PLANT INOCULATION METHOD.

Abstract

The present invention relates to a nitrogen-fixing strain of Gluconacetobacter diazotrophicus that can be obtained from a repository maintained by CABI in the United Kingdom with repository access number IMI504958, or a nitrogen-fixing variant of said strain.

Description

PLANT INOCULATION METHOD Field of invention The present invention relates to a method for inoculating plants with a nitrogen-fixing bacterium and to 5 compositions and kits suitable for use in that method. Background of the invention The nitrogen-fixing bacterium Glusonacetbacter diazotXópMóus, previously known as Acetobacter diasotropnicus (Gillis, M. et al. Int. o. Syst. Eacteriol. 39:361-364; 1999}, was originally isolated from the roots and stems of sugarcane (Cava!canterV. A, et al. (1988) Plant Soil Vol. 108, p. 23-31). It has been shown by incorporation of ί:δNχ that G. diaxotrbphicus fixes nitrogen within sugarcane plants (Sevilla, M. et al, Mol. flant Microbe Interact. 14:3S8-366; 2001; Boddey, RM et al. Pladt Solí 2:>2: 139-14 9; 200 31 and that has the ability to excrete almost half of the fixed nitrogen in a form that is potentially available to plants (Cójho, E, H et al. Fed. Eur. Microbio!. Sos. 6b 'roc:^.. Lett. 106:341-34^ 1993). The bacteria invade the cells of the mesophylls of the sugarcane root and at the points of emergence of lateral roots that are synthesized at the intercellular level, and also in the tylome, without modulation (James, E.:K'. et al. Ji Exp. Batí 52:7 47-768; 2001). The conditions in which the infection could occur have been demonstrated. intracellular Gd collation and enabling, non-nodular eridosymbiotic nitrogen fixation (EP-B-1422 997 and Couking, E1C.,. et al. (2096) In Vitro Cellular and Bialocy - Blaht Vol. 42, Noli, p:74-82). In particular, the bacteria are administered to the plant growth medium as the plant grows in germination or within 7 days of the same. Document WO2011 / 144741 suggests that bacteria such as Gd can be injected into cane cuttings to improve nitrogen fixation. Clearly, such a technique is not the only one that could be applied in any large-scale agricultural operation. Applicants have found that growing plants can be successfully inoculated with nitrogen-fixing bacteria. Summary of intent According to the present invention, a method is provided for inoculating a plant with a nitrogen-fixing bacterium, and said method comprises administering the nitrogen-fixing bacterium 20 to a lesion of a growing plant. It has been found that when applied to a lesion, particularly to the surface of a lesion, in plant tissue, subsequent plant growth is enhanced. For example, biomass or yield may be improved and / or the number of flowers may be increased. This may be due to the colonization of the plant tissue by nitrogen-fixing bacteria in a manner similar to that described, for example, in document EP-B-142.39S7, although the fact that this can occur when applied in this way is surprising. The nitrogen-fixing bacteria that have colonized within the plant tissue can provide a source of intracellular nitrogen that enhances plant growth. Therefore, the method of the invention provides a useful means of administering a plant growth-enhancing treatment to growing plants. The nitrogen-fixing bacteria must be those that can be located intracellularly within a plant cell. In one particular embodiment, these are symbiotic, nitrogen-fixing bacteria that are located intracellularly, such as the strains Oiuccuacetbucter diasetrepóicus IMI 504.99-8 (formerly IMI 501986) and OX» 50058 (formerly IMI 504853); these strains were deposited with CABI (United Kingdom) on September 21, 2012, and May 22, 2015, respectively. Such strains are novel and constitute another aspect of the invention. Alternatively, the nitrogen-fixing bacteria may be a species of Berbasplzillum. Other nitrogen-fixing bacteria include BelJer3seria, Cloátrf di tur, ÁhlzoMLW Y lipo / erum. In a particular form of reclining, the nitrogen-fixing bacteria is administered jointly or in combination with a strain of Terribadiles, as described in the applicants' co-pendant international patent application, which claims priority over British patent application No. 1400840.3. The applicants have found that a strain of this type can enhance the activity of the nitrogen-fixing bacteria. Suitable strains of Terribac 21us include terribací 1 lus sa bdhárdph i 1 as, Ter ríhaci 11 as ha 1 cph i 1 os, berro badl las goríensis or Terribáti 11na afdfngtnafs pepo in particular is a layer of Terribaci 11 as sacaharophf fus.. The bcrribacill as berribaci.^ either separately or in mixture with nitrogen-fixing bacteria <.qeuo . Ter r^ Pací 1 i un can exist in intimate mixture with nitrogen-fixing bacteria (and certainly, 1MG501985 (now IMI 50499S), has been classified as a consortium of Gd and Terrlbac.;33as) or it can be administered in the form of a co~cultivo or mixed culture. The injury may result from a nutrient or natural fallout, after which the additional availability of nitrogen may facilitate restorative growth. However, in one particular embodiment, the injury is the result of damage caused by actions such as mowing (forage, lawns), cutting (silage and forage crops), management of suckers (banana, pineapple, sugarcane, sorghum, rice, guava, cotton, abaca, ramie), pruning (fruit trees, vines), consumption by livestock, or harvesting. In some cases, other procedures such as tearing, in which plants may be unintentionally or incompletely damaged, may not be suitable. In particular, the injury will be found on a part of the plant 'above ground', such as the leaves or stems. Therefore, the method of the invention may further comprise a preliminary step of inflicting 'damage' on the plant, in particular by mowing, cutting, handling shoots, pruning, or harvesting. The nitrogen-fixing bacteria is applied appropriately within a relatively short period of time after performing these actions; for example, within 4.8 hours, for example, within 24 hours, such as within 10 hours, and appropriately within X-2 hours of inflicting damage on the plant. The delivery of the bacteria is achieved by applying a suitable formulation to the injured area, particularly to the surface of the lesion, in the form of a composition. The composition may be in the form of a liquid, a gel, or a paste, which can be applied directly or in diluted form; or it may be in the form of a solid composition. 5 as a powder or granular composition that will be dissolved in a liquid such as water before use.» In solid compositions, the bacteria will be used in dry form, for example in lyophilized form, which is reconstituted by adding water.» If desired, the bacteria can be multi-encapsulated using methods known in the art in order to maintain high viability and stability of the bacteria. In one particular embodiment, the composition is in a form suitable for spraying onto plants and is therefore suitable for dilution, which may be in the form of a liquid or a solid, particularly in the form of a liquid, or may comprise a diluted aqueous composition that can be sprayed directly. Alternatively, the composition may be one in which the surface of a plant lesion can be immersed by sinking, for example. The amount of nitrogen-fixing bacteria administered in any particular case will depend on factors such as the type of seed being treated, the specific strain of nitrogen-fixing bacteria used, the level of germination improvement required, and the method of administration, as well as the desired effect. Typically, however, a solution containing 1 to 1 x 10⁷ bacteria per milliliter of applied compound, for example, 10⁻¹⁵ bacteria per milliliter of compound, for example... 5S-2ÓQ^ bacteria· per millil-itra;ds composition.,· such meat 190 bacteria per milliliter of composition, c.^· djr..\s'ir. to the lesions of a plant. A solution of this type can be obtained by cultivating the bacteria to an easily detectable level, for example by examining the optical density and then diluting the solution to the appropriate ppm. Applicants have found, for example, that in the case of certain bacteria, the effects on a property such as biomass are affected by the number of bacteria stacked in a dose-dependent manner. This means that different doses can be administered depending on the goal of the treatment. In the case of grasses, for example, it may be required that biomass be as high as possible, whereas in the grass, slow growth may be preferable. In such cases, the number of bacteria administered will be selected to provide optimal biomass production for the desired grass species, as exemplified below. In one particular embodiment, the composition further comprises an aerant for the nitrogen-fixing bacteria, for example the composition may comprise 3% w / v sucrose, as described in document EPB-14229S7. The bacteria: which fix nitrogen can be the only active component of the composition that can be combined with other active chemical components, such as insecticides, fungicides or plant growth regulators, as required. The composition may also include: additives to excipients such as thickening agents, dispersants, diluents, wetting agents, solid carriers, etc. as known in the art, 1:@ In a particular form: the composition further comprises a polysaccharide or a surfactant acceptable in agriculture or a combination of these. In a :&»' of part-realization:lar, the composition also includes a surfactant acceptable in agriculture. The presence of a surfactant that competes with it, they use to be able to flow relatively freely over the entire surface of the lesions to facilitate the entry of nitrogen-fixing bacteria. Suitable surfactants or detergents include 20 non-ionic detergents such as those sold under the trade name 'Tween1^', for example Twen SO. Tween 8Q is a non-ionic detergent; composed of 70% oleic acid and the remainder a combination of linoleic, palmitic, and stearic acids. The pH of a 25% solution is in the range of 5.5–7.2. It is used in medicinal and food products. It has little or no activity as an antibacterial agent (Dawton et al. (1986) Data from Biochemical Analysis, 3rd ed., Oxford). University Press (New York, NY: liátk p, 289). The amount of surfactant administered to the plant lesion must be sufficient to produce an enhanced effect on plant growth when in combination with nitrogen-fixing bacteria (and optionally also a polysaccharide as further described below). This will vary depending on several factors such as the particular surfactant, the type of plant being treated, the nature of the lesion, the particular strain of nitrogen-fixing bacteria employed, and the method of administration. However, it is not usually a composition comprising 0.0005 to 10% v / v, such as 0.0005 to 0.3% v / v, for example 0.0005 to 1% v / v, inclusive from 0.0005 to 0.21% v / v, for example from 0.0005 to 0.15% v / v, such as approximately 0.1% v / v. 20: In another embodiment, the composition comprises a polysaccharide. Suitable polysaccharides for the composition include hydrocolloid polysaccharides derived from vegetable, animal, or microbial sources. In particular, these include polysaccharides such as gum arabic, ghatti gum, karáya gum and tragacanth gum, derivatives of ulose such as cardoclino, feti feelulo sathidr oxypfefe 1 a Lasa, h idr ofepccpx 1 celulose or celulose fecundum, starches and derivatives which include, for example, malt starch, cassava starch, potato starch, arica starch, wheat starch and modified versions of starches such as latinized starch, oxidized starch, ethyl starch, starch dextrin or. maltodeferin, pectin, polysaccharides derived from algae, such as agar, alginates, carrageenan and fucelarafe, seeds such as guar gum and locust bean gum, polysaccharides derived from microbial fermentation such as xautan gum and galane gum, and nitrogen-containing polysaccharides such as gitosan, or other compounds. }uu·,^ fe ' - i Ara m ~ ud ' / ai, '1 pfe is an exuded gamma polysaccharide such as gum arabic, ghatti gum, karaya gum, or tragacanth gum. A particular example of the polysaccharide is gum arabic. Gum arabic is a natural gum collected as exudates from different species of acacia trees (Fang et al. 2010; Bfemoleefe.es: U, 1398-1405); its complex polysaccharide has been extensively used in a wide range of industrial sectors including paints, glues, pharmaceuticals, textiles, and food. It is believed that gum arabic from the acacia tree is a branched polymer of galactose, rhamnose, arachidonic acid, and glucuronic acid as calcium, magnesium, and potassium salts with a molar weight of approximately 250.00. It has been shown (Badar, KV et al. (2011) Recent Research in Science and Technology 3 (5) 6-7) that it has a protective effect on the germination of seeds when the seeds of these plants are... soaked in 1% solutions of the arable gum for 24 hours before germination. In addition, document WO02 / 05S466 reports that certain compositions comprising... combines oi enea de.Polysaccharides and peptides can enhance the yields of 1 os or 11 i vos. The amount of polysaccharide administered to the plant lesion must be sufficient to produce an enhanced nitrogen fixation effect when given in combination with nitrogen-fixing bacteria and orally also a surfactant. This will depend on various factors such as the particular polysaccharide used, the type of plant being treated, the natural lesion, the particular strain of nitrogen-fixing bacteria employed, and the method of administration. However, a composition comprising from 0.1 to 1% w / w is normally used, for example, from 0 to 0.5% w / w, such as approximately 0.3% w / w of polysaccharide. In one embodiment, the composition comprises both a polysaccharide and an agriculturally accepted surfactant. It has been found that, in some circumstances, these components enhance the effect of nitrogen-fixing bacteria and appear to work synergistically together to produce a more significant improvement. Plants treated with a composition comprising these components may exhibit increased growth, as evidenced by the increased dry weight of the treated plants. The novel compositions comprising the 10 components already mentioned form another aspect of the invention:, therefore, in another aspect: the invention provides an acceptable composition in agriculture comprising a nitrogen-fixing bacterium, in particular Cluecas retobee Car dlamtrophious, and a polysaccharide, a surfactant or a combination thereof. The nitrogen-fixing bacteria are as previously described and are particularly recruceous, which are present in adequate amounts as previously described. Similarly, the 20-phosphate is a polysaccharide as previously described, such as an exuding gum polysaccharide, for example, gum arabic, and is included in the composition in an amount as previously described, for example, at a concentration from 0.1 to 1% w / w of polysaccharide 1. Additionally, the 25-surfactant is adequately a surfactant as previously described. 1Θ described atoes, tal domo un detergent non ionic., for example un surfactante que este compuesto en el 70% de ácido grasa oleic ácido y el resto de un gummo inación de aceites linoleic, palmitico y stearic. In a particular embodiment, the composition will comprise from 0.0005 to 10% v / v of surfactant, for example from 0.0805 to 0.2% v / v of surfactant.In another aspect, the invention provides a kit for preparing an agriculturally acceptable composition comprising a nitrogen-fixing bacterium. In such kits, the nitrogen-fixing bacteria, and in particular the GI toOtoto tobac tez di acto repto cas, can be kept separate from other components of the composition, for example in separate containers or in a two-part package or container. The nitrogen-fixing bacteria can be liquefied. The other components can be in the form of a concentrate, for ease of storage or transport, ready for dilution with water, for example, at the point of use. Concentrates of this nature will contain the same components as the listed compositions, but generally at higher levels. Therefore, for example, a concentrate may contain from 1 to 10% ρ / ρ, for example from 1 to 5% w / w, such as approximately 3% ρ / ρ of p©lís <toárido#y una dilución de 10 toces: que.will result in the appropriate compaction for use in the method of the invention, for example, similarly, the surfactant may be present in an amount from 0.005 to 2% w / v in the concentrate. Other components such as, for example, a nutrient for nitrogen-fixing bacteria. are appropriately included in the concentrate at the required concentration, Kits of this type can be used to produce a composition of the invention, which can be used directly. In particular, any concentrate will be diluted with water to an appropriate volume, after which nitrogen-fixing bacteria will be added. The invention makes it possible to apply and deliver nitrogen-fixing bacteria at the intracellular level to a wide range of crops. In particular, these can be perennial, biennial, or persistent annuals, including but not limited to fruit trees and shrubs (e.g., blueberries, raspberries, and tea plants), grapevines, forage crops (alfalfa and grass for silage, hay, or direct consumption by livestock), and pasture. 2ü entertainment and hedges, silviculture, horticulture and herbs.· (for example spring onion, asparagus, eggplant) < It has been previously reported that Gd can improve the yield of sucrose-rich crops such as sugar beets or sugar cane (WO2310 / 022517). However, the applicants have found that using the treatment of the invention results in improvements in crops that are not sucrose-rich, and this constitutes a particular form of realization of the invention. In one particular embodiment, the method and composition of the invention are applied to grass, such as lawn or pasture, immediately or shortly after mowing. This treatment results in improved grass growth, evidenced by an increase in the dry weight of the inoculated grass compared to the uninoculated grass. It appears that nitrogen-fixing bacteria are able to pass into the grass through the lesions resulting from the mowing procedure and colonize the grass plants at the cell level, leading to improved growth characteristics. Furthermore, Gd colonization has been found to increase chlorophyll levels in plants, particularly in grass species such as pasture, recreational, or lawn grasses. Since increased chlorophyll is linked not only to nitrogen content but also to the greenness of the plants, this property is highly desirable in applications such as recreational or lawn grasses where high levels of greenness are beneficial. ~ 16 ” Detailed description of the invention The invention will now be described by means of examples with reference to the accompanying diagrams, in which: Figure 1 is a graph showing the average dry weights (g) of a cut grass not inoculated to inoculated; Figure 2 is a graph showing the aboveground dry weights of inoculated cut grass treated with Gd and sucrase, Tween and / or gum arabic ©, or combinations thereof; Figure 3 illustrates an example of the preparation of vegetative propagation of tea, where (A) illustrates the removal of the cuttings and (B) is a schematic representation of subsections of each cutting taken for ADK isolation; Figure 4 shows an image: of a gel of products of PCR obtained from samples of tea plants that had been inoculated with Gd; all bands in the control plants were sequenced and confirmed as non-specific linkage. The sequenced bands from the inoculated plants were confirmed as Glucose acetobacter diphasatrophieus; Figure 5 is a graph that shows the effects of various factors on the biomass of cut grass; Figure 6 shows the results of an experiment to determine the effect of Gd on the number of buckets of grass flowers; and Figure 7 is a graph showing the results of treatments with various site conditions in the cut pasture biomass. However, to someone skilled in the art, it will be evident that the specific details required to practice the invention are not necessary. The following descriptions of specific embodiments of the present invention are presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, other modifications and variations are possible in view of the foregoing teachings. The embodiments are shown and described in order to best explain the principles of the invention and its practical applications, so as to enable other persons skilled in the art to make the best use of the invention and the various embodiments with the various modifications as they are suitable for the particular use contemplated. Example 1 Óplicaeigú a pastó cortado detodogia Culture of G, diasotrqpMóus: The G. dfasubrqpñfcus strain IMI 501986 (now ΧΜΪ 50998) plasmic pRGS.561 expressing GUS, was grown in medium Ate'OS, [0.8% (ρ / ν)' agar, yeast extract (2.7 g Ite, glucose (2.7 g Ite), mannitol (1.8 g Ite) , MES regulator (4.4 g Ite , K2HPO4 (4.8 g Ite, and KHteCu (0.65 g Ite) , pH 6.5) as required. The expression of the β-glucuronicase (gusA) 5 gene was assayed by plating on ATSU5 medium containing XGlnc (cyclohexylammonium salt of β-glucuronic acid) 1 50 mg Ite; the formation of dark blue colonies indicated the expression of the gusA gene. Inoculation procedures: An aqueous suspension of A. diaxotropedcus was prepared to give an optical density at 60Q nm of 1-1, o. 10 colony-forming units (CFU) per milliliter. The amount of CFU was determined by serial dilution, placing on plates in ATGUS medium (with antibiotics as appropriate) and counting the bacterial colonies after 13 days of incubation on Petri dishes (25°C, in the dark). The suspension was diluted to 10⁴ to produce a solution containing approximately 100 bacteria per milliliter ready for spraying as described below. A standard weight of Θ.5 g of seeds was sown Casal. appeals perennial vanity grass in John's compost seedling trays. Monday to.ly were lightly covered with the compost. The ideal trays were placed in larger trays and adequate water was added. - 19 of growth to a cycle of: 210C / 15aC day / night: 16 / 8h for 20 days. After which the grass was cut to a height of 2 cm above the ground level using scissors (the cuttings were removed) and the following were applied: Experiment 1. Treatments Water control 4 3% sucrose Gd to water r 3: % sucrose Experiment 2 Treatments '10 Gd to water Gd 1 water + 3% sucrose Gd + water + 0.13 Txeen Gd * water + 0.3% gum arabic Gd and water 4 3% sucrose 4 0«1 % Tween .5 Gd- + water + 3% sucrose * 0.3% gum arabic Gd to water 4 3% sucrose 4 Ote % Tesen + 0.3% gum arabic Weight: dry weight of germinated seedlings The seedlings were removed from the agar with forceps and all remaining agar was washed off the seedlings. Each seedling was placed in a paper bag and placed in an 80° oven for 48 minutes and then weighed. The results of experiments 1 and 2 are shown in figures 1 and 2, respectively. The results in Figure 1a show a significant increase of 25% in the mean dry weight of the grass (0g for the uninoculated section and 0.1276g for the inoculated section). These dry weights were significantly different. <O.G1. Por lo tanto, la inoculación de asta manera conduce claramente á un mejoramiento significativo del crecimiento. releo eou?a' oiet . \s u. K nmn 2 show a significant difference (F<Ó . 0'01) between Gd / S / T / GA and the next highest dry weight (Gd / T) and Gd / S / T, which demonstrates an anergic effect of the combination of the three components. Gd and Gd / S are not significantly different at 10 ?«€.O5. Example 2 Colonization of tea CCamellfa siren¿s) by Gluccaacstoñ^^ di aao t. r cpó leus (A io tu cvs) Vegetative reproduction from a stem cutting The standard method for vegetative propagation of tea clones is a single leaf cutting. From larger stems comprising approximately four to six nodes and a shoot tip, stem and leaf cuttings were selected based on tissue health (i.e., free from insects and diseases). The section chosen for the cutting was between red and green wood (as recommended by Yamasaki et al., Soil and Crop Management, (2009) SCM-23). ​​Newly matured shoots containing slightly reddened bark have been found to be suitable. adjacent to the mature leaves, axillary buds that break, actively, can give rise to the best success of ar rs · g,.?, From the preferred sections, a sample was selected comprising a 3-5 cm section of the stem and a healthy leaf. Each stem section was removed using a diagonal cut (Q) approximately 0.5 cm above the leaf (2) and another diagonal cut below the leaf around an internode (3), which avoided pinching or bruising the side of the lesion (see Figure 3A). The background: each tea stem cutting was dipped in a 1% solution of indole-3-butyric acid and placed in individual pots; the cutting was planted with the stem straight at a slight inclination so that the leaf did not touch the soil. Each pot contained sand and a John Innes No. 1 cutting mix in a 4:1 ratio, saturated, with water. 20 µL of water or 20 µL of Gd at 2.5 x 10⁻⁵ cfu / ml in water were applied to the upper cut surface of the cane cutting, and the moisture of each sample was maintained by covering each pot with a plastic film and lightly spraying it with water. After 3 months of growth, and in order to confirm successful colonization of the stem cuttings with Sd, uninoculated and inoculated stem cuttings were removed from the pot. Each cutting was divided into sections that were (a) the top of the shoot, including the inoculation site.(4) in (Figure 3B), (b) the nodular section (5) in figure 3B, and (c) the lower detailed section that includes any root tissue (5) in figure 3B. These sections were frozen in liquid nitrogen until they became brittle. The isolation of ABN from each cutting section (i.e., 4, 5, and 6 in Figure 3) was carried out using tri.w1 reagent according to the manufacturer's protocol and the RCR performed. The RCR carried out was a two-step reaction, as described by Tian et al. (2009); and the first step used GDI~25F (5^ TAGTGGCGG.AGGGGTGAGTAACG^ and GDI-923® <S*~ CCTTGCGGGl^ amplified a 399bp product containing the primer amplicon G-DI139F (5AGAGTAÁCGCGTAGGGA^^) and GDX91SR (5'15 GGAAACAGCOT vd·' n'Lr.' muLV.d? .u rDNA sequence information 165, available from the GenBanl database. After an initial denaturation step at 95°C for 3 minutes, the following temperature profile was run 32 times: denaturation for 20 seconds at 95°C, renaturation for 45 seconds at 55°C, and extension for 20 seconds at 72°C, with a final extension step of 5 minutes at 72°C. A sample of this PCR product was then taken and used as a template for the second PCR step using GDI39F and GDI916R. The modifications to the parameters in the second round included increasing the temperature of the reaction to €2 ®C for 15 seconds, and increasing the number of cycles 39.The PCR amplification products were analyzed on a 1% agarose gel, stained with ethyl bromide, as well as 5 sequentially to confirm the identity of the product. Interestingly, scGl was not detected in section 4 of the tea cutting, suggesting that Azdd moved spontaneously from the lesion site after inoculation and was detected in sections 5 and 6 respectively. Sequencing and subsequent BLAS results provided confirmation that the plants seen in section 1 of the control plants were the result of nonspecific inoculation of the first sets used, and the 4 bands observed in sections 2 and 3 of the inoculated tissue were identified as Gluconacetobacter diastrophiosis (10.0% identification, 86% coverage, and an E value of 700). The results suggest that Gd, although at a low copy number in the inoculated tissue, successfully colonized Camellia sporangia after inoculation of the lesion site. This is possibly the first example of colonization of a perennial plant by Gd. Example 3 Investigation of the treatment effect on grass biomass. Grass was grown in a plant growth chamber (Fitotron) (230 / 150 at 65% humidity) in seed trays using compost OPhn No. 1, for 2 weeks. Short grass was grown to a height of 8 cm and immediately sprayed with 10 ml of the treatment as described below using a domestic sprayer. Treatments 1® 1. Water 2. 31 saccharin 4 0.1% Tucen to 0.3% Gum arabic 3. Water at Gd (2.5x13® cfu / ml) 4. Water 4 3% sucrose + 0.1% Tween + 0.3% Gum arabic 4 Gd (2.5x1O3efu / ml) 5. Water e 31 sucrose + 0.1%. Tw^ne 0.11 Gum Arabic + Gd (2.5x10 cfu / ml) 6. Water at 3%. sucrose + Q. 15 Twen r δ. 3% Gum Arabic 4 gg (2.5xlOscfu / ml) 7. Water· t 35 sucrose + 0.1¾. Tween a 0.3% Gum arabic * Gd 20 .(2.5x15® ctuMl) 8. Water 4· 3% s^cathose + 0.1% Tween 4 0.3% Gum arabic + Gd (2.5x10 cfu / mi> The pasture was returned to the Phytotron for another 2 weeks under similar growing conditions. Five plants were cut from the 25 selected by plowing at ground level to form a sample and were passed on. This was repeated five more times to give a total of six master plants for each treatment. The samples were oven-dried for 48 hours and weighed. The results are shown in Figure 5. These results show that as long as some sucrose is present to support Gd growth, grass biomass increases with the addition of Gd, depending on the formulation. Furthermore, the increase is dose-dependent, with optimal growth observed at 2.5 x 10⁻⁵ cfu / ml. Therefore, a dose of this type can be beneficial if the treated grass is pasture grass, where maximizing biomass is advantageous. However, if the treated grass is recreational turf or lawn grass, the lower biomass will result in less greenness. 15: Increased dosage can be beneficial as it can improve appearance without increasing the need for further cutting or pruning. In this case, a dose of less or more than 2.5 x 10⁻¹·µL / ml can be used. Example 4 Field trial A formulation comprising water + 33 sucrose * 0.1 1 Tucen -í- 0.33 gum arabic -t Gd (2.5x10® cfu / ml) was applied to 1 nd of cut grass plot only (established perennial Lolium grass) relative to 1 nv of uninoculated cut grass plot, treated only with water, (costee!). Using a homemade sprayer, the formulation was applied along with water within 30 ± 5 minutes of mowing the grass. The control plot was protected from the treatment plot with a plastic screen. The application was carried out in the late afternoon in the absence of wind. Plots 1 were not sub-sampled using a 10 quadrant of 20 cm square wire counting the number of heads: fully extended and fully formed in bloom. The results® from each 20 cm square within each plot were averaged: and the results are shown in Figure 6. It is clear that the treatment with Gd, applied in this way, significantly impacted the flower's erection. EJen^lo 5 Comparison of composition components The method of Example 3 was repeated using various compositions that included dual components of the composition used in that experiment. Specifically, the compositions used in this experiment were as follows: Treatments 1. Water z. Water f Gd cfu / ml) 3. Water + 3% sucrose + 0.1% Teten + 0.3% Gum arabic * Gd (2.5x10- cfu / ml) 4. 0.3% Gum Arabic c Gd (1.5x105cfu / ml) 5. 3%. Sucrose + Gd (2.5x10- cfn / ml) 6u S.1S W&en + Gd> (2:.5xlX W AberGlyn grass was grown for 2 weeks in John Iones No. 1 soil in a plant growth chamber (FitotroriX) at 23 / 13°C, 80% humidity. The grass was then cut to a height of 8 cm with scissors, the cuttings were removed, and the grass was immediately treated with 10 ml of solution using a domestic sprayer. The grass was returned to the plant growth chamber for another two weeks. Five plants were selected from the tray and combined together to make one sample and were weighed. This was repeated five more times so that a total of six samples were taken per treatment. The grass was dried for 48 hours at 80X and then weighed. 2© ​​The results are shown. This experiment shows that the component used does have an effect on grass growth. In this example, the surfactant gave the greatest increase in dry weight. The Arabica showed only a marginal improvement over the control, possibly due to the fact that the - 28 surfactant may be required to help in the diffusion of Gd over the plant helps the liquid to enter the lesions of the grass (although on this occasion the combination So showed the expected improvement), Again the water / t treatment Gd was similar to the control, which indicates that Gd needs the addition of at least some of these components to co 1o n1zar 1a s 1e si ores. I can confirm that cp:n in relation to this date, the best method known to the applicant to put into practice 1(1 the aforementioned invention is the one that results from the present description of the invention...

Claims

1. A nitrogen-fixing strain of Gliwtonet diazotrophicus that can be obtained from a repository maintained by CABI in the United Kingdom with repository accession number IMX50495S, or a nitrogen-fixing variant of said strain.

2. A nitrogen-fixing strain of *Luconacetobacter diawtrophicus* that can be obtained from a repository maintained by CABI in the United Kingdom with the deposit number 11504998, or any nitrogen-fixing variant of *Luconacetobacter diawtrophicus* strain 3. A composition comprising Gfuconaoo; -Meter di aaother oph1oas, a saoarid po1i and / or a sur fac tant.

4. . the composition according to: claim 15 3, comprising Giuconacetobacter diarotropnióus, a polysaccharide and a surfactant.

5. The composition according to claim 3, wherein the polysaccharide is a hydrocolloid polysaccharide.

6. The composition in accordance with claim 203, wherein the polyaccharide is gum arabic.

2. The composition in accordance with claim 3, wherein the solvent is a non-ionic detergent.

8. The composition according to claim 3, wherein the surfactant is composed of 70% of the fatty acid: oleic acid and the remainder is a combination of oleic, pulpic and stearic acids.

9. The composition according to claim 3, wherein the sulphate is a sulphate Tucen. 5 1Ó< the composition according to claim 9, wherein the sulphate is Tocen SO, 11. The composition in accordance with claim 3, comprising from 0.0305 to 10% v / v of surfactant.

12. The composition according to claim 1Q 3, wherein diuconaceiobactér diafeürqpfecus comprises a strain or variant that can be obtained from a repository maintained by CARI in the United Kingdom under repository accession number IIHL 504953 or repository accession number XMX 504998. 15 13. The composition in accordance with claim 3, provided in a form suitable for delivery to a plant.

14. The composition according to claim 3, which is in dry or frozen form. 20 15. The composition according to claim 3, which is in a concentrated form suitable for dilution before being supplied to a plant.

16. A composition comprising a hydrocolloid polysaccharide, a non-ionic surfactant and diazotropaccus 25.

17. A kit comprising the components of the composition according to claim 3.

18. The kit according to claim 17, wherein the components are provided separately from the surfactant and / or the polyaccharide. The kit according to claim 17, having the components in separate containers.

20. A method that supplies a plant with the nitrogen-fixing strain of diutenacetoteoter 10 diazdtrqphlcus do in accordance with claim 1.

21. A method comprising supplying a tria planta the nitrogen-fixing strain of Uteconaceroteotar teazteropdicus according to claim 2.

22. A method comprising supplying a plant with the 1,3 composition in accordance with claim ®n, claim 3.

23. A plant comprising a nitrogen-fixing strain of Glucosacetetecter otezoteqphleus according to claim 1.

24. A plant comprising a nitrogen-fixing strain of Gluconacetone according to claim 2.

23. A plant comprising the composition of claim 3.