Methylobacterium spp. strains, compositions containing same and their use as biostimulant and endophytic nitrogen fixing bacteria

Abstract

The present invention provides a strain of the genus Methylobacterium and a composition containing the same, a new species having a beneficial effect on crops and a composition comprising the same, and a strain of the genus Methylobacterium that can reduce the use of at least chemical nitrogen-based fertilizers. [Solution] The bacterial strain that achieves the above object is a strain of the genus Methylobacterium (Methylobacterium sp. nov.) deposited under accession number CECT9580. The present invention also relates to a compound containing the bacterial strain, and to uses thereof.

Description

[Technical field]

[0001] The present invention relates to the field of agriculture. In particular, Methylobacterium sp. NOV. The present invention relates to a strain of Methylobacterium sp. nov., compositions containing same and their use as plant biostimulant and endophytic nitrogen fixing bacteria. [Background technology]

[0002] Many fertilizers are known in agriculture. In particular, nitrogen-based fertilizers, such as ammonia, ammonium nitrate (NH4NO3), urea, etc., are commonly used to keep crop yields as high as possible. In fact, many biological processes in plants involve nitrogen. For example, nitrogen is involved in the production of amino acids and therefore proteins.

[0003] However, only a small portion of the nitrogen-based fertilizers applied to crops are converted into plant-based materials. Fertilizers that are not converted into plant-based materials can accumulate in soil or run off into surface and groundwater, causing water pollution.

[0004] To reduce the use of chemical fertilizers, inoculants containing nitrogen-fixing bacteria can be used to partially replace such chemical fertilizers.

[0005] The main reservoirs of methylotrophic bacteria are generally soil and water (both saline and non-saline), but these bacteria are also present in a variety of natural and man-made environments, such as dust, lake sediments, air, facial cleansers, fermented products, water supply networks, washrooms and air conditioning systems.

[0006] However, known inoculants do not provide satisfactory results in stimulating plant growth and do not allow either replacing chemical fertilizers or significantly reducing the use of such chemical products.

[0007] Most of the known microorganisms that act as growth stimulants or nitrogen fixers cannot achieve high nitrogen content and thus cannot significantly reduce the nitrogen units required for plant growth. These known microorganisms produce some types of auxins, such as indole acetic acid, which promote rooting rather than nitrogen fixation. [Patent Document 1] US2015 / 101373A1 [Patent Document 2] KR2007 0106867A [Patent Document 3] US2018 / 168167A1 [Patent Document 4] WO2015 / 000612 [Patent Document 5] WO2015 / 000613 [Non-Patent Document 1] SI-W ON LEE ET AL: "Methylobacterium dankookense sp. nov. isolated from drinking water" THE JOURNAL OF MICROBIOLOGY, vol. 47, no. 6 (2009-12-01), pages 716-720. XP05535.0364, DOI: 10.1007 / S12275-009-0126-6

[0008] US Patent No. 5,999,633 discloses nitrogen fixation and plant growth promoting Methylobacterium NRRLB-5.0628 and NRRLB-5.0629. Patent Document 2 discloses Methylobacterium fujisawaense CBMB20, which promotes nitrogen fixation and plant growth. Patent Document 3 discloses the plant growth promoting fungus Glomus Iranicum var. tenuihypharum BCCM54871. Non-patent literature 1 discloses the enzymatic activity and genetic similarity of Methylobacterium dankense. Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to Methylobacterium sp. NOV. The present invention aims to provide a strain and a composition containing the same, and a new species having a beneficial effect on crops and a composition constituting the same. Another object of the present invention is to provide Methylobacterium sp. NOV. The aim of this study is to provide a strain of this fungus, which will at least reduce the use of chemical nitrogen-based fertilizers. These and other objects are achieved by the Methylobacterium according to claim 1. [Means for solving the problem]

[0010] In the following description, features of the invention are described with reference to an exemplary embodiment. However, features of the invention disclosed herein may be combined with one or more other features disclosed herein to provide a further embodiment of the invention. Accordingly, the object / subject of the present invention is the strain of the genus Methylobacterium deposited under Accession No. CECT9580.

[0011] The Methylobacterium strain of the invention, identified by the depositor under the reference number SB0023 / 3, was deposited by the applicant on 21 March 2018 at the international depository Coleccion Espanola De Cultivos Tipo (CECT), Edificio 3 CUE, Parc Cientific Universitat de Valencia, Catedratico Agustin Escardino 9, 46980 Paterna (Valencia) Espana, under the number CECT9580. Hereinafter, "Methylobacterium sp. nov. strain" may be referred to as "the strain of the present invention" or "the Methylobacterium genus strain of the present invention."

[0012] The strain of the present invention was isolated from inside a spore of the mycorrhizal fungus Glomus iranicum var. tenuihypharum, which is an arbuscular mycorrhizal fungus known in the art. This Glomus iranicum bar. tenuihaifarum was deposited by the applicant on April 19, 2013 under BCCM deposit number 54871 at the international depository Belgian Coordinated Collections of Micro-Organisms (BCCM), address: Universite Catholique de Louvain, Mycothque de I'Universite catholique de Louvain (MUCL), address: Croix du Sud 2, Box L7.05.06, 1348 Louvain-la-Neuve. Glomus iranicum bar. tenuihaifarum is disclosed in Patent Documents 4 and 5.

[0013] The present strain shows high similarity (98.7% based on 16S generation sequence) to Methylobacterium dankookense.

[0014] Phylogenetic analysis by the UBCG showed that Methylobacterium dankokensei is the closest relative to the strain of the present invention. The collected data support the creation of a new species containing the strain of the present invention, for which the name Methylobacterium symbioense nov. has been proposed.

[0015] Phenotypic and genotypic characterization was assayed to demonstrate whether it can be considered as a new species.

[0016] The strain of the present invention is Gram-negative and strictly aerobic; it has the shape of a ballius (0.8-1 μm wide, 1.2-1.6 μm long) with a lateral flagellum, and appears as individual or two pairs without sporulation. The colonies grown for 5-7 days on MMM or NFbM agar are round, pink with a clear border. This has not been described before for other similar strains. The optimum temperature is 28°C. In the presence of NaCl, the growth rate decreases to 3%, but does not grow any further.

[0017] A multi-gene genome-based phylogenetic approach was performed by using UBCG tools to more clearly identify the closest species to the present strain CECT9580. The position of the present strain was investigated in the UBCG tree, which includes 15 genomes of closely related species within the genus. The tree (Figure 13) shows a specific relationship with Methylobacterium dankookense.

[0018] Advantageously, according to one embodiment, the strain of the invention is characterized by the fact that it is an endophyte nitrogen-fixing bacteria capable of supplying nitrogen to plants (reducing the external nitrogen input by up to 60%), in other words, the strain of the invention allows a reduction of the external nitrogen input by up to 60% with respect to the external nitrogen input that would be necessary in the absence of said strain.

[0019] Advantageously, the strains of the present invention are capable of translocating plants from the roots to the leaves and vice versa.

[0020] The process of isolating the strain of the present invention comprises the following steps. (a) providing spores of Glomus iranicum var. tenuihaifarum; (b) extracting cytoplasm from the spores; (c) inoculating the extracted cytoplasm into a methanol-containing medium; (d) culturing the inoculated medium to grow the Methylobacterium sp. strain of the present invention;

[0021] According to one embodiment, the process for isolating the strain of the present invention comprises the following steps: (X) isolating spores of Glomus iranicum var. tenuihaifarum from those previously surface disinfected through the process of external disinfection cycle to obtain a spore solution; (Y) after the above step, macerating the spore solution and culturing the fungal cytoplasm in Minimum Salt Methanol Medium (hereinafter referred to as "MMM"); (Z) identifying colonies of the strain of the present invention, picking one that shows a pink color, and reisolating it from the medium;

[0022] According to one embodiment, prior to step (b), the spores may be treated with a disinfectant to disinfect the outer wall of the spores.

[0023] Disinfection of the spore outer wall can be carried out by conventional techniques. For example, disinfection of the spore outer wall can be carried out by treating the spores with sodium hypochlorite. An example of sodium hypochlorite is a 5% solution of sodium hypochlorite in a buffer solution, e.g., Tween® 80.

[0024] Extraction of the cytoplasm from spores of Glomus iranicum var. tenuihyphalum can be carried out by conventional techniques, for example by macerating the spores in a suitable buffer, preferably Ringer's solution.

[0025] The minimal salts methanol medium (MMM) of step (c) preferably has the following composition: Methanol; 20.0 ml, NaNO3; 0.5g, (NH4)2SO4; 0.5g, MgSO4-7H2O; 0.5g, K2HPO4; 1.0g, FeSO4-7H2O; 0.01g, CaCI2-2H2O; 0.01g, KCI; 0.5g, Vitamin solution; 1.0 ml; Micronutrient solution; 2.0ml, Agar; 12g, The pH is 7.20. Step (d) is preferably cultured at 28° C. for 5 days to grow the Methylobacterium strain of the present invention.

[0026] According to one embodiment, the cultivation is carried out at a temperature ranging from 25° C. to 30° C., preferably at 28° C. According to one embodiment, the cultivation is carried out at a pH range of 5 to 9 (preferably pH 7).

[0027] After step (d), colonies of the strain of the present invention are visually identified by the typical pink color development, and one or more colonies are then picked and re-isolated at least three times to ensure the absence of possible contamination. The strains of the present invention will be suitable for inclusion in compositions.

[0028] Another object of the present invention is a composition comprising the strain of the genus Methylobacterium deposited under accession number CECT9580. According to one embodiment, the compositions of the present invention may comprise an agriculturally acceptable carrier or an agriculturally acceptable co-formulant. These carriers and co-formulants are well known in the art.

[0029] According to one embodiment, the carrier or co-formulant is selected from the group consisting of talc, clay, maltodextrin, skimmed milk, glucose, tea, dried plant extracts such as cassava and quinoa, kaolin, coir, diatomaceous earth, chitin, CaCO3, alginates, carrageenan, surfactin, rhamnolipids, sophorolipids, saponin, potassium oleate and mixtures thereof.

[0030] According to one embodiment, the composition may be in the form of a solid, an aqueous liquid, an oily liquid, an emulsion, a semi-solid, or a gel. Preferably, the composition is in solid form. More preferably, the composition is in powder form. An object of the present invention is a method for producing a composition comprising the strain of the invention (i.e. the strain of the genus Methylobacterium deposited under accession number CECT9580).

[0031] According to one embodiment, the compositions of the present invention are characterized in that they are applied as a seed coating, by soil application, for example by drip irrigation systems or by drench, or by foliar application.

[0032] A method for producing the composition of the present invention comprises the following steps. (A) Providing the Methylobacterium sp. bacterium deposited under accession number CECT9580. (B) inoculating the Methylobacterium sp. into a methanol-containing liquid medium. (C) A step of culturing the Methylobacterium sp. to obtain a liquid culture containing methylbacteria. (D) drying the liquid culture to obtain a dry composition.

[0033] Advantageously, at the end of said step (C), the end product of the Mechylobacteria of the invention is verified by measuring the optical density and biomass production of the bacteria during the cultivation.

[0034] According to one embodiment, the Methylobacterium sp. of the present invention, deposited under accession number CECT9580, is isolated from the interior of a spore of the mycorrhizal fungus Glomus iranicum var. tenuihyphalum, deposited under BCCM accession number 54871. According to one embodiment, the methanol-containing medium is MMMM (ie, Minimal Mineral Medium with Methanol). According to one embodiment, the step (C) can be carried out by culturing at a temperature in the range of 25° C.-30° C. (preferably 28° C.) for 3 to 10 days (preferably 5 to 7 days).

[0035] According to one embodiment, step (A) includes (A1) recovering the strain of the present invention using a nitrogen-free medium. An example of a nitrogen-free medium is NFBG medium (Nirogen Free Bacterial Medium supplemented Glucose). This NFBG medium has the following composition: glucose, Malic acid; 5.0g, Methanol; 20.0 ml, (NH4)6MO7O 24 4H2O; 0.002g, MgSO 4; 7H2O; 0.2g, K2HPO4; 0.1g, KH2PO4; 0.4g, FeCl3; 0.01 g, NaCl; 0.1 g, KOH; 4.8g, Micronutrient Sol; 2.0 ml, Vitamin Sol; 1.0ml Bromothymol Blue Sol: 0.5% The latter three are for 2.0 ml KOH2N; pH 6.9

[0036] According to one embodiment, the step (A1) can be carried out by culturing at a temperature in the range of 25° C. to 30° C. (preferably 28° C.) for 3 to 10 days (preferably 5 to 7 days). Advantageously, the strain of the present invention forms colonies that are pinkish red, and therefore colonies of the strain of the present invention can be easily identified. According to one embodiment, the step (A) comprises: (A2) selecting colonies of the strain of the present invention; (A3) Selectively growing the strain of the present invention using a nitrogen-free, methanol-containing medium An example of a nitrogen-free methanol-containing medium is MMMNF, or Mineral Minimal Medium with Methanol but without Nitrogen.

[0037] According to one embodiment, after step (A3), a colony of the Methylobacterium of the present invention can be selected and inoculated into a liquid medium containing methanol. According to one embodiment, step (A3) can be carried out by culturing at a temperature in the range of 25° C. to 30° C. (preferably 28° C.) for 3 to 10 days (preferably 5 to 7 days).

[0038] According to one embodiment, step (C) may be carried out by fermentation. According to one embodiment, step (C) can be performed in the following first and second steps. The first step is to obtain a culture of Methylobacterium which is used as a pre-inoculum. The second step is mass propagation by fermentation.

[0039] A further object of the present invention is the use of the strain of the genus Methylobacterium according to the invention, deposited under accession number CECT9580, as a biostimulant or endophytic nitrogen fixing bacterium in plants.

[0040] Indeed, it has been surprisingly observed that the strains of the present invention, as well as compositions comprising same, when stimulated in plants, increase plant health and yield.

[0041] It has also been found that the strains of the present invention, as well as compositions containing the same, can be used to provide nitrogen to plants. In particular, it has been found that the use of chemical nitrogen fertilizers can be significantly reduced by providing plants with the strains of the present invention. For example, the use of chemical nitrogen fertilizers can be reduced by up to 60% by using the strains of the present invention or compositions containing the same.

[0042] An object of the present invention is the use of the inventive strains to reduce external nitrogen inputs, preferably by up to 60%. Advantageously, the strains of the present invention may be provided to plants alone or as a component of a composition containing other agriculturally acceptable ingredients.

[0043] It has been observed that the strains of the present invention are able to stimulate the roots or above-ground parts of plants by fixing atmospheric nitrogen. Without being bound by any particular scientific explanation, it is believed that the strains of the present invention penetrate the plant tissue and fix atmospheric nitrogen as NH4 + It has been observed that it activates the microbial enzymatic system, which contains the nitrogenase enzyme complex, which reduces NH4 + Under certain physiological conditions, it is directly incorporated into Glu (glutamic acid). This is achieved by amination of 2-oxoglutarate via mitochondrial Glu dehydrogenase (NADH-GDH) and then incorporated into glutamine by cytosolic glutamine synthetase 1 (GS1). Glutamine oxoglutarate aminotransferase, abbreviated as GOGAT, is an enzyme that generates glutamic acid from glutamine and α-ketoglutarate and, together with glutamine synthetase, plays a central role in the regulation of nitrogen assimilation in photosynthesis by plants.

[0044] Advantageously, it has also been observed that the strains of the present invention are capable of producing auxins and carotenes which increase the solubility of phosphorus, which is particularly useful at the seedling stage, and stimulate the development of secondary roots.

[0045] Advantageously, the strains of the present invention may be applied to a variety of crops, and are particularly useful when applied to horticulture, grass cultivation, citrus cultivation, drupe cultivation, vine cultivation, and forestry.

[0046] Another advantage of the strains of the present invention is that they promote photosynthesis in the host plant, thus promoting an increase in plant biomass. [Brief description of the drawings]

[0047] [Figure 1] The alveoli in the growth chamber show growing plants. [Diagram 2] Vegetable tissue samples (root (a), stem (b), and leaf (c)) exposed to specific media after disinfection for the growth of methylbacteria are shown. [Diagram 3] Shown are images of plant tissue samples from plants treated with the strains of the invention, exposed to a specific nutrient medium for Methylobacteria, by seed dressing (ac), irrigation (df) and foliar spray (gi). [Figure 4] Figure 2 shows distribution images of trials with the strain of the invention in treatment 1 and control treatment 2. a shows farm 1, b shows farm 2 and c shows farm 3. [Diagram 5] 4 shows a graph of SPAD measurements after 30 days of application. [Figure 6] The NDVI index for Farm 1 (a), Farm 2 (b), and Farm 3 (c) are shown. [Figure 7] The combined CRI index for Farm 1 (a), Farm 2 (b) and Farm 3 (c) is shown. [Figure 8] Figure 2 shows distribution images of the trial with treatment 1 using the strain of the invention and treatment 2 using the control. Farm 1 (a), Farm 2 (b). [Figure 9] 4 shows a graph of SPAD measurements after 30 days of application. [Figure 10] NDVI index for Farm 1 after 45 days of treatment measured every 15 days. [Figure 11]NDVI index for Farm 2 after 45 days of treatment measured every 15 days. [Figure 12] FIG. 12A shows the nebulizer at the time the treatment was applied, and FIG. 12B shows the particle size at the time the treatment was applied. [Figure 13] A phylogenetic tree generated by UBCG using amino acid sequences is shown. Numbers at nodes indicate gene support index (maximum value is 92). Genome accession numbers are shown in parentheses. 0.05 substitutions per position.

[0048] Experiment description The strain of the present invention was obtained from spores of Glomus iranicum var. tenuihypharum. The strain of the present invention was isolated from the inside of the spores through a process of disinfecting the outer wall of the spores several times. This process was carried out by washing with 0.5% sodium hypochlorite and Tween 80 for 5 minutes and performing 5 disinfection cycles. After that, the spore solution was macerated and the fungal cytoplasm was inoculated into MMM (the composition of which is as described in paragraph 0025) at 28°C for 5 days. Five different colonies were identified from this medium, and the colonies that showed pink color were picked up and re-isolated at least three times to ensure that there were no other contaminations.

[0049] Example 1 Test 1. Application route of the strain of the present invention. Experiments have been carried out in a laboratory by applying the strain of the present invention, namely the Methylobacterium sp., deposited under accession number CECT9580, by foliar application, soil application and seed coating (or seed dressing). The presence of the strain of the invention was evaluated in leaves, stems and roots. The migration time of the strain in the plant, i.e. from root to leaf and from leaf to root, as well as the penetration of the strain through root stomata and seeds, were also evaluated.

[0050] To confirm the penetration of Methylbacteria into plants and the colonization of different tissues, a test was carried out in which seeds were inoculated into alveoli and culture pots were inoculated with maize plants. In this case, three treatments were applied: seed coating, application by irrigation and foliar application. The presence of colonies of the strain of the invention was analyzed in the roots, stems and leaves of three biological replicas at different times and days ("hai" and "dai") after inoculation.

[0051] material and method Plant material and microorganisms used. Corn seeds (Zea mays L.), which were coated with the fungicides Fludioxonil and Mefenoxam, were used as the plant material. The strain of the present invention and bacteria belonging to Herbaspirillum seropedicae (hereinafter also referred to as "nitrogen fixing bacteria") were used as the microbial inoculant.

[0052] Corn growing conditions. Small plastic containers were used to grow the corn plants. The containers were filled with a sterile substrate based on a 2:1 mixture of silica sand and coconut fiber and placed in a growth room at 24 °C and 60% relative humidity (RH) in the presence of light for 4 weeks (Figure 1). The plants were watered twice a week with sterile decalcified water. 15 days after sowing, the plants were watered once with a 0.33% nutrient solution (ratio N:P:K: 4:17:4.5) and a solution of micronutrients.

[0053] Treatment of maize plants with the strain of the invention. Different treatments were performed on maize plants with the strains of the invention by seed coating (i.e. seed dressing), irrigation and foliar application (i.e. foliar route), the inoculum was used in solid form (powder). The bacterial concentration in this powder was 1x10 9 Each treatment was compared to untreated plants as a negative control. 6 CFU / g of nitrogen fixing bacteria was used as a positive control in the experiment.

[0054] a) Seed Coating Corn seeds were coated with a powder inoculum of the strain of the present invention.

[0055] b) Irrigation Route Four days after sowing, the strain of the present invention and nitrogen fixing bacteria were added at 1-1.2x10 6 The inoculation was carried out by irrigation at a concentration between 0.01 and 0.1 CFU / g. For this purpose, the bacterial inoculum in solid form was diluted with water and further processed.

[0056] c) Foliate root When the plants had 2-3 true leaves, they were inoculated via the leaf route with the strain of the present invention, classified as BBCH12-13, and nitrogen fixing bacteria. The solid bacterial inoculum was diluted with water to give a concentration of 1-x10 per plant. 6 The concentration of CFU / g was adjusted. Prior to this, the medium was covered with plastic to prevent contamination by the treatment. The bacterial treatment was carried out by spraying the leaves with an atomizer.

[0057] * Detection of colonies of the strain of the invention in roots, stems and leaves. After treatment with the strain of the invention, the presence of this bacterium was analyzed together with its positive control nitrogen fixing bacteria in different plant tissues. These were surface disinfected to avoid false positives. For this, they were immersed in a solution of ethanol (70%) and sodium hypochlorite (1%) for 30 seconds or 2 minutes and then washed twice with distilled water. After disinfection (roots and leaves), longitudinal and transverse incisions were made in the stems and they were transferred to a specific medium for Methylobacteria. Figure 2 shows the plant tissue samples that were disinfected and exposed to a specific medium for the growth of Methylobacteria. In particular, panel "a" of Figure 2 shows a root sample, panel "b" a stem sample and panel "c" a leaf sample. The tissue samples placed on the medium were incubated at 28°C for 7 days. The colony growth was observed with a Leica Wetzlar stereo microscope and photographed with a Leica Wetzlar camera. Photographs were taken with an MC170H. Plants were harvested for analysis between 1-24 h on day 1 and between 2-10 days after inoculation (irrigation and foliar) or emergence (coating). Three biological replicates were used for each of the three treatments and times analyzed. It was done.

[0058] *Statistical analysis of the results obtained To obtain a statistical analysis of the experimental results, the method used was a simple ANOVA, followed by Fisher's exact test of least significant difference at a significance level of p<0.05. The software STATGRAPHICS Centurion18 was used, and Microsoft Excel was used for data processing and graphic presentation.

[0059] *result Effect of application of the strains of the invention: Infection and colonization of plants.

[0060] a) Seed dressing The first evaluation to detect bacterial colonies on the treated plants inoculated by coating was performed at 48 hours, which coincided with the time of seed germination. Analysis showed that after 2 days the strains of the invention were present in the internal tissues, after 4 days they were in the stems, and after 6 days they appeared on the leaves, thus passage of the bacteria into new structures occurred as the plant developed.

[0061] b) Irrigation In the case of inoculation by irrigation, the bacteria were present in the inner tissues of the roots at 1 h, ascended to the stem between 4 and 8 days after treatment, and reached the leaves by 10 days, although it should be emphasized that this was not uniform across all replicas.

[0062] c) Foliate root Following the leaf route, bacteria were found in the inner leaf tissues 1 h after inoculation and in both stems and roots 6 h after treatment. Figure 3 shows images of tissue samples of plants treated with the strains of the invention and exposed to specific nutrient media for Methylobacteria, showing seed dressing (ac), irrigation (df) and foliar spray (gi).

[0063] Table 1 shows data relating to the detection of colonies of the strains of the invention in different parts of the plant over a period of 10 days. TIFF0007644934000001.tif71135 Legend: H: time, d: day, bacterial presence is + for 20-40%, ++ for 50-80%, +++ for 90-100%, Stem A: apical stem, Stem B: basal stem.

[0064] *Conclusion In this test, the penetration of the microorganisms is determined to be effective both in the root zone and above ground parts, and therefore colonies of the strain of the invention were identified in plants treated with any method, indicating that the endophyte micrograms are transported from the roots to the leaves and vice versa.

[0065] Example 2 Test 2: Effectiveness of using the strain of the present invention in reducing nitrogen fertilizer by 40% in long-rotation corn cultivation. *the purpose. To know the effectiveness of the strain of the present invention on the development of corn (Zea mays) on a 40% reduction in nitrogen cover fertilization in conventional growing conditions through the evolution of leaf green color (SPAD), vegetation index, nitrogen levels in plants, microbial concentration in leaves and production.

[0066] *material and method Testing station and fertilization. The trial was carried out on three farms in Huesca, Spain. The first farm, located in Montesusin, Huesca, is located at 41°52′12.7″N and 0°23′11.1″W. The second farm, located in Belver de Cinca, Huesca, is located at 41°40′47.6″N and 0°17′09.5″W. The third farm, located in Tamarite de litera, Huesca, is located at 41°47′58.1″N and 0°23′56.0″W.

[0067] The recommended fertilization regime was the conventional application plus a treatment with 40% reduction in N. For this purpose, fertilizer was distributed by a fertilizer spreader in the amounts specified in Table 2. Table 2 shows the amount, type and nitrogen fertilizer units (NFU) of fertilizer used for each fertilizer in each plot. TIFF0007644934000002.tif62127

[0068] *Distribution of treatments on farm. The areas of the farms were Farm 1 (5.43 ha), Farm 2 (3.28 ha) and Farm 3 (2.34 ha). Two adjacent areas of 16 m width were established for the evaluation of the parameters. The area marked "1" is the treated area and the area marked "2" is the control area. Figure 4 shows the distribution of tests for treatment 1 (area 1) with the strain of the invention and treatment 2 (area 2) which is the control. In particular, panel "a" of Figure 4 shows Farm 1, panel "b" shows Farm 2 and panel "c" shows Farm 3.

[0069] *Cultivation and fertilization characteristics of the present strain in long-cycle maize. Maize seeds (Zea mays) were selected for grain of variety "40F". The planting rate was 300 kg / ha, sowing was performed at a depth of 3 cm, with 75 cm between rows and 15 cm between identical rows. The width of each sowing lane is 16 m between sprinklers. Sowing dates were between 1 and 5 May 2018, with stalks emerging 5 days later, and the date of spraying of the strain of the invention was 12 June 2018, 40 days after sowing. They were sown using a seed drill in farms (fields indicated by farmers) in a representative area of ​​maize cultivation in Spain. A random design was performed selecting the farm plots for each treatment.

[0070] The strain of the present invention was applied at a yield of 333 g / ha (3x10 7 CFU / g) and reduced nitrogen fertilization by 40%. Results were compared to conventional fertilization treatments. Foliar applications were made with a sprayer spraying 300-400 litres of broth per hectare (ha). This was done when the plants were at stage 13-14 on the BBCH scale, with an air temperature of 17°C, relative humidity of 52%, wind speed of 2.3 mps from the north, no dew on the leaves and 60% cloud cover.

[0071] *Parameters and evaluation methods Counting colony forming units per gram of leaves (CFU / g) This parameter was measured to see how much of the microorganism was present in the leaves throughout the entire culture cycle. It was measured by serial dilution of 1 gram of leaves from the fourth leaf of less than 20 plants by plating them on selective medium without nitrogen and containing methanol as carbon source.

[0072] SPAD (Leaf chlorophyll level) This parameter is an indicator of a good nutritional status of the plants and is closely related to the levels of nitrogen and iron. For this, they used a parameter known as SPAD (Soil Plant Analysis Development), measuring 100 times the fourth leaf of less than 100 plants in each treatment.

[0073] Nitrogen in leaves, stems, and cobs of grain This parameter is an indicator of the amount of nitrogen stored in each plant tissue at harvest. Leaf, stem and cob grains from 100 plants were analyzed for total nitrogen by Kjeldahl's method.

[0074] Drone photos (flying a drone to get different indices) NDVI (Normalized Difference Vegetation Index): A measure of the red and near infrared bands of light reflected by plant leaves. It is a common indicator of canopy density and is often used to distinguish between live green vegetation and soil, which appears bluer due to its higher chlorophyll content. Composite CRI (Color Infrared): A composite infrared color that combines the NIR, red, and green bands. Healthy vegetation reflects high levels of NIR and appears red. Latent (dormant) vegetation is often green or bronze in color. Sandy soils are light brown, and clay soils are dark brown or blue-green.

[0075] yield. To assess the harvest yield, six blocks are randomly taken and a surface area of ​​30 square meters is harvested. The resulting grain is weighed, discounting the loss due to humidity in each zone.

[0076] Statistical analysis. The factors (SPAD and performance) and their interactions were analyzed by ANOVA for each parameter, which was performed using Statgraphics Plus software for Windows 5.1 (Manugistics, Inc. USA).

[0077] *result. Counting colony forming units (CFU). Table 3 shows data related to colony forming units (CFU) per gram of leaves and in the spray tanks after treatment (June 4, 2018) and one month after treatment (July 4, 2018). In the spray tank, there was a homogenous mixture of product, ensuring correct application to the plants. After one month of spraying on the corn foliage, populations were maintained and increased growth was seen in all plots. TIFF0007644934000003.tif57124

[0078] SPAD. FIG. 5 shows the SPAD measurements 30 days after application of the strain of the invention. One month after application, plants maintained SPAD levels comparable to the control without significant differences in all fields. We confirmed that: The reduction in chemical nitrogen fertilizer does not have a negative effect on the plants, but this is due to the contribution of bacteria.

[0079] Nitrogen in leaves, stems and cobs Table 4 presents data on the nitrogen concentrations (g / plant) found in various tissues of the maize plants at harvest. TIFF0007644934000004.tif61125 Drone photo (45 days) Figure 6 shows the drone photographs used to determine the NDVI index for Farm 1 (panel “a”), Farm 2 (panel “b”), and Farm 3 (panel “c”). Figure 7 shows the drone photographs used to determine the combined CRI index for Farm 1 (panel “a”), Farm 2 (panel “b”) and Farm 3 (panel “c”). Considering the NDVI and composite CRI indices, no differences were found in plant canopy density, chlorophyll, and plant health (blue and red intensity) in any of the farms (except farm 3) compared to the control. Farm 3 had problems with irrigation water pressure and color differences were observed in the crops in places where the sprinklers did not water properly.

[0080] yield Table 5 shows yield data for each farm. TIFF0007644934000005.tif50118In Table 5, means followed by different letters are significantly different from each other. On farm 1, the control yield was 16881 kg / ha and the application of the strain of the invention gave a yield of 17511 kg / ha, which represents a 4% increase in production. On farm 2, the control yield was 14 346 kg / ha and the application of the strain of the invention gave a yield of 18 335 kg / ha, which represents a 28% increase in production. On farm 3, the control yield was 14312 kg / ha and the application of the strain of the invention gave a yield of 17539 kg / ha, which represents an increase of 22.5%. Table 6 gives data on the nitrogen balance: nitrogen provided by fertilization (input) (column 1), nitrogen assimilated by the plant after extraction (column 2), balance of inputs to the plant and assimilation extracted by the plant (column 3), nitrogen requirement per tonne of grain (nitrogen input required) (column 4) and nitrogen extraction per tonne of grain (column 5). TIFF0007644934000006.tif49121

[0081] conclusion The strains of the present invention have an efficient application in corn cultivation and microbial persistence on the plant, ie, fertilization. The strains of the present invention maintain similar levels of SPAD (chlorophyll), plant health and plant canopy density (NDVI index and composite CRI index) as conventional fertilization where higher amounts of nitrogen fertilizer are used. The strain of the present invention provides 40% of the total nitrogen fertilizer units, which reduces cover fertilization and increases yields by between 4-27%. Since the plants only had one cob per plant, this increase is related to grain density or fertilization frequency. Observing the unit balance of nitrogen fertilizer due to the fertilization carried out and the nitrogen extraction of the plants, there is an increase of 66 to 111 kg of nitrogen per hectare. This means that several tens of kg of nitrogen were fixed by the strain of the invention during the cultivation cycle. This result is indirectly observed in the nitrogen requirement per ton of grain produced in the treated plants with the strain of the invention. This is because the nitrogen requirement to produce one ton of grain is less than in the control. It should also be noted that the nitrogen requirement to produce one ton of treated grain is lower than the nitrogen extraction carried out to produce one ton of grain. This is the opposite in the control, since more nitrogen is needed to produce the same amount of control grain.

[0082] Example 3 Test 3. Effectiveness of using the strain of the present invention in reducing nitrogen fertilizer by 30% in short cycle maize cultivation. *the purpose. To know the effectiveness of the strain of the present invention on the development of corn (maize) for a 30% reduction in nitrogen cover fertilization under conventional cultivation conditions through the evolution of leaf green color (SPAD), i.e. vegetation index, nitrogen level in the plant, microbial concentration in the leaves, and production.

[0083] *material and method Testing station and fertilization. The trial was carried out on two farms in Spain. The first farm, located in Pomar de Cinca, Huesca, is located at 41°50′13.8″N and 0°05′38.0″W. The second farm, Albacete Finca Oran, is located at 38°49′55.3″N 1°50′27.1″W.

[0084] The recommended fertilization was a conventional fertilization treatment with a 30% reduction in N. For this purpose, fertilizer was distributed by fertilizer spreader in the amounts specified in Table 7. Table 7 shows the amount, type and nitrogen fertilizer units (NFU) of fertilizer used for each fertilizer in each plot. TIFF0007644934000007.tif45129

[0085] *Distribution of treatments on farm. The area of ​​the farms was Farm 1 (74 ha) and Farm 2 (3.67 ha). Two adjacent areas of 16 m width were established for the evaluation of the parameters. The area marked "1" is the treated area and the area marked "2" is the control area. Figure 8 shows the distribution of tests for treatment 1 (area 1) with the strain of the present invention and the control treatment 2 (area 2). In particular, panel "a" of Figure 8 shows farm 1 and panel "b" of Figure 8 shows farm 2.

[0086] *Cultivation and fertilization characteristics of the strain of the present invention. Maize seeds of the variety "Guasi" were selected for farm 1 and the variety "DKC5032YG" for farm 2. The planting rate was 300 kg / ha, with sowing at a depth of 3 cm, 75 cm between rows, and 15 cm apart in the same row. The width of each sowing lane is 16 m. The sowing dates were 20 June 2018 for farm 1 and 30 June for farm 2. Stalks emerged 10 days later, and the application dates of the strain of the present invention were 12 July (farm 1) and 20 July (farm 2) of the same year, 22 and 20 days after sowing, respectively.

[0087] The strain of the present invention was applied at a yield of 333 g / ha (3x10 7 The samples were sprayed at a dose of 100 mg / g (CFU / g). Foliar applications were made with a sprayer spraying 300-400 litres of broth per hectare (ha) when the plants were at stages 13-14 on the BBCH scale, with air temperatures of 22°C and 20°C, relative humidity of 72% and 58%, respectively, wind speeds of 2.3mps and 12mps from north and north-west directions, no dew on the leaves and 0% and 15% cloud cover.

[0088] *Parameters and evaluation methods Counting colony forming units per gram of leaves (CFU / g) This parameter was measured to see how much of the microorganism was present in the leaves throughout the entire culture cycle. This was measured by serial dilution of 1 gram of leaves taken from the fourth leaf of less than 20 plants by plating them on selective medium without nitrogen and containing methanol as a carbon source.

[0089] SPAD (Leaf Chlorophyll Levels) This parameter is an indicator of a plant's good nutritional status and is closely related to nitrogen and iron levels. For this, they used a parameter known as SPAD, measuring 100 times on the fourth leaf of less than 100 plants in each treatment.

[0090] Nitrogen in leaves, stems, and cobs of grain This parameter is an indicator of the amount of nitrogen stored in each plant tissue at harvest. Leaf, stem and cob grains from 100 plants were analysed for total nitrogen by the Kjeldahl method.

[0091] Satellite imagery Photos from the ESA Copernicus satellite were downloaded and processed in the Qgis program to obtain the NDVI index. NDVI: A measure of the red and near infrared bands of light reflected by plant leaves. It is a common indicator of canopy density and is often used to distinguish between living green vegetation and soil, which appears bluer due to the high amount of chlorophyll.

[0092] yield. To assess the harvest yield, six blocks are randomly taken and a surface area of ​​30 square meters is harvested. The resulting grain is weighed, discounting the loss due to humidity in each zone.

[0093] Statistical analysis. The factors (SPAD and yield) and their interactions were analyzed by ANOVA for each parameter, which was performed using Statgraphics Plus software for Windows 5.1 (Manugistics, Inc. USA).

[0094] *result. Colony forming unit (CFU) count. Table 8 shows data related to colony forming units (CFU) and colony forming units (CFU) per gram of leaves in the spray tank (12-20 / 07 / 2018) and one month after treatment (15 / 8 / 2018). TIFF0007644934000008.tif48105In the spray tank, there was a homogenous mixture of products, which allowed for correct application to the plants. After one month of spraying on the corn leaves, populations were maintained and increased growth was seen in all plots.

[0095] SPAD FIG. 9 shows the SPAD measurements 30 days after application. One month after application, plants maintained SPAD levels comparable to the control without significant differences in all fields. We confirmed that: The reduction in chemical nitrogen fertilizer does not have a negative effect on the plants, but this is due to the contribution of bacteria.

[0096] Nitrogen in leaves, stems and cobs Table 9 presents data on the amount of nitrogen (g) in various tissues of the corn plant at harvest. TIFF0007644934000009.tif61122

[0097] Satellite photo. FIG. 10 shows the satellite images used for the NDVI index determination (measured every 15 days) of Farm 1 after 45 days of treatment. FIG. 11 shows the satellite images used for the NDVI index determination (measured every 15 days) of Farm 2 after 45 days of treatment. Uniformity in NDVI (green colour intensity) values ​​was observed throughout the cultivation cycle comparing the control and the treatment of the strain of the present invention (with reduced fertiliser).

[0098] yield Table 10 shows the yield data for each farm. TIFF0007644934000010.tif38125In farm 1, the control yield was 10,515 kg / ha, but application of the strain of the present invention resulted in a yield of 11,828 kg / ha, improving productivity by 12.5%. In farm 2, the control yield was 12,525 kg / ha, but application of the strain of the present invention resulted in a yield of 12,863 kg / ha, an increase in productivity of 2.7%. Table 11 gives data on the nitrogen balance: nitrogen provided by fertilization (column 1), nitrogen assimilated by the plant after extraction (column 2), balance of plant inputs and assimilation extracted (column 3), nitrogen requirement per tonne of grain (column 4) and nitrogen extraction per tonne of grain (column 5). TIFF0007644934000011.tif42131

[0099] conclusion The strains of the present invention have an efficient application in corn cultivation and microbial persistence on the plant, ie, fertilization. The strains of the present invention maintain similar levels of SPAD (chlorophyll), plant health and plant canopy density (NDVI index) as conventional fertilization (using large amounts of nitrogen fertilizer). In terms of yield, the strain of the invention provides 30% of the total nitrogen fertilizer units, reducing cover fertilization and increasing production by between 2.7-12.5%. This increase is related to the specific grain weight or number of fertilization runs, since the plants only had one cob per plant. Observing the unit balance of nitrogen fertilizer due to the fertilization carried out and the nitrogen extraction of the plants, an increase of 43-57 kg of nitrogen per hectare is observed. This means that this amount of about several tens of kg of nitrogen was fixed by the strain of the invention during the cultivation cycle. This result is indirectly observed in the nitrogen requirement per ton of grain produced in the treated plants with the strain of the invention. This is because the nitrogen requirement to produce one ton of grain is less than in the control. It should also be noted that the nitrogen requirement to produce one ton of treated grain is lower than the nitrogen extraction carried out to produce one ton of grain. This is the opposite in the control, since more nitrogen is needed to produce the same amount of control grain.

[0100] Example 4 Trial 4 - Evaluation of the strain of the present invention in strawberry cultures for different doses of nitrogen fertilization. *the purpose. The purpose of this study was to determine the effectiveness of the strain of the present invention on strawberry crops, which were fertilized with different doses of nitrogen, i.e., 0%, 25%, 50%, 75% and 100% of the conventional fertilization, and evaluated in terms of leaf greenness (SPAD) development, nitrogen level development, leaf microbial concentration and production.

[0101] material and method Testing station and fertilization. The tests were carried out on a farm located in the experimental farm of Szymbord (applicant), in Los Martinez del Puerto, Murcia, at latitude 37°48′59.6″N and longitude 1°05′43.6″W. The farm is 192 m 2 It is 8m wide and 24m long. The irrigation and fertilization regime was different for all treatments. The mineral fertilization program consisted of five solutions with different nitrogen contents. Table 12 shows the various fertilization treatments measured by UFN. TIFF0007644934000012.tif49107

[0102] process Table 13 shows ten treatments (T1-T10) based on five levels of fertilization, which are then divided into those treated with the strains of the present invention (T6-T10) and those that were untreated (T1-T5). TIFF0007644934000013.tif91111

[0103] Characteristics of the culture and application of the strain of the present invention. plant material. Bare-root strawberry plants of the cultivar "Fortuna" were provided by the nursery.

[0104] Plant size and density. The planting size was 2m between rows and 0.1m between plants, and the plants were arranged in quincunx on coconut fibre sacks measuring 1mx0.18mx0.15m. This resulted in 100,000 plants per hectare or 1m 2 10 plants per farm, 192 m 2 This results in 1920 plants in the entire study.

[0105] Dates of planting and removal of crops. The strawberry transplants were done on February 11, 2017, and the crop was removed on January 6, 2018.

[0106] water design A 3.5kv electric pump was used, connected to a 32mm secondary pipe which in turn was connected to a 16mm hose with a 3 litre / h self-compensating external dripper.

[0107] Irrigation water The irrigation water used to carry out the trials was supplied from a desalination plant, with 130 litres of water applied per plant throughout the trial, at a concentration of 0.1 grams of nitrate per litre (13 grams per plant). The water characteristics listed above are summarised in Table 14. Table 14 presents data on irrigation water characteristics. TIFF0007644934000014.tif80111 The strain of the present invention can be applied to the leaves at a concentration of 333 g / ha (3x10 7 The samples were sprayed at a dose of 100 mg / g (CFU / g). Foliar applications were performed with a spray backpack at a rate of 200 litres per hectare when plants were at stage 13-14 on the BBCH scale, under conditions of 20°C temperature, 50% relative humidity, no condensation on the leaves, and 15% cloudiness.

[0108] *Parameters and evaluation methods Counting colony forming units per gram of leaves (CFU / g) This parameter was measured to ascertain how abundant the microorganisms were in the leaves throughout the culture cycle. This was measured by a serial dilution method by taking 1 gram of leaves from 50 plants and plating them on selective medium without nitrogen and containing methanol as a carbon source.

[0109] Nitrogen in leaves This parameter is an indicator of the amount of nitrogen accumulated in each plant tissue at the time of analysis. Leaves, stems and fruits from 100 plants were analysed for total nitrogen by the Kjeldahl method.

[0110] yield. To assess crop yield, 30 crops were randomly selected from each treatment and harvested over the crop cycle. Statistical analysis. The factors (yield) and their interactions were analyzed by ANOVA for each parameter, which was performed using Statgraphics Plus software for Windows 5.1 (Manugistics, Inc. USA).

[0111] *result. Colony forming unit (CFU / g) count. Table 15 shows the results in terms of colony forming units per gram of leaves measured monthly from the time of application. TIFF0007644934000015.tif66122By colony sampling, persistence of the present strain is observed throughout the plant culture cycle.

[0112] Total Nitrogen Table 16 shows data relating to nitrogen concentration (g / 100g plant), total nitrogen (g / plant) and fresh weight of plant (g) for the strawberry crop. Table 16 shows that as nitrogen fertilization increases, the nitrogen concentration increases, both in the control and in the treatments with the strains of the present invention. An increase in total nitrogen was observed in plants treated with the strain of the present invention until the nitrogen fertilizer reached 75%. The highest value was observed at a nitrogen concentration of 3.28, which is the highest nitrogen concentration in plants treated with the strain of the present invention. When the fertilization with the strain of the present invention reaches 100%, the amount of nitrogen in the treated plants decreases. TIFF0007644934000016.tif96116

[0113] production From the moment production began, 30 plants were harvested each week, randomly selected from each treatment throughout the culture cycle. Table 17 shows the yield in kg per plant for each treatment. TIFF0007644934000017.tif8986The treatment with the highest yield was T9 (75% nitrogen + application of the strain of the present invention), with 1.4 kg per plant, which was significantly different from the other treatments. The treatment with a UFN of 0 was the treatment with the lowest strawberry production (kg) per plant, and the difference was significant. Table 18 shows data on the nitrogen balance: the amount of nitrogen provided by fertilizers, the amount of nitrogen assimilated by the plant after extraction, and the balance of assimilation between the amount input to the plant (amount provided) and the amount extracted. TIFF0007644934000018.tif49119

[0114] conclusion Colony forming units per gram of leaves (CFU / g of leaves) Persistence of the microorganisms in the leaves is observed throughout the strawberry growth cycle. Total Nitrogen Plants treated with the strain of the present invention, treatment T9, have the highest nitrogen concentration in the leaves compared to all control treatments. production Treatment T9 (503 UFN), which is the application of the strain of the present invention, showed a 21% increase in production over the control treatment T5 (671 UFN) and a 33% increase in production over the congener fertilized treatment T4 (503 UFN).

[0115] Example 5 Test 5 - Field-based validation test in viticulture *the purpose. The aim of the study was to find out the effectiveness of the strain of the invention on the development of grapevines, which were fertilized with conventional doses of nitrogen, as determined in terms of leaf green evolution (SPAD), microbial concentration in the leaves and production.

[0116] material and method Testing station and fertilization. Tests were conducted at two test sites, Jumilla (located at 38°33′13.9″N, 1°21′16.5″W) and Tomelloso (located at 39°11′22.4″N, 2°59′15.8″W). The recommended fertilization is conventional fertilization with a background application of 700 kg NPK (15-15-15), which is equivalent to 105 UFN over the entire cultivation cycle.

[0117] Distribution of treatments on farm. The farm sizes were as follows: Farm 1 (1 ha), Farm 2 (2.20 ha), and a 16 m wide block (i.e. area) between the two adjacent was taken for the evaluation of parameters.

[0118] Cultivation and application characteristics of the strain of the present invention. The grape variety on farm 1 was petit verdot and on farm 2 was Grenache. The application dates of the strains of the invention were May 10, 2018 (farm 1) and May 15, 2018 (farm 2). The strain of the present invention was applied at a yield of 333 g / ha (3x10 7 The doses were determined using a 100% ELISA kit (Table 1). Foliar applications were made with a nebulizer at 350 litres of broth per hectare when the plants were at the peppercorn stage and under the following climatic conditions: air temperatures of 17°C and 20°C, relative humidity of 20% and 38%, wind speeds of 5.4 mps and 9 mps from the south and northwest, no dew on the leaves, and 10% and 25% cloudiness. FIG. 12A shows the nebulizer upon application of the treatment, and FIG. 12B shows the particle size upon application of the treatment.

[0119] Parameters and evaluation methods yield To assess crop yield, six blocks were randomly sampled, each with an area of ​​30 m2, and the grapes obtained on the plants were weighed. Statistical analysis. The factors (yield) and their interactions were analyzed for each parameter. This was done by ANOVA using Statgraphics Plus software.

[0120] *result yield Tables 19A and 19B show the results in terms of yield (kg) per plant for each treatment. TIFF0007644934000019.tif56109 conclusion On farm 1, application of the strain of the invention increased the yield by 18% over the untreated control. On farm 2, application of the strain of the invention increased the yield by 41% over the untreated control.

[0121] Example 6 Trial 6 - Evaluation of the strain of the invention on the yield of escarole crop (Cichorium endivia latifolia, cultivar Lempika). The following tests were performed by an independent accredited entity for GEP testing. The design of this study, the analysis and reporting of the results were carried out, wherever possible, in accordance with EPPO guidelines PP1 / 152 ( 4 ), PP135 ( 4 ) and PP1 / 181 ( 4 ).

[0122] General information about the experiment The experiment started on August 17, 2018 in Vila Sacra, Girona, Spain, and ended on November 12 of the same year. The transplantation date was August 21, 2018. Conventional fertilization was performed on August 23, 2018, and the application of the strain of the present invention was on September 28, 2018, after the cultures had reached the appropriate developmental stage (BBCH17-19). Study design The trial consisted of a randomized block system with four treatments and five replications. Each plot was 2.5 m wide, 7 m long and had an area of ​​17.5 m. 2 The rows of plants were spaced 0.56 m apart, with 0.35 m spacing between plants within a row, for a total of 80 plants in 4 rows.

[0123] Cultivation The effect of the strain of the invention was studied on endive crops (Cichorium endivia latifolia, cultivar Lempika). Previous crops on the local farms were onion in 2018 and sunflower and lettuce in 2017.

[0124] soil The soil showed good levels of fertilization. %MO:2.02 Texture: clay loam pH: 7.70 CEC;0.22 Soil drainage: Good

[0125] maintenance Throughout the cultivation cycle, necessary phytosanitary treatments were applied to maintain the health of the plants.

[0126] Statistical Comments Statistical analysis and reporting was realized using the software Agricultural Research Manager (ARM) (developed by Gylling Dates Management Inc.). The evaluated data were analyzed using one-way analysis of variance (ANOVA) combined with Student-Newman-Keuls at α = 5% for comparison of means. Means followed by the same letter are not significantly different.

[0127] Purpose of the experiment To evaluate the effect of the strain of the present invention on crop performance and yield of escarole crops applied with normal or reduced fertilization rates. To compare the effectiveness of standard commercial fertilization with the strains of the present invention. To evaluate the unexpected effects of the strains of the present invention on target crops.

[0128] Overview of the study The purpose of this study was to determine the effect of the strain of the present invention (3x10 7 The objective of this study was to evaluate the effect of 100 mg of 1000 CFU / g on the yield of escarole crops. The study was carried out in an open field in the city of Vila Sacra (Girona), north-east of Spain, on a small area typical for vegetable cultivation. This study was carried out in accordance with the principles of Good Experimental Practice as laid down in European Union Regulation 1107 / 2009.

[0129] The strain of the present invention was tested at the same rate (300 gfp / ha) in three areas with different nitrogen fertilization rates (100% (normal), 60%, and 40%). It was evaluated against an "untreated" control, which received 100% nitrogen (N) fertilization and no application of the strain of the present invention. Fertilization was performed before transplanting (August 23, 2018). The products were applied at BBCH stages 17-19 (September 28, 2018) using a knapsack-type calibrated sprayer (HONDA WRJ2525).

[0130] These assessments were made in terms of percentage of crop vigour during crop development. They were recorded at or near harvest. Yield parameters were assessed on 20 plants each in the two central rows of the plot, except for the two border rows, which were not assessed. Plant diameter (cm), number of plants present (distinguishing between marketable, non-marketable, dead and live plants), plant weight and yield (2 central rows (7.84 m2)). 2 The number and weight (kg) of marketable plants per hectare were assessed. Yield per hectare, number of marketable plants and percent increase compared to the standard unfertilized treatment (treatment 1) were calculated automatically using ARM software. Phytotoxicity and vigor assessments were performed throughout the trial. Table 20 shows the processing list. TIFF0007644934000020.tif11386 The following abbreviations are used in Table 20: UTC: untreated control UF: Normal fertilization F: Fertilization Ms: Methylobacterium sp. strain, i.e., the strain of the present invention

[0131] conclusion Product Benefits According to the test conditions, it can be concluded that the strain of the present invention, which was applied with a reduced dose of nitrogen, obtained a numerical increase in the average plant weight (kg) and commercial yield (number of plants per hectare) compared with only one application of conventional fertilizer (without the strain of the present invention), which means that the strain of the present invention was able to provide the nitrogen required for the normal development of plants.

[0132] Treatments 3 and 4 achieved a yield increase of 3.8% and 13.89%, respectively, compared to treatment 1, with statistically significant differences. There were no significant differences in the rest of the evaluated parameters between the strains of the invention treated with different doses of nitrogen, although the results obtained suggested the best results with higher nitrogen reduction.

[0133] The strain of the present invention produces a yield of 300 g / ha (3x10 7 cfu / g). The strain of the present invention is effective in reducing the fertilization rate of 300g / ha (3x10 7 cfu / g) showed the best results of the test with an increase in yield of 13.89%, which is statistically significant. Regarding the other parameters evaluated, there are no significant statistical differences between the treatments, indicating that the strains of the present invention provide the nitrogen necessary for the normal development of the plants.

[0134] The use of the bacterial strains of the present invention reduces the amount of nitrogen fertilization required for crops, as shown by results for the parameters evaluated that were similar or even better than those obtained with conventional fertilization.

[0135] Example 7: Methods of Producing Exemplary Compositions According to the Invention Collect Methylobacterium sp. strain NFBG medium (nitrogen-free bacterial medium supplemented with glucose). Growth parameters Growth temperature: 20-35℃ Culture period: 5~7 days

[0136] An isolated pink-red colony is selected and re-plated onto MMMNF (nitrogen-free minimal medium supplemented with methanol). Growth parameters Growth temperature: 20-35℃ Culture period: 5~7 days

[0137] An isolated pink-red colony is selected and re-seeded in MMM (methanol-containing minimal medium), grown in liquid culture and used as a pre-inoculum. Fermentation parameters Growth temperature: 20-35℃ Stirring speed: 50-250 rpm Fermentation period: 5 to 7 days

[0138] Inoculation of the entire bioreactor by pre-inoculation with microorganisms. Fermentation parameters Growth temperature: 20-35℃ Stirring speed: 50-250 rpm Aeration rate: 0.3-3.2N*m 3 / h Fermentation period: 80-130 hours

[0139] Mix with carrier and dry by spray drying One liter of the fermentation broth containing the microorganism is mixed with an agriculturally acceptable carrier. The mixture was then spray dried using the following drying parameters: Feed flow: 7-11ml / l Compressed air flow: 225-375l / h Dry air flow: 35-50m 3 / h Inlet temperature: 100-180℃ Cyclone performance: 25-40%

[0140] Blending of products for final product formulation Each 1 g of dry product (powder + microorganisms) is mixed with the next formulation until 101-550 g of final product is obtained, which is used to fertilize 1 ha. *1-50 g of agriculturally acceptable co-formulation. *100-500g of agriculturally acceptable carrier. Table 21 shows the composition in g / L of the media used throughout the production process. Filter sterilized components are added after autoclaving and once the medium has cooled to below 60°C. TIFF0007644934000021.tif160123 Table 22 shows the amounts in grams of micronutrients and vitamins. TIFF0007644934000022.tif109110

Claims

1. A strain of Methylobacterium sp. nov. deposited under accession number CECT9580.

2. A composition comprising the strain of Methylobacterium sp. NV deposited under accession number CECT9580.

3. Contains either an agriculturally acceptable carrier or an agriculturally acceptable co-formulant, or both The composition according to claim 2 .

4. The carrier or co-formulant may be talc, clay, maltodextrin, skim milk, glucose, dried plant extracts, extracts from tea, cassava, quinoa, kaolin, coir, diatomaceous earth, chitin, CaCO 3 , alginates, carrageens, surfactin, rhamnolipids, sophorolipids, saponin, potassium oleate and mixtures thereof. The composition according to claim 3.

5. The composition is in a form selected from a solid form, an aqueous liquid form, an oily liquid form, an emulsion form, a semi-solid form, or a gel form. The composition according to claim 2 .

6. The composition is in solid form. The composition according to claim 5 .

7. The composition is in powder form. The composition according to claim 6.

8. Applied as a seed coating, by soil application, drip irrigation systems, drench, or foliar application The composition according to claim 2 .

9. 3. The method for producing the composition according to claim 2, (A) providing a strain of Methylobacterium sp. NV deposited under Accession No. CECT9580; (B) inoculating the strain of Methylobacterium sp. NV in a liquid medium containing methanol; (C) culturing the inoculated strain in the liquid medium to produce a liquid culture comprising the strain; (D) drying the liquid culture to obtain a dry composition; 3. A method for making the composition of claim 2, comprising:

10. The strain was isolated from the interior of a spore of the mycorrhizal fungus Glomus iranicum var. tenuihypharum.

10. The method of claim 9.

11. (E) adding an agriculturally acceptable carrier to the liquid culture produced in step (C) prior to step (D).

10. The method of claim 9, further comprising:

12. (F) adding an agriculturally acceptable co-formulation to the dry composition produced in step (D).

10. The method of claim 9, further comprising:

13. (G) adding the agriculturally acceptable co-formulation to the dry composition produced in step (D). The method of claim 12 further comprising:

14. 2. Use of the strain of Methylobacterium sp. NOV according to claim 1 as a biostimulant in plants.

15. The plant is any one of a horticultural crop, a herbaceous crop, a pasture crop, a timber crop, a grape, a berry, and a legume.

15. The use according to claim 14.

16. Increase the yield of the plant 15. The use according to claim 14.

17. 2. Use of the strain of Methylobacterium sp. NOV according to claim 1 for reducing external nitrogen input by up to 60%.

18. 3. Use of the composition according to claim 2 as a biostimulant in plants.

19. The plant is any one of a horticultural crop, a herbaceous crop, a pasture crop, a timber crop, a grape, a berry, and a legume.

20. The use according to claim 18.

20. 20. The use according to claim 18 for increasing the yield of said plant.

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