Pseudomonas shirazensis nfv3 and the metabolites thereof vehiculised in silver nanoparticles as plant growth promoters, stimulants of adaptation under water stress conditions and stimulants of secondary metabolism of pharmacological and food interest

Abstract

The present invention relates to a bacterial strain of Pseudomonas shirazensis CECT 31128, a microorganism of the group of Gram-negative bacteria, of the genus Pseudomonas, which is isolated from the rhizosphere of Nicotiana glauca, its total metabolic liquid (TML) and its metabolic liquid vehiculised in silver nanoparticles (ML-AgNP), stimulants of growth and yield in crop production, stimulants of nitrogen, phosphorous, potassium, sulphur, calcium, magnesium, boron, manganese, zinc and iron absorption in plants, stimulants of adaptive metabolism against abiotic stress and enhancers of photosynthesis, under normal conditions and under water stress conditions, as well as stimulants of secondary metabolism of terpenes and phenolic compounds of pharmacological and food interest. Both the bacterial strain and the metabolic products thereof can be used as the basis for various preparations intended to enhance plant growth and production, as well as within the field of organic biofertilisers to improve mineral nutrition with respect to the indicated nutrients and improve adaptation to abiotic stress.

Description

[0001] DESCRIPTION

[0002] Pseudomonas shirazensis NVF3 and its metabolites delivered in silver nanoparticles as plant growth promoters, stimulants of adaptation to water stress situations, and secondary metabolism of pharmacological and nutritional interest.

[0003] The present invention relates to a strain of Pseudomonas shirazensis (NVF3, internal laboratory code) isolated from the rhizosphere of Nicotiana glauca, with biochemical characteristics that make it suitable for use in the treatment of plants in order to improve nutrition and plant growth under stress conditions, as well as to stimulate the secondary metabolism of terpenes and phenols in rosemary and olive, which are plants of pharmacological and food interest; and the invention also relates to two metabolic products of this strain with the same activities and applications, the total metabolic liquid (TML), with all the metabolites released by NVF3 during growth into the culture medium, and this metabolic liquid carried in silver nanoparticles (TML-AgNP), a formulation that offers greater efficacy with less environmental impact.

[0004] This strain has been deposited for patent purposes in the Spanish Type Culture Collection (CECT 31128), dated September 18, 2024, where it has been assigned deposit number 31128. The CECT is based in the research building of the University of Valencia, located on the Burjassot campus (DP 46100 - Valencia, Spain).

[0005] SCOPE OF APPLICATION. -

[0006] The invention falls within the fields of agri-food biotechnology and nanotechnology, specifically in the area of ​​biostimulation, since the bacterial strain NVF3 and its metabolic derivatives LMT and LM-AgNP in the form of silver nanoparticles can serve as a basis for the preparation of different types of products designed to increase production, both under normal conditions and under water stress. These products will improve the adaptation of plants to various abiotic stress conditions that compromise agricultural production.

[0007] And also in the field of organic biofertilizers, since these bacterial products can be used to improve the mineral nutrition of plants in nutrients essential for their development. STATE OF THE ART.

[0008] Plants have different mechanisms to adapt to situations of water stress and salinity in the environment and improve their mineral nutrition.

[0009] Photosynthesis is a physiological process that transforms light energy into chemical energy according to the following equation: H₂O + CO₂ + E (Av) to produce C₆H₁₂O₆ + O₂, where water is absorbed by the roots and CO₂ by the leaves. It occurs 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. This process requires photosynthetic pigments for energy capture and generates significant oxidative stress (free radicals - ROS).The plant has systems for eliminating free radicals to keep them within physiological limits, since certain free radicals, such as H2O2, are physiological signals necessary for the plant's proper functioning. Adequate water availability and good plant hydration are absolutely fundamental to this entire process. The uptake of CO2 through stomata is vital for photosynthesis, which creates a problem that contradicts the established requirements for good photosynthesis, as it is the pathway for water loss (transpiration), a process that accelerates under adverse temperature and humidity conditions.

[0010] Thus, the plant needs to be hydrated; the parameter that defines the plant's water status is the water potential, which, in short, reflects the balance between water content (pressure potential) and solutes (osmotic potential). To achieve this, the plant is able to generate a negative osmotic potential with respect to the soil, so that water enters carried 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 keeps the plant turgid. Nutrient absorption occurs through specific pumps, and once absorbed, nutrients accumulate in vacuoles and / or are translocated to the aerial parts via transpiration, where they are incorporated into organic structures. 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 a negative osmotic potential and maintain hydration, keeping stomata open longer; if the drought is severe, the stomata close. When plants are in saline soils, they tend to accumulate sodium ions (Na+). + which, in high concentrations, are toxic to the plant. One of the adaptation mechanisms is to pump Na ions. + to the exterior to prevent its accumulation. This response involves the generation of ROS, which activates the hormonal stress response (activation of specific protein synthesis and increase in Ethylene (Et), Jasmonic Acid (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, prioritizing stomatal closure when water is limited. In addition to stomatal closure, the plant, as previously mentioned, also has mechanisms to retain water, increasing the internal concentration of solutes (osmolytes, ions, and other organic molecules), and antioxidant systems to ensure overall health with efficient photosynthesis, keeping free radicals at physiological levels.

[0013] This succinct description of the plant's mechanisms for adapting to stressful situations highlights the numerous potential areas for improvement in agricultural production.

[0014] In this sense, the use of plant growth promoting bacteria (PGPBs) to improve the plant's ability to adapt to water stress and salinity conditions is a field of research in constant development.

[0015] There is a growing body of scientific evidence showing that beneficial bacteria (PGPBs) are capable of modifying several plant targets simultaneously; that is, they use more than one mechanism of action, which also varies according to the plant's needs and is always aimed at improving the plant's 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 and indirect.In the case of direct mechanisms, bacteria or their metabolites alter the plant's metabolism (hormonal activity, stimulation of adaptive mechanisms), with the secondary metabolism of certain compounds with bioactive effects on human health being of particular interest. Indirect mechanisms encompass the synthesis of compounds that facilitate nutrient uptake or mobilization or prevent the growth of pathogenic microorganisms on the plant, without altering the plant's metabolism; this is not an exhaustive list. In the case of this patent, both are of interest. The plant possesses a highly inducible adaptive metabolism related to its adaptation to adverse situations it faces during its life, and this metabolism is susceptible to modification by PGPBs.

[0016] Furthermore, it has been demonstrated that certain molecules of bacterial origin produced during fermentation are capable of activating plant metabolism (Algar et al., 2012, dx.doi.org / 10.1021 / jf303334q; Ramos Solano et al., 2008 10.1094 / PHYTO-98-4-0451; Gutierrez-Mañero et al., 2012. 10.3109 / 13880209.2012.664150; Martin Rivilla et al., 2020 10.3390 / plants9081020). These molecules, called elicitors, have different chemical natures and are capable of activating physiological processes in plants.

[0017] The genus Pseudomonas encompasses an extraordinarily diverse group of microorganisms. It is one of the largest genera among Gram-negative bacteria (Girard et al., 2021. 10.3390 / microorganisms9081766) and is common among soil bacteria. Genomic analysis techniques have enabled the discovery and description of new species within the genus, with genetic analysis criteria prevailing over the traditional taxonomic criteria of Bergey's Manual. Currently, taxonomic criteria for bacteria have been standardized in the Genome Taxonomy Database (GTDB). According to the GTDB, the Pseudomonadaceae family currently includes 18 genera, among them Azomonas, Azotobacter, Entomomonas, Oblitimonas, Pseudomonas, Pseudomonas_B, Pseudomonas_E, Thiopseudomonas, and Ventosimonas (https: / / gtdb.ecogenomic.org / tree7M Pseudomonadaceae - last accessed September 3, 2024). The genera Pseudomonas and Pseudomonas_E contain the most species.

[0018] The NVF3 strain of Pseudomonas of the present invention falls within the Domain Bacteria, Phylum Proteobacteria, Class gamma-proteobacteria, Order Pseudomonadales, Family Pseudomonaceae, Genus Pseudomonas_E.

[0019] The genus Pseudomonas exhibits great metabolic versatility due to a large number of plasmids containing inducible operons for the synthesis of specific enzymes that catabolize compounds present in the environment, conferring a high capacity for survival in hostile environments. It is very abundant in the soil system and has been repeatedly described as a protective bacterium against various plant diseases. Some bacteria in this group produce fluorescent, yellow-green pigments that are easily soluble in water. Among other functions, these pigments act as siderophores (molecules capable of capturing iron from the environment for the microorganism's metabolism). The production of cyclic lipopeptides (PCL - Pseudomonas cyclic lipopeptides) has also recently gained importance (Geudens et al., 2018 doi.org / 10.3389 / fmicb.2018.01867). Furthermore, the presence of Pseudomonas sp.In the rhizosphere of different plants, it beneficially affects their physiology, indicating that it is very possible for the plant to select it at the rhizospheric level.

[0020] With regard to the genus Pseudomonas, there are several publications that describe their ability to produce siderophores and modulate the metabolism involved in oxidative stress (Bar-Ness et al., 1991, Journal Iron nutrition and interactions in plants, 271-281; ​​Weger et al., 1988, DOI: 10.1128 / jb.170.10.4693-4698.1988; Ghosh et al., 2018, DOI: https: / / doi.org / 10.1007 / s13213-018-1366-7; Vives-Peris et al., 2018, DOI: https: / / doi.org / 10.1007 / s00299-018-2328-z), or by improving the production of secondary metabolites (Gutierrez-Albanchez et al., 2019 doi: 10.1002 / jsfa.9507; Martin-Rivilla et al 2021 doi.org / 10.1002 / jsfa.10632), protection against pathogens (García-Villaraco et al, 2021 doi.org / 10.3390 / plants10020331) or adaptation to abiotic stress (Yasmin et al 2022, doi.org / 10.1111 / ppl.13497; 10.3389 / fmicb.2023.1198131). However, since Pseudomonas shirazensis is a recently described species (Girard et al, 2022), there are no publications or patents for any P. strain.shirazensis as a stimulator of the adaptive metabolism of plants in the face of biotic or abiotic stress, improver of plant nutrition or secondary metabolism.

[0021] The P. shirazensis NVF3 strain is capable of exerting beneficial effects on plants under both normal conditions and water stress, improving fruit production. It enhances CO2 fixation, keeping stomata open for longer, while simultaneously increasing water use efficiency (WUE). This reflects stimulation of the metabolism involved in adaptation, preventing the formation of free radicals and improving their elimination. This promotes the plant's adaptation to these conditions through a redox metabolic homeostasis mechanism, resulting in a greater capacity to withstand water stress. Furthermore, it improves plant nutrition, enhancing growth and yield. It also stimulates the plant's secondary metabolism, increasing the polyphenol and terpene content both in the living plant and post-harvest.Furthermore, the cell-free metabolic liquid obtained by fermentation of NVF3 reproduces the effects observed by inoculation of cell-free cells from the metabolic liquid, demonstrating that in this system the bacterial metabolites responsible for the biological effect appear.

[0022] Nanotechnology is a relatively young science that deals with the creation, manipulation, and use of materials at the 1–100 nm scale. At this scale, significant differences occur in the properties of certain materials compared to the same material at larger scales. The synthesis of nanomaterials, specifically metallic nanoparticles, began with energy-intensive chemical or physical synthesis processes that had a negative environmental impact. However, the convergence of nanotechnology with biology has given rise to a new field called nanobiotechnology, which incorporates the use of biological materials in the nanoparticle synthesis process (Shah et al. 2015;10.3390 / ma8115377).

[0023] The synthesis process of nanoparticles (NPs) involves the reduction of a metal, which crystallizes to form a nanomaterial with its own structure and characteristics depending on the synthesis conditions. The formation of NPs using biological methods reduces the metal with organic compounds. Plant extracts rich in antioxidant compounds were the first to be successfully used, and this process was termed "green synthesis." However, other materials such as algae, bacteria, or fungi, their metabolites, or their extracts are capable of forming NPs with biological activity to stimulate plants or inhibit the growth of pathogenic microorganisms, among other applications (Ibrahim et al., 2019, 10.1039 / c9ra04246f; Gosh et al., 2021 10.3389 / fmicb.2021.638068).

[0024] In the case of NVF3, its biologically active bacterial metabolites are able to reduce metal ions (Ag) forming nanoparticles (NP), which are coated with molecules with biological activity exclusive to NVF3, thus preserving their effect. Furthermore, they have greater activity at very low concentrations due to their size, which has suggested the use of NP to improve the plant's ability to adapt to water stress and salinity conditions.

[0025] Furthermore, the formulation of the metabolic liquid using NP technology replicates the effects of adaptation to water stress and stimulation of secondary metabolism in plants. It is also capable of stimulating secondary metabolism when applied post-harvest. Patents exist that protect metabolites produced by species of the genus Pseudomonas under certain conditions, which have applications as plant protection products for disease control, as in the case of the US patents with publication numbers US20230165260-A1 and US20200178541-A1, or for stimulating secondary metabolism of pharmaceutical and industrial interest (Spanish patent ES2902976-E), but in no case are these metabolites produced by P. shirazensis.

[0026] There are also patents on nanoparticles, specifically silver nanoparticles, and the synthesis method, such as US20200268807-A1; the synthesis of AgNP with other strains of the genus Pseudomonas (10.3390 / molecules27217589) with antimicrobial potential is also published, but in no case is the biological activity attributed to the AgNP of P. shirazensis reported.

[0027] THE INVENTION.

[0028] The invention for which, in view of the aforementioned state of the art, a patent right is sought, relates to three interrelated products of a recently discovered species of bacteria of the genus Pseudomonas: (1) A specific strain of Pseudomonas shirazensis, to which the internal laboratory code NVF3 has been assigned, with capabilities promoting mineral nutrition and plant growth, also capable of stimulating the secondary metabolism of plants of pharmacological and nutritional interest; (2) the total metabolic liquid (TML) of this strain, with all the metabolites generated in the fermentation process, which reproduces the effects of the bacteria and can be used for the same purposes; and, especially due to its greater advantages, (3) this metabolic liquid carried in silver nanoparticles (TML-AgNP).

[0029] As the first inventive component, the isolation and characterization of the bacterial strain P. shirazensis with deposit number CECT 31128 is claimed, a microorganism of the Gram-negative bacteria group, genus Pseudomonas, for its demonstrated ability to enhance, under different abiotic stress conditions, the mineral nutrition of plants, by stimulating the assimilation of nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, boron, manganese, zinc and iron from the soil, as well as the adaptive metabolism to said conditions, by modulating the oxidative response through induction of the metabolism involved in the elimination of free radicals (ROS) and the accumulation of osmolytes, and the photosynthetic process, by stimulating the fixation of CO2 from the air and transpiration, and optimizing the capture of energy through photosynthesis, increasing CO2 uptake and water use efficiency (WUE).All of which makes it useful in the treatment of horticultural and woody plants to improve growth and production, under normal conditions and in the face of water stress, whether due to lack of water (reduced irrigation) or salinity.

[0030] This strain is also capable of stimulating the secondary metabolism of terpenes and phenolic compounds from plants of pharmacological and food interest, such as rosemary or olive.

[0031] The physiological characteristics and genetic analysis of this strain have allowed it to be unequivocally identified as P. shiraziensis, differentiating it from other species of the genus Pseudomonas.

[0032] After genetic characterization, various in vitro tests were performed to demonstrate the biochemical activities indicative of its potential nutrient mobilization and plant growth promotion capabilities. These included auxin production, 1-aminocyclopropane-1-carboxylate degradation, phosphate solubilization, and the production of siderophores and chitinases. The test was positive for siderophore production and phosphate solubilization.

[0033] The second component of the invention relates to the metabolites released by the new NVF3 strain during its fermentation process in the culture medium, both the total cell-free, liquid metabolic LMT, which reproduces the effects of the bacteria and can be used for the same purposes, and the liquid metabolic LM-AgNP formulated in silver nanoparticles, obtained by taking advantage of the ability of the metabolites to reduce the metal ions of NOsAg added to the medium.

[0034] Once the effect of the metabolites contained in the NVF3 metabolic fluid was demonstrated, this metabolic fluid was used to synthesize silver nanoparticles using the bacterial metabolites as reducing agents of the metal, so that the NPs are coated with these metabolites.

[0035] NVF3 metabolites formulated as nanoparticles exhibit greater PGPR activity at very low concentrations due to their size, allowing for lower doses. Furthermore, the nanoparticle formulation is an environmentally friendly, bio-based synthesis. The biological effects of the three resulting bacterial products: P.The three products *Agnus shirazensis* NVF3, LMT, and LM-AgNP, which exhibit the aforementioned improvements in plant growth, have been tested on tomato and blueberry plants. This testing involved measuring the nutrient content (N, P, K, S, Ca, Mg, B, Mn, Zn, Fe) of treated plants under various abiotic stress conditions, as well as parameters related to adaptive metabolism (malondialdehyde (MDA), H2O2 as a representative of reactive oxygen species (ROS), and osmolytes), and photosynthetic efficiency (photosynthetic pigment concentration, CO2 fixation, transpiration, water use efficiency (WUE), fresh weight, dry weight, and plant height). The effects of these three products on the secondary metabolism of rosemary plants during the growth cycle, and on rosemary and olive trees post-harvest, were also tested, determining the concentrations of metabolites with pharmacological activity (phenols and terpenes).

[0036] Specifically, a greenhouse experiment was carried out on tomatoes, with direct application of Pseudomonas shirazensis NVF3 cells to the roots, subjecting them to an osmotic stress shock (PEG), detecting an improvement in photosynthesis, increasing CO2 fixation (Pn), an increase in water use efficiency (WUE), the concentration of photosynthetic pigments and osmolytes (glycine-betaine and proline), and a decrease in oxidative stress.

[0037] In another experiment conducted with the strain on tomatoes grown outdoors at very high temperatures, an improvement in photosynthesis was also detected, with an increase in production and an improvement in nutrient absorption.

[0038] Another experiment similar to the previous one, in tomatoes, outdoors, but maintaining strong water stress, due to limited water supply (drought) throughout the growth cycle, revealed the ability of NVF3 to increase water use efficiency (WUE), decreasing photosynthetic pigments and increasing non-enzymatic antioxidants (polyphenols) and osmolytes (proline and soluble sugars), as well as an improvement in nutrient absorption.

[0039] In other experiments conducted on intensively cultivated blueberries with direct inoculations of P. shirazensis NVF3 during the production cycle, NVF3 has been found to increase blueberry yield by stimulating CO2 fixation and increasing water use efficiency (WUE), thus improving nutrient concentration in leaves (N, P, K, Mg, Ca, Fe). Furthermore, it improves fruit quality by significantly increasing flavonoid concentration. The same effects are observed when the experiment is conducted with a 33% reduction in water and nutrient supply during the plant's production cycle.

[0040] In another experiment, conducted on tomatoes grown in greenhouse pots, applying a culture of NVF3 cells to the roots, then withholding irrigation for one week, and finally applying rescue irrigation seven days later, it was found that NVF3 cells stimulate growth (height, fresh weight, dry weight) and nutrition (N, P, Cu). The improved recovery capacity after the stress period was evidenced by the higher transpiration and CO2 fixation values ​​seven days after irrigation was restored.

[0041] In another experiment with tomatoes under the same conditions as the previous one, but applying cell-free NVF3 metabolic liquid (MLT) to the roots, the result was the same. The metabolic liquid was found to stimulate nutrient assimilation (N, P, Cu) and growth (height, fresh weight, dry weight); an increase in photosynthetic pigments and transpiration was observed, along with a decrease in oxidative stress.

[0042] In another experiment with tomatoes under the same conditions, but applying the NPs at two concentrations via foliar spraying, the results were also the same. It was found that the NPs stimulate nutrient assimilation (N, P, K, S, Cu, Fe, Mn, Zn) and growth (height, fresh weight, dry weight). They also stimulate photosynthesis and transpiration; increase oxidative stress and ROS scavenging systems; and demonstrate a faster recovery of photosynthetic activity after rescue irrigation.

[0043] Experiments have also been carried out demonstrating the ability of the NFV3 strain and its derivative products LMT and LM-AgNP to stimulate the secondary metabolism of phenols and terpenes in plants of pharmacological and nutritional interest.

[0044] In rosemary, grown in experimental plots, a culture of NFV3 cells has been applied to the roots, several applications, evaluating the effect on the content of secondary metabolites with bioactive effect, finding that the strain induces an increase in the concentration of phenols and terpenes.

[0045] In another experiment carried out on rosemary, the bacterium Pseudomonas shirazensis NVF3, the bacteria-free metabolic liquid and the NPs were applied post-harvest, and a stimulation of secondary metabolism was found, both by the NPs and by the metabolic liquid and by the bacterial cells, increasing the concentration of phenols and terpenes, which are metabolites with pharmacological activity.

[0046] In olive trees, it has been applied by applying NP in post-harvest, resulting in a stimulation of secondary metabolism, increasing the concentration of oleuropein and triterpenes, with pharmacological activity.

[0047] The aforementioned experiments on the capabilities of P. shirazensis NVF3 and its metabolites, unformulated or formulated as nanoparticles (NP), demonstrating the aforementioned benefits for plants, are presented at the end of this report, within the section on method of implementation.

[0048] The purpose of this invention, which constitutes its technical advantage, is, firstly, to have a new bacterium of a recently discovered species, P. shirazensis, to improve nutrition, growth, and agricultural production, both without stress and under water stress conditions (osmotic and / or drought), and to optimize the concentration of secondary metabolites with pharmacological activity in plants of interest to the industry; and secondly, to have the metabolites of the strain's metabolic liquid, LMT, formulated in metallic nanoparticles, LM-AgNP, which is an added advantage, since this presentation of the metabolic product offers greater efficacy of action on plants and is environmentally friendly.

[0049] Accordingly, this patent application claims the use of the Pseudomonas shirazensis NVF3 strain and its metabolic fluid, either unformulated or formulated as nanoparticles (NP), for application in any type of plant species, as part of any preparation, either individually or in combination with other organisms, in order to improve mineral nutrition and the adaptation of plants to any abiotic stress condition, increasing agricultural production under normal conditions, without stress, or in the face of water stress, whether natural, osmotic and / or drought, or due to lack of irrigation, and also for application in crops of interest to the pharmaceutical and food industries, in order to improve the concentration of bioactive compounds with pharmacological activity.

[0050] FIGURES AND GRAPHS. - At the end of this descriptive report are included figures with images and graphs of the bacteria, the process of obtaining the metabolic nanoparticles, and results of the experiments carried out.

[0051] Figure 1 is a TEM image of the morphology of P. shirazensis NVF3 after 24 h of incubation at 28° on standard methods agar (PCA)

[0052] Figure 2 includes images and graphs of UV spectra resulting from the synthesis of nanoparticles (NPs) under different pH conditions and different volumes of LMT-AgNOs 1mM mixture

[0053] Figure 3 shows the image (TEM) and histogram of the mean size distribution of the two resulting NPs

[0054] Figure 4 shows the XRD spectrum with the typical Bragg refraction peaks of the NPs formed

[0055] Figure 5 shows the graphs resulting from the characterization of the surface compounds of AgNPs.

[0056] Figure 6 shows two graphs that show the results of the analysis of phenols and flavolones by colorimetry after applying NVF3, LMT and LM-AgNP to rosemary pruning remains, and Figure 7 shows two graphs of the concentration of the phenolic compound rosmarinic acid and the terpene carnosol (mg / g dry weight) in rosemary plants in post-harvest (experiment 10).

[0057] METHOD OF IMPLEMENTATION. -

[0058] The strain of the genus Pseudomonas described here was isolated by studying the phytosphere of natural populations of Nicotiana glauca along a transect of the Mediterranean coast of Almería. This plant species (Nicotiana glauca) was selected because it belongs to the Solanaceae family, has an active secondary metabolism, and has proven to be a good source of microorganisms with biological activity of interest in agriculture.

[0059] The biosphere samples for bacterial strain isolation were collected from natural populations of Nicotiana glauca on the coast of Almería. As a result of this sampling, 960 strains were collected, among which P. shirazensis (NVF3, internal laboratory code) was found. This strain was isolated on nutrient agar (PCA).

[0060] In the laboratory, this microorganism is maintained with a high survival rate in 20% glycerol in nutrient broth (Pronadisa) at -80°C or in 15% glycerol in water at -20°C and is easily recovered in the culture medium used for isolation in both solid and liquid phases at 28°C.

[0061] Morphological, biochemical and genetic characteristics of P. shirazensis NVF3.-

[0062] For the characterization of the strain, different phenotypic characteristics were considered, which are detailed in this report: (i) colony morphology, (ii) cell morphology, (iii) growth conditions, (iv) biochemical characteristics, (v) comparative analysis of the genome of the P. shirazensis NVF3 strain. (i) The morphology of the colonies after 24 h of incubation at 28° on agar for standard methods (PCA) is specified in Table 1:

[0063] Table 1

[0064] i) The morphological characteristics of P. shirazensis NVF3 after 24 h of incubation at 28°C on PCA (Standard Methods Agar) correspond to a Gram-negative bacillus, with average dimensions of 1.888 m x 882.3 nm, and lophotrichous flagella, as shown in Figure 1. iii) Growth conditions. This strain can grow in a medium with 6% NaCl and tolerates pH values ​​between 6 and 8, with no growth observed at pH 5 or above pH 8. It can grow in a temperature range of 4 to 30°C, losing viability above this value. iv) Once isolated and characterized, with internal reference code NVF3, 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), degradation of 1-aminocyclopropane-1-carboxylate (Glick et al., 1995. http: / / doi.org / 10.1139 / m95-070), phosphate solubilization (De Freitas et al., 1997. Biol. Fértil. Soils, 24(4), 358-364), siderophore production (Alexander and Zuberer, 1991. Biol. Fértil. Soils, 12(1), 39-45), and chitinases (Frándberg and Shunürer, 1998 Can.J. Microbiol. 44: 121-127). It tested positive for siderophore production and phosphate solubilization. v) The genome of strain NVF3 was first analyzed using tools available on EzBioCloud. First, the EzBioCloud identification service provides validated similarity-based searches in quality-controlled databases of 16S rRNA sequences. After that, ANI (average nucleotide identity), AAI (average amino acid identity), dDDH, and TETRA analyses were performed. The Type (Strain) Genome Server (TYGS) was used to analyze the entire genome.Based on these analyses, the highest similarity was found with the strain Pseudomonas shirazensis SWRI56, exhibiting ANI and AAI values ​​of 99.04% and 98.53%, respectively; an intergenomic distance of 0.0095; a digital DNA hybridization (dDDH) value of 92.04%; and a guanine-cytosine (G+C) content of 0.21%. The tetranucleotide frequency correlation coefficient (TETRA) was 0.99964. Based on the ANI, AAI, dDDH, and TETRA results, it can be concluded that the strain corresponds to the species Pseudomonas shirazensis. The 16S rDNA gene sequence is deposited in the GeneBank under deposit number PQ284154.

[0065] Metabolic fluid (MFL) obtaining process.-

[0066] The process for obtaining the NVF3 metabolic liquid (MLL) consists of fermentation at a constant temperature (28°C-32°C), with agitation, for 24 to 72 hours. The cells are separated by centrifugation and filtration through a 0.25 µm filter. The culture medium used is a general laboratory medium called nutrient broth. The characterization of the organic matter present in the MML is performed by FTIR and is shown in Figure 5. Synthesis and characterization process of silver nanoparticles using NVF3 metabolic liquid as an Ag+ reducing agent (ML-AgNP).

[0067] The indicated metabolic fluid was used to synthesize silver nanoparticles using bacterial metabolites as reducing agents for the metal, such that the nanoparticles were coated with these metabolites. The synthesis process consists of using the NVF3 metabolic fluid obtained as described above. The cell-free metabolic fluid (CFMF) was adjusted to a pH between 7 and 9, mixed with 1 mM NChAg in appropriate proportions, and the mixture was incubated for 24 h with shaking (250 rpm) at 28–37°C under constant illumination. The most suitable CFMF:1 mM NChAg ratios were 5:1 v / v (S1) and 2:4 v / v (S4). Once the process was complete, the nanoparticles were precipitated by centrifugation, washed with distilled water to complete the nucleation process, and lyophilized to prepare a stock solution of known concentration.

[0068] The formation of NP is confirmed by a color change from light yellow to brown, and an increase in absorbance at 420 nm, characteristic of Ag°.

[0069] Figure 2 shows images of test tubes that are visual confirmation evidence of nanoparticle synthesis under pH 7 and 9 conditions by mixing different volumes of LMT and 1mM AgNOs, and the corresponding UV spectrum graphs showing the maximum absorption of reduced silver between 420-440.

[0070] Under the microscope, the NPs are spherical; the S1s have a mean diameter of 14-2 nm and the S4s have a mean diameter of 16.68 nm (TEM), as can be seen in the image and histogram of the mean size distribution of S1 and S4 particles in Figure 3 (Inset bar = 200 nm).

[0071] The XRD spectrum in Figure 4 shows the typical Bragg refraction peaks at 20° with values ​​of 28°, 32°, 32°, 46°, 54° and 57°, revealing a typical cubic structure of AgCl crystals (JCPDS Card no. 31-1238) from the NP.

[0072] The FTIR analysis performed on the crude culture medium (without bacterial growth), the supernatant (LMT, after bacterial growth, cell-free) and the NPs indicates differences between the three products, reflecting the changes that have taken place in the culture medium as a result of bacterial growth, characterizing the metabolic fluid with effect, and the change operated in its composition when the NPs are formed.

[0073] The graphs in Figure 5 show the result of this characterization of the surface compounds of the AgNPs, culture media (nutrient broth - blue) and metabolic liquid used for the synthesis of NP (green) and AgNPs S1 (A-orange) and S4 (B-red).

[0074] The profiles show similarities, indicating that some molecules were incorporated during NP synthesis. There is a peak exclusive to S4 NPs at 806 cm⁻¹ -1 (Figure 5).

[0075] The transmittance values ​​indicative of organic matter on the surface of the NPs appear in Table 2. This table shows the most relevant XRD identifying peaks for each type of nanoparticle (Ferro et al. 2019 https: / / doi.org / 10.3390 / molecules24183237; Pernas-Pleite et al. 2022, https: / / doi.org / 10.3390 / molecules27217589).

[0076] Table 2

[0077] Demonstrative experiments of the capacity of P. shirazensis NVF3 and its metabolites, complete or formulated in NP, as an enhancer of plant nutrition and production and of secondary metabolism of pharmacological and food interest.

[0078] 1 oOsmotic Stress Protection Experiment in Tomato. An elicitation experiment was conducted on Solanum lycopersicum var. Casillas plants using direct application of bacterial suspensions to the roots. The objective was to demonstrate plant protection under osmotic stress conditions. A bacterial suspension of the NFV3 strain (density 10⁸ CFU / mL) was inoculated into the roots of tomato seedlings twice: the first time one month after sowing and the second time one week later. One week after the second inoculation with NFV3, the plants were subjected to osmotic stress by watering them with 10 mL of a 10% polyethylene glycol 6000 (PEG 6000) solution. Three days later, parameters related to photosynthetic capacity were measured, and the plants were harvested.The following parameters were analyzed: photosynthetic pigments, malondialdehyde (MDA) as a marker of cellular oxidative stress, and H₂O₂ as a representative of reactive oxygen species (ROS); osmolytes (proline and glycine betaine). Plants under stress and treated with NFV₃ increased net photosynthesis (net CO₂ fixation) by 37% compared to the stressed control, without altering transpiration, which increased water use efficiency (WUE) by 29%. Furthermore, an increase in photosynthetic pigments (65–70%) was detected. Plants treated with NFV₃ exhibited lower oxidative stress, evidenced by a decrease in MDA (12%) and a 40% decrease in H₂O₂ content. Compatible osmolytes used by the plant as a defense mechanism against osmotic stress also increased in plants treated with NFV3 (35% in proline and 4.5% in glycine betaine) compared to the stressed control.

[0079] 2 oExperiment to improve production and mineral nutrition in tomatoes. The experiment was conducted with Solanum lycopersicum var. Alcolea plants by direct application of bacterial suspensions (density 10⁸ CFU / mL) to the roots of plants in 10 L pots located in open-air experimental plots, maintaining the soil water potential between -200 and -300 mbar, values ​​corresponding to well-hydrated soil. The experiment was carried out from May to July 2023, under sustained very high temperatures (minimum 22°C, maximum 42°C).A bacterial suspension of the NVF3 strain was inoculated into the root of tomato plants 6 times, the first time at transplanting, and the others every 15 days, with harvesting starting 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), transpiration and 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.A significant increase in CO2 fixation (63%) and transpiration (58%) was observed, along with a consequent increase (2.9%) in water use efficiency (WUE); a decrease in photosynthetic pigments (6% chlorophylls, 1.5% carotenes), reduced oxidative stress (4% MDA), an increase in H2O2 (8.8%) as a signaling metabolite; and a decrease in the analyzed osmolytes, specifically proline (62%) and glycine-betaine (5%). Nutrient content increased in plants treated with NVF3, particularly K, Ca, Mn, and Zn (Table 3). Production increased in fruit weight (51%), number of fruits (30%), and average fruit weight (18%).

[0080] Table 3 shows the nutrient content in tomato leaves after 3 applications under the described conditions.

[0081] Table 3.

[0082] 3 oAn experiment was conducted to protect tomatoes from drought and high-temperature stress by improving mineral nutrition. This experiment was carried out in parallel with Experiment 2, but with limited water application, maintaining soil water potential between -400 and -500 mbar, values ​​corresponding to moderate soil water stress. The parameters detailed in Experiment 3 were analyzed during the interim sampling. A significant increase (96%) in water use efficiency (WUE) was observed, along with a decrease in photosynthetic pigments (30% chlorophylls, 17% carotenes), an increase in H₂O₂ (3%) as a signaling metabolite, and an increase in the analyzed osmolytes: proline (x3) and soluble sugars (x2). Nutrient content increased in plants treated with NVF₃ (P, K, Ca, Mn, Zn) (Table 4). Yield remained the same as the control.

[0083] Table 4 shows the nutrient content in tomato leaves after 3 applications under the described conditions (reduced irrigation).

[0084] Table 4

[0085] 4 oBlueberry production improvement experiment in a tunnel greenhouse. Direct inoculation experiment with Pseudomonas shirazensis NVF3 in blueberries (Vaccinium corymbosum var. Cupla). The experiment was conducted 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), using a randomized block design. Cell suspensions of the strain (density 10E8 CFU / mL) were applied to the roots from July to February, twice a month. Total production was collected, and a sampling point was determined after the summer stress period, in September, when photosynthetic parameters of CO2 fixation were determined. An improvement in CO2 fixation (+77%) was recorded, decreasing transpiration and, therefore, increasing water use efficiency (WUE) (84%).Furthermore, it improves mineral nutrition, increasing the content of N, K, Mg, Ca, B and Fe, as indicated in Table 5. With all this, an increase in fruit production is recorded (+5.5%).

[0086] Table 5 shows the nutrient content in blueberry leaves from control and inoculated with NVF3.

[0087] Table 5.

[0088] 5 oBlueberry production improvement experiment in a tunnel greenhouse with irrigation and nutrient limitations. Direct inoculation experiment with Pseudomonas shirazensis NVF3 in blueberries (Vaccinium corymbosum var. Cupla). The experiment was conducted 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), using a randomized block design. Cell suspensions of the strain (density 10E8 CFU / mL) were applied to the roots twice a month from July to February; water and nutrient application via irrigation were limited by 33% throughout the cycle. Total production was collected, and a sampling point was determined in September, after the summer stress period, when CO2 fixation photosynthetic parameters were measured.An increase in fruit production (+2%) was recorded, along with an improvement in CO2 fixation (x2) by decreasing transpiration, and therefore increasing (x2.2) water use efficiency (WUE). Nutrient uptake (P, Mg, Ca, Cu, and Fe) was also improved (Table 6). Table 6 shows the nutrient content in control plants and plants inoculated with NVF3, under water and nutrient limitation (R).

[0089] Table 6

[0090] 6 o Demonstration experiment of the ability of NVF3 to improve drought adaptation in tomato, carried out in pots in a greenhouse. Tomato plants Lycopersicum esculentum var. Razymo Rz. were used. The seeds were germinated in pots (6x6x6 cm). 3The plants grew for 4 weeks after the first inoculation with metabolite-free NVF3 cells (density 10⁸ CFU / mL) in the roots, while maintaining an uninoculated control (n=3, 24 plants per replicate). Two further inoculations were performed 5 and 2 days after the first. Irrigation was then withheld for one week, followed by a rescue irrigation on day 7, and the plants were harvested one week later. NVF3 cells were found to stimulate growth (height 7%, fresh weight 22%, dry weight 20%). The improved recovery capacity after the stress period was evidenced by the higher transpiration (34%) and CO₂ fixation (10%) values ​​7 days after irrigation was restored. Nutrient uptake (N, P, S, Cu, Fe) was also improved (Table 6).

[0091] Table 7 shows the nutrient content in tomato leaves, control and inoculated with NVF3 cells, free of culture medium, 7 days after rescue irrigation, and increases over the control.

[0092] Table 7 7 oA demonstration experiment was conducted on the ability of NFV3 to stimulate the secondary metabolism of phenols and terpenes in rosemary plants (Salvia rosmarinus, Rosmarinus officinalis) in experimental plots. Rosemary plants (Rosmarinus officinalis) were used. Rosemary seedlings were transplanted to the plots, and NFV suspensions (density 10⁸ CFU / mL) were applied to the roots every 15 days for 8 months, maintaining uninoculated controls (n=12). Branches were cut, dried at 40°C, and extracted with 75% ethanol for 15 h, followed by centrifugation for 10 min. This process was repeated four times, and the supernatants were combined, filtered (using 0.4 µm cellulose acetate filters), and dried. After resuspending the dry residue in ethanol, it was filtered again and the rosmarinic acid and carnosic acid content was analyzed by HPLC, and a metabolomic analysis was also performed by HPLC-MS.HPLC analysis revealed an increase in the concentration of rosmarinic acid (87%) and carnosic acid (49%). HPLC-MS analysis revealed increases in certain phenols (luteolin, quinic acid, and rosmadial) and terpenes (diterpenes: 12-methoxy-carnosic acid; triterpenes: asiatic acid), as shown in Table 8.

[0093] Table 8

[0094] 8 o Demonstration experiment of the capacity of NVF3 metabolic fluid (MTF) to improve drought adaptation in tomato, carried out in pots in a greenhouse. Tomato plants Lycopersicum esculentum var. Razymo Rz. were used. The seeds were germinated in pots (6x6x6 cm). 3The plants grew for 4 weeks after the first application of NVF3 metabolic liquid to the roots, while maintaining an uninoculated control (n=3, 24 plants per replicate). Two further applications were made 5 and 2 days after the first. Irrigation was then withheld for one week, followed by a rescue irrigation on day 7, and the plants were harvested one week later. The metabolic liquid was found to stimulate growth (height 14%, fresh weight 57%, dry weight 49%); an increase in photosynthetic pigments and transpiration (8%) and CO2 fixation (3%) were also observed. It improved the uptake of nutrients: N, P, Cu, and Mn. Table 9 shows the nutrient content in tomato leaves of control plants and those inoculated with cell-free NVF3 metabolic liquid (MLT) 7 days after the rescue irrigation, and the increases over the control.

[0095] Table 9

[0096] 9 oExperiment to improve drought adaptation using NPs in potted tomatoes grown in a greenhouse. Tomato plants of *Lycopersicum esculentum* var. *Razymo* Rz. were used. Two NP concentrations, 30 and 60 g / mL, were tested as a foliar application. Seeds were germinated in 6x6x6 cm pots. 3The plants grew for four weeks after the first application of NVF3 NP, via foliar spray, while maintaining an uninoculated control (n=3, 8 plants each). Two further applications were made 5 and 2 days after the first. Irrigation was then withheld for one week, followed by a rescue irrigation on day 7, and the plants were harvested one week later. Photosynthetic parameters analyzed included fluorescence (F0, Fv / Fm, PSII, NPQ), transpiration and CO2 fixation, photosynthetic pigments, malondialdehyde (MDA) as a marker of cellular oxidative stress, and H2O2 as a representative of reactive oxygen species (ROS); and nutrient content. Transpiration and CO2 fixation were analyzed before the rescue irrigation, at 15 hours, and on day 7. Results: At harvest time, a significant increase in height (h), fresh weight (FW) and dry weight (DW) was observed, both in NP30 (h = 15%, FW = 53%, DW = 54%) and in NP60 (h = 11%, FW = 46%, DW = 50%).In CO2 fixation and transpiration, the lowest dose (NP30) was more efficient, increasing both parameters (36% and 10%, respectively), while the highest dose only increased transpiration (26%). Higher concentrations of the signal metabolites MDA (6% and 14%, respectively) and H2O2 (11% and 18%, respectively) were detected. The ROS scavenging system was selectively modified, with SOD activity increasing with NP60 (48%), APX activity increasing with both doses (50% and 17%, respectively), and catalase activity decreasing in both cases. Nutrient content increased in plants treated with NP30 and NP60, with increases in N, Cu, and Fe evident in both cases, and P, S, and Zn only in NP60. Table 10 shows the nutrient content in control plants and plants inoculated with AgNP at two doses NP30 and NP60, 7 days after rescue irrigation, and increases of each treatment over the control.

[0097] Table 10

[0098] 10. Experiment to improve the polyphenol and terpene content in rosemary pruning waste. Treatments were applied by spraying freshly pruned rosemary branches, ensuring contact of all plant material with the treatment. The treatments were: 1) NVF3 suspension (10E8 cfu / mL), 2) LMT, 3) NVF3 AgNP (60 ppm), 4) mM AgNO3, and 5) water. The branches were allowed to dry, and leaf extracts were prepared following the procedure described in Experiment 7. Total phenols and total flavonols were analyzed by colorimetry, and rosmarinic acid and carnosic acid were quantified by HPLC. Total phenols increased with NFV3 (46%), LMT (53%) and NP (28%), as well as total flavonols, NFV3 (34%), LMT (48%), NP (25%), as shown in the graphs in Figure 6.

[0099] HPLC analysis of the extracts revealed an increase in phenolic compounds and terpenes. The graphs in Figure 7 show the increase in concentration (mg / g dry weight) of rosmarinic acid (phenolic compound) and carnosol (terpene) in post-harvest rosemary plants after applying the three bacterial products. Rosmarinic acid increased by 84% with NVF3, LMT by 122%, and NP by 32% compared to the control. In the case of NP, the increase is ruled out as being due to silver, since the effect of this treatment (4) only induced a 4% increase compared to the control. Regarding carnosic acid and its derivatives, increases were detected with NVF3 (25%), LMT (31%), and NP (4%). 11th Experiment: Improvement of polyphenol content in olive pruning residues. An NVF3 LM-AgNP suspension was applied to newly pruned Arbequina olive branches.The leaves were dried and the extracts were prepared by sonicating the powdered leaves in 80% ethanol for 30 min; the process was repeated until all the material was used; the supernatants were combined, dried, resuspended in methanol using HPLC, filtered (0.2 µm nylon filters), and the polyphenol content was analyzed by HPLC. An increase in oleuropein (63%), a terpene-phenol with an iridoid nucleus exhibiting anti-inflammatory activity, among other properties, was found.

Claims

CLAIMS 1. Bacterial strain Pseudomonas shirazensis with deposit number CECT 31128, microorganism of the Gram bacteria group genus Pseudomonas, characterized by its ability to stimulate the assimilation of nutrients in horticultural and woody shrub plant species: N nitrogen, P phosphorus, K potassium, S sulfur, Ca calcium, Mg magnesium, B boron, Mn manganese, Zn zinc and Fe iron; to stimulate the adaptive metabolism to water stress; and to stimulate the secondary metabolism of terpenes and phenolic compounds in rosemary and olive plants.

2. Total metabolic liquid (TML) of the bacterial strain Pseudomonas shirazensis with deposit number CECT 31128, characterized by a obtaining process consisting of fermentation in general laboratory culture medium at constant temperature (28°C-32°C) under agitation (24-72 h.) and separation of the cells by centrifugation and filtration through 0.25 microns, with the capacity to stimulate the assimilation of nutrients in horticultural and shrubby woody plant species: N nitrogen, P phosphorus, K potassium, S sulfur, Ca calcium, Mg magnesium, B boron, Mn manganese, Zn zinc and Fe iron, to stimulate the adaptive metabolism to abiotic stress, and to stimulate the secondary metabolism of terpenes and phenolic compounds in rosemary and olive plants.

3. Metabolic liquid carried in silver nanoparticles (ML-AgNP) of the bacterial strain Pseudomonas shirazensis with deposit number CECT 31128, characterized by a process of obtaining it from the total metabolic liquid (TML) consisting of bringing the TML to pH 7-9, mixing with 1mM AgNOs in LML:AgNOs ratios of 5:1 v / v (S1) and 2:4 v / v (S4), incubating the mixture for 24h with shaking (250 rpm) at 28-37°C under constant illumination, precipitation of the nanoparticles by centrifugation, washing with distilled water, lyophilization and preparation of a stock solution of known concentration, with the capacity to stimulate the assimilation of nutrients in horticultural and shrubby woody plant species: N nitrogen, P phosphorus, K potassium, S sulfur, Ca calcium, Mg magnesium, B Boron, manganese (Mn), zinc (Zn), and iron (Fe) stimulate adaptive metabolism to abiotic stress and stimulate the secondary metabolism of terpenes and phenolic compounds in rosemary and olive plants.

4. Use of the bacterial strain of the species Pseudomonas shirazensis with CECT deposit number 31128, or of the total metabolic fluid (TMF), or of the metabolic fluid carried in silver nanoparticles (TMF-AgNP), according to claims 1, 2 or 3, in any species of horticultural plants, such as tomato plants, to improve mineral nutrition in N nitrogen, P phosphorus, K potassium, S sulfur, Ca calcium, Mg magnesium, B boron, Mn manganese, Zn zinc and Fe iron in the face of water stress, whether natural, osmotic and / or drought, or due to lack of irrigation, or under conditions of absence of water stress.

5. Use of the bacterial strain of the species Pseudomonas shirazensis with deposit number CECT 31128, or of the total metabolic liquid (TML), or of the metabolic liquid carried in silver nanoparticles (TM-AgNP), according to claims 1, 2 or 3, in any species of shrubby woody plants, such as blueberry plants, to improve mineral nutrition in N nitrogen, P phosphorus, K potassium, S sulfur, Ca calcium, Mg magnesium, B boron and Fe iron against water stress, whether natural, osmotic and / or drought, or due to lack of irrigation, or under conditions of absence of water stress.

6. Use of the bacterial strain of the species Pseudomonas shirazensis with deposit number CECT 31128, or of the total metabolic liquid (TML), or of the metabolic liquid carried in silver nanoparticles (TM-AgNP), according to claims 1, 2 or 3, in any species of horticultural plants, such as tomato plants, or woody shrubs, such as blueberry plants, in order to improve the adaptation of the plants to any water stress condition.

7. Use of the bacterial strain of the species Pseudomonas shirazensis with CECT deposit number 31128, or of the total metabolic liquid (TML), or of the metabolic liquid carried in silver nanoparticles (TM-AgNP), according to claims 1, 2 or 3, in any species of horticultural plants, such as tomato plants, or woody shrubs, such as blueberry plants, to improve plant growth and production.

8. Use of the bacterial strain of the species Pseudomonas shirazensis with CECT deposit number 31128, or of the total metabolic liquid (TML), or of the metabolic liquid carried in silver nanoparticles (TM-AgNP), according to claims 1, 2 or 3, in any species of horticultural plants, such as tomato plants, or woody shrubs, such as blueberry plants, to improve plant growth and production under conditions of water stress, due to drought or reduced irrigation water.

9. Use of the bacterial strain of the species Pseudomonas shirazensis with CECT deposit number 31128, or of the total metabolic fluid (TMF), or of the metabolic fluid carried in silver nanoparticles (TMF-AgNP), according to claims 1, 2 or 3, in any type of crops of agronomic or nutritional interest, such as olive or rosemary, in order to increase the content of terpenes and phenolic compounds.

10. Use of the bacterial strain of the species Pseudomonas shirazensis with deposit number CECT 31128, or of the total metabolic liquid (TML), or of the metabolic liquid carried in silver nanoparticles (TM-AgNP), according to claims 1, 2 or 3, in any type of olive or rosemary crop of pharmacological interest, in order to increase the content of terpenes and phenolic compounds.

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