Peribacillus aracenensis bbb004 stimulator of plant adaptive metabolism against water stress, enhancer of plant nutrition and polyphenol content

Abstract

The invention relates to a bacterial strain Peribacillus aracenensis (CECT 30655), a microorganism of the Gram-positive group of bacteria, genus Peribacillus, stimulator of plant adaptive metabolism against water stress, enhancer of plant production and nutrition, and enhancer of polyphenol content. This strain, isolated from the rhizosphere of Pinus pinaster, in nutrient agar (PCA), has been characterised from a morphological, biochemical and genetic point of view by sequencing its entire genome, being identified as a new species. It can be used to increase production under conditions of water stress, due to its ability to increase CO2 fixation and transpiration, optimising energy capture through photosynthesis and reducing oxidative stress. It can also be used as a biofertiliser, due to its ability to enhance nutrient absorption both in the presence and in the absence of water stress, as well as to enhance the quality of fruit, due to its ability to increase antioxidant polyphenols in plant species of pharmacological and food interest.

Description

[0001] DESCRIPTION

[0002] Peribacillus aracenensis BBB004 stimulates adaptive plant metabolism in response to water stress, improving plant nutrition and polyphenol content.

[0003] The present invention relates to a strain of Peribacillus aracenensis (BBB004, internal code of the laboratory), as a new species of the genus Peribacillus, for use in the treatment of plants with the aim of improving production under conditions of water stress, increasing CO2 fixation and transpiration, optimizing energy uptake through photosynthesis and decreasing oxidative stress, with the consequent increase in growth and production. Furthermore, it is capable of stimulating the absorption of various nutrients and increasing the polyphenol content, both under normal conditions and under said stress conditions, which is why it is also of interest as a biofertilizer for organic and conventional agriculture, and to increase the polyphenol content in plant species of pharmacological and food interest, to improve the quality and durability of food.

[0004] This strain has been deposited for patent purposes with the Spanish Type Culture Collection (CECT) on June 27, 2022, where it has been assigned deposit number 30655. The CECT is headquartered in the Research Building of the University of Valencia, located on the Burjassot campus (DP 46100 - Valencia, Spain).

[0005] SCOPE.-

[0006] The invention falls within the fields of agro-food biotechnology, specifically within the field of plant growth regulating bacteria, since the bacterial strain can serve as a basis for the preparation of different types of products intended to increase production under conditions of water stress, especially due to lack of water or osmotic stress, improving adaptation to sustained or punctual stress, increasing CO2 fixation, optimizing energy capture through photosynthesis and reducing oxidative stress. These products will improve the adaptation of plants to different conditions of abiotic stress such as water deficit or excess soil salinity, improving production under conditions of low water input.It also falls within the field of organic blofertillzantes, since the strain can be used to improve the nutrition of the plant in general, both in any bacterial formulation and with bacterial metabolites obtained under certain conditions.

[0007] STATE OF THE ART.-

[0008] Plants have various mechanisms to adapt to situations of water stress and salinity.

[0009] Photosynthesis is a physiological process that involves transforming light energy into chemical energy according to the following equation: H2O+CO2+E (Av) to produce C6HI2O6+O2, where water is absorbed by the roots and CO2 by the leaves. It develops in two main stages, i) the absorption of light energy and its transformation into chemical energy (ATP and NADPH), and ii) its subsequent use for CO2 fixation, which is used to build organic carbon skeletons. It is a process that requires photosynthetic pigments for energy capture and generates great oxidative stress (free radicals - ROS).The plant has free radical elimination systems to keep them within physiological limits, since, in turn, certain free radicals, such as H2O2, are physiological signals necessary for the proper functioning of the plant. For this entire process, adequate water availability and good hydration of the plant are absolutely essential. The uptake of CO2 through stomata is vital for the proper functioning of photosynthesis, which generates a problem that contradicts the requirements established for good photosynthesis since it is the exit route for water (transpiration), a process that is accelerated under adverse conditions of temperature and humidity.

[0010] Thus, the plant needs to be hydrated; the parameter that defines the plant's water status is its water potential, which, in summary, represents the balance between water content (pressure potential) and solutes (osmotic potential). To achieve this, it is capable of generating a negative osmotic potential with respect to the soil, such that water is carried in by the solutes (ions) absorbed from the soil. These ions accumulate in the vacuoles, in turn forcing water into the vacuole, which maintains a water reservoir and the plant's turgidity. Nutrient absorption occurs through specific pumps, and once absorbed, they accumulate in vacuoles and / or are translocated to the aerial parts through transpiration, where they are incorporated into organic skeletons. Therefore, mineral nutrition requires water loss through transpiration.

[0011] Water stress can be caused by a lack of water (drought) or an excess of salts (salinity). When it is due to a lack of water, the plant increases the synthesis of compatible solutes (organic molecules) to generate the negative osmotic potential and remain hydrated, keeping stomata open longer; if the drought is severe, it closes them. When plants are in saline soils, they tend to accumulate Na+ ions, which, in high concentrations, are toxic to the plant. One of the adaptation mechanisms is to pump Na+ ions to the exterior to prevent their accumulation. This response involves the generation of ROS, which activates the hormonal stress response (activation of specific protein synthesis and increased ethylene (Et), jasmonic (JA), and abscisic acid (ABA); ABA is responsible for stomatal closure.

[0012] Thus, the plant must maintain a balance between the opening and closing of stomata to allow the exchange of CO2 and water, delaying stomatal closure when water is limited. In addition to stomatal closure, the plant, as previously mentioned, also has mechanisms to retain water, increasing the concentration of internal solutes (osmolytes, ions, and other organic molecules), and antioxidant systems to ensure a good overall condition with efficient photosynthesis, keeping free radicals at physiological levels.

[0013] This brief description of plant adaptation mechanisms to water and salinity stress highlights the numerous potential opportunities for improving agricultural production; in particular, the use of PGPB (Plant Growth Promoting Bacteria) to enhance the plant's ability to adapt to these adverse conditions.

[0014] There is a growing body of scientific evidence showing that beneficial bacteria are capable of modifying multiple plant targets simultaneously, that is, they use more than one mechanism of action, which also varies according to the needs of the plant and is always aimed at improving plant adaptation (Llagunamaran and Smith, 2017, doi: 10.3389 / fpls.2017.01768). The mechanisms of action of plant growth-promoting bacteria can be summarized into two types: direct, when the bacteria or their metabolites alter plant metabolism (hormonal activity, stimulation of adaptive mechanisms), and indirect, when they synthesize compounds that facilitate the uptake or mobilization of nutrients or prevent the growth of pathogenic microorganisms in the plant, without altering plant metabolism; this is not an exhaustive list. In the case of this patent, both are of interest.The plant has an adaptive, highly inducible metabolism, related to the adaptation of the plant to adverse situations that it has to face during its life and is susceptible to modification by PGPB.

[0015] The genus Bacillus encompasses an extraordinarily diverse group of microorganisms currently comprised of 239 species / subspecies, ranging from pathogens to strains extremely useful in agriculture (Patel and Gupta, Int. J. Syst. Evol. Microbiol. 2020; 70:406-438, DOI 10.1099 / ijsem.0.003775). Following the taxonomy of the Bergeys Manual, 2009 edition, this bacterium falls within the Domain Bacteria, Phylum Firmicutes, Class Bacilli, Order Bacillales, 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). Recently, based on an exhaustive analysis of Bacillus genomes, the existence of six new genera has been described: Peribacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen.Nov, which allow a more appropriate classification of the species previously assigned to the genus Bacillus (Patel and Gupta, 2020, doi 10.1099 / ¡jsem.0.003775). The genus Peribacillus groups rod-shaped cells, Gram-positive or Gram-variable, aerobic or facultative aerobic, capable of forming endospores under conditions of environmental or nutritional stress; most have been isolated from soil or animal intestines. The type strain is Peribacillus simplex.

[0016] Since Patel and Gupta described the genus Peribacillus in 2020, 8 new species have been described within this genus, P. simplex, P. muralis, P. butanilovorans, P. louselieroae, P. gossypii, P. asahii, P. psychrosaccharolyticus and P. castrensis. The latter (Rodríguez et al. 2022, doi: 10.3389 / fpls.2022.896728), with beneficial effects for agriculture due to its ability to stimulate growth and its biocontrol capacity, that is, involving mechanisms external to the plant.

[0017] In the case of the present patent application, new technologies have made it possible to determine a new species, Peribacillus aracenensis (BBB004, internal laboratory code), based on its unique genetic characteristics, which differentiate it from other Peribacillus strains when the genome of P. aracenensis BBB004 is compared with those of the currently available Peribacillus and Bacillus genomes.

[0018] The parameters that allow to identify a new species are some of the following: i) average nucleotide identity (ANI) and average amino acid identity (AAI), being the limit values ​​to define a new species less than 95%; i) DNA-DNA digital hybridization (dDDH), with values ​​below 70%; ii) guanine-cytosine content (G+C), values ​​above 1%; iv) tetranucleotide usage frequency correlation index (TETRA), with values ​​below 0.99; v) multilocus sequence analysis (MLSA), with values ​​below 97%.

[0019] The genome of P. aracenensis BBB004 was first analyzed using tools available in EzBioCloud. First, the EzBioCloud Identification service provides verified similarity-based searches against quality-controlled databases of 16S rRNA sequences. This was followed by ANI (Average Nucleotide Identity), AAI (Average Amino Acid Identity), dDDH, and TETRA analyses. The Type (Strain) Genome Server (TYGS) was used to analyze the whole genome.

[0020] First, the 16s gene of P. aracenensis BBB004 was analyzed in the EzBlocloud, detecting the most similar species within the genus Peribacillus Brevibacterium frigotolerans (reclassified as P. frigotolerans in 2020. https: / / dol.Org / 10.1099 / ljsem.0.005389); P. simplex, P. muralis and P. butanolivorans with values ​​less than 99.99% pairwise similarity. The highest similarity values ​​were found with the first two (99.93 and 99.65%, respectively).

[0021] When comparing the P. aracenensis BBB004 genome with the genomes of the 4 most similar species according to 16s, the following results were obtained: With P. simplex, the ANI and AAI values ​​were 93.54% and 94%, respectively, both below the 95% threshold, the DNA-DNA digital hybridization (dDDH) values ​​were 60.6%, below the 70% threshold and the guanine-cytosine (G+C) content was 0.12%, below the 1% threshold. The tetranucleotide usage frequency correlation index (TETRA) values ​​were 0.99822, below the 0.99 threshold. With P. frigoritolerans the following values ​​are obtained: ANI and AAI 93.96 and 93.26%, respectively, both below 95%; DNADNA digital hybridization (dDDH) 59.2%, below 70%; and the difference in guanine-cytosine (G+C) content 0.62, below 1%; finally, the tetranucleotide usage frequency correlation index (TETRA) values ​​were 0.99704, equal to or greater than the reference 0.99%.Based on the results obtained from ANI, AAI and dDDH, we can affirm that P. aracenensis BBB004 is different from the two closest strains in the phylogenetic tree.

[0022] Therefore, based on the polyphasic approach, a new species has been described, for which the name Peribacillus aracenensis sp. nov has been proposed (CECT Deposit No. 30655). The physiological characteristics and genetic analysis of this strain allow its unequivocal identification, differentiating it from other species of the genus Peribacillus.

[0023] The P. aracenensis BBB004 strain is characterized by its beneficial effects on plants under water stress conditions, improving production: it improves CO2 fixation, keeping stomata open for longer, reflecting a stimulation of the metabolism involved in adaptation. It prevents the formation of free radicals and facilitates their elimination, improving the plant's adaptation to these conditions through a redox metabolism homeostasis mechanism. It also increases the plant's water potential under these conditions, a clear indicator of this strain's ability to induce water balance, resulting in a greater capacity of the plant to withstand water stress conditions. Moreover, it is capable of improving plant nutrition, improving its growth and production.

[0024] The fact that Peribacillus is a genus of bacteria discovered recently, in 2020, may explain why there are no known published patents for the described species of this genus on any of the reported effects, so the present application constitutes, a priori, the first invention of a bacterial strain of the genus Peribacillus as PGPB.

[0025] THE INVENTION.-

[0026] The object of the invention described herein and which, in view of the prior art, is understood to meet the conditions of novelty and inventive activity necessary to be worthy of the patent right, is the isolation and characterization of the bacterial strain Peribacillus aracenensis BBB004, with deposit number CECT 30655, which is a microorganism of the group of Gram + bacteria, genus Peribacillus, with a demonstrated capacity to stimulate the metabolism of adaptation to water stress in plants, reducing oxidative stress, allowing greater fixation of atmospheric CO2 and greater transpiration, optimizing energy uptake and increasing plant growth and production under said conditions.It is also capable of enhancing the absorption of nutrients, particularly nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, boron, manganese, and zinc, improving mineral nutrition, and increasing the concentration of polyphenols, improving the antioxidant and preservative capacity of food, both under normal and stress conditions.

[0027] The physiological characteristics and genetic analysis of this strain, with a genome sequenced according to the WIPO ST.26 standard that is attached to this specification via an XML file, allow it to be identified unequivocally, differentiating it from other species of the Peribacillus genus.

[0028] In a bacterial screening carried out in the rhizosphere of Pinus pinaster, a strain belonging to the genus Peribacillus was isolated, whose genetic analysis did not allow it to be placed in any of the known species of this genus.

[0029] Once isolated, a characterization of the repertoire of unique genes of P. aracenensis BBB004 was performed. In the comparative analysis against the genomes of Peribacillus and Bacillus, the BPGA program identified a total of 389 genes present exclusively in P. aracenensis BBB004, of which 324 genes were associated with a specific subsystem. Among the 133 unique genes of P. aracenensis BBB004, those involved in copper transport and the synthesis and degradation of polyamines (which participate in processes such as nucleic acid packaging, modulation of membrane receptors and ion channels, regulation of gene expression and cell signaling) and nutrient absorption pumps stand out due to their abundance. These 324 genes correspond to SEQ ID NO: 1 to 324 of the sequence listing.

[0030] After its characterization, various tests were performed to reveal biochemical activities that indicated its potential capacity for nutrient mobilization and plant growth promotion. These were auxin production, 1-amylclopropane-1-carboxylate degradation, nitrogen mobilization, phosphate and calcium solubilization, and production of siderophores and chilnases, resulting positive for siderophore production. An API 50 CHB / E Medium was performed, resulting positive for the degradation of carbon sources, L-Arabinose, Beta-Methyl-D-Xylose, Glucose, and L-Fucose. Initially, experiments consisting of the direct inoculation of the strain in a model plant (Arabidopsis thaliana) were carried out, where it induced a stimulation of the plant's adaptive metabolism that involves the signal transduction pathway mediated by ABA, JA and ET, activating the transcription of the PR1 and LOX2 genes.An increase in CO2 fixation and an increase in transpiration were found in both cases.

[0031] Subsequently, another greenhouse experiment was conducted on tomatoes, inducing water stress (osmotic) conditions for six weeks. An improvement in photosynthesis was detected, with a decrease in the concentration of photosynthetic pigments and oxidative stress, and an increase in water potential.

[0032] Another greenhouse experiment was conducted on tomatoes, inducing the plant with the bacteria and subjecting it to osmotic stress. An improvement in photosynthesis was detected, with a decrease in the concentration of photosynthetic pigments and oxidative stress.

[0033] Another experiment was conducted on tomato, under open-air conditions, maintaining severe water stress by limiting water supply (drought). This experiment revealed the ability of P. aracenensis BBB004 to increase CO2 fixation and transpiration, decrease oxidative stress generated by photosynthesis, decrease photosynthetic pigments and free radical production without compromising photosynthetic efficiency, and increase non-enzymatic antioxidants (polyphenols) and osmolytes (soluble sugars). When water supply is not limited, earlier flowering and increased CO2 fixation are detected. Increased production was detected under limiting water conditions, as well as improved nutrient uptake.

[0034] Another greenhouse experiment was conducted on rice, inducing the plant with the bacteria and subjecting it to osmotic stress. P. aracenensis BBB004 is able to enhance CO2 fixation and transpiration, decreasing the concentration of photosynthetic pigments and oxidative stress in the absence of stress. When osmotic stress is applied, the effect on transpiration and pigments is maintained, but oxidative stress increases, indicating the activation of protective systems.

[0035] Another greenhouse experiment was conducted on rice, inducing the plant with the bacteria and subjecting it to a water stress shock (drought) and assessing its recovery capacity. P. aracenensis BBB004 is able to improve CO2 fixation and transpiration, decreasing the concentration of photosynthetic pigments and oxidative stress, indicating the immediate activation of protection systems against water shortage, which does not involve the accumulation of osmolytics.

[0036] Another experiment was conducted on olive trees using one-year-old seedlings under controlled conditions, outdoors, maintaining high water stress, limiting water supply (drought) and in a saline (osmotic) soil. This experiment revealed the ability of P. aracenensis BBB004 to reduce oxidative stress generated by photosynthesis, increasing non-enzymatic antioxidants (polyphenols) and osmolytics (protein and soluble sugars), and modifying the expression of genes involved in maintaining ionic homeostasis.

[0037] On the other hand, elimination experiments have been carried out with P. aracenensis BBB004 in olive trees, limiting the water supply during the production cycle of the plant in order to increase production in well-established intensive cultivation and in full production (drought). In particular, the application of the aforementioned bacterial strain in olive trees has been tested, limiting the water supply during the summer months in order to increase production. It has been found that P. aracenensis BBB004 increases production by stimulating transpiration, decreasing oxidative stress, decreasing photosynthetic pigments and increasing osmolytics (prollna).

[0038] In another experiment with P. aracenensis BBB004, limiting water supply during the plant's production cycle in order to increase blueberry production under intensive (drought) cultivation, it was found that P. aracenensis BBB004 increased blueberry production by stimulating CO2 fixation and transpiration, decreasing oxidative stress, and decreasing photosynthetic pigments, and increasing osmolytics (proline and soluble sugars) and phenols in leaves.

[0039] Finally, an experiment was carried out on olive trees, applying P. aracenensis BBB004 on recently pruned branches, finding increases in phenols of interest for human health, specifically oleuropein and its precursor secologanin in leaf extracts, with a potential protective effect on the cardiovascular system.

[0040] The aforementioned experiments on the use of P. aracenensis BBB004 as a production enhancer under water stress conditions (osmotic and drought), as well as an enhancer of adaptation to stress and nutrition, are set forth in the present specification in the embodiment section. The purpose pursued with this invention, which constitutes the technical advantage provided herewith, is to have a bacterium that improves agricultural production under water stress conditions (osmotic and / or drought), by improving its nutrition, improving plant production due to its ability to optimize energy absorption, and increasing the efficiency of the photosynthetic process by improving carbon fixation, associated with a decrease in oxidative stress.

[0041] Consequently, this patent application claims the use of the P. aracenensis BBB004 strain for application to any type of plant species, as part of any preparation, either individually or in combination with other organisms, in order to improve plant nutrition, increasing agricultural production under conditions of water stress (osmotic and / or drought), and improving plant adaptation to stress situations. Based on these effects on plants, P. aracenensis BBB004 improves plant nutrition and production (kg / ha) under conditions of water stress.

[0042] FIGURES.-

[0043] The images in Figure 1, included at the end of this report, show the resulting appearance of rice plants in a greenhouse after the strain performance experiments, described in the following section.

[0044] METHOD OF IMPLEMENTATION. -

[0045] The strain of the genus Peribacillus described here was isolated by studying the rhizosphere of natural populations of Pinus pinaster along a transect of the Sierra de Aracena, in the province of Seville. This plant species (Pinus pinaster) was selected for its growth in nutrient-poor soils and low rainfall, distributed in occasional strong storms.

[0046] Rhizosphere sampling for bacterial strain isolation was carried out in natural populations of Pinus pinaster in October 2019. P. aracenensis (BBB004, internal laboratory code) was found. This strain was isolated from the soil using nutrient agar (PCA). In the laboratory, this microorganism maintains a high survival rate in 20% glycerol in nutrient broth (Pronadisa) at -80°C or in 15% glycerol in water at -20°C. It is easily recovered in the culture medium used for isolation, both in the solid phase and in the liquid phase at 28°C.

[0047] I. Morphological, biochemical and genetic characteristics of P. aracenensis BBB004.-

[0048] For the characterization of the strains, different phenotypic characters were considered, which are detailed in this report: (i) colony morphology (I) cell morphology (II) metabolic profile, iv) comparative analysis of the genome of the strain P. aracenensis BBB004 and the complete genomes available of the genera Bacillus and Peribacillus.

[0049] I) The morphology of the colonies after 24 h of incubation at 28° on agar for standard methods (PCA) is specified in Table 1:

[0050] TABLE 1

[0051] Growing in liquid medium (LB LURIA BROTH) the color of the medium changes to yellow from the exponential phase of growth to the stationary phase of growth.

[0052] i) The morphological characteristics of P. aracenensis BBB004 after 24 h of incubation at 28°C in agar for standard methods (PCA) correspond to a Gram-positive bacillus. ill) Once isolated and characterized, with internal reference code BBB004, various tests were performed to demonstrate the PGPB potential of this bacterium. These were auxin production (Brick et al., 1991 Appl. Environ. Microbiol., 57(2), 535; Sergeeva et al., 2007, Plant Soil, http: / / doi.org / 10.1007 / s11104-007-9314-5), 1-aminocyclopropane-1-carboxylate degradation (Glick et al., 1995. http: / / doi.org / 10.1139 / m95-070), nitrogen mobilization, phosphate solubilization (De Freitas et al., 1997. Biol. Fértil. Soils, 24(4), 358-364), calcium (Tamilselvl et al., 2016. Front, plant sel, 7, 1828), and siderophore production (Alexander and Zuberer, 1991. Biol.Fertil.Soils, 12(1), 39-45) and chitinases (Frandberg and Shunürer, 1998 Can.J. Microbiol. 44: 121-127).

[0053] This strain can grow in a medium containing 6% NaCl and tolerates pH values ​​between 5 and 7, with no growth detected at pH 4 or above pH 8. It can grow at temperatures ranging from 22°C to 40°C, but loses viability above this value.

[0054] The metabolic profile (API 50 CHB / E Medium, https: / / us.vwr.com / store / product / 29016242 / api- 50-chb-e-medlum-blomerleux) of the strain indicates that it is capable of degrading the carbon sources L-Arablnose, p-Metll-D-Xllose, Glucose, and L-Fucose. iv) Comparison with the closest complete genome, P. simplex, the ANI and AAI values ​​were 93.54% and 94%, respectively, both below the 95% threshold; DNADNA digital hybridization (dDDH) values, 60.6%, below the 70% threshold and guanine-cytosine (G+C) content 0.12%, above the threshold (1%). The tetranucleotide usage frequency correlation index (TETRA) values ​​were 0.99822, above the reference threshold (0.99). Based on the ANI, AAI, dDDH, and TETRA results, we can state that it meets the requirements for a new species.

[0055] II. Demonstrative experiments on the ability of P. aracenensis BBB004 to improve plant nutrition and production under water stress conditions.

[0056] 1 o Demonstrative experiment of the strain's ability to activate the plant's adaptive metabolism to abiotic stress conditions (salinity), in two phases.

[0057] First phase: Stress-free. Direct inoculation experiment of P. aracenensis BBB004 in a model plant (Arabidopsis thaliana), comparing it with a non-inoculated control. The experiment was carried out in an experimental chamber under controlled conditions of light, humidity and temperature on a total of 42 plants and an N = 3, with a randomized block model. A cell suspension of the strain was inoculated into pre-germinated A. thaliana seedlings at 2 and 5 weeks after germination. After 6 weeks, photosynthetic parameters were measured, recording a significant increase in CO2 fixation and transpiration; the plants were harvested, and the expression of genes involved in the activation of adaptive metabolism was analyzed by RT-qPCR, via salicylic acid (SA) and via ethylene / jasmonic acid (Et / JA).A slight increase in LOX expression was recorded, revealing an activation of the adaptation system (priming-pre challenge), which will manifest in a faster and more intense response when faced with a stress factor.

[0058] Second phase: Salt stress. A similar experiment was carried out, subjecting A. thaliana plants stimulated with P. aracenensis BBB004 and non-inoculated control plants to salt stress shock 5 weeks after germination7: a significant increase in the expression of PR1, pdf1 and LOX was recorded compared to the non-inoculated control plant subjected to stress, revealing a simultaneous activation of the adaptation system via SA and Et / JA. A significant increase in CO2 fixation and transpiration was recorded.

[0059] 2 o. Two-phase experiment on protection against sustained osmotic stress in tomato. Elicitation experiment on Solanum lycopersicum var. Casillas plants by direct application of bacterial suspensions to the root. The objective is to demonstrate improved growth and plant protection under sustained osmotic stress conditions.

[0060] First phase (stress-free). A bacterial suspension of the P. aracenensis BBB004 strain was inoculated into the roots of tomato seedlings three times, the first time when the cotyledons and a pair of leaves were present, and the following two times, every two weeks. After 6 weeks, the photosynthetic parameters of fluorescence (FO, Fv / Fm, <|JPSII, NPQ), CO2 fixation, and water potential were measured in both inoculated plants and non-inoculated controls, and the plants were harvested 5 days after the last application. The following parameters were analyzed: fresh and dry weight of the plants, photosynthetic pigments, malondialdehyde (MDA) as a marker of cellular oxidative stress and H2O2, as a representative of reactive oxygen species (ROS); osmolytes (proline, soluble sugars and glycine betaine).In non-stressed plants, an increase in dry weight (5%) of the aerial part was observed after inoculation, together with a decrease in photochemically fixed energy (PSII) and a significant increase in energy dissipation (NPQ); a significant increase in CO2 fixation, which supports the improvement in growth. In addition, a decrease in photosynthetic pigments (10%), less oxidative stress (MDA 13%) and lower osmolyte content (22% in proline and 4% in glycine betaine) were detected compared to the non-inoculated control. Second phase: Osmotic stress. Inoculated plants and non-inoculated controls were subjected to stress with a 10% polyethylene glycol (PEG) solution applied in the irrigation throughout the experiment to reproduce sustained osmotic stress conditions.An increase in fresh weight (27%) and dry weight (1%) of the aerial part was observed in inoculated plants subjected to stress compared to the non-inoculated control subjected to stress, together with an improvement in the maximum photosynthetic potential (Fv / Fm), of the energy fixed in photochemistry (<|)PSII) and a decrease in energy dissipation (NPQ), indicating an optimization of energy absorption in photosynthesis. In addition, a decrease in photosynthetic pigments (12%) and less oxidative stress (MDA, 13% and H2O2, 32%) were detected. An increase in water potential and a decrease in osmolyte content (25% proline and 7% glycine betaine) were also detected.

[0061] 3 o. Osmotic stress shock protection experiment in tomato. Elicitation experiment in Solatium lycopersicum var. Casillas plants by direct application of bacterial suspensions to the root, under controlled conditions, with the objective of demonstrating protection against osmotic stress shock in inoculated plants and their non-inoculated controls. A bacterial suspension of the P. aracenensis BBB004 strain was inoculated into the roots of tomato seedlings twice, the first time when they had cotyledons and a pair of leaves, and the second time, one week later; 7 days later, irrigation with 10% PEG was applied to apply an osmotic shock, and harvested three days later. A similar response was observed in the inoculated plants subjected to stress in the profile and intensity of biochemical modifications in photosynthesis, pigments, oxidative stress, and osmolytes.In addition to a significant increase in CO2 fixation and transpiration compared to the non-inoculated control subjected to osmotic stress.

[0062] 4 o. Experiment on protection against drought stress, improving production and mineral nutrition in tomato. The experiment was carried out with Solatium lycopersicum var. Alcolea plants by direct application of bacterial suspensions to the plant roots, in open-air experimental plots, with limited water supply, maintaining soil water potential under extreme hydration conditions (between -300 and -400 hPa). The experiment was carried out from May to August 2022, with sustained very high temperatures (minimum 22°C, maximum 42°C). A bacterial suspension of P.aracenensis BBB004 was applied to the roots of tomato plants 6 times, the first time at transplantation and the remaining times every 15 days, with the harvest beginning two months after the first application; an intermediate sampling was carried out after the third application and the photosynthetic parameters of fluorescence (F0, Fv / Fm, <|>PSII, NPQ), CO2 fixation, photosynthetic pigments, malondialdehyde (MDA) as a marker of cellular oxidative stress and H2O2, as a representative of reactive oxygen species (ROS); osmolytes (proline, soluble sugars and glycine betaine) and nutrient content were analyzed.It was observed, in treated plants compared to control plants without the strain under drought stress, a similar response in the profile and intensity of biochemical modifications: a significant increase in CO2 fixation (20%) and transpiration (26%), decrease in photosynthetic pigments (42% chlorophylls, 64% carotenes), less oxidative stress (13% MDA) and osmolyte modification, decrease in proline (26%) and increase in soluble sugars (15%). Nutrient content increased in plants treated with P. aracenensis BBB004 (Table 2). Production increased by 91%, based on an increase in the number of fruits (40%) and their weight. Table 2 shows the nutrient content in tomato leaves after 3 applications under the stress conditions described, with limited irrigation. Under normal conditions, an increase in nutrient absorption of Nitrogen, Magnesium and Sulfur is observed.

[0063] TABLE 2

[0064] 5 o . Osmotic stress protection experiment in rice. An experiment was carried out in rice (Oryza sativa var. Bahia) under controlled greenhouse conditions on a total of 300 plants, using a randomized block model. The treatments included control treatments and treatments with P. aracenensis BBB004, with and without PEG, as an osmotic stress agent. A cell suspension of the strain was inoculated into pre-germinated Oryza sativa seedlings at 4 and 5 weeks after germination. After 6 weeks, irrigation with PEG (25%) was applied in the appropriate treatments, and after 3 days, photosynthesis measurements (fluorescence and CO fixation) were taken. 2) and water potential and the plants were harvested.

[0065] In treated non-stressed plants, a significant increase in CO2 fixation (28%), transpiration (8%) and water use efficiency (25%) was recorded compared to control plants without the strain; the maximum potential efficiency of the Fv / Fm system and <|>PSII increased, along with a decrease in photosynthetic pigments (32% chlorophylls and 21% carotenes) associated with similar levels of oxidative stress (MDA), and an increase in H2O2 (32%), a priming marker that reveals an activation of the adaptation system (priming-pre challenge), which will manifest when faced with a stress factor. In addition, the osmolytes proline (11%), soluble sugars (4%) and glycine-betaine (5%) increased.

[0066] In treated plants subjected to osmotic stress, a similar response to non-stressed plants was found in fluorescence and photosynthetic pigment parameters. However, CO2 fixation decreased (50%) and transpiration increased (3%) in plants treated with P. aracenensis BBB004 compared to the control, both under stress conditions. An increase in oxidative stress (30%) was detected along with an increase in H2O2 (70%); osmolytes decreased.

[0067] 6 o. Drought protection experiment (drought period recovery capacity) in rice. In an experiment similar to experiment 5, the stress situation was applied 6 weeks after germination and after 2 inoculations, irrigation was suspended for 2 days and subsequently, irrigation was resumed and the same parameters were measured 24 hours later, when the plants had recovered (Figure 1). The behavior of photosynthesis followed the same pattern, with a decrease in photosynthetic pigments (49% chlorophylls and 40% carotenes), a decrease in oxidative stress (26% MDA) and H2O2 (60%), and a marked decrease in osmolytes, proline (50%), soluble sugars (37%) and glycine-betaine (7%). The significant increase in CO2 fixation (74%), transpiration (37%) and water use efficiency (28%) in plants treated with P. aracenensis BBB004 compared to the control, both subjected to the stress period, is very striking.Figure 1 shows the appearance of treated rice plants: A) before drought stress, in control plants without the strain and in those treated with BBB004; and B) after stress (irrigation was eliminated), after irrigation was restored, in control plants without the strain and treated with P. aracenensis BBB004.

[0068] 7 o. Experiment to improve adaptation to drought and salinity conditions in olive seedlings. An experiment was carried out on one-year-old olive seedlings var. Arbequina, in pots filled with saline soil (conductivity 6.07 dS / m), with open-air irrigation every two weeks with saline water (8.20 dS / m). A bacterial suspension of P. aracenensis BBB004 was inoculated at the root level by irrigation. Applications were made every 15 days for 1 year. The number of plants in the trial was 6 per treatment. Sampling was carried out after summer heat stress, assessing photosynthetic fluorescence parameters (F0, Fv / Fm, <|)PSII, NPQ). Photosynthetic pigments were analyzed, malondialdehyde (MDA) as a marker of cellular oxidative stress; osmolytes (proline, soluble sugars) and antioxidant polyphenols (flavonols and oleuropein).It was observed that in treated plants compared to control plants without the strain in the initial stress situation, a decrease in the energy fixed in photochemistry (<|)PSI I ) and a significant increase in energy dissipation (NPQ) were detected. In addition, an increase in photosynthetic pigments (10%), higher osmolytic content (77% in proline and 42% in soluble sugars), more antioxidant polyphenols (flavonols +47%, oleuropein +25%) and lower oxidative stress (MDA-18%).

[0069] 8°. Experiment to improve production with limited open-air irrigation in olive trees. An experiment was conducted in a super-intensive irrigated olive tree, reducing irrigation by 25% compared to usual (eliminating one irrigation out of every 4). The P. aracenensis BBB004 strain was tested in root application. 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 the two irrigation regimes (100% water and 75% water). Follow-up sampling (September) prior to harvest indicated that the P. aracenensis BBB004 strain applied at the root level caused a 9% increase in production (kg / ha) over control plants without the strain with water reduction stress, with olives treated with P. aracenensis BBB004 reaching the same weight in both irrigation regimes; The increase in fat yield was proportional (9%).In addition, the photosynthetic parameters of CO2 fixation, photosynthetic pigments, malondialdehyde (MDA) as a marker of cellular oxidative stress and osmolytics (proline, soluble sugars) of the treated plants were analyzed with respect to the control with irrigation limitation, finding an increase in CO2 fixation and transpiration (60%), decrease in photosynthetic pigments (40%), increase in water potential (13%), along with an increase in proline (87%) and decrease in soluble sugars (80%).

[0070] 9 o. Experiment to improve production with limited open-air irrigation in blueberries. Direct inoculation experiment with P. aracenensis BBB004 in blueberries (Vaccinium corymbosum var. Cupla) in production greenhouses. The experiment was carried out under real-life field production conditions on a total of 21 plants per treatment (n=3, 7 plants per replicate) for each treatment (control and bacterial), with a randomized block experimental model. Cell suspensions of the P. aracenensis BBB004 strain were applied to the root level twice a month from October to September during the 10-month production cycle; water was limited by 25% throughout the cycle. Total production was collected, and a sampling time was determined after the summer stress in September. Photosynthetic parameters of CO2 fixation and water potential were determined.Photosynthetic pigments, malondialdehyde (MDA) as a marker of cellular oxidative stress, phenolic compounds as antioxidants; osmolytics (proline, soluble sugars) were analyzed. Treated plants, compared to control plants without the strain under water stress, showed an increase in fruit production (+33%), an improvement in CO2 fixation (+8%) and transpiration; in water potential (+21%), associated with an increase in proline (85%) and soluble sugars (120%); a decrease in photosynthetic pigments (40%) and an increase in antioxidant polyphenols (+272%), associated with lower oxidative stress (-37% MDA).

[0071] 10°. Experiment to improve polyphenol content in olive pruning debris. A suspension of P. aracenensis BBB004 was applied to freshly pruned olive branches var. Arbequina. The branches were allowed to dry and leaf extracts were prepared by spraying the leaves, adding 80% ethanol, vortexing for 45 s and sonicating for 30 min, followed by 5' centrifugation; the process was repeated 4 times; the supernatants were pooled, brought to dryness, resuspended in 5 mL of methanol (HPLC), filtered (0.2 µm nylon filters), and analyzed for polyphenol content by HPLC-MS. An increase in the bioactive polyphenols oleuropein (21%), secologanlna (96%) and hydroxytyrosol (8%) was found in the treated pruning debris compared to controls without the strain.

[0072] III. Demonstrative experiment of the capacity of P. aracenensis BBB004 as a nutritional enhancer in Solanum lycopersicum in the absence of stress conditions. -

[0073] 1 oMineral nutrition improvement experiment in tomato. The experiment was carried out with Solanum lycopersicum var. Alcolea plants by direct application of bacterial suspensions to the plant roots, in open-air experimental plots in the absence of stress conditions, maintaining the soil water potential under optimal hydration conditions (between -100 and -200 hPa). The experiment was carried out from May to August 2022, with sustained very high temperatures (minimum 22°C, maximum 42°C). A bacterial suspension of P. aracenensis BBB004 was inoculated into the roots of tomato plants 6 times, the first time at transplantation, and the remaining times every 15 days. Nutrient measurements were taken in leaves, indicating an improvement in the nutrition of nitrogen, sulfur, magnesium, and zinc of the treated plants compared to the control without the strain. TABLE 3.

[0074] INDUSTRIAL APPLICATION. -

[0075] Given the above-mentioned properties of P. aracenensis BBB004, it can be used to increase plant production under water stress conditions due to its ability to increase the photosynthetic process by improving carbon fixation, increasing transpiration, decreasing oxidative stress, and improving nutrient absorption. With respect to nutrient absorption, an increase is detected both in the presence and absence of water stress. It also improves its metabolic performance, decreasing photosynthetic pigments and modifying the osmolyte profile, improving the adaptability of plants to stress. It also increases the content of phenolic compounds, whose application would be to revalue plant remains as a source of active ingredients, in addition to improving the quality and durability of food due to its antioxidant and preservative capacity.

[0076] 22

[0077] SEQUENCE LISTS

[0078] Attached XML file (WIPO Standard ST.26)

Claims

CLAIMS 1. Bacterial strain of the species Peribacillus aracenensis, with deposit number CECT 30655, microorganism of the group of Gram + bacteria, genus Peribacillus, characterized by comprising the nucleotide sequences corresponding to SEQ ID NO: 1 to 324 of the sequence lists.

2. Bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claim 1, characterized by its capacity to stimulate adaptive metabolism to water stress in plant species, increasing CO2 fixation and transpiration, decreasing the concentration of photosynthetic pigments and oxidative stress, improving plant photosynthesis, growth and plant production.

3. Bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claim 2, characterized by its ability to stimulate the adaptation metabolism to water stress by simultaneous stimulation of the signal transduction pathways mediated by SA and Et / JA.

4. Bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claim 2, characterized by improving energy uptake through photosynthesis and CO2 fixation, increasing water use efficiency (WUE) and improving plant recovery after severe water stress, reducing oxidative stress.

5. Bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claim 2, characterized by improving energy uptake through photosynthesis and CO2 fixation, increasing dry weight, both under sustained osmotic stress conditions and with osmotic shock, reducing oxidative stress.

6. Bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claim 1, characterized by its capacity to stimulate the absorption of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, boron, manganese and zinc in herbaceous and woody species, under conditions of water stress, improving mineral nutrition.

7. Bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claim 1, characterized by its absorption-stimulating capacity of nitrogen, sulfur, magnesium and zinc in herbaceous and woody species, in conditions of absence of water stress, improving mineral nutrition.

8. Bacterial strain of the species Peribacillus aracenensis - with deposit number CECT 30655 according to claim 1, characterized by its capacity to stimulate secondary metabolism under conditions of water stress, increasing the concentration of polyphenols in plant species, improving the quality and durability of food due to its antioxidant and preservative capacity.

9. Use of the bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claims 1 and 2, for its application in any type of herbaceous and woody crop, in order to improve the adaptation of the plant to any condition of water stress, whether natural, osmotic and / or drought, or due to lack of irrigation.

10. Use of the bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claims 1 and 6, for its application in any type of herbaceous and woody crop, with the aim of improving mineral nutrition in nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, boron, manganese and zinc, against water stress, whether natural, osmotic and / or drought, or due to lack of irrigation.

11. Use of the bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claims 1 and 7, for its application in any type of herbaceous and woody crop, with the aim of improving mineral nutrition in nitrogen, sulfur, magnesium and zinc in conditions of absence of water stress.

12. Use of the bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claims 1 and 8, for its application in plant species of pharmacological and food interest, with the aim of improving the concentration of polyphenols, thus improving its antioxidant and preservative capacity.

13. Use of the bacterial strain of the species Peribacillus aracenensis with deposit number CECT 30655, according to claims 9 to 12, for its application in compositions, forming part of any bacterial preparation, either individually or in combination with other organisms, and by any available means that puts the bacteria in contact with the seed, the root or aerial system of the plants.