WO2011067440A2

WO2011067440A2 - Bacterial strain cect7624, use of said strain in order to enhance the growth and water stress tolerance of a plant and xeroprotectant compound produced from same

Info

Publication number: WO2011067440A2
Application number: PCT/ES2010/000516
Authority: WO
Inventors: Maximino Manzanera Ruiz; Juan Ignacio VÍLCHEZ MORILLAS
Original assignee: Universidad De Granada; GONZÁLEZ LÓPEZ, Jesús Juan; NARVÁEZ REINALDO, Juan Jesús
Priority date: 2009-12-04
Filing date: 2010-12-03
Publication date: 2011-06-09
Also published as: WO2011067440A3; ES2389366A1; ES2362035B2; BR112012013505A2; ES2362035A1; ES2389366B2

Abstract

The invention relates to a microorganism of the Microbacterium sp. bacterial species (access number CECT7624). The invention also relates to the use of said microorganism or a population of same in order to enhance the water stress tolerance of a plant or to enhance the growth of a plant with said plant under optimum water content conditions. The invention further relates to the use of said microorganism or a population of same for the production of a xeroprotectant composition, said composition comprising trehalose, oxoglucuronic acid, lactate, glutamate, glutamine and pyruvate or also comprising fucose. Furthermore, the invention relates to a method for enhancing the water stress tolerance of a plant or producing the xeroprotectant composition.

Description

Bacterial strain CECT7624, use of said strain to increase growth and tolerance to water stress of a plant and

xeroprotector compound produced by it. The present invention relates to a microorganism of the bacterial species Microbacterium sp. with access number CECT7624. Also the present invention relates to the use of said microorganism or a population thereof to increase the tolerance to water stress of a plant or to increase the growth of a plant under optimum conditions of water content of said plant. Furthermore, the present invention relates to the use of said microorganism or a population thereof for the production of an xeroprotective composition, wherein said composition comprises trehalose, oxoglucuronic acid, lactate, glutamate, glutamine and pyruvate, or further comprises fucose. The present invention also relates to the method for increasing the water stress tolerance of a plant or for producing the xeroproctector composition of the present invention.

STATE OF THE PREVIOUS TECHNIQUE

The search for better phenotypes and genotypes of crop plants to increase production, nutritional improvements, quality in the fruit and plant, is of great interest to the scientific community. The selection of phenotypes has shown good results in obtaining better varieties, however, the role of biotechnology is especially important since the use of microorganisms to plant roots has led to great progress in understanding the effect of the microorganisms of the rhizosphere in the metabolic and genetic regulation of plants such as, for example, the use of Agrobacterium to obtain transgenic plants, or the use of nitrogen fixing bacteria associated with leguminous plants. In crop plants, the rhizosphere is one of the main sources of organic matter in soils. This rhizosphere stimulates microbial activity. Complex interactions between microorganisms and plant roots occur in the rhizosphere. The water potential of the rhizosphere is a key aspect that determines the bioavailability of water, oxygen and substrates of plants and microorganisms. Exudates of certain microorganisms can protect bacteria from water stress. In Mediterranean agriculture, water stress is one of the main factors that limit crop production.

It is known that mycorrhizal fungi colonize the roots of most plants. These fungi are obligate symbionts and receive carbon compounds in the form of energy from host plants; during this association they transfer nutrients, usually phosphorus and zinc (Ryan and Angus, 2003. Plant and Soil, 250 (2): 225-239). Mycorrhizal fungi significantly increase the surface area of absorption of the radical system of plants, improving their ability to absorb water and nutrients, maintain soil structure and increase resistance to stresses and diseases. There is evidence that indicates the influence of these fungi on the water relations of the host plants and the increase in drought resistance of them. It has been suggested that fungi could have positive effects on host plants in field conditions during periods of stress for them such as water stress (Alien and Alien, 1986. New Phytologist, 104: 559-71; Fitter, 1986. New Phytologist, 103: 767-776).

It is shown that some non-pathogenic bacterial strains are capable of stimulating the defensive metabolism of the plant in such a way that when there is a pathogen attack, the plants are protected and can survive with a much lower mortality than if not were treated with the bacteria. In some cases, the effect is very specific, and it is only effective against certain pathogens, while in others, the effect is much broader, even being effective against abiotic factors such as saline or water stress.

In this sense, the application of bacteria in certain crops is a biotechnological alternative that respects the environment since the bacteria would produce a double beneficial effect, on the one hand, it stimulates plant defenses against a pathogen, thus reducing the use of substances chemical, and on the other, it allows to obtain better yields under certain stress conditions. However, the selection of said microorganism is a complex aspect that requires the testing and characterization of numerous species and strains to obtain a microorganism capable of producing significant benefits to plants.

On the other hand, the conservation of biological materials by dehydration and osmoconcentration is a known technology. However, current conservation methods require high energy costs and generally need storage at low temperatures. Sometimes, after its conservation, the biological material has an activity and / or viability that does not reach satisfactory levels. Conservation methods, such as drying at room temperature, liquid formulations, freezing with cryoprotectants or lyophilization produce significant reductions in the activity / viability of the conserved material.

The processes currently used are slow and involve high energy consumption. In addition, lyophilization confers only a modest level of thermotolerance in the final product, and cooling is still required to reduce deterioration during storage. During evolutionary natural selection, certain species of plants and animals acquired the remarkable ability to tolerate extreme dehydration, remaining dormant in hostile environments for very long periods of time and still capable of acquiring a complete vital activity once hydrated again. Examples include the "resurrection plant" Selaginella lepidophyla, Artemia salina sea shrimp, Saccharomyces cerevisiae yeast or Macrobiotus hufelandi tardigrade. These organisms are called cryptiobiotics and the procedure by which they survive is known as anhydrobiosis. All species of animals and plants that have this capacity contain protective molecules that form amorphous crystals such as the disaccharide trehalose (aD-glucopyranosyl-aD-glucopyranoside).

The formation and use of amorphous crystals is well documented (Manzanera et al., 2002. Appl Environ Microbiol, 68: 4328-4333). Some of the preservatives that form these crystals are suitable for this type of preservation and include non-reducing carbohydrates such as trehalose, hydroxyectoin, maltitol, lactitol (4-OaD-glucopyranosyl-D-glucitol), palatinit [GPS mixture (aD -glucopyranosyl-1-6-sorbitol) and GPM (aD-glucopyranosyl-1-6-mannitol)] and its individual components GPS and GPM. Non-reducing glycosides of polyhydroxy compounds can be neotrehalose, laconeotrehalose, galactosyl trehalose, sucrose, lactosecarose, raffinose, etc. Other amorphous crystal-forming preservatives include amino acids such as hydroxyectoin.

The presence of water in the dry state is generally less than 0.2 g / g dry cell weight in most cryptobionts. These water levels are sufficient for these invertebrate organisms or microorganisms to resist extreme dehydration, high temperatures, ionizing radiation or, in some species of tardigrades, pressures of up to 600 MPa. It is known that biofilms (subaerial biofilms), formed by bacteria of the genus Rhodococcus sp among others, are capable of producing osmoprotective compounds, that is, polymeric extracellular substances (EPS) (Gorbushina, 2007. Environmental Microbiology, 9 (7): 1613 -1631). Likewise, LeBlanc (2008) (LeBlanc, 2008. Applied and environmental microbiology, 74 (9): 2627-2636), refers to the microorganism Rhodococcus jostii RHA1, an actinomycete with favorable metabolic capacities for bioremediation of contaminated soils, capable of secreting the ectoin and trehalose osmoprotectors.

EXPLANATION OF THE INVENTION The present invention relates to a microorganism of the bacterial species Microbacterium sp. with access number CECT7624. Also the present invention relates to the use of said microorganism or a population thereof to increase the tolerance to water stress of a plant or to increase the growth of a plant under optimum conditions of water content of said plant. Furthermore, the present invention relates to the use of said microorganism or a population thereof for the production of an xeroprotective composition, wherein said composition comprises trehalose, oxoglucuronic acid, lactate, glutamate, glutamine and pyruvate, or further comprises fucose. The present invention also relates to the method for increasing the water stress tolerance of a plant or for producing the xeroproctector composition of the present invention.

The ability to increase the tolerance to water stress of a plant is of importance in the agricultural sector to obtain fruits in conditions of low water content in soils or other conditions that limit the water absorption capacity of said plants. In the present invention, tools are provided to prevent or combat the negative effects suffered by plants subjected to water stress conditions. In addition, both the content and the proportion of various xeroprotective compositions obtained from cultures of strain CECT7624 are offered in the present invention.

Therefore, one aspect of the present invention relates to a microorganism of the bacterial species Microbacteríum sp. with access number CECT7624. Said microorganism is tolerant to desiccation. This strain has been deposited in the Spanish collection of type crops (CECT) on November 10, 2009 and was assigned the deposit number CECT7624. The address of said International Deposit Authority is: University of Valencia / Research building / Campus of Burjassot / 46100 Burjassot (Valencia).

The scientific classification of strain CECT7624 of the present invention is: Kingdom: Bacteria I Phylum: Actinobacteria I Order: Actinomycetales I Family: Micrococcineae I Genus: Microbacteríum.

The characteristics of said strain are:

- The substrates that the CECT7624 bacteria oxidize or ferment are: Dextrin, glycogen, manan, tween 40, tween 80, L-arabinose, D-arbulin, D-cellobiose, D-fructose, L-fucose, D-galactose, D acid -galacturonic, gentiobious, D-gluconic acid, α-D-glucose, maltose, maltotriose, D-mannitol, D-mannose, D-melezitose, 3-methyl glucose, palatinous, D-psychosa, salicin, sedoheptulose, D-sorbitol , sucrose, D-trehalose, turanosa, xylose, acetic acid, α-hydroxybutyric acid, -ketoglutaric acid, L-lactic acid, methyl pyruvate, succinamic acid, L-alaninamide, L-alanine, L-alanyl-glycine, L-aspargina , L-glutamic acid, glycyl-L-glutamic acid, L-serine, putrescine, glycerol, adenosine, ^2'- deoxyadenosine, inopin, Thymidine, adenosine-5 ^' -monophosphate, thymidine-5 ^' -monophosphate, DL-glycerolphosphate.

- The maximum temperature tolerated for the growth of this strain was between 35 ° C and 40 ° C. The minimum temperature to detect growth was between 15 ° C and 20 ° C, while its optimum growth temperature was around 35 ° C. It was unable to grow at pH equal to or greater than 13 although if it proliferated at pH 12. - The minimum pH tolerated for the growth of this strain was between pH 5 and pH 7, considering around 12 as the optimum pH for the growth of this strain. strain

- He was also able to proliferate in LB medium in the absence of NaCI although he was unable to grow in this medium with a concentration of

NaCI equal to or greater than 1.2 M, with the maximum concentration tolerated between 0.8 M and 1.2 M NaCI. This strain showed optimal growth at a concentration of 0.2 M NaCl. - Antibiotic sensitivity tests showed growth inhibition halos in the five antibiotics tested on disk: rifampin 3 ₀ (3.37 cm); Streptomycin ₂ 5 (2.07 cm); tetracycline ₂ or (1, 07 cm); Chloramphenicol ₅₀ (4.28 cm); Kanamycin ₃₀ (1, 53 cm). Likewise, the present invention also relates to a microorganism derived from the microorganism deposited with accession number CECT7624. The derived microorganism can be produced intentionally, by mutagenic methods known in the state of the art such as, for example, the growth of said original microorganism on exposure with known agents capable of forcing mutagenesis. Another aspect of the present invention relates to a bacterial population comprising the microorganism deposited with access number CECT7624. The bacterial population can be formed by other strains of microorganisms of any species. The bacterial population is a set of microorganism cells where at least one cell of said microorganism is deposited with access number CECT7624, at any stage of the development state and at any stage of growth, seasonal or stationary, regardless of the morphology that present, in the form of coconut, bacillus or intermediate morphologies of the above.

Hereinafter, the microorganism or the bacterial population may be referred to as the "microorganism of the present invention" or the "microorganism of the invention".

Another aspect of the present invention relates to the use of the microorganism of the present invention to increase the tolerance to water stress of a plant, with respect to a control. In the present invention, the control refers to a plant of the same species, subspecies or variety as the previous plant, which has not been exposed to said microorganism or to a plant that has been exposed to the rhizobacterium P. putida KT2440. The term "water stress" as understood in the present invention refers to the reduction of water content in plant tissues that causes alterations in metabolic processes, causing negative effects on plant growth and development. The magnitude of these alterations in the metabolic processes involved depends on the species, moment of the development cycle of the affected plant, the intensity and duration of the situation that causes such stress, among others factors. The techniques for estimating water stress in a plant are known by the person skilled in the art, for example, but not limited to, measure of leaf expansion, measure of stomatic closure, measure of leaf death.

Water stress can be caused by various causes, including saline stress, since it is known that the concentration of soluble salts in the soil raises the osmotic pressure of the solution of said soil. Since water tends to pass from less concentrated to more concentrated solutions, equalizing the osmotic pressures of both, when the saline concentration of the soil solution is higher than that of the plant cells, the water will tend to leave the latter towards soil solution. Therefore, in saline environments, although there is sufficient available water, the plant suffers water stress.

The characteristics of a plant that can increase its tolerance to water stress can be, among others, but if limited, the maintenance of tissue turgidity by increasing the water retention index or, maintaining or increasing the absorption of Water.

Another preferred embodiment of the present invention relates to the use of the microorganism of the invention to increase the amount of water absorbed (recovered) by a plant, with respect to a control, under conditions of water stress. In the present invention the amount of water recovered by a plant is measured by means of (see in embodiments of the invention):

- The measure of the weight of the totally turgid plant. - The measure of water recovery potential or water recovery index, which consists in calculating the difference between the water recovered by the plant and its fresh weight. - The measure of the relative water content. This data indicates the relationship between the water present in the plants at the time of extraction with respect to the total water. In this way, the maximum value of 1 would be given in the theoretical situation in which, due to its good water state, the plant could not or would not need to recover water in its totally turgid state with respect to the fresh weight.

Another preferred embodiment of the present invention relates to the use of the microorganism of the invention to increase the water retention rate of a plant, with respect to a control, under conditions of water stress. The water retention rate in a plant is the difference between the fresh weight and the dry weight of that plant.

A preferred embodiment of the present invention relates to the use of the microorganism of the invention to increase the biomass of a plant, with respect to a control, under conditions of water stress. In the present invention, the increase in biomass is determined primarily by calculating the dry weight of the plant, as well as by measuring the length of the stem and roots under conditions of water stress. The amount of water recovered by a plant, the water retention index or the increase of the biomass of said plant can be determined by means of the measurement of other parameters different from those specified above and in the section of examples of embodiment . Another aspect of the present invention relates to the use of the microorganism of the present invention to increase the growth of a plant with respect to a control. This aspect of the invention relates to conditions in which the plant does not experience water stress, that is, in optimal water conditions for the development and growth of said plant.

A preferred embodiment of the present invention relates to the use of the microorganism of the invention to increase the growth of a plant with respect to a control, where the increase in growth refers to the increase in the length of the stem or the increase in the length of the plant. root of said plant.

The term "plant" encompasses each part of it, which can be preserved or cultivated in isolation or in combination, as well as the germplasm. The germplasm is defined by that biological material that contains the intraspecific genetic variability or to the genetic materials that can perpetuate a species or a population of an organism. Thus, germplasm is the seed, tissue of any part of the plant or plants established in ex situ collections, without excluding any other material that falls within this definition.

The plant of the present invention can be, for example, but not limited to, any plant intended for human or animal consumption, plants whose fruit is of interest for its fresh or industrial processed consumption, plants that are used in the generation of beneficial products for human health, such as drugs (biofarms), plants that are used for the generation of biomass to manufacture biodiesel, plants used in the bioremediation of different types of substrates (phytoremediation). In this sense, the plant can be selected from the list that includes, but is not limited to, the genus Solanum, Cucumis, Citrullus, Cucúrbita, Capsicum, Brasica, Nicotiana, Citrullus or Lactuca.

Another preferred embodiment relates to the use of the microorganism of the invention to increase the tolerance to water stress of a plant with respect to a control, or to the use of the microorganism of the invention to increase the growth of a plant with respect to a control, where the plant It is of the genus Capsicum. The plant species of the genus Capsicum is selected from the list comprising, but not limited to, Capsicum angulosum, Capsicum annuum, Capsicum pendulum, Capsicum minimum, Capsicum baccatum, Capsicum abbreviatum, Capsicum anomalum, Capsicum breviflorum, Capsicum buforum, Capsicum brasilianum, Capsicum brasilianum, Capsicum brasilianum campylopodium, Capsicum cardenasii, Capsicum chacoense, Capsicum chínense, Capsicum chlorocladium, Capsicum ciliatum, Capsicum coccineum, Capsicum cordiforme, Capsicum cornutum, Capsicum dimorphum, Capsicum dusenii, Capsicum exile, Capsicum eximicumum Capsicumum capsicumum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum capsicum field galapagoensis capsicum, capsicum geminifolum, hookerianum Capsicum, Capsicum lanceolatum, leptopodum Capsicum, Capsicum luteum, microcarpum capsicum, capsicum minutiflorum, Capsicum mirabile, parvifolium capsicum, capsicum praetermissum, Capsicum pubescens, schottianum capsicum, capsicum scolnikianum, stramonifolium capsicum, capsicum tetragonum, Capsi cum tovarii, Capsicum villosum or Capsicum violaceum. According to a more preferred embodiment, the plant is of the species Capsicum annuum. Another aspect of the present invention relates to a method for increasing the tolerance to water stress of a plant with respect to a control, which comprises inoculating the microorganism of the invention to a plant cell, any part of a plant or to a seed. This method increases the growth of a plant that is not in conditions of water stress, with respect to control.

The term "inocular" as used in the present invention refers to introducing the microorganism of the invention into a plant by any technique known in the state of the art, such as, but not limited to, through a solution in hydroponics, through a solution applied to the soil, through the application of the microorganism of the invention by spraying any aerial part of the plant, by germinating the seeds of the plant in the presence of said microorganism, by cultivating plant material in vitro in contact with the microorganism of the invention or by microinjection of the microorganism of the invention in at least one plant cell or protoplast. A preferred embodiment of the present invention relates to the method described in the preceding paragraphs, which comprises contacting the microorganism of the invention with at least one root of said plant. A more preferred embodiment refers to the method where said microorganism or bacterial population is contacted with at least one root by means of an aqueous solution.

Another preferred embodiment refers to any method described in previous paragraphs, to increase the growth of a plant with respect to a control, where the plant is of the genus Capsicum. According to a more preferred embodiment, the plant is of the species Capsicum annuum. Another aspect of the present invention relates to the use of the microorganism of the invention for the production of an xeroprotective composition.

A further aspect of the present invention relates to the xeroprotective composition produced by the microorganism of the invention.

To refer to the xeroprotective composition of the present invention, the term Bacterial Milking Product (POB) can be used. The term "xeroprotective composition" as understood in the present invention refers to a composition that prevents the adverse effects of water stress, that decreases the effects of said water stress on a plant or that promotes the growth of a plant.

A preferred embodiment of the present invention relates to the xeroprotective composition produced by the microorganism of the invention, or to a synthetic xeroprotective composition, comprising trehalose, oxoglucuronic acid, lactate, glutamate, glutamine, fucose and pyruvate. A more preferred embodiment of the present invention relates to the xeroprotective composition produced by the microorganism of the invention, or to a synthetic xeroprotective composition, comprising a ratio of between 0.5 and 1.5 trehalose: 0.15 and 0, 45 of oxoglucuronic acid: 0.7 and 1, 7 of lactate: 0.1 and 0.2 of glutamate: 0.15 and 0.45 of glutamine: 1, 2 and 3.4 of fucose: 0.15 and 0 , 3 pyruvate. That is, a ratio (trehalose) :(oxyglucuronic acid) :( lactate) :( glutamate) :( glutamin a) :( fucose) :( pyruvate), of (0.5 to 1, 5): (0.15 to 0.45): (0.7 to 1, 7): (0.1 to 0.2): (0.15 to 0.45): (1, 2 to 3.4): (0.15 to 0.3), respectively. An even more preferred embodiment refers to the xeroprotective composition where the ratio of (trehalose) :( oxoglucuronic acid) :( lactate) :( glutamate) :( glutamine) :( fucose) :( pyruvate), is (0.7 to 1, 3): (0.25 to 0.35): (0.9 to 1, 6): (0.12 to 0.18): (0.2 to 0.4): (1, 8 to 2.8): (0.2 to 0.25). Preferably the xeroprotective composition has a ratio of (trehalose) :( oxoglucuronic acid) :( lactate) :( glutamate) :( glutamine) :( fucose): (pyruvate) of (1) :( 0.31) :( 1, 18) :( 0.14) :( 0.28) :( 2.26) :( 0.23), respectively.

Another preferred embodiment of the present invention relates to the xeroprotective composition produced by the microorganism of the invention, or to a synthetic xeroprotective composition, comprising trehalose, oxoglucuronic acid, lactate, glutamate, glutamine and pyruvate.

A more preferred embodiment of the present invention relates to the xeroprotective composition produced by the microorganism of the invention, or to a synthetic xeroprotective composition, comprising a ratio of between 0.5 and 1.5 trehalose: 0.03 and 0, 09 of oxoglucuronic acid: 0.3 and 0.9 of lactate: 0.05 and 0.15 of glutamate: 0.05 and 0.15 of glutamine: 0.1 and 0.3 of pyruvate. That is, a ratio (trehalose) :( acid

oxoglucuronic) :( lactate) :( glutamate) :( glutamine) :( pyruvate), of (0.5 to 1.5): (0.03 to 0.09): (0.3 to 0.9): (0.05 to 0.15): (0.05 to 0.15): (0.1 to 0.3), respectively. An even more preferred embodiment refers to the xeroprotective composition where the ratio of (trehalose) :( oxoglucuronic acid) :( lactate) :( glutamate) :( glutamine) :( fucose) :( pyruvate), is (0.7 at 1, 3): (0.05 to 0.07): (0.5 to 0.7): (0.07 to 0.12): (0.07 to 0.12): (0.15 to 0.25). Preferably the xeroprotective composition has a ratio of (trehalose) :( oxoglucuronic acid) :( lactate) :( glutamate) :( glutamine) :( pyruvate) of (1) :( 0.06) :( 0.62) :( 0.1) :( 0.1) :( 0.21), respectively. The term "proportion" as understood in the present invention refers to the due correspondence of the elements of the composition related to each other. That is, it refers to a mathematical relationship that links the elements of the composition. As an example, the xeroprotective composition that has a ratio of (trehalose) :( oxoglucuronic acid) :( lactate) :( glutamate) :( glutamine) :( pyruvate) of (1) :( 0.06) :( 0.62) :( 0.1) :( 0.1) :( 0.21), respectively, may have, for example, concentrations of (2) :( 0.12) :( 1, 24) :( 0 , 2) :( 0.2) :( 0.42) mg of each element respectively / ml.

Hereinafter reference may be made to any composition described in the preceding paragraphs as "composition of the present invention" or "composition of the invention".

Another aspect of the present invention relates to the use of the composition of the invention for the conservation of biological material with a residual moisture content equal to or less than 10%. The residual moisture content of the biological material may be equal to or less than 9, 8, 7, 6 5, 4, 3, 2 or 1% residual moisture. The preservation of said material can be carried out by stabilizing it. In the present invention, the term "dry material biological material" can be used to refer to this type of biological material. Under these conditions, the preservative or stabilizer coalesces to reach a non-crystalline, vitreous, and solid state (for example an amorphous crystal). The organic crystal particles that are formed by drying the biological material with the stabilizer are covered by the stabilizer that produces high stability by drastically reducing chemical reactions. In this way the dried biological material is embedded in the amorphous crystal formed by the stabilizer.

The dry biological material in the presence of the stabilizer that forms the amorphous crystal is resistant to plastics in a liquid state, while the material that is not dry in the presence of these stabilizers is not resistant to plastics in a liquid state.

The dry biological material in these forms can be in a non-particulate state and can be supplied in forms, for example, but not limited to, moldings or solids in 3 dimensions such as, but not limited to, blocks, pads, patches, sheets, balls, or seeds of dry biological material. The term "residual moisture" as used in the present invention refers to the amount of moisture a product contains after it has passed through some type of process capable of removing water from it. Residual humidity is the mass percentage of the product that corresponds to water with respect to the total mass. In other words, a residual moisture value of a product equal to 10% means that 10 g of every 100 g of the product correspond to water. Residual humidity can be measured by methods known in the state of the art such as, but not limited to, by the titrimetric method, the azeotropic method or the gravimetric method.

The term "conservation of biological material" refers to the maintenance or care of the permanence of the intrinsic characteristics of the biological material. A preferred embodiment relates to the use of the composition of the invention for the conservation of biological material in the dry state, where the biological material is an invertebrate organism, a seed, a seedling, a microorganism, an isolated organ, an isolated biological tissue or a cell. The cell can be prokaryotic or eukaryotic. The cell can be a cell of a microorganism in any state of development. The cell can be somatic or germinal, plant or animal. That cell It can come from any organism or microorganism and can occur in any state of differentiation, such as, but not limited to, from a culture of a cellular tissue or organs, sperm, ovules or embryos. The cell can be a totipotent, multipotent or unipotent stem cell. The microorganism can be unicellular or multicellular. The unicellular microorganism is selected from the list comprising, but not limited to, E. coli, S. typhimurium, P. putida., Salmonella spp, Rhizobium spp, Pseudomonas spp, Rhodococcus spp, Lactobacillus spp. or Bifidobacterium spp. The multicellular microorganism can be, for example, but not limited to, a nematode.

The cell conserved by the composition of the present invention is a viable cell that is, it is capable of performing the normal functions of the cell including cell replication and division. On the other hand, the cell may have been treated, manipulated or mutated before preservation. For example, but not limited to, a cell may have become competent for transformations or transfections, or it may contain recombinant nucleic acids. The cells that are conserved can form a homogeneous or heterogeneous population, for example, but not limited to, a library of cells in which each cell contains a variation of some nucleic acid. Preferably, the cells are non-anhydrobotic cells (desiccation-sensitive cells) such as, but not limited to, cells from non-anhydrobial prokaryotic microorganisms that are generally not sporulant.

The conservation of the microorganism can be improved by culture under conditions that increase the intracellular concentration of trehalose or other amorphous crystal-forming stabilizers. For example, but not limited, under conditions of high osmolarity (high concentration of salts) that stimulate the intracellular production of trehalose or other amorphous crystal-forming stabilizers. The invertebrate organism is, but is not limited to, an insect larva or a crustacean. Said invertebrate organisms can be preserved under drying conditions, allowing the vital activity thereof, so that, when rehydrated, said organisms have the ability to move. The seedling is a plant in its early stages of development, from germination until it develops

The composition of the invention can be used for the conservation of biological material in the dry state, where the biological material is a vertebrate organism belonging to the Tetrapoda Superclass (with four limbs),

Amphibia class (amphibians) or Reptilia class (reptiles), or any of its parts (Vernon and Jackson, 1931. The biological bulletin, 60: 80-93). Vernon and Jackson carried out a study on the Leopard frog (Rana pipiens), in which their skin, tongue, spleen, and liver naturally dries with a water loss of between 43-81% of the water content total.

An isolated organ, or an isolated biological tissue (including blood) can be preserved by the composition of the present invention. In Serrato et al. (2009) results of cryopreservation protocols of biological tissues can be observed (Serrato et al., 2009. Histology and histopathology, 24: 1531-1540). A more preferred embodiment relates to the use of the composition of the invention, where the biological material is a molecule with biological activity. The term "molecule with biological activity" as understood in the present invention refers to a biological molecule whose origin is a living organism or that has been alive, or derivatives or analogs of said molecule. The term "derivatives" refers to molecules obtained by the modification of a molecule with biological activity, which They have similar functionality. On the other hand, the term "analogs" refers to molecules that have a function similar to the molecule with biological activity. According to another even more preferred embodiment of the composition of the present invention the molecule with biological activity is an enzyme. An even more preferred embodiment of the present invention relates to the use of the composition of the invention, where the enzyme is an enzyme with lipase activity. The enzyme with lipase activity is selected from the list of enzymes with EC numbers (Enzyme Commission numbers) comprising carboxylic ester hydrolases (EC 3.1 .1) EC 3.1.1 .1 (Carboxylesterase), EC 3.1 .1.2 (Arilesterase) , EC 3.1 .1 .3 (Triacylglycerol lipase), EC 3.1 .1 .4 (Phospholipase A (2)), EC 3.1.1.5 (Lysophospholipase), EC 3.1 .1 .23 (Acylglycerol lipase), EC 3.1 .1. 24 (3-oxoadipate enol-lactonase), EC 3.1 .1 .25 (1, 4-lactonase), EC 3.1 .1 .26 (Galactolipase), EC 3.1 .1 .32 (Phospholipase A (1)), EC 3.1 .1 .33 (6-acetylglucose deacetylase), EC 3.1 .1 .34 (Lipoprotein lipase). Preferably the enzyme lipase has Triacylglycerol lipase activity (EC 3.1 .1 .3). Another aspect of the present invention relates to a method of obtaining the xeroprotective composition of the invention comprising: a) culturing the microorganism of the invention in a culture medium with glucose as a carbon source,

b) dehydrate the microorganisms obtained in the culture of step (a) until they have a residual humidity equal to or less than 10%,

c) rehydrate the dehydrated microorganisms of step (b) in a hypotonic medium and

d) select the liquid fraction of the product obtained in step (c) comprising the xeroprotective composition. The culture medium is any culture medium known in the state of the art for the growth of a microorganism of the present invention, for example but not limited to, the M9 mineral medium. Rich media such as Luria Bertani (LB) medium in which xeroprotectors, or natural osmolytes are not present, because the bacteria would prefer to take them from outside to synthesize them.

A preferred embodiment of the present invention relates to the method of obtaining the xeroprotective composition, where the culture medium of step (a) is solid. The term "solid" as understood in the present invention refers to a gelled culture medium to a greater or lesser extent, that is, comprising agar to facilitate its gelation or any gelling compound. Dehydration of the microorganisms in step (b) is carried out by means of any technique known in the state of the art. Another preferred embodiment of the present invention relates to the method, where the dehydration of the microorganisms according to step (b) is carried out by means of a hypertonic solution or by means of an air current. Preferably the hypertonic solution or the air stream have sterility conditions. The hypertonic solution is a solution that has a higher concentration of solute in the external environment than in the cytoplasm of the cells of the microorganism of the present invention, therefore, the cell releases water, that is, it is dehydrated.

The rehydration of the microorganisms, described in step (c), is carried out in a hypotonic medium. The hypotonic medium or hypotonic solution is a solution that has a lower concentration of solute in the external medium than in the cytoplasm of the cell of the microorganism of the present invention, therefore, the cell recovers water, that is, it is rehydrated. A further preferred embodiment refers to the method, where the hypotonic medium for the rehydration of the microorganisms according to step (c) it is water, partially or totally distilled, partially or totally deionized or partially or totally demineralized. The cells and the hypotonic medium must be in contact for at least 5 minutes. Preferably they will be in contact for at least 20 minutes with stirring. Obtaining the medium containing the stabilizing substances will be carried out by any method that guarantees the separation of cells from the liquid content, preferably by gentle centrifugation, followed by a filtration step. Preferably 0.4 micrometer pore diameter filters will be used.

Another preferred embodiment relates to the method, where, in addition, the liquid fraction of step (d) is dehydrated until the xeroprotector product has a residual humidity equal to or less than 10%.

The xeroprotector product of the invention can be separated from the culture medium by any method of concentration. Preferably, lyophilizer-type dryers that produce the stabilizer in the dry state will be used. The stabilizing molecules may be dissolved or dispersed in a proportion of between 10 and 30%. This solution or dispersion is added to the biological material that is to be preserved and will be dried. Another preferred embodiment of the present invention relates to the method of obtaining the xeroprotective composition, where the dehydration of the microorganisms according to step (b) is carried out by means of a hypertonic solution or by means of an air current. Another preferred embodiment of the present invention relates to the method of obtaining the xeroprotective composition, where the hypotonic medium for the rehydration of the microorganisms according to step (c) it is water partially or totally distilled, deionized or demineralized.

Another preferred embodiment of the present invention relates to the method of obtaining the xeroprotective composition, where in addition, the liquid fraction of step (d) is dehydrated until the xeroprotector product has a residual humidity equal to or less than 10%.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES With the intention of complementing the description that has been carried out, as well as helping to better understand the characteristics of the invention, according to some examples made, they are shown here, for illustrative and non-limiting purposes. , the following figures: FIG. 1. Shows the viability of the different bacterial isolates selected by exposure to chloroform after 24 hours of air drying.

Error bars show the standard deviation of at least three replicas. The microorganism strains represented in this figure are:

1 J3A, 1 J14, 2J2, 2J8A, 2J12B, 2J15B, 2J16A, 2J30, 3J18, 6J30, Acitenobacter calcoaceticus, Pseudomonas putida, in addition to the Microbacterium sp. 3J1.

FIG. 2. Shows the lipase activity (in percentage relative to 100% positive control activity) after drying of the lipase enzyme in the presence of the different components of 10% bacterial milking products (w / v).

The positive control is trehalose at 10%. The negative control (-) corresponds to the absence of any compound as an additive prior to the drying of the enzyme. 3J1 is the value of lipase activity recorded after drying stabilization and subsequent reconstitution of the enzyme in the presence of POB extracted from strain 3J1 by hyper / hypoosmotic shock respectively. 3J1 D is the lipase activity recorded after drying stabilization and subsequent reconstitution of the enzyme in the presence of POBSIA (Bacterial Milking Product extracted by Drying by Air Incubation) extracted from strain 3J1 by drying and subsequent hydration treatment.

FIG. 3. Shows the state of the plants inoculated with Microbacterium sp. 3J1, P. putida KT2440 and non-inoculated plants after 33 days without irrigation.

Two plants of each type of sample are shown at the last sampling point that include non-inoculated plants (Water); inoculated with P. putida and inoculated with 3J1. FIG. 4. Shows the Fresh Weight of the plants inoculated with each of the strains at the different times of the test after the cessation of irrigation. In ordinates the weight in mg of the plants is shown. In abscissa the inoculums used are indicated. The colors of the bars refer to the sample time, with the white corresponding to day 7 of sampling, light gray, at day 14, dark gray at 20 and black at day 33. FIG. 5. Shows the Dry Weight of the plants inoculated with each of the strains at the different times of the test after the cessation of irrigation.

In ordinates the weight in mg of the plants is shown. In abscissa the inoculums used are indicated. The colors of the bars refer to the sample time, with the white corresponding to day 7 of sampling, light gray, at day 14, dark gray at 20 and black at day 33.

FIG. 6. Shows the Totally Turbid Weight of the plants inoculated with each of the strains at the different times of the test after the cessation of irrigation.

FIG. 7. Shows the Relative Content of Water in plants inoculated with the different strains at the different times of the test after the cessation of irrigation. In ordinates show the dimensionless values of the CRA. In abscissa the sampling times are indicated.

FIG. 8. Shows the water retention potential of the plants inoculated with each of the strains at different test times after the cessation of irrigation.

In ordinates the water structurally retained by the plants in mg is shown. In abscissa the inoculums used are indicated. The colors of the bars refer to the sample time, with the white corresponding to day 7 of sampling, light gray, at day 14, dark gray at 20 and black at day 33.

FIG. 9. Shows the stem height of the plants inoculated with each of the strains at the different times of the test after the cessation of irrigation.

In ordinates the height in cm of the plant is shown. In abscissa the inoculums used are indicated. The grayscale of the bars refers to the sample time, with the target corresponding to day 7 of sampling, light gray, day 14 being dark gray at 20 and black at day 33.

FIG. 10. Shows the root length of the plants inoculated with each of the strains at the different times of the test after the cessation of irrigation.

In ordinates the height in cm of the plant is shown. In abscissa the inoculums used are indicated. The grayscale of the bars refers to the sample time, with the target corresponding to day 7 of sampling, light gray, day 14 being dark gray at 20 and black at day 33. FIG. 11. Shows the stem height of the plants inoculated with each of the strains at the different times of the test in humid conditions. In ordinates the height in cm of the plant is shown. In abscissa the inoculums used are indicated. The colors of the bars refer to the sample time, with the white corresponding to day 7 of sampling, light gray, at day 14, dark gray at 20 and black at day 33. FIG. 12. Shows the root length of the plants inoculated with each of the strains at the different test times in humid conditions.

In sample ordinates the length in cm of the roots. In abscissa the inoculums used are indicated. The colors of the bars refer to the sample time, with the white corresponding to day 7 of sampling, light gray, at day 14, dark gray at 20 and black at day 33.

FIG. 13. Shows the Fresh Weight of the plants inoculated with each of the strains at the different times of the test in humid conditions.

FIG. 14. Shows the Dry Weight of the plants inoculated with each of the strains at the different times of the test and grown in humid conditions. In ordinates the weight in mg of the plants is shown. In abscissa the inoculums used are indicated. The colors of the bars refer to the sample time, with the white corresponding to day 7 of sampling, light gray, at day 14, dark gray at 20 and black at day 33.

FIG. 15. Shows the Totally Turbid Weight of the plants inoculated with each one of the strains at the different times of the test and cultivated in humid conditions. In ordinates the weight in mg of the plants is shown. In abscissa the inoculums used are indicated. The colors of the bars refer to the sample time, with the white corresponding to day 7 of sampling, light gray, at day 14, dark gray at 20 and black at day 33. FIG. 16. Shows the water retention potential of the plants inoculated with each of the strains at the different times of the test and grown in humid conditions.

EXAMPLES

The invention will now be illustrated by illustrative and non-limiting tests carried out by the inventors who describe the isolation of the strain of the present invention as well as its uses. EXAMPLE 1. Isolation of the microorganism belonging to strain 3J1, extraction of POB (Bacterial Milking Products).

1.2. Isolation of strain 3J1 (CECT7624).

1 g of dry soil was homogenized, from Granada (37,182 Latitude N and 3,624 Longitude O), not exposed to rain or irrigation, for a period exceeding three months. The soil was taken from the area surrounding oleander roots (Nerium oleander). The samples were homogenized to obtain a fine earth grain that would guarantee their contact with the chloroform. The earth was deposited in glass vials to which 3 ml of pure chloroform was added. After the addition of the chloroform, they were incubated at room temperature for 30 minutes with sporadic agitation to maximize contact of the sample with the solvent. To remove the chloroform from the samples after the contact time had elapsed, the soil samples were deposited in a sterile glass petri dish without a lid until the evaporation of the chloroform was completed. Once the soil samples were dried, they were resuspended in 10 ml of TSB. Serial dilutions were made with the soil suspensions and seeded in TSA plates that were incubated 48 hours at 30 ° C. After this time, the number of colony forming units (CFU) per milliliter of each sample was quantified. Thus, 2-10 ⁵ CFU / g of soil was detected in the untreated samples, while these were reduced to 1, 3-10 ⁵ CFU / g of soil after 30 minutes of treatment.

36 strains were randomly selected and to identify the strains that produced spores, a test was conducted based on the different sensitivity of the vegetative cells with respect to the spores to heat treatment and based on the method described by Vilchez et al. (Vilchez et al. , 2008. Extremophiles, 12: 297-299). In this way independent colonies from TSA plates were taken of at least 24 hours of growth. These colonies were resuspended in 1 ml of M9 solution in sterile microtubes of 1.5 ml. Then 10 μΙ of this suspension was seeded in TSA. The rest of the suspension was then incubated in Mixing Block MB-102 thermoblock at 72 ° C for 30 minutes. Again 10 μΙ of each sample was taken and seeded on TSA plate. Those strains capable of tolerating heat treatment were considered sporulants, while those that did not tolerate heat treatment were considered non-sporulants. As positive and negative controls, colonies of Bacillus pumilus and Burkholderia cepacia were used respectively. The proportion of sporulant strains went from levels below 40% for untreated samples to levels above 50% after 30 min of exposure of soil samples to chloroform. In order to analyze the ability to tolerate desiccation of isolated non-sporulant strains, an air drying study was conducted. In this trial, a strain of Acinetobacter calcoaceticus (PADD68) isolated from the Tabernas desert (Almería) identified as tolerant to desiccation was included in a previous study and was used as a positive control. In addition, Pseudomonas putida KT2440 cells were also included in the study, which were used as negative controls as a strain sensitive to desiccation (Antheunisse et al., 1981. Antonie Leeuwnhoek, 47: 539-545; Manzanera et al., 2002. Appl. Environ. Microbiol., 68: 4328-4333). Independent colonies of the 17 strains identified as non-sporulants isolated in the previous study and from 72-hour TSA plates were used for this test to calculate their level of drying tolerance as indicated below. To calculate the tolerance to desiccation, we started from isolated colonies from TSA plates with 48 hours of growth. Using a Sterile handle was taken a single colony that was resuspended in 1 ml of sterile M9 solution. Starting from this cell suspension, serial dilutions were made that were seeded on TSA plates in order to identify the starting CFU / ml number. On the other hand, 100 μΙ of each suspension was taken and deposited in 5-10 μΙ drops on sterile petri dishes without medium. The microplates were placed under a stream of sterile air in a laminar flow hood for 24 hours. The plates remained dry after 2-3 hours of incubation. After 24 hours of incubation, they were resuspended in 1 ml of sterile M9 solution. Starting from this solution, serial dilutions were made and seeded on TSA plates. From the CFU / ml count of this second sowing, survival rates were calculated in reference to the first count. These tests were performed in triplicate. The results were expressed as the average of the three trials in percentage, using the wet data as a reference of 100%. The standard deviation to the mean allowed the calculation of the student's T to study if the differences in survival were significant. 17 strains were isolated under these conditions with tolerance levels significantly higher than those of the positive control Acinetobacter calcoaceticus (PADD68). One of the isolated strains was named as 3J1 and belongs to the genus Microbacterium sp. This strain was characterized by sequencing the 16S rDNA and comparing the sequence with those present in the databases, as well as by BIOLOG metabolic studies and DNA-DNA hybridization with the nearest species. FIG. 1 shows the tolerance values of the isolated strains.

1.2. Extraction of products from bacterial milking.

For the extraction of the products of the bacterial milking and in order to identify the accumulated molecules with the capacity to protect biomolecules sensitive to desiccation, a strategy was used Based on three steps. In a first step an extraction of the accumulated molecules was carried out by means of a variation of the technique known as "bacterial milking" generated by the inventors. In a second step an xeroprotection test was carried out with the substances obtained to identify their capacity to protect enzymes against desiccation.

Given that the production and accumulation of xeroprotective substances was carried out in a medium with minerals, we proceeded to identify the most appropriate carbon sources for the cultivation of the strains in M9 mineral medium. To obtain substances with xeroprotective capacity, the cells of said strain were grown in mineral medium with the appropriate carbon source (glucose) until obtaining a sufficient biomass. After this accumulation, the cells were deposited on plates of the same medium with agar for the production of solid medium. A sterile 0.4 micron filter was deposited between the cells and the medium to allow the passage of nutrients to the cells and their subsequent collection and handling. The plates with filters and cells were subjected to sterile air drying for 24 hours. Halomononas elongata was included as a positive control, since it is a strain recognized as a halotolerant (Sauer and Galinski, 1998. Biotechnol. Bioeng., 57: 306-313) and P. putida KT2440 as a halosensitive strain (De Castro et al., 2000 Appl. Environ. Microbiol., 66: 4142-4144). To this end, the selected strain (3J1), hypertolerant at drying, was inoculated. In addition, H. elongata was included as a positive control since it had already been used by Sauer and Galinski to obtain hydroxyectoin, and as a negative control, E. coli was included as an example of a halo and xerosensitive strain. Said inoculums were incubated with shaking for 48 hours. The samples were then centrifuged for 10 minutes at 10,000 rpm in a Beckman Avanti-J25 centrifuge and removed the supernatant fraction, resuspending the bacterial sediment in 20 ml of sterile M9 solution and deposited on filters. After 24 hours, the filters, subjected to desiccation by a stream of sterile air, were separated from the medium and deposited in tubes with sterile distilled water allowing to incubate 20 minutes at 30 ° C and 150 rpm. Consecutively the same centrifugation process was repeated, the bacterial precipitate was discarded and the supernatant was filtered (using a 0.22 pm filter). The filtrate of each sample was divided into two fractions, one of which was used to determine the xeroprotective activity of the supernatant (milking fraction) and the other for the identification and characterization of the compounds present in this fraction. Both fractions were subjected to a drying process using a lyophilizer (Labconco Freezone 6) for 48 hours, obtaining a sediment that was resuspended in 100 μΙ of sterile milliQ water. Once lyophilized, the Bacterial Milking Product (POB), it was solubilized in 100 microliters of water. These POB solutions were used in xeroprotection studies.

Table 1 shows the compositions of the bacterial milking products of strain 3J1 after extraction by hyper / hypoosmotic shock (3J1), or after extraction by drying by air incubation (3J1 D).

Table 1. Composition of the bacterial milking product (POB) of strain 4J27.

3J1 3J1D

Trehalosa 1 Trehalosa 1

Ac. oxoglucuronic 0.31 Ac. oxoglucuronic 0.06

Lactate 1, 18 Lactate 0.62

Glutamate 0.14 Glutamate 0.1

Glutamine 0.28 Glutamine 0.1 Fucose 2.26 - -

Pyruvate 0.23 Pyruvate 0.21

3J1 is the composition of the POB of said strain after its extraction by hyper / hypoosmotic shock. 3J1 D is the composition of the POB of said strain after extraction by drying by incubation in air.

EXAMPLE 2. Enzyme xeroprotection assay. The objective of this test was to determine the ability of the bacterial milking product fraction to protect enzymes from drying out. For this, the enzyme lipase was used. Starting from 1 μΙ containing 0.00554 units of Burkholderia cepacia lipase (Sigma-Aldrich 62309-100 mg), 15 μΙ of the product fraction of the bacterial milking to study were added. As a positive control, 15 μΙ of a 10% solution of trehalose was added to 1 μΙ (0.00554 U) of lipase solution and as a negative control 15 μΙ of water was added to 1 μΙ (0.00554 U) of the solution of lipase The 16 μΙ mixtures with lipase were deposited in a 2 ml capacity microtube and dried at 50 ° C for 120 minutes. Once dried, they were incubated at 100 ° C for 5 minutes. They were finally stored in a desiccator at room temperature for 24 hours. After the incubation time, the reactions were resuspended in 50 μΙ of a TrisHCI solution (50 mM) and transferred to a microtube together with 950 μΙ of Tris HCI (50 mM) pH8 and 1 ml of Substrate Solution. The xeroprotective capacity determination of each bacterial milking product fraction (POB) was determined by the lipase activity measurement test. For the measurement of lipase activity, a variation of the method described by Gupta et al. (2002) consisting of the spectrophotometric quantification of p-nitrophenol released by the enzyme lipase of Burkholderia cepacia (Sigma-Aldrich 62309-100 mg) from the p-nitrophenol palmitate (pNPP) substrate (Gupta et al., 2002. Analytical Biochemistry, 31 1: 98-99). For this, 1 ml of cell-free medium (985 μΙ of 0.05M Tris-HCI was used together with 15 μΙ of POB obtained by the "bacterial milking" method) mixed with 1 ml of substrate solution of a stationary phase culture . This test mixture was incubated at 30 ° C for 30 minutes in sterile 2 ml microtubes. The reaction was stopped by incubation at 100 ° C for 4 minutes in thermoblock and 2 minutes at -20 ° C. The absorbance was measured on a Hitachi U-2000 spectrophotometer at a wavelength of 410 nm. The substrate solution (SS) was prepared by mixing 10 ml of solution A (30 mg of pNPP in 10 ml of isopropanol) with 90 ml of solution B (0.1 g of gum arabic and 0.4 ml of Triton X-100 in 90 my 50mM Tris-HCI buffer pH8). The mixture of solution A and B was gently stirred until completely dissolved. FIG. 2 shows the protection values of the lipase enzyme on drying generated by the POBs and POBSIAs produced by the strain.

EXAMPLE 3. Test of the increased tolerance to water stress of plants.

The capacity of strain 3J1 on plants to protect them against drought conditions and / or promote their growth was studied. In this first phase of experimentation, sweet pepper plants (Capsicum annuum) were used.

14 days after the germination of the seeds of these plants, the irrigation of all the pots was stopped to begin to cause drought stress conditions in the plants considering this moment as the time 0. From this moment, samples were taken at 7, 14, 20 and 33 days, by taking three plants inoculated with the bacterial strain Microbacterium sp. 3J1. As a positive control, plants inoculated with the rhizobacterium P. putida KT2440 were used and as a negative control plants were used to which the equivalent volume of inoculum was added as water and therefore not inoculated. In each extraction the Fresh Weight (PF) parameters were taken as the weight of the newly extracted plant; Totally Turbid Weight (PTT) as the weight of the plant after 48 hours incubated in darkness and at a humidity of 100% right after its extraction; Dry Weight (PS) as the weight after incubating the plant for 48 hours at 75-80 ° C and the Relative Water Content (CRA) defined through the equation CRA = (PF-PS) / (PTT-PS) . The data obtained are broken down below. In general, after 33 days in the absence of irrigation, the plants inoculated with Microbacterium sp. 3J1 tolerated drought better by showing a better appearance and size than plants inoculated with P. putida KT2440 or than non-inoculated plants as can be seen in FIG. 3.

Thus, the Relative Water Content (ARC) of the study plants was the numerical value that indicated the degree of resistance to drought stress that the plants have with their respective treatments. Although other calculations were also carried out with the parameters initially taken to study all aspects that reflected the effects of water scarcity for plants. Thus, the Water Retention Index (IRA) determined as the difference between the Fresh Weight (PF) and Dry Weight (PS) as well as the Water Recovery Potential (PRA) for the plants given by the difference between Totally Turgid Weight (PTT) and Fresh Weight (PF). As can be seen in FIG. 4 Fresh Weight (PF) of the plants inoculated with each of the strains, suffered a regular increase in all conditions during the first sampling points corresponding to days 7 and 20. In the samples taken on day 33 only the plants inoculated with Microbacterium sp. 3J1 showed a fresh weight greater than that recorded on day 20. Thus, in plants inoculated with Microbacterium sp. 3J1, their PF values were four times higher than those of P. putida KT2440 and those of non-inoculated plants.

Similarly, the Dry Weight (PS) of the plants was measured on days 7, 14, 20 and 33 after the irrigation ceased. The PS of the different plants was much lower than the PF, with values always below 12 mg. As shown in Fig. 5, the dry weight of the different inoculated plants increased slightly more or less regularly in all conditions during the first sampling points. Again the plants inoculated with Microbacterium sp. 3J1 showed the highest values, doubling at 33 the final values of the plants inoculated with both P. putida KT2440, as well as those of the non-inoculated plants.

The values corresponding to the fully turgid weight (PTT) of the plants were also taken on days 7, 14, 20 and 33 after the cessation of irrigation. As shown in Fig. 6, the totally turgid weight of the plants inoculated with the different inoculums followed a pattern similar to that of the fresh weight, but with higher units. Thus, it increased regularly in all conditions during the sampling points of days 7, 14 and 20. At the sampling point of day 33 it is when differences began to be appreciated, since from it only inoculated plants with Microbacterium sp. 3J1 increased their PTT, reaching triples after 33 days to those of P. putida and those of non-inoculated plants.

A more representative data to know the state of water stress of plants is the Relative Water Content (CRA), since it indicates the relationship between the water present in the plants at the time of extraction with respect to the total water. In this way, the maximum value of 1 would be given in the theoretical situation in which, due to its good water state, the plant could not or would not need to recover water in its totally turgid state (PTT) with respect to the Fresh Weight (PF).

In general, it is observed that for the sampling point on day 14, in all the inoculated plants, except for those not inoculated, the CRA parameter decreased although slightly compared to the value recorded on day 7 although this parameter increased again between days 14 and 20. Regarding plants inoculated with Microbacterium sp. 3J1, the value of CRA increased between days 14 and 20. For plants inoculated with P. putida KT2440, the value of CRA decreased slightly between days 14 and 20 (less than 5%) and increased somewhat more than 5% between days 20 and 33. For non-inoculated plants, the value of CRA was reduced very slightly between days 7 and 14, and remained practically stable between days 14 and 20. However, at day 33, only the value of CRA in plants inoculated with Microbacterium sp. 3J1 showed a tendency to continue increasing, generating values around 0.95. It should be noted that the increase in the value of ARC for plants inoculated with Microbacterium sp. 3J1 occurred on both day 20 and day 33. The results can be seen in FIG. 7.

In order to quantify the structural water retention index (ARI) in the different plants, the difference between PF and PS was calculated, although this value is not as representative as the CRA, it also allows to know the water status of plants. Thus, as can be seen in FIG. 8, the highest values were in the plants inoculated with Microbacterium sp. 3J1. For plants inoculated with P. putida KT2440 and non-inoculated plants, a marked decrease was also observed after day 20, reaching day 33 about half the value they had on day 20.

In this way, the IRA values of the plants inoculated with Microbacterium sp. 3J1 for day 33 of sampling were approximately five times higher than those of plants inoculated with P. putida as well as those of non-inoculated plants.

EXAMPLE 4. Test of the increase in biomass of plants inoculated with Microbacterium sp under conditions of water stress.

In order to identify if the bacterial strain 3J1 had some kind of growth promoting effect on plants under conditions of water stress, it was decided to quantify the length of the plants and their roots. Thus, for the height data, the length in cm was taken from the whitish stem base or the point where the first roots were found, to the horizontal plane formed by the first pair of leaves (seminal leaves). For the roots, the length in cm was taken from the whitish stem base or the point where the first roots were found, to the tip of the longest radical apex without forcing the same.

For the treatment of plant height data, we chose to calculate the difference in height from an initial point (time 0). These plant height increases were made at each sampling point at the time of their extraction at the same time as I took the fresh weight and comparing the height at that sampling point with the initial height of the plant.

In FIG. 9 it can be observed how, in the conditions of water deficit, the inoculated plants reached greater height than the non-inoculated plants. Plants inoculated with Microbacterium sp. 3J1 grew steadily even up to 33 days after starting the water deficit process, which was not observed in plants inoculated with P. putida as well as in non-inoculated plants. In the latter cases, it was observed that in both the plants inoculated with P. putida KT2440 and in the non-inoculated plants the growth was very reduced and even reduced at the last sampling point (day 33). Thus, on day 33 of the test, the height difference of the plants inoculated with Microbacterium sp. 3J1 was more than three times higher than the values of plants inoculated with P. putida as well as those of non-inoculated plants.

For the treatment of root length data, we chose the measurement of the root lengths of each plant inoculated at the sampling points on days 7, 14, 20 and 33. The measurement was performed as in the case of the height of the plants at the time of the extraction of the plants for the measurement of the fresh weight. The data obtained are shown in FIG. 10. The length of the roots of the plants inoculated with Microbacterium sp. 3J1 increased to the last point (day 33), while in plants inoculated with P. putida strain and in non-inoculated plants, growth was even reduced at this same sampling point.

EXAMPLE 5. Test of the increase of the growth of the plants inoculated with Microbacterium sp in optimal hygienic conditions. In this trial the same methodology used with the previous ones was applied with the exception that irrigation was maintained periodically (40ml every 48-72 hours) from t = 0. From this moment, samples were taken at 7, 14, 20 and 33 days, by taking three plants inoculated with the bacterial strain Microbacterium sp. 3J1. As a positive control plants inoculated with the rhizobacterium promoting growth of P. putida KT2440 plants were used and as a negative control plants were used to which the equivalent volume of inoculum was added as water and therefore not inoculated.

For the height data, the length in cm was taken from the whitish stem base or the point where the first roots were found, to the horizontal plane formed by the first pair of leaves (seminal leaves). For the roots, the length in cm was taken from the whitish stem base or the point where the first roots were found, to the tip of the longest radical apex without forcing the same.

For the treatment of plant height data, we chose to calculate the difference in height from an initial point (time 0). These increases in plant height were made at each sampling point at the time of their extraction at the same time as the fresh weight was taken and comparing the height at that sampling point with the initial height of the plant.

As can be seen in FIG. 1 1, the heights reached by the plants inoculated with strains were in all cases greater than those obtained by the plants without inoculation on day 33 of the experiment. The greatest increases in height occurred between days 20 and 33 of the trial, becoming almost 4 times for plants inoculated with Microbacterium sp. 3J1. This increase was not significant for the plants not inoculated. On day 33 of the experiment, the values obtained by the plants inoculated with Microbacterium sp. 3J1 became 1.5 times higher than those obtained by plants inoculated with P. putida KT2440, and 6 times higher than those obtained by non-inoculated plants. The values obtained by the plants inoculated with P. putida KT2440 on day 33 of the test became three times higher than those obtained by non-inoculated plants.

For the treatment of root length data, we chose the measurement of the root lengths of each plant inoculated at the sampling points on days 7, 14, 20 and 33. The measurement was performed as in the case of the height of the plants at the time of the extraction of the plants for the measurement of the fresh weight. The data obtained are shown in FIG. 12.

Only the data obtained by the plants inoculated with Microbacterium sp. 3J1 were statistically superior to the rest of the values obtained by the plants with another inoculum. Thus, the values obtained by the plants inoculated with Microbacterium sp. 3J1 were almost 1.5 times higher than the values obtained by the plants inoculated with P. putida KT2440, as well as those obtained by the plants without inoculation.

As indirect parameters related to the size of the plant, measures of Fresh Weight (PF), Dry Weight (PS), Totally Turgid Weight (PTT) were taken, as well as their combination to obtain the parameters of Relative Water Content ( CRA), Water Recovery Potential (PRA) and Water Retention Index (IRA), also described for the previous trial. As can be seen in FIG. 13, the Fresh Weight (PF) of the plants inoculated with each of the strains, underwent a regular increase in all conditions during the first sampling points corresponding to days 7 and 20. In the samples taken on day 33, the plants inoculated with Microbacterium sp. 3J1 showed a fresh weight greater than the rest, around 10% higher than that of the plants inoculated with P. putida KT2440 and almost double that of the non-inoculated plants.

Similarly, the Dry Weight (PS) of the plants was measured at days 7, 14, 20 and 33. The PS of the different plants was much lower than the FP, with values always below 12 mg. As can be seen in FIG. 14, the dry weight of the different inoculated plants increased slightly more or less regularly in all conditions during the first sampling points. On day 14 of the test the plants inoculated with Microbacterium sp. 3J1 had a slight decrease in their PS values. Plants inoculated with Microbacterium sp. 3J1 and with P. putida KT2440 showed higher values than those obtained by non-inoculated plants, becoming at day 33 of the test around 40% higher.

The values corresponding to the fully turgid weight (PTT) of the plants were also taken at days 7, 14, 20 and 33. As can be seen in FIG. 15, the totally turgid weight of the plants inoculated with the different inoculums followed a pattern similar to that of the fresh weight, but with higher units. Thus, it increased more or less regularly in all conditions during the sampling points on days 7, 14 and 20.

At the sampling point on day 33, the highest value in this growth was achieved in plants inoculated with Microbacterium sp., Reaching this is about 10% higher than that obtained by the plants inoculated with P. putida, and around 75% with respect to the non-inoculated plants.

Regarding the structural water retention index (ARI) in the different plants, it shows that the water retention increased very slightly between days 1 and 20 of the experiment in all plants, but that on day 33 there was an increase in Structural water retention in plants inoculated with Microbacterium sp. 3J1 and with P. putida KT2440 of between 25 and 50%. There is no such increase in the value of ARI for non-inoculated plants, the ARI value of them being even half that of the plants inoculated with Microbacterium sp. 3J1. The data can be seen in FIG. 16.

Claims

one . Microorganism of the bacterial species Microbacterium sp. with access number CECT7624.

2. Bacterial population comprising the microorganism according to claim 1.

3. Use of the microorganism according to claim 1 or the bacterial population according to claim 2 to increase the tolerance to water stress of a plant, with respect to a control.

4. Use of the microorganism according to claim 1 or of the bacterial population according to claim 2 to increase the biomass of a plant, with respect to a control, under conditions of water stress.

5. Use of the microorganism according to claim 1 or the bacterial population according to claim 2 to increase the amount of water absorbed by a plant, with respect to a control, under conditions of water stress.

6. Use of the microorganism according to claim 1 or the bacterial population according to claim 2 to increase water retention rate of a plant, with respect to a control, under conditions of water stress.

7. Use of the microorganism according to claim 1 or the bacterial population according to claim 2 to increase the growth of a plant with respect to a control.

8. Use according to claim 7, wherein the increase in growth refers to the increase in the stem length of said plant.

9. Use according to claim 7, wherein the increase in growth refers to the increase in the root length of said plant.

10. Use according to any of claims 3 to 9, wherein the plant is of the genus Capsicum.

eleven . Use according to claim 10, wherein the plant is of the Capsicum annuum species.

12. Method for increasing the tolerance to water stress of a plant with respect to a control, which comprises inoculating the microorganism according to claim 1 or the bacterial population according to claim 2 to a plant cell, to any part of a plant or to a seed.

13. Method according to claim 12, wherein the microorganism according to claim 1 or the bacterial population according to claim 2 is contacted with at least one root of said plant.

14. A method according to claim 13, wherein said microorganism or bacterial population is contacted with at least one root by means of an aqueous solution.

15. Method according to any of claims 12 to 14, wherein the plant is of the genus Capsicum.

16. Method according to claim 15, wherein the plant is of the Capsicum annuum species.

17. Use of the microorganism according to claim 1 or the bacterial population according to claim 2 for the production of an xeroprotective composition.

18. Xeroprotective composition produced by the microorganism according to claim 1 or by the bacterial population according to claim 2.

19. Composition according to claim 18 comprising trehalose, oxoglucuronic acid, lactate, glutamate, glutamine, fucose and pyruvate.

20. Synthetic xeroprotective composition comprising trehalose, oxoglucuronic acid, lactate, glutamate, glutamine, fucose and pyruvate.

twenty-one . Composition according to any of claims 19 or 20 comprising a proportion of trehalose: oxoglucuronic acid: lactate: glutamate: glutamine: fucose: pyruvate, between (0.5 and 1.5): (0.15 and 0.45) : (0.7 and 1, 7): (0.1 and 0.2): (0.15 and 0.45), (1, 2 and 3.4), (0.15 and 0.3) respectively.

22. Composition according to claim 21, wherein the ratio of trehalose: oxoglucuronic acid: lactate: glutamate: glutamine: fucose: pyruvate is between (0.7 and 1, 3): (0.25 and 0.35): ( 0.9 and 1, 6): (0.12 and 0.18): (0.2 and 0.4): (1, 8 and 2.8): (0.2 and 0.25), respectively .

23. Composition according to claim 18, comprising trehalose, oxoglucuronic acid, lactate, glutamate, glutamine and pyruvate.

24. Synthetic xeroprotective composition comprising trehalose, oxoglucuronic acid, lactate, glutamate, glutamine, fucose and pyruvate.

25. Composition according to any of claims 23 or 24, comprising a proportion of trehalose: oxoglucuronic acid: lactate: glutamate: glutamine: pyruvate, between (0.5 and 1.5): (0.03 and 0.09 ): (0.3 and 0.9): (0.05 and 0.15): (0.05 and 0.15), (0.1 and 0.3), respectively.

26. Composition according to claim 25, wherein the ratio of trehalose: oxoglucuronic acid: lactate: glutamate: glutamine: pyruvate is between (0.7 and 1, 3): (0.05 and 0.07): (0, 5 and 0.7): (0.07 and 0.12): (0.07 and 0.12): (0.15 and 0.25), respectively.

27. Use of the composition according to any of claims 19 to 26 for the conservation of biological material with a residual moisture content equal to or less than 10%.

28. Use according to claim 27, wherein the biological material is an invertebrate organism, a seed, a seedling, a microorganism, an isolated organ, an isolated biological tissue or a cell.

29. Use according to claim 27, wherein the biological material is a molecule with biological activity.

30. Use according to claim 29, wherein the molecule with biological activity is an enzyme.

31. Use according to claim 30, wherein the enzyme is a lipase.

32. Method for obtaining the xeroprotective composition according to claim 18 comprising: a) culturing the microorganism of claim 1 or the population of claim 2 in a mineral medium with fructose as a carbon source,

c) rehydrate the dehydrated microorganisms of step (b) in a hypotonic medium, and

d) select the liquid fraction of the product obtained in step (c) comprising the xeroprotective composition.

33. A method according to claim 32, wherein the dehydration of the microorganisms according to step (b) is carried out by means of a hypertonic solution or by means of an air current.

34. A method according to any of claims 32 or 33, wherein the hypotonic means for rehydration of the microorganisms according to step (c) is water partially or totally distilled, deionized or demineralized.

35. Method according to any of claims 32 to 34, wherein, in addition, the liquid fraction of step (d) is dehydrated until the xeroprotective product has a residual humidity equal to or less than 10%.