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    • 专利摘要:
    Klebsiella michiganensis strain to be used as a plant growth stimulant, as a means of biological control and as a rooting agent. The present invention refers to a bacterial strain of the species Klebsiella michiganensis CECT 9633 and its use as a plant growth stimulant, as a means of biological control and/or as a rooting agent. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2783098A1
    申请号:ES201930234
    申请日:2019-03-13
    公开日:2020-09-16
    发明作者:Cano José Manuel Hernandez
    申请人:Hernandez Cano Jose Manuel；
    IPC主号:
    专利说明:

    [0002] FIELD OF THE INVENTION
    [0004] The present invention falls within the field of plant physiology. It particularly refers to a bacterial strain of the species Klebsiella michiganensis CECT 9633 and its use as a plant growth stimulant, as a means of biological control and / or as a rooting agent.
    [0006] STATE OF THE ART
    [0008] The current market trend is to produce natural (organic) products that replace or reduce in the medium term the excessive use of chemical fertilizers and their negative effects on the environment, for example, the contamination of groundwater, rivers and lakes. It is known and studied that in the soil there are a diversity of microorganisms interacting positively with plants and are capable of enhancing their growth through activities known as PGPR (Plant Growth Promoting Rhizobacteria), such as fixation. of nitrogen in free life, solubilization of phosphate, production of siderophores, synthesis of ACC deaminase to inhibit the synthesis of ethylene, production of phytohormones that develop root elongation, etc.
    [0010] The most well-known PGPRs activities are those that have been detected in this work, within the many beneficial interactions between plant-microorganisms, we focus on:
    [0012] • Production of indole acetic acid (IAA): among the different compounds produced by the soil-plant interaction are phytohormones, simple organic molecules that act as chemical messengers, control the growth and development of the plant, respond to environmental changes and regulate the expression of certain genes of the plant. IAA is an auxin natural present in most plants, which, among other effects, influence growth, cell division and root formation
    [0013] Nitrogen fixation: nitrogen is the most important element in agriculture, in fact, the main products are called NPK (Nitrogen + Phosphorus + Potassium) that are added artificially to the soils so that the plant assimilates these compounds, the Fixation of atmospheric nitrogen (N2) is an essential process for the development of plants since it is part of molecular structures such as proteins, nucleic acids, hormones, etc. There are bacteria that use enzymes capable of capturing nitrogen from the atmosphere and passing it to the plant.
    [0014] ACC-deaminase activity: the activity of the deaminase enzyme of 1-aminocyclopropane-1-carboxylic acid (ACC) or ACC deaminase is a mechanism used by some rhizobacteria to promote plant growth, said activity results in the decrease of the concentrations of ethylene. in the plant and in the increase in the availability of ammonia in the rhizosphere. Ethylene stops the elongation of the root, induces the production and elongation of adventitious roots, increases the speed of flower senescence and promotes the absorption of fruits, which causes the premature death of the plant. The intervention of microorganisms plays a fundamental role in reducing these amounts of ethylene through ACC deaminase activity.
    [0015] Production of Siderophores: iron is an essential nutrient for most living organisms, involved in different functions such as enzyme cofactor, oxygen transport, DNA synthesis, nitrogen fixation, etc. In the soil, most of the iron is found in the form of ferromagnesium silicates, iron hydroxides or oxides, forms that are not easily assimilated by plants. Rhizobacteria that act like PGPR are capable of producing siderophores that keep the metal in solution making it bioavailable to plants.
    [0016] Biocontrol: siderophores are a mechanism for the biocontrol of pathogens since there are elements that, due to their low availability, represent a competition between plants, fungi and bacteria. In order to acquire these elements, bacteria use the production of siderophores as a mechanism for their incorporation.
    [0017] • Phosphate solubilization: phosphorus (P) is, after N, the inorganic nutrient most required by plants and microorganisms and, in addition, in the soil it is the limiting factor of plant development because, generally, it is present in non-organic forms. available for plants. There is a large amount of P in the soil, approximately between 400 and 1,200 mg / kg of soil, but its bioavailability is scarce since it is found as part of minerals such as apatite or in organic form such as phytate, phosphomonoesters and phosphotriesters. Bacteria capable of solubilizing phosphates play a key role in optimizing the availability of P to the plant, which could result in increased crop yields.
    [0019] Biofertilizers used for plant development, and application in cultivation, are based on finding those microorganisms whose PGPR activities are the most suitable for developing a fertilizer based on rhizobacteria. Main problems of microbial inoculants for their use are susceptibility to the environment, soil conditions, pH changes, protozoan predation and competition with native bacteria.
    [0021] Thus, the success of the use of these biopreparations resides in the study of compatible strains that are often specific to a crop and the ecological conditions of the soil.
    [0023] In particular, in the present invention, the microorganisms from the Capsicum annuum rhizosphere have been isolated, based on the different PGPRs activities, in order to prepare a biofertilizer that can be used as a plant growth stimulant, a biological control medium and / or as a rooting agent.
    [0025] DESCRIPTION OF THE INVENTION
    [0027] Brief description of the invention
    [0029] In order to obtain a product capable of stimulating plant growth, in the present invention, bacteria were isolated from the soil, specifically from the area of influence of the root, and their PGPRs properties were studied to produce a product. final based on crop deficiencies. In this case, the best results were obtained with the bacterium Klebsiella michiganensis CECT 9633 that promotes the rooting of the plant and presents qualities as a biostimulant.
    [0031] Said strain Klebsiella michiganensis CECT 9633 was deposited in the Spanish Type Culture Collection (CECT) on March 15, 2018.
    [0033] Thus, within the present invention, an inoculant has been developed from a rhizobacterium isolated in the rhizosphere of the red pepper variety Billis ( Capsicuum annuum) capable of stimulating the growth of roots through the production of IAA. IAA is a natural auxin secreted by bacteria associated with the root of the crop that, among other effects, influences growth, cell division and root formation. The composition developed in the present invention contains a bacterial formulation that emits high amounts of IAA, and outperforms many bacterial species described in the literature. After carrying out tests under different conditions and culture media for the development of said bacterium, it has been observed that it has a wide growth range without affecting its IAA production capacity. The results in laboratory and field tests with different varieties of crops allow us to conclude that their activity favors the formation of roots in a short period of time, allowing the elaboration of a rooting agent that is useful for all types of greenhouse crops.
    [0035] Furthermore, as shown in the examples, the strain of the invention can be used as a plant growth stimulant, a biological control medium, as well as as a rooting agent.
    [0037] Therefore, the first aspect of the present invention refers to a bacterial strain of the species Klebsiella michiganensis with deposit number in the Spanish Collection of Cultures Type CECT 9633 or strain whose DNA sequence of the 16S rRNA gene has a percentage of identity of at least 95%, 96%, 97%, 98%, or 99%.
    [0039] The second aspect of the present invention refers to a culture medium for microorganisms (liquid or solid) comprising said bacterial strain and any molecule, metabolite or substance secreted into the medium by said strain.
    [0040] The third aspect of the present invention refers to a composition comprising said bacterial strain or said culture medium.
    [0042] The fourth aspect of the present invention refers to the use of said strain, culture or composition to stimulate plant growth, for the biological control or elimination of phytopathogenic organisms (bacteria, insects, fungi and / or nematodes) or as a rooting agent for stimulation of root growth.
    [0044] The fifth aspect of the present invention refers to a method to stimulate plant growth, for the biological control of phytopathogenic organisms or to stimulate rooting, which comprises putting said strain, culture or composition in contact with the plant to be treated.
    [0046] The sixth aspect of the present invention refers to the use of the Capsicuum annuum rhizosphere for obtaining and isolating Klebsiella michiganensis CECT 9633.
    [0048] For the purposes of the present invention, the following definitions are incorporated:
    [0050] • The term "comprising" is understood to include, but is not limited to, what follows the word "comprising". Therefore, the use of the term "comprising" indicates that the listed items are required, but other items are optional and may or may not be present.
    [0051] • By "consisting of" is meant to include, and is limited to, what follows the phrase "consisting of". Therefore, the phrase "consisting of" indicates that the listed items are required, and that no other items may be present.
    [0053] Description of the figures
    [0055] Figure 1 . Photograph with the optical magnifying glass of the bacterium of the invention Klebsiella michiganensis CECT 9633.
    [0057] Figure 2 . Production of IAA in different culture media (Bergensen, DF-salts and Nutritive Solution "SN"). The "Y" axis data are expressed in ug of IAA / ml, with the highest production of IAA being 57.85 ug / ml in Bergensen medium.
    [0058] Figure 3 . Color changes in Bergensen medium due to AIA production. From left to right A) Negative control, medium without bacteria; B) Klebsiella michiganensis CECT 9633; C) Brevibacillus Brevis BEA1 and D) Azospirillum oryzae COC8 (T). The figure below represents IAA production in different bacteria described as PGPR.
    [0059] Figure 4 . Color halo after 10 days of incubation. The halo represents the siderophores exuded by the bacteria. The culture medium of the Petri dish is solid CAS medium. The image corresponds to a Petri dish where Klebsiella michiganensis CECT 9633 was grown where "A" represents the iron chelation halo of the culture medium due to the presence of siderophores, and "B" the growth of Klebsiella michiganensis CECT 9633 in the center of the plate. This image makes it possible to measure the radius of the halo of siderophore production and the radius of bacterial growth in centimeters, being able to establish a percentage of siderophore production efficiency.
    [0060] Figure 5 . From left to right the different colorations due to the production of siderophores are observed. When adding the CAS reagent, if the iron in the culture medium has been chelated, it changes the dark green color to an orange, the intensity of the color depends on the amount of iron chelated. The cuvettes correspond to A) Control (without bacteria) B) Achromobacter xylosoxidans NBRC 15126 (T) C) Klebsiella michiganensis CECT 9633 D) Psudomonas aeruginosa JCM 5962 (T). In addition, the amount of siderophores produced is represented in the lower graph, the units of siderophores produced per mg of protein are represented (Y-axis).
    [0061] Figure 6 . Color changes in the medium due to the production of ACC deaminase. From left to right A) Control (without bacteria), B) Psudomonas aeruginosa JCM 5962 (T) 1 C) Achromobacter sp D) Klebsiella michiganensis CECT 9633. In addition, the data is shown in the bar graph, where the X axis represents bacteria with the ability to produce ACC, while the Y axis represents the amount of the ACC-deaminase enzyme produced, expressed in ((nmol to ketobutyrate / mg * protein * h).
    [0062] Figure 7. Color change in the PVK culture medium as a consequence of the decrease in pH. As examples are shown from left to right: A) Control, B) Achromobacter xylosoxidans NBRC 15126 (T), C) Azospirillum oryzae COC8 ( T) and D) Klebsiella michiganensis CECT 9633.
    [0063] The lower graph shows the amount of soluble phosphate in the culture medium, the "Y" axis shows the amount of PO4 "dissolved in the culture medium, expressed in | ig PO4" x mg protein "1.
    [0064] Figure 8 . Development of secondary roots seen through the optical magnifying glass after plant growth at 10 days of incubation. Image A) shows the seed treated with water, image B) shows the seed treated with Klebsiella michiganensis CECT 9633.
    [0065] Figure 9 . Red pepper growth at 4 months, the plants were inoculated with PGPRs from different bacteria. From left to right: Klebsiella michiganensis CECT 9633; Azospirillum oryzae COC8 ( T), Achromobacter xylosoxidans NBRC 15126 (T); all together and finally the control (Without bacteria).
    [0066] Figure 10 . Growth measurements at 4 months of inoculation: A) Control (No bacteria), B) Azospirillum oryzae COC8 ( T); C) Klebsiella michiganensis CECT 9633; D) Achromobacter xylosoxidans and E) All strains mixed.
    [0068] Figure 11 . Greenhouse rooting effect on C. annuum plants at different concentrations: A) plant without rooting, B, C and D different concentrations in increasing order of Klebsiella michiganensis CECT 9633.
    [0070] Figure 12 . Tomato growth at 4 months, the plants were inoculated with PGPRs from different bacteria. From left to right: Control (No bacteria); Azospirillum oryzae COC8 ( T) Achromobacter xylosoxidans NBRC 15126 ( T), Klebsiella michiganensis CECT 9633 and lastly all strains together.
    [0072] Figure 13 . Growth measurements at 4 months of inoculation, from left to right, A) Control (No bacteria), B) Azospirillum oryzae COC8 ( T); C) Klebsiella michiganensis CECT 9633, D) Achromobacter xylosoxidans NBRC 15126 ( T), and E) all strains together.
    [0074] Figure 14 . Effect of the field application of the bacteria as a rooting agent, after several months of inoculation a great abundance of roots was observed in the treated tomato plants.
    [0076] EXAMPLES
    [0078] Example 1. Materials and Methods
    [0080] 1.1 Sampling
    [0082] In the greenhouse, the plants with the best condition were randomly selected. With the help of a shovel the surface soil was removed up to 12 cm approximately, the secondary roots were cut off and placed in a pot with a damp paper. They then moved to the laboratory.
    [0084] 1.2. Isolation of bacteria from the rhizosphere of C. annumm
    [0086] After removing most of the soil adhering to the roots, these were cut into pieces of about 0.5 cm to weigh 0.25 g and were washed with saline solution by vortex. The supernatant was collected and transferred to a 2 ml Eppendorf tube where the heavier pieces of soil were allowed to separate by decantation.
    [0088] Serial dilutions were made from the supernatant, and they were inoculated into Petri dishes (100 µl) containing different culture media and left to incubate at 30 ° C until the appearance of the bacteria. For the isolation of nitrogen-fixing bacteria, the procedure described by [Castellano-Hinojosa et al 2016. Isolation of N2-fixing rhizobacteria from Lolium perenne and evaluating their plant growth promoting traits was used; Antonio Castellano-Hinojosal, David Correa-Galeote, Josep Palau and Eulogio J. Bedmar; J. Basic Microbiol. 2016, 56, 85-91].
    [0090] After independent colonies appeared in the different culture media where the rhizobacteria from C. annuum were isolated, the colony-forming units (CFU) were morphologically characterized. For this, the morphology of the same was observed by optical microscopy ( Figure 1 ). With the help of a sterile toothpick, the different colonies were picked and seeded in TSA medium for 24 hours at 30 ° C to observe in isolation if they presented the same morphology among those selected.
    [0092] 1.3. Culture media
    [0093] 1.3.1. Tryptic Soy Broth / Agar Medium (TSB / TSA). Medium rich in nutrients routinely used for the growth and preservation of bacteria:
    [0098] Casaminoacid medium (CAS), It has been used for the qualitative determination of the production of siderophores. It is a culture medium that allows, through coloration, to know if the bacteria is capable of chelating iron and making it available to the plant.
    [0099] Culture medium Succinate Medium (SM), is a poorly nutritious medium used for the quantitative production of siderophores, it allows bacterial growth under minimal conditions and thus quantifies the siderophores formed when it is difficult to grow.
    [0100] Potate Dextrose Agar (PDA), a culture medium used for the growth of fungi, was used in the biocontrol tests.
    [0101] Culture media DF Salts (DF Salts), developed by Dworkin and Foster, hence its name, is a medium with the basic nutrients for the growth of bacteria, it is a defined or minimal medium, it was used to determine the basal capacity to produce ACC deaminase. ACC deaminase is an enzyme produced to inhibit the synthesis of ethylene, and therefore avoid or reduce stress in plants.
    [0102] Pikovskaya medium (PVK), culture medium used to verify the ability to solubilize tricalcium phosphate in bacteria, bacteria that are capable of growing in this poorly nutritious medium exude acidic compounds that reduce the pH of the medium and change its color, indicating that the insoluble phosphate (Tricalcium Phosphate) is being solubilized and therefore in a form available to plants.
    [0103] Bergersen culture medium, culture medium used to check the ability to produce indole acetic acid in bacteria, it is a minimum medium with sufficient nutrients for the growth of bacteria, in such a way that the indole acetic acid obtained is the one that the bacterium produces in basal conditions.
    [0104] 1.4. Production of Indole Acetic Acid (IAA)
    [0106] To determine AIA production, the methodology described by [Gravel et al. (2007). Gravel V, Antoun H, Tweddell RJ. (2007). Growth stimulation and fruit yield improvement of greenhouse tomato plants by inoculation with Pseudomonas putida or Trichodermaatroviride: possible role of indole acetic acid (IAA). Soil Biology and Biochemistry. 39: 1968-1977].
    [0108] • Each rhizobacteria was cultured in 3 ml of Bergersen minimal culture medium (between 1 and 2 days depending on each strain) until a bacterial density of approximately 108 cells / ml was reached. The culture was centrifuged for 3 minutes at 13,000 rpm.
    [0109] • Cell pellet was separated and stored to determine protein concentration. 0.5 ml were taken from the supernatant and transferred to a 1.5 ml microtube.
    [0110] • 1 ml of Salkowski's reagent was added [Gordon and Weber, 1951) Gordon SA, Wirber RA. (1951). Colorimetric estimation of indolacetic acid. Plant Physiol.
    [0111] 26: 192-195], and was mixed by inversion. 3 replicates per strain were used.
    [0112] • It was transferred to 4 ml spectrophotometry cuvettes and allowed to incubate for 20 minutes in the dark at room temperature.
    [0113] • To determine the intrinsic production of IAA production, the absorbance at 535 nm was determined. Using as a reference 0.5 ml of Bergensen liquid culture medium, and 1 ml of Salkwoski reagent was added.
    [0114] • Skawolski reagent colors range from pastel pink to dark orange due to reaction with reagent and IAA.
    [0115] • For this test, Brevibacillus brevis [Moreno et al.
    [0116] 2009). Solvent tolerance acquired by Brevibacillus brevis during an olive-waste vermicomposting process, Beatriz Moreno Astrid Vivas Rogelio Nogales Emilio Benitez, Ecotoxicology and Environmental Safety Volume 72, Issue 8, November 2009, Pages 2109-2114].
    [0117] • To purchase the absorbance data, a standard curve was established.
    [0118] • A stock of (2 mg / 20 ml) of IAA was used, from which different concentrations were taken to establish a standard curve from 0 to 20 p, g / ml of IAA.
    [0119] 1.5. Qualitative determination of siderophore production
    [0121] It was carried out in the manner described by [Schwyn, B, Neilands, JB. (1987). Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry. 160: 47-56]. The process comprises the following steps:
    [0123] • Cultivate each rhizobacteria, independently, in TSB medium for approximately 24 h, under constant shaking at 120 rpm until reaching an optical density of approximately 108 cells / ml.
    [0124] • Pour the CAS medium into Petri dishes, 3 replicates per bacteria, and wait for it to solidify.
    [0125] • Inoculate 4 pl of culture in the central zone of the Petri dish.
    [0126] • Incubate for 21 days at 30 ° C.
    [0128] Every two days, the halo of siderophore production and the size of the colonies were measured in cm. In this way, the efficiency of the production of siderophores (EPS) was determined using the following equation:
    [0133] Where dS = diameter of the siderophore and dC = diameter of growth of the halo
    [0135] Fe 3+ stains the agar blue. When the siderophores are combined with Fe 3+ there is a color change from blue to orange. Therefore, the orange halos around the colonies indicate the excretion of siderophores.
    [0137] 1.6. Quantitative determination of siderophore production
    [0139] The methodology described by [Sayyed et al. (2004), Sayyed R, Badgujar MD, Sonawane HM, Mhaske MM, Chincholkar SB. (2005). Production of microbial iron chellators (siderophores) by Pseudomonas fluorescent. Indian Journal of Biotechnol.4: 484-490] as follows. The procedure comprises the following steps: • Cultivate each rhizobacterium independently in test tubes with 3 ml of SM liquid medium, allow to grow between 24 and 72 hours (depending on each rhizobacterium), under constant stirring at 120 rpm until reaching an optical density of approximately 108 cells / ml (three replicates per rhizobacteria).
    [0140] • Centrifuge the cultures for 3 minutes at 13,000 rpm. to separate the cell pellet from the supernatant.
    [0141] • Mix 0.5 ml of CAS reagent with 0.5 ml of the supernatant and save the cell pellet to determine protein.
    [0142] • After mixing the CAS reagent with the supernatant, determine the absorbance at a wavelength of 630 nm.
    [0143] • As blank, use 1 ml of SM medium and 0.5 ml of SM culture medium and 0.5 ml of CAS reagent as reference.
    [0144] • To analyze the data obtained, the production of siderophores is calculated from the following equation:
    [0145] Production of siderophores
    [0146] Where AR = reference absorbance at 630 nm and AS = sample absorbance at 630 nm.
    [0148] 1.7. Phosphate solubilization
    [0150] To establish the solubilization efficiency of the insoluble phosphate, 2 experiments were carried out. In both cases, tricalcium phosphate (Ca3 (PO4) 2 was used as a source of phosphate.
    [0152] -Qualitative determination of inorganic phosphate solubilization:
    [0154] It was carried out according to the methodology described by [Peix A, Rivas-Boyero AA, Mateos PF, Rodríguez-Barrueco C, Martínez-Molina E, Velázquez E. (2001). Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobiummediterraneum under growth chamber conditions. Soil. Biol.Biochem. 33: 103-110] using the YED-P culture medium. The procedure comprises the following steps:
    [0155] • Cultivate the bacteria independently from glass tubes, in 3 ml of TSB liquid culture medium, for 24 hours, at 120 rpm at 28 ° C, until reaching a battery suspension of approximately 108 cells / ml.
    [0156] • Once the rhizobacteria have grown, wash the culture in saline solution at 13,000 rpm for 3 minutes. Remove the supernatant and resuspend the pellet in 1 ml of saline. Repeat twice.
    [0157] • Place 3 µl of the supernatant in the center of a Petri dish with YED-P.
    [0158] • Incubate at 28 ° C for 12 d.
    [0159] • Every 2 days the growth diameter (DC) of the bacteria and the diameter of the solubilization halo (DS) were determined.
    [0161] Azospirillum brasilense was used as a control strain (Cárdenas et al. 2010). The formula used to calculate the efficiency of the solubilization of phosphate (E) was that indicated by Nguyen et al. (1992):
    [0166] -Quantitative determination of inorganic phosphate solubilization:
    [0168] The procedure comprises the following steps:
    [0170] • Cultivate the bacteria, independently, in 3 ml of TSB for 24 h at 120 rpm until obtaining an approximate growth of 108 cells / ml.
    [0171] • Inoculate the bacteria (1: 1000, v / v) into 100 ml flasks with 50 ml of PVK liquid medium.
    [0172] • Every five days take 5 ml samples from the flask and centrifuge at 500 rpm for 30 seconds.
    [0173] • Take the supernatant and centrifuge for 3 minutes at 12,500 rpm.
    [0174] • Store the cell pellet at -20 ° C to determine protein.
    [0175] • Determine the pH and phosphate content of the supernatant.
    [0177] The determination of soluble phosphate was carried out as indicated by [Murphy and Riley (1962): Murphy J, Riley JP. (1962). A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27: 31-6].
    [0178] -Micromethod to determine phosphates (PO4) -2. The procedure comprises the following steps:
    [0180] • Place 30 µl of reagent mix on a microplate. Next, 250 µl of sample is added.
    [0181] • Incubate at room temperature for 10 minutes (color stable up to 2 hours).
    [0182] • Read absorbance at 655 nm on a microplate reader.
    [0184] -Preparation of the reactive mixture:
    [0186] The reaction mixture is based on the mixture of 4 solutions:
    [0188] • Solution A: Weigh out 1.5 g of ammonium heptamolybdate [(NH4) 6Mo7O24 x 4 H2O] and dissolve it in 50 ml of deionized water. Store in plastic containers.
    [0189] • Solution B: place 14 ml of concentrated sulfuric acid (density = 1.82) in 90 ml of deionized water, and allow the mixture to cool to avoid spontaneous reaction when mixed with the other solutions. Store in a glass container.
    [0190] • Solution C: Dissolve 2.7g of ascorbic acid in 50 ml of deionized water. • Solution D: dissolve 0.034 g of antimony potassium tartrate in 25 ml of deionized water. Store in a plastic container.
    [0192] The procedure includes the following steps: mix 10 ml of solution A with 25 ml of solution B, then mix with 10 ml of solution C and, finally, with 5 ml of solution D. The mixture is stable for 5- 6 months.
    [0194] -Preparation of the standard curve of (PO4) -2:
    [0196] The procedure comprises the following steps: Dissolve 0.0816 g of KH2PO4 in 100 ml of deionized water. Add 1 ml of chloroform and store in a container Dark. The concentration (PO4) -2 in this solution is 6 mg / l, from which dilute solutions of different concentrations are prepared.
    [0198] The rationale for this test is that the decrease in pH due to phosphate production by bacteria is associated with the change from purplish to blue color of bromophenol.
    [0200] 1.8. Acetylene to Ethylene Reduction
    [0202] The procedure comprises the following steps: growing the rhizobacteria independently in glass tubes, in 3 ml of TSB liquid culture medium, for 24 hours, at 120 rpm at 28 ° C, until reaching a battery suspension of approximately 108 cells / ml.
    [0204] Once the rhizobacteria have grown, wash the culture in saline at 13,000 rpm for 3 minutes. Remove the supernatant and resuspend the pellet in 1 ml of saline. Repeat twice.
    [0206] Inoculate in a nitrogen-free medium (Burk) until reaching an OD 0.03-0.05. The test tube is covered with a septum and left to incubate at 30 ° C under constant agitation of 120 r.p.m for the time necessary to reach an OD of 0.3-0.5 nm.
    [0208] After reaching the desired O.D., 10% of the atmosphere in the tube is replaced by acetylene. Samples of the gas (250 µl) are analyzed for 24 h - 10 days using a Hewlett Packard gas chromatograph equipped with a flame detector and a metal column (180x3.2 mm), respectively.
    [0210] Nitrogen was used as a carrier gas. Ethylene concentration was calculated by interpolation with respect to a standard curve using pure ethylene.
    [0212] 1.9. Determination of ACC deaminase production
    [0214] The determination of ACC deaminase activity was carried out as follows:
    [0215] -Qualitative:
    [0216] • Seed the rhizobacteria independently in Petri dishes, with TSA medium and incubate for 24 h at 30 ° C.
    [0217] • Seed the bacteria with the platinum loop in a single Petri dish with solid DF Salts medium supplemented with sterile 3mM ACC as the sole nitrogen source. Incubate at 30 ° C for 2 days.
    [0218] • Bacterial growth is indicative of ACC deaminase activity.
    [0220] -Quantitative:
    [0221] -Step A: ACC deaminase activity
    [0222] Take a Colony Forming Unit (CFU) with the seeding loop and inoculate into test tubes with liquid DF Salts medium (e.g. 3 ml) supplemented with 3 mM ACC. The procedure comprises the following steps:
    [0223] • Incubate for 24 hours at 30 ° C with constant shaking (120-150 rpm) until an optical density of 0.3-0.5 is reached at 600 nm absorbance.
    [0224] • Inoculate (1: 100; v: v) Falcon tubes with 25 ml of DF salts medium supplemented with 3 mM ACC and incubate again at 30 ° C (120-150 rpm).
    [0225] • After growth, centrifuge the Falcon tubes at 6000 rpm for 10 min at 4 ° C and remove the supernatant.
    [0226] • Resuspend the cell pellet in 5 ml of 0.1 M Tris-HCL at pH 7.6.
    [0227] • Centrifuge the Falcon tubes at 6000 rpm for 10 min at 4 ° C and remove the supernatant
    [0228] • Wash the cells again in 5 ml of 0.1 M Tris-HCL at pH 7.6.
    [0229] • Wash the cells again in 5 ml for one last time in 0.1 M Tris-HCL at pH 7.6.
    [0230] • Store the Falcon tubes with the cell pellet at -20 ° C until later use.
    [0232] -Step B: Determination of ACC deaminase activity based on acetobutyrate production.
    [0233] Once we have the cell pellet, three replicates per strain, the following steps were performed:
    [0234] • The cell pellet obtained in the previous process is resuspended in 1 ml of 0.1M Tris-HCL at pH 7.6 and transferred to a 1.5 ml eppendorf tube.
    [0235] • Centrifuge at 13,000 rpm for 3 minutes and remove the supernatant.
    [0236] • Resuspend the cell pellet obtained in 600 µl of 0.1 M Tris-HCL at pH 8.5, and add 30 µl of toluene.
    [0237] • Resuspend with the help of a micropipette and vortex for 30 s. • Transfer 200 µl of the toluenized cells to 1.5 ml microtubes and add 20 µl of sterile 0.5M ACC. Vortex and incubate at 30 ° C for 15 minutes.
    [0238] • Add 1 ml of 0.56 M HCl, vortex briefly and centrifuge at 1300 rpm for 3 minutes.
    [0239] • Take 1 ml of the supernatant (the rest is saved for protein determination) and add 800 µl of 0.56 M HCL. Shake manually (use test tubes to subsequently measure absorbance).
    [0240] • Add 300 µl of 2,4-dinitrophenylhydrazine (0.2% of 2,4-dinitrophenylhydrazine in 2M HCl) and incubate at 30 ° C for 30 minutes.
    [0241] • Prepare the blank for absorbance determination by mixing 600 µl of 0.1 M Tris-HCL at pH 8.5, 30 µl of toluene, 1 ml of 0.56 M HCl and 300 µl of 2.4-dinitrophenylhydrazine.
    [0242] • Add 2 ml of 2 N NaOH. Mix and determine the absorbance of the mixture at 540 nm.
    [0244] To normalize the absorbance data, a standard curve was obtained from which the results were obtained.
    [0246] 1.10. Genomic DNA extraction
    [0248] The rhizobacteria were cultured in 3 mL of TSB liquid medium at 120 rpm, the adequate time for each of them to reach the final phase of logarithmic growth (24 h). The bacterial cultures were centrifuged at 12,000 rpm for 3 minutes and the pellet obtained was kept until use. To obtain genomic DNA, used the commercial Real PureGenomic DNA Extraction Kit, from Durviz, as follows:
    [0250] • Resuspend the cell pellet from the culture in 500 µl of extraction buffer®.
    [0251] • Add 100 µl of PVP® solution and vortex for 20 s.
    [0252] • Add 60 µl of lysis solution and 3 µl of RNase. Vortex the mixture vigorously and incubate at 37 ° C for 30 min.
    [0253] • Cool the samples to room temperature and add 250 µl of protein precipitation buffer®. Vortex vigorously for 20-30 s, and incubate at -20 ° C for 10 minutes.
    [0254] • Centrifuge at 13,000 r.p.m. for 5 minutes. Check the appearance of protein precipitate.
    [0255] • Collect the supernatant, approximately 600 µl, containing the DNA, in a microtube with 600 µl of isopropanol previously cooled to -20 ° C. Mix • gently by inversion and centrifuge again at 14,000 r.p.m. for 3 minutes.
    [0256] • Remove the supernatant and wash the pellet with 600 µl of 70% ethanol.
    [0257] Mix again by inversion. Centrifuge at 14,000 r.p.m. for 2 minutes.
    [0258] • Eliminate ethanol and air dry by inverting the tube on absorbent paper for 15 minutes.
    [0259] • Resuspend the precipitate in 20 µl of sterile distilled water. Favor the
    [0260] • DNA suspension by immersion of the tube in water at 65 ° C for 1 hour.
    [0262] Shake periodically by hand to ensure complete resuspension of the DNA
    [0264] 1.11. DNA quantification
    [0266] A NanoDrop spectrophotometer, model ND1000 (Thermo Fisher Scientific, USA) was used. The DNA concentration (se ng / pl) and the purity of the preparations were estimated by determining the ratios of the optical densities at 260nm / 280nm and 260nm / 230nm 1.12. 16S rRNA gene amplification
    [0268] The 16S rRNA gene was amplified by PCR using the primers fD1 and rD1 described by [Weisburg WG, Barns SM, Pelletier DA, Lane DJ. (1991). 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173: 697-703].
    [0270] The mixture that was used for the reaction was that described in Table 1 :
    [0272] Table 1
    [0274] R E A C T IV O V O L U M E N (m i) C O N C E N T R A T IO N F IN A L B u ffe r (10X re a tio n T am p on) 2.5 IX
    [0275] M g C E (50 m M) 1.5 1.5 m N
    [0276] d N T P s (1 O m M) 1 200 p M
    [0277] f D l (15 p m o l / p l) 1 0.6 p ic o m o l / p l r D l (15 p m o l / p l) 1 0.6 p ic o m o l / p l
    [0278] T a q A D N p o lim era sa (S ig m a) 0.2 0.08 U
    [0279] A D N 1 20 - 100 ng
    [0280] A g u a M Q (M illip o re) Up to 25
    [0282] The amplification reaction was carried out in a thermal cycler (Eppendorf) operating under the conditions indicated in Table 2 :
    [0284] Table 2
    [0287] Initial D esaturation 1 95 9 minutes D esaturation 95 60 sec on two Alignment 30 60 60 sec on two Elongation 72 2m inutes E xtension 1 72 7 m inutes M anten im inet QO 4 QO The PCR products (5 pl) were supplemented (4: 1 v / v) with loading solution (SC, 40% sucrose and 0.25% bromophenol blue) and were used to load 0.7% agarose gels Prepared in TBE buffer solution (trizma base, 10 g / l; boric acid 5.5 g / l and EDTA, 0.90 g / l, pH 8.5). Electrophoresis was carried out using a direct current of 80 V. The gels were stained with a GelRed ™ solution (1 plGelRed ™ / 1ml distilled water) and photographed under UV light. Molecular Marker III (Roche) was used as molecular size marker. The gels should be checked for the appearance of a DNA band with a molecular size of approximately 1500 base pairs (bp).
    [0289] 1.13. Purification of amplification products
    [0291] Amplified DNA products were purified using the commercial QIAquik PCR Product Purification KIT (QIAGEN®). For it:
    [0293] • Add 5 volumes of PB® solution to the PCR product. Mix by manual inversion.
    [0294] • Transfer the mixture to a purification column®, adapt the receiving tube and centrifuge at 13,000 r.p.m. for 1 minute.
    [0295] • Remove the liquid from the bottom of the receiver tube.
    [0296] • Add 570 µl of PE® solution previously supplemented with 220 µl of ethanol. Centrifuge at 13,000 r.p.m. for 1 minute.
    [0297] • Remove the liquid from the bottom of the receiver tube. Centrifuge again at 13,000 r.p.m. for 1 minute.
    [0298] • Place the column on a new microtube, add 20-50 μl of sterile distilled water in the center of the column, wait 1 minute and, finally, centrifuge at 13,000 r.p.m. for 1 minute.
    [0299] • The purified DNA was quantified in a NanoDrop spectrophotometer, model ND1000 (Thermo Fisher Scientific, USA).
    [0301] 1.14. 16S rRNA gene sequencing
    [0303] 0.5 ml microtubes were taken with 20-80 ng of DNA, the amount recommended for the sequencing of amplification products between 1000 and 2000 bp. 1 pl of the specific primer (6.4 pmoles) and MQ water to a final volume of 12pl The sequencing reactions were carried out in the DNA Sequencing Service of the Zaidín Experimental Station (CSIC State Agency, Granada) using a Perkin Elmer equipment, ABI PRISM 3130xl model, provided with 16 capillaries 80 cm long, using the commercial product AbiPrism (Perkin-Elmer) and Amplitaq FS DNA polymerase. The determination of the sequence was carried out by means of the system of terminators labeled with fluorochromes
    [0305] 1.15. Reading and identification of sequences
    [0307] The sequences received from the Sequencing Service were analyzed using the CHROMAS bioinformatics program and compared with the sequences deposited in the GenBank databases through the BLAST online program of the National Center for Biotechnology Information (NCBI) (http: //blast.ncbi.nlm .nih.gov / Blast.cgi). The Geneious 8.0.4 program was used to assemble the sequences and the EZTaxon server (http://www.ezbiocloud.net/eztaxon) was used to compare the sequences obtained with those of the corresponding type strains.
    [0309] 1.16. Seed tests
    [0311] To demonstrate the rooting capacity of the bacteria, the following test was carried out: red pepper seeds were used, and 3 were placed per Petri dish with a filter paper base.
    [0313] The bacteria were cultured 24 h in TSB culture medium until reaching the desired concentration. 1ml of bacteria was inoculated on the seeds and they were incubated at 30 ° C in the dark. During the first 10 days the length of the root was measured, and the development of root hairs of the seeds was observed with the magnifying glass. As a negative control, the culture medium without bacteria and sterile tap water were used.
    [0315] 1.17. Greenhouse Trials
    [0317] To check the promoting effect of the bacteria, a test was carried out under greenhouse conditions. For this, soil from an agricultural farm was used and it was used as substratum. 22 cm diameter pots were prepared and filled with approximately 15 kg of substrate. The pepper seeds were inoculated in the pots and it was waited until the plants reached about 4 cm in length for the inoculation of the bacteria. In this test, different bacteria with plant growth promoting properties were used, among which was the bacterium to be patented.
    [0319] To prepare the strains, a bacterial preinoculum was started, and it was allowed to grow until obtaining 109-108 CFU. They were washed in saline solution. And they were inoculated in the plants. With the help of a pipette, 1ml of each bacteria was taken and spread over the part between the stem and the root.
    [0320] It was grown for 4 months. Irrigation was carried out in the pot plate, depending on the needs of the crop.
    [0322] 1.18. Field trials
    [0324] In both greenhouse and field conditions, 2L of the bacterium was used per bacterium in rich culture medium and it was diluted in a 1000L tank with water, the inoculation was done by means of a drip irrigation system, through of 4mm diameter drippers.
    [0326] Example 2. Results
    [0328] 2.1. Indoleacetic acid production (IAA)
    [0330] The bacterium's ability to produce IAA was demonstrated from the synthesis of tryptophan, the bacterium has the ability to convert this tryptophan into a phytohormone capable of stimulating the radical development of the plant.
    [0332] After inoculating the bacteria in different culture media, its IAA production capacity was observed. Figure 2 shows the IAA produced by the bacteria, where the highest production value corresponds to the bacteria inoculated in Bergensen 57.85 | ig / ml medium of IAA, this is a minimum medium, therefore, the values obtained represent the basal production of IAA by the bacteria.
    [0333] Brevibacillus brevis BEA1 was used as a control strain, which produced an amount of 16.60 ug / ml of IAA. By way of example, Figure 3 shows the color changes in Bergensen medium as a function of the difference in AIA concentration and its representation on a bar graph.
    [0335] Notably, the inventive strain Klebsiella michiganensis CECT 9633 showed sufficiently high IAA concentrations to have a direct influence on plant growth.
    [0337] In addition, the growth of Klebsiella michiganensis CECT 9633 in all the culture media used allows us to ensure its development in any type of soil, since the composition of agricultural soils varies depending on the area, this strain will be able to easily obtain tryptophan from the soil and contribute it as IAA to the plant. Therefore, no matter how poorly the soil is in nutrients, the bacterium of the invention is capable of producing high amounts of IAA.
    [0339] Thus, the present invention demonstrates the intrinsic capacity of Klebsiella michiganensis CECT 9633 for the production of this phytohormone, since tryptophan was not added in the culture medium where they were inoculated.
    [0341] The results of this activity allow us to conclude that Klebsiella michiganensis CECT 9633 developed the elongation of the root part of the plant, the formation of secondary roots and therefore its extension in the soil, being able to capture the most distant nutrients and develop much more than other strains .
    [0343] Compared to other strains previously studied for their ability to produce IAA, our bacterium Klebsiella michiganensis CECT 9633 is significantly superior to other bacteria such as Bacillus methylotrophicus CKAM, with 34 | ig / ml of IAA production and used as PGPR in the agricultural industry or Azospirillum brasilense Az39, with a production of IAA (13.16 | ig / ml) known for its ability to stimulate plant growth.
    [0345] Furthermore, it has been reported in previous studies that the amount of bacterial IAA may be an important factor in promoting growth. Production of the phytohormone in the most active strains it is between 80 to 100 pg / ml, but the excess of IAA may cause negative effects to the plant due to the high concentrations of IAA. Our strain Klebsiella michiganensis CECT 9633 emitting 57.85 gg / ml does not pose a risk to the plant.
    [0347] 2.2. Qualitative determination of siderophore production
    [0349] After 12 days of growth in solid CAS medium, Klebsiella michiganensis CECT 9633 showed siderophore production. With a production percentage of 70% (see Figure 4 ) that reached its maximum value after ten days of cultivation.
    [0351] It is generally very abundant in soils, but its predominant chemical species is the Fe + 3 ion, a form that reacts to give oxides and hydroxides that are insoluble and therefore inaccessible to plants and microorganisms.
    [0353] The production efficiency of siderophores (EPS) was demonstrated, which allows to know qualitatively that Klebsiella michiganensis CECT 9633 has the ability to chelate the iron in the soil, which is predominantly found in the Fe + 3 ion, this Fe + 3 is a A form that reacts to give oxides and hydroxides that are insoluble and therefore inaccessible to plants and microorganisms. Thanks to the siderophores production capacity of Klebsiella michiganensis CECT 9633 is able to attract iron to the rhizosphere where it can be absorbed by the plant
    [0355] Furthermore, thanks to the rapid growth of Klebsiella michiganensis CECT 9633 in a poorly nutritious culture medium (Succinate Medium), we know that this bacterium will adapt to any environment and will be able to establish itself in the root zone of influence with ease, without being displaced. by the bacteria present in the soil, and once "hosted" it will be able to produce siderophores that benefit the development of the plant.
    [0357] Despite the absorption capacity of iron, the siderophores formed and exuded to the medium affect the competitiveness for nutrients by limiting or inhibiting the growth of other microorganisms, preventing the development of pathogens. Which tells us that Klebsiella michiganensis CECT 9633 can be used for the preventive biocontrol of phytopathogenic fungi that kill crops such as Fusarium sp, Phytoctora sp. etc.
    [0359] 2.3. Quantitative determination of siderophore production
    [0361] The strain produced 68.26 siderophore units and 355.31 siderophore units / mg ml'1 protein (see Figure 5 ). The strains isolated in this study produce with superiority the amount necessary for the stimulation of plant growth, and inhibition of pathogenic fungi.
    [0363] Compared with other strains of the genus Bacillus sp Paenibacillus and Burkholderia. , currently known and used for growth stimulation and pathogen biocontrol, Klebsiella michiganensis CECT 9633, showed a better ability to use these low molecular weight molecules to influence the uptake of iron and other metals thanks to this PGPR activity.
    [0365] With these results, it is evident that the Klebsiella michiganensis CECT 9633 strain meets all the requirements to stimulate plant growth in soils with a deficiency of Iron, and other heavy metals, and will also be able to prevent fungal infections of the crop in which apply.
    [0367] 2.4. ACC deaminase activity
    [0369] The ACC deaminase activity was qualitatively demonstrated in 16 bacteria isolated from C. annuum.However, in the quantitative test, only 3 rhizobacteria were shown to have ACC deaminase capacity ( Figure 6 ), Klebsiella michiganensis CECT 9633 showed 41.72 | imol-1 a mg protein -1 h-1 respectively. See Table 3 where the ACC deaminase activity of the strains isolated from the roots of C.annuum is shown.
    [0371] The strains were cultured in DF-Salts medium for 3 days. For the statistical analysis, the Kruska-Wallis test was used (P <0.05; n = 3). For the same column, the values followed by the same letter are not statistically significant. Values followed by the same letter are not statistically different. The strains are ordered according to the results of the statistical test, from lowest to highest ACC deaminase activity.
    [0372] Table 3
    [0374] Strain ACC deaminase activity (nmolacetobutyrate / mg * protein * h) Psudomonas aeruginosa JCM 5962 (T). 13.54c Achromobacter sp 23.52b
    [0375] Klebsiella michiganensis CECT 9633 41.72a
    [0377] Within the range of values obtained, Klebsiella michiganensis CECT 9633 can act as PGPR. The production of ACC deaminase allows the bacteria to inhibit the ethylene produced by the plant due to environmental stresses such as drought, salinity, etc., and therefore reduce its premature senescence.
    [0379] It is the first time that the ability of Klebsiella michiganensis CECT 9633 to produce ACC-deaminase has been shown.
    [0381] The amount of ACC produced in our studies allows us to know that Klebsiella michiganensis CECT 9633 is capable of adapting to areas where the plant is subjected to stress and inhibiting the production of abundant ethylene that can cause poor fruit development, elongation insufficient root, and premature death of the plant.
    [0383] 2.5 Biocontrol
    [0385] The bacterium Klebsiella michiganensis CECT 9633 shows the ability to carry out biocontrol (preventing or controlling the development of phytopathogenic organisms such as fungi) due to its ability to produce siderophores. Klebsiella michiganensis CECT 9633 is able to compete for soil nutrients thanks to its ability to produce siderophores, preventing the development of phytopathogens such as fungi. In addition, t he metabolites produced by Klebsiella michiganensis CECT 9633 inhibit the growth of fungi.
    [0386] 2.6 Quantitative determination of inorganic phosphate solubilization
    [0388] All strains solubilized phosphate, since a color change was observed due to the decrease in pH in the PVK medium during 15 days of culture ( Figure 7 ).
    [0390] The determination of soluble phosphate in the medium showed that only Klebsiella michiganensis CECT 9633 was able to solubilize phosphate in the medium. As a positive control, it is observed that A. brasilense produces 371.05 | ig PO4- x mg protein-1 ( Table 4 ) at 12 days of the experiment. Phosphorus is an essential element in the first development of plants. Thus, from this test it can be deduced that Klebsiella michiganensis CECT 9633 is capable of stimulating plant growth in its early stages, since to use it as a rooting agent, in addition to providing phytohormones to the crop, it is necessary that the insoluble phosphate that exists in the soil becomes soluble. Klebsiella michiganensis CECT 9633 demonstrates rooting ability through phosphate solubilization. Table 4 shows the soluble phosphate concentration (^ g PO4- x mg protein-1) in the supernatant of the bacteria isolated from the roots of C.annuum. The bacteria are incubated in PVK medium for 12 days. For statistical analysis, the Kruskal-Wallis test (P <0.05, n = 3) was used. For the same column, the values followed by the same letter are not statistically different.
    [0392] Table 4
    [0393] Strain Day 12 Achromobacter xylosoxidans NBRC 15126 (T) 42.79c Klebsiella michiganensis CECT 9633 313.69a Azospirillum oryzae COC8 (T) 71.66b Azospirillum brasilense 371.05a
    [0395] P is the second main element for plants, all fertilizers today have an NPK composition in different concentrations, thanks to this test we deduce that by applying Klebsiella michiganensisCECT 9633, the amount of P contributed to the crop can be reduced, and even substituted to chemical fertilizers and still the plant will obtain a greater development. Not only improving the solubility of phosphate in the soil and its bioavailability, but also lowering costs in the fertilization campaign.
    [0396] 2.7. Nitrogen Fixation
    [0397] Because the growth of bacteria in nitrogen-free media does not guarantee the ability of the strains to reduce atmospheric N2, the ability of the strains isolated in Burk medium (specific medium to isolate nitrogen-fixing microorganisms) to reduce acetylene to ethylene was confirmed. . Klebsiella michiganensis CECT 9633 showed a concentration of 58.65 nmol of ethylene x mg-1x protein-1.
    [0399] In comparison with other studies where PGPRs bacteria were isolated and different types of Azospirillum sp are studied, it allows us to deduce that Klebsiella michiganensis CECT 9633 has the ability to promote plant growth and provide nitrogen (the first essential element) to the plant.
    [0401] Therefore, we know that Klebsiella michiganensis CECT 9633 can be used to decrease or replace the nitrogen supplied as a chemical synthesis fertilizer.
    [0403] 2.8. Seed tests
    [0404] After the inoculation of the different treatments in the seeds, it was observed that the elongation of the main root was similar in the different treatments in the first ten days ( Table 5 ). Which, on the one hand, rules out the phytotoxicity of the bacteria for the seeds or, as in other cases, a delay in germination. Table 5 shows the root development after 10 days of treatment, the seeds were incubated in 1% water agar medium for 10 days. For statistical analysis, the Kruskal-Wallis test (P <0.05, n = 3) was used. For the same column, the values followed by the same letter are not statistically different.
    [0406] Table 5
    [0408] Length growth treatment
    [0409] root (cm)
    [0410] H 2 O 1.3a
    [0411] Klebsiella michiganensis CECT 9633 1.7a
    [0412] Culture medium 1.6a
    [0413] On the other hand, a significant change was observed in the production of secondary root hairs, which leads to think that the AIA produced was just for the production of new secondary roots instead of lengthening the taproot, since the secondary roots are the younger and more active. This allowed the roots to obtain more water from their environment ( Figure 8 ). This test demonstrates the ability to stimulate plant cell division, which affects the formation of root hairs, showing the rooting capacity of Klebsiella michiganensis CECT 9633.
    [0415] 2.9 Greenhouse test
    [0417] After demonstrating the efficacy of Klebsiella michiganensis CECT 9633 for rooting, this test demonstrated the ability to stimulate plant development using the bacterium to be patented as biostimulant / rooting agent.
    [0419] For this test, 5 treatments were used ( Figure 9 ) that demonstrated the efficacy of promoting plant growth of Klebsiella michiganensis CECT 9633. On the one hand, Azospirillum sp was used , as a positive control, Klebsiella michiganensis CECT 9633, Achromobacter xylosoxidans NBRC 15126 (T ), Azospirillum oryzae COC8 (T), and an uninoculated plant.
    [0421] After 4 months, weight and length measurements were carried out ( Figure 10 ) where the ability to promote plant growth was once again demonstrated.
    [0422] The ability of Klebsiella michiganensis CECT 9633 to improve the development of the red pepper plant was demonstrated in all cases.
    [0424] Klebsiella michiganensis CECT 9633 was the one that obtained the highest value in all the parameters measured, surpassing the other strains selected for this study. Compared with other studies with PGPRs, (Purple corn-associated rhizobacteria with potential for plant growth promotion; A. Castellano-Hinojosa, V. Pérez-Tapia, EJ Bedmar and N. Santillana; Journal of Applied Microbiology ISSN 1364-5072; 2018 , In comparison with other tests with PGPRs in horticultural crops, it is seen that Klebsiella michiganensis CECT 9633 is capable of increasing the growth rate in 4 months, reaching values equal to or higher than other tests with PGPRs.
    [0426] Furthermore, the plant growth promoting effects in this trial reinforce the elaboration of a biofertilizer with Klebsiella michiganensis CECT 9633, either as a single microorganism, or in consortium with other microorganisms that reinforce its deficiencies.
    [0428] If we look at the results of fresh root weight, they far exceed control (A), which again reinforces the elaboration of a rooting agent with this bacterium (see Figure 11 ).
    [0430] In addition, its PGPR effect was verified in another horticultural crop such as tomato, the results were similar to those of the pepper, the ability to promote plant growth under greenhouse conditions was demonstrated ( Figure 12, Figure 13 ).
    [0432] Therefore it can be deduced that this strain adapts with more than one type of culture to enhance development
    [0434] 2.10. Field test
    [0436] For this test, open field facilities were used, where 2L of product were mixed, for every 1000L of water, in an irrigation tank. Irrigation was opened for 1 hour. Irrigation was done by fertigation / drip so that the biostimulant seeps into the soil and reaches the roots, where the bacteria associated with the plant.
    [0438] Several plants were removed to see the rooting effect, and indeed the weight of the roots was significantly higher in those plants treated with Klebsiella michiganensis CECT 9633. In Figure 14 , the effect of the IAA contributed by the bacteria to the plant can be observed, and the response of the crop producing abundant secondary roots that allow the uptake of nutrients from more remote areas and their growth.
    [0439] 2.11. 16S rRNA gene amplification
    [0441] The partial sequencing amplification of the 16S rRNA gene allowed the comparison of its sequences with those of the type strains deposited in the Databases (BLAST) and in the EzTaxon-e. In this way the strain of the genus Klebsiella was identified . Table 6 identifies the strain isolated from the rhizosphere of Red pepper var. billis ( Capsisum annuum).
    [0442] Table 6
    [0444] Associated species according to the 16S rRNA gene% Identity Sequence length (bp) Klebseilla. michiganensis W14 (T) CECT 9633 99.80
    [0445] Klebsiella oxytoca JCM 1665 (T) 99.72
    [0446] 1.439 Citrobacter europaeus 97/79 (T) 98.67
    [0447] Raoultella ornithinolytica JCM 6096 (T 98.67
    权利要求:
    Claims (12)
    [1]
    1. Bacterial strain of the species Klebsiella michiganensis with deposit number in the Spanish Collection of Cultures Type CECT 9633 or strain whose DNA sequence of the 16S rRNA gene has a percentage identity of at least 95%, 96%, 97%, 98% or 99% with the CECT 9633 strain.
    [2]
    2. Microorganism culture medium comprising the strain of claim 1 and any molecule or substance secreted into the medium by said strain.
    [3]
    3. Composition comprising a bacterial strain according to claim 1 or a culture medium according to claim 2.
    [4]
    4. Use of the strain, culture or composition of claims 1, 2 or 3 to stimulate plant growth.
    [5]
    5. Use of the strain, culture or composition of claims 1, 2 or 3 for the biological control or elimination of phytopathogenic organisms.
    [6]
    6. Use according to claim 5, wherein the phytopathogenic organisms are bacteria, insects, fungi and / or nematodes.
    [7]
    7. Use of the strain, culture or composition of claims 1, 2 or 3 as a rooting agent or for stimulation of root growth.
    [8]
    8. Process for stimulating plant growth comprising contacting the strain, culture or composition of claims 1, 2 or 3 with the plant to be treated.
    [9]
    9. Method for the biological control of phytopathogenic organisms, comprising contacting the strain, culture or composition of claims 1, 2 or 3 with a plant affected by the phytopathogenic organism.
    [10]
    10. Process for stimulating the rooting of plants comprising contacting the strain, the culture or the composition of claims 1, 2 or 3 with the plant to be treated.
    [11]
    11. Process according to any one of claims 8 to 10, wherein the strain, culture or composition is applied by blanket irrigation, spraying or dripping.
    [12]
    12. Use of the rhizosphere of Capsicuum annuum to obtain and isolate Klebsiella michiganensis CECT 9633.
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    CN108587970A|2018-05-07|2018-09-28|长治学院|A kind of acid-producing Klebsiella bacterium and its application in promoting RADIX CODONOPSIS PILOSULAE from Shanxi of China's growth|
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    ES201930234A|ES2783098B2|2019-03-13|2019-03-13|KLEBSIELLA MICHIGANENSIS STRAIN TO BE USED AS A PLANT GROWTH STIMULANT, AS A MEANS OF BIOLOGICAL CONTROL AND AS A ROOTING|ES201930234A| ES2783098B2|2019-03-13|2019-03-13|KLEBSIELLA MICHIGANENSIS STRAIN TO BE USED AS A PLANT GROWTH STIMULANT, AS A MEANS OF BIOLOGICAL CONTROL AND AS A ROOTING|
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