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    • 专利摘要:
    Strain of rhizobium leucaenae and its use as biofertilizer. The present invention relates to a strain of rhizobium that, when administered to a plant, has the ability to interact with it and promote its growth, thus increasing both the leaf and root development thereof. Therefore, the invention describes a strain of rhizobium leucaenae deposited in the spanish type culture collection with the deposit number cect 8306 with capacity to increase or promote the growth of the plants, the composition comprising it and its use as a biofertilizer for increase or promote the growth of plants. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2583202A1
    申请号:ES201530349
    申请日:2015-03-18
    公开日:2016-09-19
    发明作者:Lorena CELADOR LERA；José David FLORES FÉLIX；Pedro Francisco MATEOS GONZÁLEZ；Eustoquio MARTÍNEZ MOLINA；Encarna VELÁZQUEZ PÉREZ；Raúl RIVAS GONZÁLEZ
    申请人:Universidad de Salamanca；
    IPC主号:
    专利说明:

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    Rhizobium leucaenae strain and its use as a biofertilizer
    DESCRIPTION
    The present invention relates to a strain of Rhizobium leucaenae CECT8306 that, when administered to a plant, has the ability to interact with it and promote its growth, thereby increasing both foliar and root development thereof. Therefore, the strain described in the present invention has its application in the field of agriculture as a biofertilizer.
    STATE OF THE TECHNIQUE
    One of the main objectives of the current agriculture is the reduction of the use of chemical fertilizers, due to both the increasing expense that they imply and the reduction of environmental impacts minimizing the emission of greenhouse gases derived from their use and avoiding the contamination of ecosystems . However, the methodology used by conventional agriculture to meet this challenge is only able to meet it through the application of techniques that either imply a reduction in production or an increase in costs. For this reason, the use of biofertilizers is an efficient, cheap and sustainable alternative capable of addressing this objective in addition to making efficient use of the natural resources of the soil. The group of bacteria called plant growth promoting bacteria or PGPR exhibit a series of mechanisms that influence the balance of nutrients available to the plant, in addition to increasing their resistance to different types of stress and as pathogen resistance elicitors, among others. Among these mechanisms we find the biological fixation of nitrogen, both in symbiosis and in free life, the solubilization of phosphate either through the production of acids or the production of specific enzymes such as phytases and phosphatases, the production of siderophores that increase the iron available for the plant and the production of phytohormones.
    The plant-microorganism interaction becomes more direct and efficient when the bacteria that show the mechanisms of plant growth promotion are able to colonize the interior of the plant, occupying the intercellular spaces of ralz, stem and leaves, behaving like an endophyte organism, establishing a close relationship with the plant without harming or triggering the mechanisms of
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    defense of it. At the same time, a necessary requirement for the formulation of biofertilizers is the use of harmless bacteria for humans, since some pathogenic bacteria such as Escherichia coli, Klebsiella pneumoniae or Burlkolderia cepacia have qualities to promote plant growth but trigger another series of problems In the health. Associated with the species of the Rhizobium genus, mechanisms for the promotion of plant growth have been described, such as the solubilization of phosphate, the production of siderophores and the production of various phytohormones such as indole acetic acid, cytokinins and gibberellins (Mehbood, I. et al., 2009. Critical Reviews in Plant Science, 28: 432-456) making these species suitable for use as mobilizers, producers or facilitators of the absorption of nutrients for the plant. In addition, it has been shown that the symbiotic characteristic is not only given between Rhizobium-Leguminosa, where it forms nitrogen-fixing nodules, but also between non-leguminous plants, since complex and specific molecular machinery have been described that allow it to interact with plants of different types. genres, colonizing and entering into plants such as rice and lettuce, promoting plant growth and even increasing production yield.
    It is known that plants and animals are normally associated with various microorganisms. In the intestine, bacteria have a prominent role to stimulate immunity and development. In the same way that plant bacteria stimulate their defense responses, bacteria from the roots and the rhizosphere benefit from the exudates from the roots. In addition, some bacteria and fungi are able to enter the plant as endophytes without causing damage and can establish mutual associations. Plants constitute large niches of various endophyte organisms. Endophytic bacteria have been isolated from a great diversity of plants. Both populations of endophytic and rhizospheric bacteria are conditioned by biotic and abiotic factors, but endophytic bacteria may be better protected from biotic and abiotic stress than rhizospheric bacteria. This is due to the fact that the interior of the plant is a much more stable and homogeneous environment over time, independent of the fluctuations in humidity, salinity or pH that may occur in the rhizosphere.
    The Spanish patent application ES2402039 describes a strain of the Rhizobium genus, specifically the Rhizobium leguminosarum strain CECT7758, which has the ability to promote growth in non-leguminous plants.
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    Due to this reason, the use of bacteria of the Rhizobium genus is presented as a basic and interesting tool for the development and utilization of future biofertilizers, since its use does not represent any danger to human health since within this genus they have not described pathogenic species of humans and yet, if it presents positive connotations in agriculture and the economy. Therefore, there is a need to provide alternative fertilizers to those existing in the state of the art that do not represent a threat to the environment or to human health.
    DESCRIPTION OF THE INVENTION
    The authors of the present invention have isolated a strain of Rhizobium, in particular Rhizobium leucaenae which, surprisingly, has the ability to promote plant growth (both follicular and radial) and thereby improve crop yield. The strain isolated by the inventors is the Rhizobium leucaenae strain CECT8306. The inventors isolated said strain from the root of Zea mays and by various tests (see Example 1), they verified that the plant growth of plants is increased when they grow in the presence of Rhizobium leucaenae CECT8306 compared to control plants (see Figures 5 to 10).
    Based on this new strain, a series of inventive aspects have been developed that will be described in detail below.
    Strain and composition of the invention
    As described at the beginning of this description, the authors have isolated a strain of Rhizobium leucaenae that has the ability to promote and / or increase plant growth of plants.
    Thus, in one aspect, the invention relates to a strain of Rhizobium leucaenae deposited in the Spanish Type Culture Collection with the deposit number CECT 8306, hereinafter referred to as "strain of the invention".
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    The strain of the invention was isolated from Zea mays as described in Example 1 and deposited on June 19, 2013 under the Budapest Treaty in the Spanish Type Crops Collection as an International Deposit Authority (Building 3 CUE, Parc Cientlfic University of Valencia, Professor Agustln Escardino, 9, 46980 Paterna (Valencia) SPAIN). The deposit number assigned was CECT 8306. The depositor was Mr. Raul Rivas Gonzalez, with direction Department of Microbiology and Genetics, Lab 210, Biologla Department Building, Miguel de Unamuno Campus, 37007 Salamanca. The scientific classification of strain CECT 8306 of the present invention is: Kingdom: Bacteria, Phylum: Proteobacteria, Class: alpha-Proteobacteria, Order: Rhizobiales, Family: Rhizobiaceae, Genus: Rhizobium, Species: Rhizobium leucaenae.
    The analysis and study of the strain of the invention showed that it was capable of producing siderophores, solubilizing phosphates in the form of Ca3 (PO4) 2), producing indole acetic acid or IAA (auxin that promotes the growth of meristematic tissues of plants), colonize the root of the plants and interact with them, produce cellulose and form biofilms. In addition, it was observed that the strain has an insert in the 16S gene of approximately 70 base pairs that is absent in the rest of the Rhizobium species. On the other hand, seeds inoculated with the strain of the invention gave rise to plants that showed a greater growth of the aerial part and a greater thickness and pilosity of the root in comparison with plants obtained from seeds not inoculated with the strain of the invention. Therefore, the strain of the invention is capable of promoting or increasing the growth of plants.
    Within the present invention, those microorganisms or bacteria that derive from the strain of the invention and that retain the ability to increase or promote plant growth are also contemplated. Examples of strains or microorganisms derived from the strain of the invention may be mutants that have variations in their genome with respect to the genome of the strain of the invention but that do not affect the ability of the strain to increase or promote plant growth. Thus, mutant strains derived from the strain of the invention that retain the ability to increase or promote plant growth are also contemplated within the present invention. Therefore, in another aspect, the present invention also relates to a strain derived from the strain of the invention capable of increasing or promoting plant growth.
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    The strain derived from the strain of the invention can be produced naturally, or intentionally by methods of mutagenesis known in the state of the art such as, for example, without being limited to, the growth of the original strain in the presence of mutagenic agents or stressors, or by genetic engineering aimed at the modification of specific genes. Thus, genetically modified mutants derived from the strain of the invention that retain their ability to increase or improve plant growth are also contemplated within the present invention.
    On the other hand, within the present invention, cellular components, metabolites and molecules secreted by the strain of the invention or by the strain derived from the strain of the invention are also contemplated, as well as the compositions comprising said components and uses of them to increase or promote the growth of plants. Among the cellular components of the bacterium could be included the components of the cell wall (such as but not limited to, peptidoglycan), nucleic acids, membrane components, or others such as proteins, carbohydrates and carbohydrates and their combinations , such as lipoprotelnas, glycolipids or glycoprotelns. The metabolites include any molecule produced or modified by the bacteria as a result of its metabolic activity during its growth, its use in technological processes or during product storage. Examples of these metabolites are, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoprotelns, glycolipids, glycoproteins, vitamins, salts, metals or nucleic acids. Secreted molecules include any molecule exported or released abroad by the bacteria during its growth, its use in technological processes (for example food or drug processing) or product storage. Examples of these molecules include, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoprotelns, glycolipids, glycoproteins, vitamins, salts, metals or nucleic acids.
    In the present invention it is understood that a strain (or compound derived from it) has the ability to "increase" or "promote" plant growth when said strain is capable of increasing the amount of biomass produced by a plant in unit time. Thus, in equality of time and conditions, the biomass produced by a plant comprising the strain of the invention will be greater than the biomass
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    produced by a plant of the same species that does not comprise the strain of the invention (or that has not been contacted with the strain or composition of the invention).
    In another aspect, the invention relates to a biologically pure culture of the strain of the invention. In the present invention, it is understood that a culture is biologically pure when at least 95% of the microorganisms present in the culture correspond to the strain of the invention.
    The strain of the invention can be grown in any culture medium that is suitable for Rhizobia. Examples of culture medium suitable for the cultivation of the strain of the invention include, but are not limited to, YMA and TY media. However, the preferred culture medium is YMA medium. In the present invention, "YMA medium" or "MYA culture medium" is understood as that medium consisting of K2HPO4, MgSO4, NaCl, yeast extract and agar, preferably in the following concentrations: K2HPO4 at a concentration of approximately 0, 2 g / L, MgSO4 at a concentration of about 0.2 g / L, NaCl at a concentration of about 0.1 g / L, yeast extract at a concentration of about 2 g / L, and agar at a concentration of approximately 20 g / L. The composition of the TY medium comprises yeast extract from 0.2 to 0.4%, tryptone from 0.3 to 9.5% and agar from 1.5 to 2%.
    As the person skilled in the art understands, the strain of the invention or the strain derived from it or the components derived therefrom may be forming part of a composition. Thus, in another aspect, the present invention relates to a composition comprising the strain of the invention, the strain derived from it and / or the components derived therefrom.
    The composition, defined in general, is a set of components that is formed at least by the strain of the invention in any concentration. It should be noted with respect to the strains used in the present invention, it is interesting that said strains are in exponential phase of growth, since in this way they are in a better metabolic disposition for their development and promote the beneficial effects within the plants. However, in a particular embodiment, the concentration of the strain of the invention in liquid medium is 1x108 CFU / mL and 3.8x108 CFU / mL, in particular between 1.8x108 CFU / mL and
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    3.6x108 CFU / mL, more particular, between 2x108 CFU / mL and 3x108 CFU / mL, even more particular, 2.5x108 CFU / mL. In solid vehicles or media the concentration of the strain of the invention is between 1.8x108 CFU / g and 3.6x1011 CFU / g.
    Accompanying the strain of the invention, the composition may comprise other organisms that may be beneficial for plants, such as bacteria (including actinomycetes), fungi, algae and protozoa. Examples of such microorganisms include, but are not limited to, fungi of the genus Trichoderma (such as T. harzianum Rifai, T. viride Pers., T polysporum Link fr, T. reesei EG Simmons, T. virens, T. longibrachatum Rifai, T. parceromosum, T. pseudokoningii, T. hamatum, T. lignorum, T. citroviride, etc.), mycorrhizae (Glomus sp., etc.), Rhizobium bacteria (such as R. cellulosilyticum, R. daejeonense, R. etli , R. gallicum, R. hainanense, R. indigoferae, R. leguminosarum, R. loessense, R. lusitanum, R. mongolense, R. phaseoli, R. rhizogenes, R. sullae, R. tropici, R. yanglingense , etc.), of the genus Neorhizobium (N. galegae, N. huautlense, etc.), Allorhizobium (A. undicola), Pararhizobium (P. giardinii), Mesorhizobium (M. loti, M, ciceri, M. mediterraneum, M huakuii, M. tianshanense, etc.), Ensifer (E. fredii, E. meliloti, etc.), Bradyrhizobium (B. japonicum, B. lupini, etc.), bacteria that colonize internal plant tissues (such as Azospirillum sp ., Herbaspirillum sp ., Gluconacetobacter diazotrophicus, Paenibacillus sp, etc.), seaweed (such as Ascophyllum nodosum, Fucus serratus, Laminaria sp., Etc.), etc. Therefore, in another particular embodiment, the composition of the invention comprises a bacterium of the Rhizobium genus different from the strain of the invention, preferably said Rhizobium genus bacterium is Rhizobium leguminosarum CECT 7759 or Rhizobium leguminosarum CECT 7758.
    Additionally, the composition of the invention may comprise compounds that participate in plant development, such as phytohormones. Phytohormones are substances produced by plant cells that intervene in a multitude of biological processes and are capable of regulating the physiological phenomena of plants. Examples of phytohormones that can be employed in the composition of the invention include, but are not limited to, auxins, gibberellins, cytokinins and ethylene.
    Auxins are a group of phytohormones that function as regulators of plant growth (essentially cause elongation of cells) and can be natural (synthesized by a plant cell in a wild way) or artificial
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    (synthesized in the laboratory). Examples of natural auxins include, but are not limited to, indole-3-acetic acid (AIA), 4-chloroindole-3-acetic acid (4-Cl-IAA) and phenylacetic acid. Examples of artificial auxins include, but are not limited to, indole butyric acid (AIB) and naphthalenacetic acid (ANA).
    Gibberellin is a phytohormone produced in the apical zone, fruits and seeds, whose main functions are the interruption of the dormancy period of the seeds, making them germinate, the induction of the development of buds and fruits and the regulation of the longitudinal growth of the stem. Examples of gibberellins include, but are not limited to, gibberellic acid (GA3), gibberellin A1 (GA1) and gibberellin A4 (GA4).
    Cytokinins are phytohormones that promote cell division and differentiation. Examples of cytokinins include, but are not limited to, cis- and trans-zeatin, isopentenyladenine, dihydrozeatin (with their respective glycosylated derivatives), benzyladenine, kinetin and topoline. Other compounds of non-plant origin and synthetic derivatives of diphenylurea such as CPPU and thidiazuron (TDZ), which act as structural analogs of the natural molecule, are also considered cytokinins.
    As the person skilled in the art understands, macro and microelements that provide nutrients to plants can also be included in the composition of the invention. Thus, in a particular embodiment, the composition of the invention further comprises macroelements and / or microelements.
    Macroelements are those elements that are expressed as% in the plant or g / 100g. The main ones are nitrogen, phosphorus, potassium, calcium, magnesium and sulfur. The microelements are expressed as ppm (part per million) = mg / Kg = mg / 1000g, and the main ones are iron, zinc, copper, mobdylene manganese, boron and chlorine. It is known that there are about 27 chemical elements that have functions in the plant that, because the plant requires them at certain concentrations, must be added to the soil. There are also other beneficial nutrients such as silicon, sodium and cobalt that strengthen some characteristics of plants in different species. The person skilled in the art knows which are the macro- and micronutrients suitable to add to the plant of interest to cover its needs.
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    Thus, in a particular embodiment, the macroelements of the composition of the invention are selected from the group consisting of nitrogen salts, phosphorus salts, potassium salts, calcium salts, magnesium salts, sulfur salts and combination thereof.
    In another particular embodiment, the microelements of the composition of the invention are selected from the group consisting of iron salts, zinc salts, copper salts, manganese salts, molybdenum salts, boron salts, chlorine salts and combinations of they.
    The composition of the invention may additionally comprise the seed of a plant whose growth is to be promoted. In this way, the seed is in contact with the strain of the invention (and with other components that can be included in the composition of the invention) which allows a better development of the seed and the plant derived from it and finally, Obtaining a plant with optimal characteristics. The seed can belong to any plant, both crop plants, as well as ornamental and forage plants. Preferably, the seed belongs to a non-leguminous plant, more preferably, to a carrot plant, a broccoli plant or a malz plant.
    The strain or composition of the invention can be found in any form of presentation suitable for administration or application to the soil surrounding the plant, to the plant itself or to the seed. In this way, it can be found, for example, but not limited to solid or liquid form. Liquid presentation forms are suitable for spraying on soil, plant or plant material, or for creating a solution in which plants or plant material are submerged. Alternatively the compositions can be found in solid form by lyophilization and subsequent pelletization thereof, which can be applied directly to the soil or resuspended in solutions, preferably aqueous.
    Uses of the composition of the invention
    The present invention relates to a strain of Rhizobium leucaenae CECT8306 which, when administered to a plant, has the ability to interact with it and
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    promote its growth, increasing both foliar and root development of it.
    Therefore, in another aspect, the invention relates to the use of a strain of Rhizobium leucaenae deposited in the Spanish Type Culture Collection with the deposit number CECT 8306, or the composition comprising it, as a biofertilizer.
    In the present invention, "biofertilizer" is understood as the composition that is intended to supply and supply the plant with the necessary components for its growth and development, being among said components microorganisms (or substances produced by them) that provide or improve the Nutrient availability when applied to the plant favoring its development and growth. In the present invention, the microorganisms of the composition comprise at least one microorganism of the strain Rhizobium leucaenae CECT 8306.
    Biofertilizers can be presented in liquid form or in solid form, depending on the form of administration to be used. They can be applied directly to the soil before or after planting the crop, by spraying or in the planting groove or over the entire surface. Another possibility is to apply the biofertilizer to the seed before being sown. The doses and times of application of the biofertilizer during the crop cycle will depend on the concentration of microorganisms and / or the type of culture. Techniques on how to administer biofertilizers and the amount to be administered are widely known in the state of the art and their use is routine practice for the person skilled in the art. Preferably, in a particular embodiment, the concentration of the strain of the invention is 1x108 CFU / mL and 3.8x108 CFU / mL, in particular between 1.8x108 CFU / mL and 3.6x108 CFU / mL, more particular, between 2x108 CFU / mL and 3x108 CFU / mL, even more particular, 2.5x108 CFU / mL. In solid vehicles or media the concentration of the strain of the invention is between 1.8x108 CFU / g and 3.6x1011 CFU / g.
    In the present invention, the biofertilizing capacity is analyzed by analyzing the difference in the dry weight of the treated plants with respect to the control plants, assuming a greater dry weight a greater development as a result of a greater number of structures or a greater development of the same. Other methods
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    known in the state of the art could be used to determine the fertilizer capacity, for example, the determination of biomass or the measurement of plant height with respect to controls.
    Based on the ability of the strain of the invention to increase and / or promote plant growth, in another aspect the invention relates to the use of a strain of Rhizobium leucaenae deposited in the Spanish Type Crop Collection with the number of deposit CECT 8306, or the composition that comprises it, to increase or promote the growth of plants.
    As explained in previous paragraphs, it is understood that a strain (or strain or compound derived from it) has the ability to "increase" or "promote" plant growth when said strain is capable of increasing the amount of biomass produced by a plant in unit of time.
    The strain of the invention or composition comprising it can be applied to any plant whose growth is to be increased, mainly, plants with some commercial interest, thus optimizing their production and yield. In a particular embodiment, the plants are selected from crop plants and ornamental plants.
    Examples of crop plants include, but are not limited to, cereals (such as rice, maize, wheat, spelled, barley, oats, quinoa, wheat, rye, spelled, etc.), vegetables (such as carrots, chard, spinach, beets , celery, turnip celery, chirivla, parsley, eggplant, potato, pepper, tomato, garlic, ascalonia, onion, asparagus, leek, chard, borage, endive, escarole, canonigo herb, lettuce, champignon, pea, bean, judla green, zucchini, squash, cucumber, artichoke, sweet potato, broccoli, white cabbage, Chinese cabbage, Brussels cabbage, Milan cabbage, Lombard cabbage, cauliflower, kohlrabi, kohlrabi, turnip, radish, etc.), tobacco plants and plants sunflower. Another type of plants whose growth can be increased are fruit trees, such as orange, apple, pear, etc., or berry fruit, such as strawberries, raspberries, blueberries, currants, etc. Examples of ornamental plants include, without limitation, carnations, roses, tulips, easter flowers and geraniums.
    In a particular embodiment, the crop plants are non-leguminous plants. In the present invention, "non-leguminous plant" means all those plants that do not belong to the Fabaceae or Leguminosae family within the order Fabales.
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    Among the non-leguminous plants, one of the ones with the greatest agronomic interest is the solanaceas family, which includes tomatoes and peppers. However, in another more particular embodiment, the non-leguminous plant is selected from the group consisting of a carrot plant, a broccoli plant and a maize plant.
    Method to increase or promote plant growth
    In another aspect, the present invention relates to a method for increasing or promoting the growth of a plant, hereinafter referred to as the "method of the invention" which comprises:
    (a) contacting a plant or the seed of said plant with the strain or with the composition of the invention, and
    (b) develop the seed or plant of stage (a).
    The expression "increase or promote" the growth of a plant has been previously defined in the present description. Likewise, plants on which the method of the invention can be applied have also been mentioned above. Thus, in a particular embodiment, the plants they are selected among crop plants and ornamental plants, which in another more particular embodiment, are non-leguminous crop plants, which in another even more particular embodiment, the non-leguminous crop plant is selected from the group consisting of a carrot plant, a broccoli plant and a malz plant.
    In a first stage, the method of the invention comprises contacting a plant or seed from said plant with the strain or composition of the invention. The plant or seed thereof can be contacted with the strain or composition of the invention by any known technique, such as, but not limited to, through hydroponics, through a solution applied to the soil, by means of applying the strain or composition by spraying, spraying, coating, fumigation or impregnation of any aerial pate of the plant or seed, by introducing the strain or the composition of the invention in the irrigation water, by the germination of seeds of the plant in the presence of said strain or composition, by cultivation of plant material in vitro in contact with the strain or composition of the invention, or by immersion of the root of the plant in a solution comprising the strain or The composition of the invention.
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    Thus, in a particular embodiment, the contact between the plant or the seed coming from it and the strain or composition of the invention is carried out by means of irrigation water, spraying, spraying, coating, fumigation, immersion. or impregnation.
    In a second stage, the method of the invention comprises developing the seed or plant of stage (a). This development includes providing the plant or seed with the right conditions of light, temperature, humidity, nutrients, etc. for the plant to grow or for the seed to germinate and give rise to a plant. Suitable growth conditions for different crop plants can be found in manuals widely known to those skilled in the art and available to the public.
    In another aspect, the present invention relates to a seed comprising the strain of the invention, hereinafter referred to as "seed of the invention." The seed may come from any plant of those mentioned above. As understood by the person skilled in the art. In view of the present invention, the plant from the seed of the invention will show greater growth and development than plants that come from seeds that have not been in contact and that does not comprise the strain of the invention.
    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 characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1 is a fluorescence image showing a biofilm formed on the slide after having had it and seen through ultraviolet illumination.
    Figure 2 shows an image obtained by fluorescence microscopy showing the strain R. leucaenae CECT8306 colonizing the surface of the root of Zea mays. In
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    light gray (dots) R. leucaenae CECT8306 cells are observed and in dark gray the vegetable cells had with a solution of propidium iodide (5 mM).
    Figure 3 is an image obtained by fluorescence microscopy showing the strain R. leucaenae CECT8306 colonizing the surface of the root of Daucus carota. In light gray (dots) the cells of R. leucaenae CECT8306 are observed and in dark gray the vegetable cells had with a solution of propidium iodide (5 mM).
    Figure 4 is an image obtained by fluorescence microscopy showing the strain R. leucaenae CECT8306 colonizing the surface of the broccoli root (Brassica oleracea italica). In light gray (dots) R. leucaenae CECT8306 cells are observed and in dark gray the vegetable cells had with a solution of propidium iodide (5 mM).
    Figure 5 is a photograph of the maize plants grown in the greenhouse at 15 days after planting. On the right is the control malz plant without
    inoculate and on the left the maize plant inoculated with R. leucaenae CECT8306
    where the height difference is clearly observed in the first stages of growth.
    Figure 6 is a photograph of the maize plants grown in the greenhouse at 40 days after planting. On the right is the control malz plant without
    inoculate and on the left the maize plant inoculated with R. leucaenae CECT8306
    where the height difference is clearly observed in the first stages of growth.
    Figure 7 is a photograph of the maize plants grown in the greenhouse at 70 days after planting. On the right is a control maize plant without inoculation and on the left a marsh plant inoculated with R. leucaenae CECT8306 where the height difference between the two is clearly observed.
    Figure 8 is a photograph of the maize plants grown in the greenhouse. On the left is a control maize plant without inoculation and on the right a maize plant inoculated with R. leucaenae CECT8306 where the difference in root surface is observed.
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    Figure 9 is a photograph of carrot plants in the greenhouse. On the left is a control plant without inoculation and on the right a plant inoculated with R. leucaenae CECT8306.
    Figure 10 is a photograph of broccoli plants in the greenhouse. On the left is a control plant without inoculation and on the right a plant inoculated with R. leucaenae CECT8306.
    EXAMPLES
    Next, the invention will be illustrated by tests carried out by the inventors, which shows the effectiveness of the product of the invention.
    Example 1
    Isolation of Rhizobium leucaenae strain CECT8306 (strain CRMZ151) and its use as a promoter of plant vegetative growth
    I - MATERIALS AND METHODS
    Strain Isolation
    The strain under study was isolated from the inside of ralz de Zea mays. In order to assimilate the endophyte microorganisms, the surface root is sterilized. To do this, the ralz was washed with sterile water to remove any remaining soil. Next, small pieces of roots of about 5 mm in length were cut, which were treated in tubes with 70% ethanol for two minutes, then they were introduced into a 20% commercial bleach solution (1% active chlorine) during 5 minutes and finally again in 70% ethanol for 1 minute. Then, and maintaining the aseptic conditions, four washes were carried out with sterile water to remove the remains of ethanol and lejla. Then, the ralces were crushed with the help of a sterile glass rod, with which the crushing was sown in Petri dishes with YMA culture medium (Vincent, JM 1970. A Manual for the Practical Study of Root Nodule Bacteria. Oxford: Modified Blackwell Scientific (10 gL "1 mannitol, 1 gL" 1 yeast extract, 0.2 gL "1 K2HPO4, 0.2 gL" 1 MgSO4.7H2O, 0.5 gL "1 NaCl, 20 gL" 1 agar ). The plates were grown at 28 ° C for 4 days.
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    The isolated object of study was named as strain CRMZ151 (R. leucaenae CECT8306). The 16S rRNA gene of strain CRMZ151 was analyzed as described in Rivas et al., 2007. The sequence obtained was identified by comparison with the type strains using the Eztaxon 2.0 program (Chun et al., 2007. Int J Syst Evol Microbiol 57: 2259-2261).
    Tests used to characterize strain CRMZ151
    The ability of the strains to solubilize phosphate in vitro was performed in YED-P (Peix et al., 2003. Int J Syst Evol Microbiol 53: 2067-2072). after being incubated at 28 ° C for 7 days. Sideroforo production was evaluated in the M9-CAS-AGAR medium (Schwyn and Neilands, 1987. Anal Biochem 160: 47-56) modified with HDTMA, a cationic solvent that stabilizes the Fe-CAS complex, giving it a characteristic color ( Alexander and Zuberer, 1991. Biol Fertil Soils 12: 39-45). The production of indole acetic acid was carried out in the JMM medium (O'hara et al., 1989. Appl Emviron microbiol 55, 1870-1876) supplemented with 0.17g / L tryptophan. After 7 days, supernatants were recovered by centrifugation and filtration using 0.22 pm Millipore filter (Millipore Co., Amicon, USA). Then, 1mL of Salkowsky agent was added for every 2 mL of supernatant. The resulting solution was measured by spectrophotometer at a wavelength of 550 nm using an ATI Unicam 8625 spectrophotometer (Mattson, USA) (Khalid et al., 2004. J Appl Microbiol 96: 473-480). The labeling of the strain CRMZ151 was performed using a biparental conjugation, using the Escherichia coli S17.1 strain transformed with the plasmid pHC60 (Cheng and Walter, 1998. J Bacteriol 180: 5183-5191). From fresh cultures of the recipient and donor strains, the strains were mixed on YMA plates and incubated 24 hours at 28 ° C. The conjugates were selected in Minimal Medium (O’Gara and Shanmugan, 1976. Biochim Biophys Acta 451: 342-352) supplemented with tetracycline at a concentration of 10pg / mL. Once the pure culture of strain CRMZ151 transformed with the plasmid pHC60 was obtained, they were grown in YMA medium supplemented with tetracycline (10 pg / mL).
    For the study of plant colonization, maize, carrot and broccoli seeds were superficially sterilized by introducing them 30 seconds in 70% ethanol and sodium hypochlorite for 5 minutes, in the case of marshmallow 20 minutes. After performing 5 washes with sterile water, they were placed in plates with water agar on a filter paper. The seeds were inoculated with strain CRMZ151
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    wild and the differences that appear between those inoculated and those that do not speak have been inoculated.
    Similarly, maize, broccoli and carrot seedlings were inoculated with the strain transformed with the plasmid pHC60 and were observed by fluorescence microscopy using a NIKON eclipse 8Oi microscope at different times.
    The ability to produce cellulose was evaluated using the conventional YMA culture medium with 25 mg / L of Congo Red. Congo Red is a compound capable of binding to 1,4-p glucose polymers and is commonly used for cellulose detection. On the surface of the medium 5pl of bacterial suspension was added, incubating the plates for 3 days at 28 ° C.
    On the other hand, tests related to the formation of biofilms were carried out according to (Fujishige, N.A. et al. 2006. FEMS Microbiol Ecol 56: 195-206) with some modifications. A pre-inoculum was prepared by incubating strain CRZM151 at 28 ° C with orbital agitation at 180 rpm, for 2-3 days, until reaching the latency phase (optical density at 600 nm, about 2.0). When I reach this stage, it is centrifuged and washed with sterile distilled water. The pellet was then resuspended in the same medium at a D.O. 600 nm of 0.2 (approximately 1x107 cells / mL) and stored for later use. On the other hand, a previously sterile slide was introduced into a 50 mL falcon tube, 25 mL of culture medium for the microorganism was added, in this case YMB (10 gL-1 mannitol, 1 gL-1 yeast extract, 0.2 gL-1 K2HPO4, 0.2 gL-1 MgSO4.7H2O, 0.5 gl-1 NaCL, 20 gL-1) and inoculated with 10 pL of bacteria. It was then incubated at 150 rpm at 28 ° C for 3 days. The biofilm formation was observed under a microscope. To do this, the slide was carefully removed from the falcon tube and immersed in a calcofluor blank solution. Next, in a 10% KOH solution, it was observed on which side of the slide the bacterium had been best adhered, choosing this to see it under a fluorescence microscope and the other side was cleaned well avoiding any remaining dye. Finally, plants of all species were placed on substrate and inoculated with strain CRMZ151 to determine if it was capable of promoting plant growth under microcosm conditions.
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    II - RESULTS
    The strain CRMZ151 was isolated from the interior of Zea mays root grown in soil from the town of Ciudad Rodrigo, located in the southwest of the province of Salamanca.
    The sequence obtained from the 16S rRNA gene of strain CRMZ151 was analyzed and compared in GenBank using the BLASTN programs (Alschul et al., 1990. J Mol Biol 215: 403-410) and EzTaxon (Chun et al., 2007. Int J Syst Evol microbiol 57: 22592261). The sequence was aligned using Clustal_X software (Thompson et al., 1997. Nucleic Acid Res 25: 4876-4882). The comparison of the sequence of the 16S rRNA gene of strain CRMZ151 in the EzTaxon database showed that our isolate had a 100% similarity with respect to the Rhizobium leucanae type strain. It is the first time that this species is found in Spanish soils and as an endophyte of malz. The strain CRMZ151 was deposited with the Spanish Society of Crops type with the registration number CECT8306.
    The strain CRMZ151 showed that it was capable of producing siderophores, solubilizing phosphate in the form of Ca3 (PO4) 2 and producing 0.362 g / L of indole-acetic acid when these growth promotion mechanisms are tested in vitro.
    The first stage in the study of the plant-microorganism interaction was the inoculation of Zea mays, Daucus carota and Brassica oleracea italica seeds. In this trial it was observed that the Zea mays seeds inoculated with the strain under study showed a higher growth than the negative control and a greater thickness of the secondary root, so that it would provide it with greater anchorage and fixation of the plant to the soil, in addition of the support, having more surface also absorbs more water and mineral nutrients dissolved in it, causing greater growth. Similarly, it is related to a greater capacity to withstand environmental fluctuations because the root reaches deeper layers of the soil and is less sensitive to situations of hydric or nutritional stress. The inoculated Zea mays seeds also had roots with a more hairy appearance, probably related to the production of indolacetic acid that stimulates their development. This character plays in favor of the inoculated seeds since the absorption surface of the plant is increased. The inoculated Daucus carota plants showed an earlier development of the root than those that do not speak inoculated and a greater elongation of the stem than in the negative control, this is a benefit since if the root is longer, there will be a greater support so that
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    produce symbiotic associations with microorganisms. In addition, a larger stalk implies that the competition for the light factor be decanted towards those inoculated plants. Finally, the seeds of broccoli (Brassica oleracea italica) inoculated show an earlier development of the root, which provides greater support as well! as an earlier onset of development, and more surface to absorb nutrients, in addition to confer greater stress resistance. These results have a close relationship with the production of indole acetic acid, since it has been described that the inoculation of seeds and seedlings with bacteria producing indole acetic acid is related to an increase in early stage development and a greater number of radical hairs .
    In order to compare the cellulose production of strain CRZM151, control strains (negative and positive) that were already standardized in the laboratory were used. Once the experiment was over, it was observed that the strain was capable of producing a large amount of cellulose. Cellulose production by plant growth promoting bacteria may be an essential factor in the formulation of bioinoculants, since these endophytes are able to adhere to plants through cellulose production, subsequently forming biofilms that contribute to growth. , development and defense of the plant.
    When it was analyzed whether the strain was capable of forming in vitro biofilm-like structures, it was observed that strain CRZM151 was capable of forming this type of biofilm (Figure 1). It is very important, therefore, a good colonization by bacteria, since its formation in three-dimensional structures, such as biofilms, protects microorganisms from the action of adverse agents, increases the availability of nutrients for growth , facilitates the use of water, reducing the possibility of dehydration and enables the transfer of genetic material. In addition to an increase in resistance, the presence of an extracellular matrix protects the constituent cells from external aggressions and acts as a barrier against the diffusion of small molecules. In this way it is clear that the formation of biofilms is an adaptive strategy of microorganisms, which allows them to establish positive and beneficial interactions with the plant they colonize.
    The labeling with plasmid pHC60 allowed fluorescence microscopy studies. In the first place, the study of the interaction between
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    CRMZ151 and Zea mays seedlings. The colonization observed was optimal, the bacteria growing mainly in the upper part of the root but spreading over the entire root surface over time (Figure 2). In this plant an intercellular colonization pattern was observed, where the bacteria colonized the intercellular spaces forming a mosaic on the root surface. When colonization was studied in the oldest parts of the root, that is, those closest to the seed, a large number of bacteria were observed growing between the root root hairs, covering the root surface and forming three-dimensional structures attached to the radical hairs where the extracellular polymers secreted by the bacteria formed large aggregates similar to biofilms. With reference to this fact, it has already been mentioned that CRMZ151 was able to produce cellulose in vitro, so both results must be related since cellulose is one of the main components of Rhizobium exopolysaccharide, directly involved in the formation of biofilms . In the case of corn, it was observed that colonization was highly directed and located mainly in the upper areas where it is able to reproduce, but also the growth zones are also colonized although in a much more diffuse way, restricting only the intercellular spaces.
    Daucus carota seedlings inoculated with the strain CRMZ151 labeled with plasmid pHC60 were observed under a fluorescence microscope showing good colonization. When studying the roots of this plant a continuous colonization was seen and slightly dispersed by the root surface, showing an outstanding predilection for the upper areas and decreasing to the root elongation zone (Figure 3). A direct relationship was seen between the areas where radical hairs abounded and colonization by the strain CRMZ151, so that the areas where there was a greater amount of radical hair were colonized to a greater extent and the areas where there were fewer radical hairs or these were of a smaller size, the number of bacteria that colonized the root was significantly smaller. A curious fact observed was the adhesion of this bacterium during the first days after inoculation to the radical hairs and that was not observed again during the study, appearing with time more bacteria in the valleys between the cells of the surface of the root. The most rational explanation to this fact is that during the formation of radical hairs there are important changes in the cell wall of plants, excreting substances that could be captured by bacteria as indicators of penetration sites
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    towards the interior of the plant demonstrating that the endophytic bacteria have a special capacity to interact with the host plants and respond to their stimuli.
    The last of the inoculated plants was broccoli (Brassica oleracea italica). In it, the images obtained allowed us to verify how from the first days after inoculation there is a very active colonization of the root surface. The bacterium was able to colonize the entire root surface forming a homogenous but not excessive tapestry (Figure 4). With the passage of time since inoculation, the disposition of the bacteria evolved showing an important predilection for the valleys or depressions formed between the cells of the root. In other cases, as of the seventh day of inoculation the strain CRZM151 was observed, in addition to showing a characteristic disposition on the root, it was also arranged around the emergency locations of radical hairs. This is a strategy followed by some species of the Rhizobium genus when it comes to forming nitrogen-fixing nodules and also by many endophytes. In this way, the microorganism takes advantage of the weakest places on the root surface to colonize the interior of the plant, occupying a space where it can develop and interact with it. Another of the highlights observed during the study of the colonization of broccoli ralces was the formation of biofilms or protobiopellules around the surface of the root. These data reaffirm the results obtained when evaluating the ability to form biofilms in vitro. This quality brings one more benefit to the plant since the bacterium is able to increase the surface of contact with the environment, increasing the useful surface for the absorption of nutrients. It should also be noted that this polysaccharide can protect the plant against environmental fluctuations due to its greater capacity for water retention.
    Finally, the capacity of this strain to promote the development in Zea mays, Daucus carota and Brassica oleracea italica plants was analyzed.
    The microcosm tests were carried out in a greenhouse with temperature and controlled light to avoid stress situations for the plants. A mixture of peat and vermiculite, in proportion 3: 1, was selected as a substrate to develop the experiment. The ability to promote growth in the early stages of plant development was evaluated for that purpose plants were grown from each of the crops chosen for inoculation and other plants as a negative control that would not be inoculated (Figures 5, 6 and 7) .
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    The first of the plants evaluated was corn. The corn inoculated with CRZM151 showed a 40% increase in the root surface compared to the control without inoculation, which means an increase in the capacity of the plant to absorb nutrients and water by greatly increasing the useful surface of absorption of the root (Figure 8). The central stem of the corn is an axis formed by knots and internodes whose number and length vary significantly in the inoculated plants. The internodes have an active zone in which new tissues are produced, in this way, the stem of the corn can increase rapidly in length during the growth period, that is why the greater number of knots is of great importance, than in the present case It was 30% higher in the inoculated plant. The stem size of the inoculated plants was 10% higher, which gave them greater bearing and plant resistance. It was also observed that the air part of the inoculated plants was much greater in the early stages of growth.
    The roots of the carrots inoculated with strain CRZM151 had a length greater than the roots of the control carrots that did not speak were inoculated. Specifically, the length of the roots of the inoculated carrots was 15% greater than those that do not speak have been inoculated. These carrots also showed an increase in the contour of the ralz compared to the uninoculated carrots, whose average diameter was lower (Figure 9). In addition, control carrots have a lower air bearing than those other carrots treated with strain CRZM151, in which a 10% increase in leaf length was observed, increasing the photosynthetic surface and therefore the ability to synthesize organic matter. This fact is related to the increase in dry weight observed in the inoculated carrots corresponding to 10% with respect to the control carrots.
    Finally, in the case of broccoli, a larger size of the leaves was observed in the inoculated plants, with a leaf area superior to the control plants without inoculation. They also presented superior root development than negative controls without inoculation, since it directly influences a greater capacity to absorb nutrients (Figure 10).
    III - CONCLUSION
    The strain CRZM151 {Rhizobium leucaenae CECT8306) is an endosymbiotic bacterium of Zea mays capable of promoting foliar and root development in plants of Zea mays, Brassica oleracea italica and Daucus carota, actively colonizing the rhizosphere of plants and acting as an endophyte promoter of plant growth in 5 of them, so it is likely to be used as a biofertilizer in crops of these plants.
    权利要求:
    Claims (19)
    [1]
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    1. Rhizobium leucaenae strain deposited in the Spanish Type Crop Collection with the deposit number CECT 8306.
    [2]
    2. A strain derived from a strain according to claim 1, capable of increasing or promoting plant growth.
    [3]
    3. A biologically pure culture of the strain according to claim 1 or 2.
    [4]
    4. A composition comprising a strain of Rhizobium leucaenae according to claim 1 or 2.
    [5]
    5. Composition according to claim 4, wherein the concentration of the Rhizobium leucaenae CECT 8306 strain is 1x108 CFU / mL and 3.8x108 CFU / mL, in particular between 1.8x108 CFU / mL and 3.6x108 CFU / mL, more particular, between 2x108 CFU / mL and 3x108 CFU / mL, even more particular, 2.5x108 CFU / mL.
    [6]
    6. Composition according to claim 4 or 5, further comprising another endophthetic organism, preferably, a bacterium of the Rhizobium genus different from the Rhizobium leucaenae strain deposited in the Spanish Type Culture Collection with the CECT deposit number 8306.
    [7]
    7. Composition according to any of claims 4 to 6, further comprising macroelements and / or microelements.
    [8]
    8. Composition according to claim 7, wherein the macroelements are selected from the group consisting of nitrogen salts, phosphorus salts, potassium salts, calcium salts, magnesium salts, sulfur salts and combination thereof.
    [9]
    9. Composition according to claim 7 or 8, wherein the microelements are selected from the group consisting of iron salts, zinc salts, copper salts, manganese salts, molybdenum salts, boron salts, chlorine salts and combinations of them.
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    [10]
    10. Use of a composition according to any of claims 4 to 9 as a biofertilizer.
    [11]
    11. Use of a strain according to revindication 1 or 2, or a composition according to any of claims 4 to 9, to increase or promote plant growth.
    [12]
    12. Use according to revindication 11, in which the plants are selected from crop plants and ornamental plants.
    [13]
    13. Use according to revindication 12, in which the crop plants are non-leguminous plants.
    [14]
    14. Use according to revindication 13, in which the non-leguminous cultivation plant is selected from the group consisting of a carrot plant, a broccoli plant and a malz plant.
    [15]
    15. Method to increase or promote the growth of plants comprising:
    (a) contacting a plant or the seed of said plant with a strain of Rhizobium leucaenae according to revindication 1 or 2, or with a composition according to any of claims 2 to 7, and
    (b) develop the seed or plant of stage (a).
    [16]
    16. Method according to revindication 15, wherein the contact between the plant or seed and the Rhizobium leucaenae strain according to revindication 1 or 2, or the composition according to any of claims 4 to 9, is carried out through irrigation, spraying, spraying, coating, fumigation, immersion or impregnation water.
    [17]
    17. Method according to revindication 15 or 16, in which the plants are selected from crop plants and ornamental plants.
    [18]
    18. Method according to revindication 17, in which the crop plants are non-leguminous plants.
    [19]
    19. Method according to claim 18, wherein the non-leguminous crop plant is selected from the group consisting of a carrot plant, a broccoli plant and a malz plant.
    5 20. A seed comprising a strain according to claim 1 or 2.
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