plant has mechanisms to eliminate these free radicals; these processes are inducible and essential to maintain them within adequate physiological levels and prevent them from causing structural damage at the cellular level, which leads to a loss of performance in all the plant's vital processes.

[0011] A critical aspect of this entire process is the adequate availability of water and proper hydration of the plant, since CO2 uptake necessarily involves water loss through the stomata (transpiration). This transpiration process tends to accelerate under adverse temperature and humidity conditions. Therefore, there is a contradiction in the requirements of photosynthesis, since good CO2 uptake through the stomata is essential, but at the same time, it can result in significant water loss under unfavorable conditions.

[0012] Thus, it is essential that the plant is adequately hydrated. The key indicator of a plant's water status is its water potential, which reflects the balance between water content (pressure potential) and solute concentration (osmotic potential). The plant has the ability to generate a negative osmotic potential relative to the soil, thereby forcing water uptake associated with nutrient uptake. These ions accumulate in the plant's vacuoles, which in turn favors the entry of water into these vacuoles, thus maintaining a water reservoir and preserving the plant's turgor. Proton pumps are essential in this process since both nutrient uptake and their accumulation in the vacuole occur through specific transporters, which require the generation of an electrochemical proton gradient, a process that relies on proton pumps (H +-ATPase). Thus, there are recent studies on the H pump + -Plasma membrane (PM) ATPase, a prominent proton pump found in the plant cell membrane. This pump plays a crucial role in generating an electrochemical gradient by pumping protons outward, an essential process in generating the energy needed for nutrient and water uptake leading to cell growth. Activation of H + The modification of the MP ATPase domain involves the modification of its autoinhibitory terminal domains, suggesting a possible regulation of proton pumping by physiological factors interacting with these regulatory terminals. Furthermore, various signals that affect plant growth have been shown to also interfere with the post-translational regulation of this enzyme (Zhang et al., 2021).

[0013] In another recent study, overexpression of a single rice gene, known as Oryza sativa plasma membrane (PM) H+-ATPase 1 (OSA1), was shown to improve ammonium uptake and assimilation in roots, while increasing stomatal aperture. This resulted in a 33% increase in grain yield and a 46% improvement in overall nitrogen use efficiency. Since H + -MP ATPase is highly conserved in plants, this enzyme appears as a promising target inducible by microbial factors to achieve more effective nitrogen absorption and carbon sequestration, constituting an essential tool to promote food security and sustainability in agriculture (Falhof et al., 2016).

[0014] Once these nutrients have been absorbed, they are stored in vacuoles or transported to the aerial parts of the plant through transpiration. These nutrients are subsequently incorporated into organic compounds (assimilation), essential compounds for plant growth. Therefore, it is important to emphasize that the mineral nutrition of the plant requires the loss of water through the process of transpiration.

[0015] Thus, the plant must maintain a balance between stomatal opening and closure to facilitate the exchange of carbon dioxide and water, prioritizing stomatal closure when water becomes scarce. In addition to regulating stomatal opening, as previously mentioned, the plant has other mechanisms to conserve water by increasing the internal concentration of solutes, including osmolytes and other organic molecules. It also has antioxidant systems to maintain a healthy overall state and ensure efficient photosynthesis, keeping free radical levels within physiological limits. Having demonstrated the role of proton pumps in improving plant nutrition using genetic modification techniques, it is worth asking whether a similar effect can be achieved without genetic modification.In this regard, there is a growing body of scientific evidence demonstrating that some plant growth-promoting bacteria (PGPB) have the ability to simultaneously influence various aspects of plant physiology, including the activity and efficacy of the pumps responsible for nutrient uptake. In other words, they employ multiple mechanisms of action that vary according to the plant's needs, always with the aim of improving its adaptation (Llagunamaran and Smith, 2017). The mechanisms of action of PGPB bacteria can be summarized into two groups: direct, when the bacteria or their metabolic products affect plant metabolism (including altering hormonal balance and stimulating adaptive mechanisms); and indirect, when they synthesize metabolites that facilitate nutrient uptake or mobilization or prevent the growth of pathogenic microorganisms in the plant without altering its metabolism.It should be noted that this list is not exhaustive. In the context of this patent, both types of mechanisms are of great interest. Plants have an adaptable and highly inducible metabolism related to their ability to cope with adverse situations throughout their life, and this metabolism can be modified by PGPB or their metabolic products, where we can find elicitor molecules, capable of activating plant metabolism by interacting with plant receptors, or enzymes capable of mobilizing nutrients from the soil or inhibiting the growth of pathogens.

[0016] Among the PGPB bacteria are numerous representatives of the genus Bacillus, which currently comprises a total of 239 species and subspecies, ranging from pathogens to extremely beneficial strains in agriculture (Patel and Gupta, 2020). Following the taxonomic classification of the 2009 Bergeys Manual, the genus Bacillus is located in the Domain Bacteria, the Phylum Firmicutes, the Class Bacilli, the Order Bacillales and the Family Bacillaceae (Logan NA, De Vos P. Genus Bacillus Cohn 1872. In: De Vos P, Garrity M, Jones D, Krieg R and Ludwig W (editors). Bergey's Manual of Systematic Bacteriology. New York: Springer; 2009. pp. 21-128). This genus is organized into operational groups, one of which is Bacillus amyloliquefaciens.

[0017] Recent advances in genomic analysis have increased interest in the biotechnological potential of bacterial species of the Bacillus amyloliquefaciens operative group, some of whose members are recognized as plant growth-promoting bacteria (PGPB) due to the ability of certain isolates to perform nitrogen fixation, solubilize phosphate, produce siderophores and phytohormones, and synthesize antimicrobial compounds. Furthermore, they are known for their production of various enzymes such as α-amylase, protease, lipase, cellulase, xylanase, pectinase, aminotransferase, barnase, peroxidases, and laccase. These bacteria also produce antimicrobial compounds, including non-ribosomal peptides and polyketides, capable of inhibiting the growth of pathogens.Genomic analysis has also revealed potential applications for members of the Bacillus amyloliquefaciens operative group in the elimination of organophosphate (OP) pesticides (Ngalimat et al., 2021). Regarding the Bacillus amyloliquefaciens operative group, innovative technologies have enabled the description of novel impacts on plant nutrition and carbon dioxide capture by utilizing an isolated strain of Bacillus nakamurai, or metabolic compounds produced during the growth of the strain; this bacterium belongs to the Bacillus amyloliquefaciens operative group.

[0018] Currently, since Bacillus nakamurai is a very recent species, there is no description of PGPB potential under water stress conditions, nor are there any published patents on strains of this species for application in plants under such conditions.

[0019] THE INVENTION.-

[0020] The invention for which, in view of the commented state of the art, the patent right is requested, has as its object two types of interrelated products of a PGPB of the Bacillus genus, a specific strain of Bacillus nakamurai, which is a recently discovered species, and the metabolites generated in its fermentation process, both with capacities to promote mineral nutrition and plant growth.

[0021] As a first inventive component, the isolation and characterization of the bacterial strain Bacillus nakamurai with deposit number CECT 30581 is claimed, which is a microorganism from the Gram + bacteria group, due to its demonstrated ability to enhance the mineral nutrition of plants by stimulating the absorption of nitrogen, phosphorus, potassium and manganese from the soil, as well as the photosynthetic process, by stimulating the fixation of CO2 from the air and transpiration, optimizing energy uptake and efficiency in the use of water, which translates into an improvement in crop growth and production, even under conditions of water stress. And as a second inventive component, the separation / extraction of the Total Metabolic Fluid (TMF) from the strain and the identification of the metabolites responsible for its effects; metabolites that will serve as an unequivocal identifying profile of the TMF for its different uses.

[0022] Once isolated, the physiological characteristics and genetic analysis of B. nakamurai CECT 30581 allowed it to be unequivocally identified, differentiating it from other species of the Bacillus genus, and from other subspecies of the Bacillus amyloliquefacien operational group.

[0023] To study and characterize the metabolites generated during bacterial growth, which form a complex mixture collectively known as Total Metabolic Fluid (TMF), a multistage fractionation process was carried out based on the distinct polarity of the compounds. First, the TMF was extracted by partitioning, obtaining a fraction known as the HF, which represents the "Hexane Fraction," in which the hexane-soluble compounds are separated from the aqueous culture medium. However, due to the complexity and diversity of compounds present in the TMF and TMF, it was necessary to continue the separation process to elucidate the exact composition of the metabolites, performing a more detailed and specific analysis on an even more refined fraction, called SH, which corresponds to the "Hexane Subfraction."This SH subfraction was obtained by subjecting the FH to a new fractionation process based on the polarity and size of the molecules.

[0024] Due to its low polarity nature, the composition of the SH extract was examined using gas chromatography-mass spectrometry, and to ensure that all compounds were detected, an analysis was carried out using the extract dehvatized with BSTFA (N,O-trimethylsilyl bis-th-fluoroacetamide). This compound was used to transform the polar hydroxyls into silicon ethers, which are less polar and volatile, facilitating analysis by mass chromatography. The result was the identification of the following metabolites generated during the growth of B. nakamurai CECT 30581 , largely indicative of an intense terpenic metabolism:

[0025] - Glycerol;

[0026] - Prenol;

[0027] - Nonanoic acid;

[0028] - Diethylene glycol; - Ethoxybenzoate;

[0029] - Dodecan;

[0030] - Dodecanoic acid;

[0031] - Triethylglycol;

[0032] - Myristic acid;

[0033] - Manopyrasone;

[0034] - Talopyrasone;

[0035] - Palmitic acid;

[0036] - Stearic acid;

[0037] - Squalene;

[0038] - Hexadecane and other hydrocarbons.

[0039] It is important to highlight that composition analysis focuses primarily on the HS, as it represents a more manageable and specific sample of the metabolites of interest. Once the indicated compounds were identified and characterized in the HS, their presence was confirmed in both the LMT and the FH. This rigorous analytical approach is essential to unravel the chemical complexity of the metabolites in the culture broth and understand their potential biological impact.

[0040] The biological effects of the four resulting bacterial products: B. nakamurai, LMT, FH and SH, which imply the commented improvement in mineral nutrition and photosynthetic efficiency of plants, have been tested in different plant species, such as tomato, rice or strawberry plants, by measuring the levels of nitrogen, phosphorus, potassium and manganese of the treated plants, as well as parameters related to photosynthetic efficiency, net photosynthesis rate, photosystem efficiency ¡i (éPSR), water use efficiency (WUE), dry weight, fresh weight and plant height. They have also been tested under water stress conditions, created by reducing irrigation water, in plants such as olive or blueberry, an increase in production (kg / ha) is found.

[0041] Initially, experiments were carried out on tomato plants where the three types of substances: LMT, FH, and SH were applied at the root level (Tests 1 and 2 of the Method of Execution section). A significant increase in the photosynthesis rate was observed, which is an important indicator of the plant's efficiency in converting sunlight into chemical energy, which in turn can have a positive effect on biomass production and crop quality. In fact, significant increases were recorded in several growth-related parameters, such as height, fresh weight, and dry weight compared to the control group. A notable increase in the concentrations of nitrogen, phosphorus, potassium, and manganese in the aerial parts of the plants was also detected. These findings suggested that the application of the new metabolic products has a positive effect on the growth and nutrition of the treated plants.

[0042] Similar trials were then conducted in rice, applying LMT, FH, and SH at the root level (Trials 3 and 4). A significant increase in the net photosynthetic rate was also observed. Furthermore, increases were recorded in photosystem II efficiency (ePSR), a key indicator of photosynthesis, and in water use efficiency (WUE), meaning that plants are more effectively utilizing solar energy and water for growth and development. In terms of growth, plants experienced significant increases in height and fresh weight, and also in nitrogen, phosphorus, potassium, and manganese, all of which confirms that the application of these three metabolic products has a positive effect on plant growth and nutrition.

[0043] In another experiment with rice plants, a study was conducted on the efficient concentration of the hexane fraction and subfraction (Trial 5). FH and SH were applied at the root level, using three different concentrations of these extracts. First, it was observed that certain specific concentrations of both had a significant impact on the net photosynthesis rate, indicating an improvement in the plants' ability to convert sunlight into useful energy, thus improving their growth and development. Furthermore, a substantial increase in the levels of nitrogen, phosphorus, potassium, and manganese in the aerial part was also recorded, suggesting that the application of both extracts to the roots promotes the absorption and mobilization of these nutrients from the soil, essential for their healthy development, and that their availability at high levels may contribute to better crop yield and quality.

[0044] To validate and reinforce the results obtained in the previous trial, a second experiment was conducted in which the hexane fraction and subfraction were again applied at the root level to the same rice plants, using three different concentrations (Trial 6). This second trial yielded data that support and expand the previously obtained conclusions, as significant improvements in transpiration and net photosynthesis were detected, as well as significant increases in plant height and fresh weight. These results indicate a positive impact on overall plant growth and development, which could be of great relevance in agricultural and crop applications. Significant increases in the levels of nitrogen, phosphorus, potassium, and manganese in the aerial parts of the plant were also observed.

[0045] In another experiment carried out on tomato, B. nakamurai CECT 30581 was applied at the root level (Test 7), observing a significant increase in the height of the treated plants and in the levels of the indicated nutrients.

[0046] The bacteria isolated from olive trees has also been tested, with and without water supply limitation during summer (Test 8), finding that the bacteria increases production and fat yield both when water supply is reduced and under normal conditions, although the effect is much more pronounced when water supply is limited.

[0047] In another trial with B. nakamurai during the plant's production cycle with the aim of increasing blueberry production under water-limiting conditions (25% reduction in water input), in intensive cultivation, it has been found that the bacteria increases blueberry production (Trial 9).

[0048] With B. nakamurai and its LMT, another trial was carried out on strawberries in intensive production, during the plant's growth cycle with the aim of increasing production, confirming increases in production with both products (Trial 10).

[0049] In short, the technical advantage provided by the present invention is the availability of a strain of B. nakamurai and its identified metabolic products, contained in the LMT and in the FH and SH fractions in a higher concentration, for use as biofertilizers for plants in essential nutrients such as nitrogen, phosphorus, potassium and manganese, and as bacterial products that promote photosynthesis and plant growth (PGPB), even under conditions of water stress, whether due to drought or reduction of irrigation water.

[0050] Consequently, the present patent application claims the use of the strain B. nakamurai CECT 30581 and its LMT, FH and SH, for its application in any type of plant species, forming part of any bacterial preparation, either individually or in combination with other organisms, and by any available means that puts the bacteria or its metabolites in contact with the seed, the radical or aerial system of the plants, in order to improve plant nutrition and agricultural production. In addition to its impact on plant mineral nutrition, the bacteria and its LMT also aim to provide benefits in situations of water stress. Drought or water scarcity conditions represent a constant challenge in agriculture, as they can have significant negative effects on crop production.In this context, the bacteria and their metabolic products seek to act as agents that allow plants to better resist and adapt to conditions of water scarcity, improving their ability to survive and maintain production even under these unfavorable conditions.

[0051] FIGURES AND GRAPHICS. -

[0052] At the end of this descriptive report, figures with a process diagram and graphs of the experimental results obtained are included.

[0053] Figure 1 shows the scheme of the production process of the hexane fraction (FH) and hexane subfraction (SH) of the strain culture broth

[0054] The graph in Figure 2 is the gas chromatogram of the hexane subfraction SH, and the graph in Figure 3 is the mass spectrophotometry of that same sample.

[0055] METHOD OF IMPLEMENTATION. -

[0056] A bacterial screening conducted in the rhizosphere of Pinus pinaster isolated a strain belonging to the genus Bacillus. After being identified by genetic analysis of the 16S gene using the tools available in Ezbiocloud, it was found to be 100% identical to B. nakamurai. Table 1 shows the results of this analysis.

[0057] TABLE 1 I. Production of LMT, FH and SH.-

[0058] B. nakamurai CECT 30581 can be cultivated in shake-fermentation flasks on a small or large scale (in the laboratory or industrial fermentation chambers) using a suitable medium and under conditions that allow cell growth. The culture must contain a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available commercially or can be prepared according to published compositions. After fermentation, a supernatant, filtrate and / or extract of, or derivative of, said B. nakamurai CECT 30581 can be used as an active ingredient to be applied directly to plants, or in compositions to formulate an organic biofertilizer.

[0059] The concept of TML, or "Total Metabolic Fluid," encompasses all the metabolites generated by the bacteria during their growth process in the appropriate thermoregulator and culture medium. To produce this TML, we start with the liquid resulting from the thermoregulator once the bacteria have completed their growth cycle.

[0060] The process for obtaining TML involves several steps. First, the bacteria are separated from the liquid using a highly efficient centrifugation process. This process eliminates more than 99% of the bacteria present in the liquid. A second phase of the process then involves the use of a 0.2-micrometer filter. This filter is capable of retaining even the smallest bacteria, ensuring that any remaining bacteria are completely eliminated from the liquid. Once this second step is completed, a pure liquid is obtained that contains only the molecules and compounds generated by the bacteria during its growth phase. This liquid is called TML (Total Metabolic Fluid).

[0061] The production of the Hexane Fraction (HF) is an essential process carried out from the Total Metabolic Fluid (TMF). This process is based on separating the molecules present in the TMF according to their affinity for the solvent hexane compared to their affinity for water. The procedure begins with the addition of 10% of the total volume of the TMF in hexane, an organic solvent with a low affinity for water. The resulting mixture is then subjected to stirring at 3000 rpm for a period of 5 minutes. This stirring allows the molecules present in the TMF to distribute between hexane and water selectively, based on their polarity. After completing the stirring, the mixture is left to stand for a period of 24 hours. During this time, the molecules that are more soluble in hexane will dissolve in this phase, while those with a greater affinity for water will remain in the aqueous phase.After this resting time, the two phases are separated, yielding the hexane fraction (HF). In this context, the HF represents a highly enriched fraction of molecules that have demonstrated greater affinity for hexane compared to water. This polarity-based separation is crucial for selectively isolating and concentrating the compounds of interest present in the LMT, facilitating their subsequent study and characterization.

[0062] The production of the Hexane Subfraction (HS) is a process that begins with the Hexane Fraction (HF). This procedure is carried out using a molecular separation technique based on the polarity and size of the molecules, using a column packed with silica gel. First, the hexane is removed from the HF through an evaporation process. Then, 0.1% of its volume of chloroform is added to resuspend the molecules present in the solution. This mixture is then carefully applied to the silica gel column. A crucial step in the process involves the subsequent elution from the column. At this point, the same volume of hexane that had previously been removed from the HF is introduced. This has a specific purpose: the hexane acts as an entrainment agent, carrying with it the molecules that had been suspended in the chloroform during the previous step. These molecules pass through the silica gel column along with the hexane.Finally, the liquid obtained at the end of the silica gel column contains the hexane-soluble molecules that were able to pass through the column. This resulting liquid corresponds to the Hexane Subfraction (HS), which is now ready for use or further processing.

[0063] Figure 1 shows schematically this process of production of LMT, FH and SH for the isolated bacterial strain.

[0064] II. SH Analysis.-

[0065] The chromatographic profile of the sample, reflected in the gas chromatogram in Figure 2, reveals the presence of long-chain compounds (fatty acids, alcohols), sugars, phenolic compounds, and mostly hydrocarbons. Approximately 35 peaks can be identified in the chromatogram. A detailed analysis of the predominant peaks was carried out using mass spectroscopy. Fourteen compounds were identified by comparing the data with the NIST library database and using the Kovats index. These results are summarized in Table 2.

[0066] The analytical conditions have been carried out in a PREM EI&CI equipment, 436GC chromatograph, Rxi-5Sil MS column 30 x 0.25 mm, 0.25um, flow: 1.2 ml / min in a temperature range of 40-300oC in 60 minutes, electronic ionization: 70eV from 40 to 650 urns.

[0067] TABLE 2

[0068] The hydrocarbons were identified by their fragmentation pattern, which generated an ion at 57 m / z with successive losses of the methyl group CH3 (15 u). The graph in Figure 3 shows the mass spectrum obtained for hexadecane.

[0069] III. Demonstrative tests of the capacity of B. nakamurai, LMT, FH, and SH as enhancers of mineral nutrition (nitrogen, phosphorus, potassium and manganese), and stimulants of the photosynthetic process, promoting an increase in growth in terms of dry weight and improving the efficiency of water use. - Test 1: Solanum lycopersicum var. San Pedro, LMT and FH. - Direct inoculation at the root level of LMT and FH (Control, LMT, FH) in tomato (Solanum lycopersicum var. San Pedro). Experiment carried out in an experimental chamber under controlled conditions of light, humidity and temperature on a total of 84 plants and N = 3, with a randomized block model. LMT and FH were inoculated in tomato plants at 4a week of germination. At 6 weeks, photosynthetic parameters were measured, registering a significant increase in net photosynthesis in LMT-treated plants compared to control plants. A biometric analysis recorded a significant increase in height, dry weight, and fresh weight in LMT- and FH-treated plants compared to control plants. Finally, a nutritional analysis was performed, registering a significant increase in nitrogen, phosphorus, potassium, and manganese in LMT- and FH-treated plants compared to control plants.

[0070] Trial 2: Solanum lycopersicum var. San Pedro, SH.- Direct inoculation of SH (Control & SH) at the root level in tomato (Solanum lycopersicum var. San Pedro). The experiment was carried out in an experimental chamber under controlled conditions of light, humidity and temperature on a total of 24 plants and N = 3, with a randomized block model. Two inoculations of the hexane subfraction were carried out on tomato plants, 2 and 3 weeks after germination.After 4 weeks, photosynthetic parameters were measured, registering a significant increase in the efficiency of photosystem II (ePSR) in SH-treated plants compared to control plants; a biometric analysis recorded a significant increase in dry weight in SH-treated plants compared to control plants; finally, a nutritional analysis was performed, registering a significant increase in SH-treated plants compared to control plants in nitrogen, phosphorus, potassium, and manganese levels.

[0071] Trial 3: Oryza sativa var. Bomba, LMT, and FH.- Direct inoculation of LMT and FH at the root level in rice (Oryza sativa var. Bomba). The experiment was carried out in an experimental chamber under controlled conditions of light, humidity, and temperature on a total of 450 plants and N = 3, using a randomized block model. Rice plants were inoculated with LMT and its fraction in the first and second weeks after germination.At 3 weeks, photosynthetic parameters were measured, registering a significant increase in LMT-treated plants relative to control plants in net photosynthesis, photosystem II efficiency (ePSR), and water use efficiency (WUE); a biometric analysis recorded a significant increase in LMT-treated plants relative to control plants in height and fresh weight; finally, a nutritional analysis was performed, registering a significant increase in LMT-treated plants relative to control plants in nitrogen, phosphorus, potassium, and manganese levels.

[0072] Essay 4: Oryza sativa var. Bomba, SH.- Direct inoculation of SH at the root level in rice (Oryza sativa var. Bomba). The experiment was carried out in an experimental chamber under controlled conditions of light, humidity, and temperature on a total of 300 plants and an N = 3, with a randomized block model. Two inoculations of the hexane subfraction were carried out on rice plants, at 2 and 3 weeks after germination. After 4 weeks, photosynthetic parameters were measured, without observing significant changes in the treated plants with respect to the control plants. A biometry analysis recorded a significant increase in fresh weight in the plants treated with SH compared to the control plants. Finally, a nutritional analysis was performed, recording a significant increase in the plants treated with SH compared to the control plants in the levels of nitrogen, phosphorus, potassium, and manganese.

[0073] English:Trial 5: Oryza sativa var. Bomba, FH and SH, efficient concentration study. - Direct inoculation at the root level in rice (Oryza sativa var. Bomba) at the root level of FH and SH with three different concentrations (Control, FH ([1:1], [1:10], [1 :100]) and SH ([1 :1], [1 :10], [1:100]) hexanic, v:v). Experiment carried out in an experimental chamber under controlled conditions of light, humidity and temperature on a total of 105 plants and N = 3, with a randomized block model. Two inoculations of SH were carried out in rice plants, at 2 and 3 weeks after germination.At 5 weeks, photosynthetic parameters were evaluated, observing significant changes in plants treated with FH [1:10] and SH [1:10] with respect to the control plants in net photosynthesis; a biometry analysis did not observe significant changes in treated plants with respect to the control plants; finally, a nutritional analysis was performed, recording a significant increase in plants treated with FH [1:10] and SH [1:10] with respect to the control plants in the levels of nitrogen, phosphorus, potassium, and manganese.

[0074] English:Trial 6: Oryza sativa var. Ricetech, FH and SH optimal concentration.- Direct inoculation at the root level in rice (Oryza sativa var. Ricetech) at the root level of FH and SH with three different concentrations (Control, fraction ([1 :1], [1:10], [1:100]) and subfraction ([1:1], [1 :10], [1 :100]) hexane). Experiment carried out in an experimental chamber under controlled conditions of light, humidity and temperature on a total of 105 plants and N = 3, with a randomized block model. Two inoculations of SH were carried out in rice plants, 2 and 3 weeks after germination.At 5 weeks, photosynthetic parameters were evaluated, observing significant changes in plants treated with FH [1:1] and SH [1:1] with respect to control plants in transpiration and net photosynthesis; a biometry analysis revealed significant changes in plants treated with FH [1:1] and SH [1:1] with respect to control plants in height and fresh weight; finally, a nutritional analysis was performed, recording a significant increase in plants treated with hexane FH [1:1] and SH [1:1] with respect to control plants in the levels of nitrogen, phosphorus, potassium, and manganese.

[0075] Essay 7: Solanum lycopersicum var. San Pedro, B. nakamurai CECT 30581.- Direct root inoculation of B. nakamurai CECT 20581 in tomato (Solanum lycopersicum var. San Pedro). The experiment was carried out in an experimental chamber under controlled conditions of light, humidity, and temperature on a total of 42 plants, N=3, using a randomized block model. Two inoculations of B. nakamurai were carried out at 2 and 4 weeks after the appearance of the first true leaf. After 8 weeks, biometric parameters (height) were evaluated, observing significant increases in both parameters in treated plants compared to control plants. Finally, a nutritional analysis was performed, recording a significant increase in treated plants compared to control plants in the levels of nitrogen, phosphorus, potassium, and manganese.

[0076] Trial 8: Olea europaea var. Arbequina, B. nakamurai CECT 30581, with and without water restriction.- Direct root inoculation of B. nakamurai CECT 20581 in olive trees (Olea europaea var. Arbequina). Applications were made every 15 days from April to September (12 applications). The number of plants in the trial was 20 per treatment plus the control, with both irrigation regimes (100% water and 75% water). In the normal conditions section of the trial, it was observed that B. nakamurai caused a 4% increase in production (kg / ha) over the control, and an increase in oil yield (0.4%) was also recorded. In the part referring to water limitation during the summer months (25%) it was observed that B. nakamurai caused an increase in production (kg / ha) of 20.7% over the control, an increase in fat yield was also recorded (17.7%).

[0077] Trial 9: Vaccinium corymbosum var. Cupla B. nakamurai CECT 30581.- Direct inoculation of B. nakamurai CECT 20581 at the root level in blueberry (Vaccinium corymbosum var. Cupla). Production improvement experiment with limited open-air irrigation in blueberry. Experiment carried out under real field production conditions on a total of 21 plants per treatment (n=3, 7 plants per replicate) for each treatment (control and bacteria), with a randomized block experimental model. Cell suspensions of the strain were applied to the root level during the 10-month production cycle, twice a month, from October to September; water was limited by 25% throughout the cycle. Total production was harvested. An increase in fruit production (+11%) was recorded in treated plants compared to the control.

[0078] Trial 10: Fragaria vesca var. Fortuna, B. nakamurai CECT 30581 and LMT.- Direct inoculation at the root level of B. nakamurai CECT 20581 and LMT on Fragaria vesca var. Fortuna. Experiment focused on intensive strawberry production, with the aim of boosting production. In this study, the effects of the bacteria B. nakamurai CECT 20581 and its LMT have been evaluated through monthly applications at the root level, carried out from October to May. The experiment was carried out in production greenhouses, using 60 plants per treatment. The results obtained reveal a notable increase in strawberry production, observing a 138% increase in plants treated with LMT, while those treated with the B. nakamurai strain experienced an increase of 72%.

[0079] References cited:

[0080] Falhof, J., Pedersen, J. T., Fuglsang, A. T., & Palmgren, M. (2016). Plasma membrane H+- ATPase regulation in the center of plant physiology. Molecular plant, 9(3), 323-337.

[0081] Ilangumaran, G., & Smith, D. L. (2017). Plant growth promoting rhizobacteria in amelioration of salinity stress: a systems biology perspective. Frontiers in Plant Science, 8, 1768.

[0082] Ngalimat, M. S., Yahaya, R. S. R., Baharudin, M. M. A. A., Yaminudin, S. M., Karim, M., Ahmad, S. A., & Sabri, S. (2021). A review on the biotechnological applications of the operational group Bacillus amyloliquefaciens. Microorganisms, 9(3), 614.

[0083] Patel, S., & Gupta, R. S. (2020). A phylogenomic and comparative genomic framework for resolving the polyphyly of the genus Bacillus: Proposal for six new genera of Bacillus species, Penbacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen. nov. International journal of systematic and evolutionary microbiology, 70(1), 406-438.

[0084] Zhang, M., Wang, Y., Chen, X., Xu, F., Ding, M., Ye, W., ... & Zhu, Y. (2021). Plasma membrane H+-ATPase overexpression increases rice yield via simultaneous enhancement of nutrient uptake and photosynthesis. Nature Communications, 12(1), 735.

Claims

CLAIMS 1. Bacterial strain of the species Bacillus nakamurai with deposit number CECT 30581, a microorganism from the Gram-positive bacteria group, genus Bacillus, characterized by its ability to stimulate the absorption of nitrogen, phosphorus, potassium and manganese in plants under water stress conditions, and to optimize energy uptake and water use efficiency under these conditions.

2. Total metabolic fluid (TMF), from the fermentation in the culture medium of the bacterial strain Bacillus nakamurai with deposit number CECT 30581, with the following component metabolites: glycerol, prenol, nonanoic acid, diethylene glycol, ethoxy benzoate, dodecane, dodecanoic acid, triethyl glycol, myristic acid, mannopyrasone, talopyrasone, palmitic acid, stearic acid, squalene, hexadecane and other hydrocarbons, obtained by a process of separation of the bacteria from the fermentation supernatant by two operations: (1) centrifugation and (2) filtration with a 0.2 pm filter, characterized by its capacity to stimulate the absorption of nitrogen, phosphorus, potassium and manganese in plants under conditions of water stress, and to optimize energy uptake and water use efficiency under these conditions.

3. Hexane fraction (FH) of the Total Metabolic Fluid (TMF) from the fermentation in the culture medium of the bacterial strain Bacillus nakamurai with deposit number CECT 30581, with the following component metabolites: glycerol, prenol, nonanoic acid, diethylene glycol, ethoxybenzoate, dodecane, dodecanoic acid, triethyl glycol, myristic acid, mannopyrasone, talopyrasone, palmitic acid, stearic acid, squalene, hexadecane and other hydrocarbons, obtained by a process comprising four operations: (1) addition of 10% of the total volume of the TMF in hexane until the mixture is complete, (2) stirring at 3000 rpm for 5 minutes, (3) resting for 24 hours and (4) separation of the FH from the aqueous phase, characterized by its capacity to stimulate the absorption of nitrogen, phosphorus, potassium and manganese in plants under water stress conditions, and to optimize energy uptake and water use efficiency under these conditions.

4. Hexane subfraction (HS) of the Total Metabolic Fluid (TMF) from the fermentation in the culture medium of the bacterial strain Bacillus nakamurai with deposit number CECT 30581, with the following component metabolites: glycerol, prenol, nonanoic acid, diethylene glycol, ethoxybenzoate, dodecane, dodecanoic acid, triethylglycol, myristic acid, mannopyrasone, talopyrasone, palmitic acid, stearic acid, squalene, hexadecane and other hydrocarbons, obtained from the hexane fraction (FH) of the LMT by a process comprising five operations: (1) removal of hexane from the FH in a rotary evaporator, (2) resuspension in chloroform, adding 0.1% of its volume of chloroform, (3) application of the resulting mixture on a silica gel column, (4) elution of the column with the addition of hexane, in the same volume removed from the FH, and (5) extraction of the liquid corresponding to the SH contained at the end of the column, characterized by its ability to stimulate the absorption of nitrogen, phosphorus, potassium and manganese in plants under water stress conditions, and to optimize energy uptake and water use efficiency under these conditions.

5. Use of the bacterial strain of the species Bacillus nakamurai with deposit number CECT 30581, or of the total metabolic fluid (TMF), the hexane fraction (HF) or hexane subfraction (SH) of the culture medium of the strain, according to claims 1 to 4, for its application in any species of plants, such as olive or blueberry plants, to improve plant growth and production under conditions of water stress, due to drought or reduction of irrigation water.

6. Use of the bacterial strain of the species Bacillus nakamurai with deposit number CECT 30581, or of the total metabolic fluid (TMF), the hexane fraction (HF) or hexane subfraction (SH) of the culture medium of the strain, according to claim 5, forming part of any bacterial preparation, either individually or in combination with other organisms, and by any available means that puts the bacteria or its metabolites in contact with the seed, the radical or aerial system of the plants.

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