Preparations for enhanced biocontrol

Abstract

To provide nitrogen in a form which is available for an extended period of time.SOLUTION: The invention includes a composition, such as a liquid seed coating composition, which comprises at least one macronutrient and at least one microbe, which macronutrient comprises nitrogen in the form of a basic L-amino acid coupled with phosphorus. Further, the invention includes a seed treated to extend the duration of the nitrogen effect without lethal effects on surrounding microbes, and a method of treating a seed to achieve the same objective.SELECTED DRAWING: Figure 1

Description

Detailed Description of the Invention

[0001] FIELD OF THEINVENTION The present invention relates to the field of plant cultivation, in particular the cultivation of plants that utilize biological materials such as microorganisms to directly or indirectly enhance their growth. Preparations according to the invention include compositions in which seeds are combined with microorganisms such as bacteria, as well as granules containing bacteria together with binders, buffers, etc. Other preparations include compositions containing microorganisms such as bacteria and suitable for seed coating.

[0002] 2. Background of the Invention Management of soils and growth substrates as living systems, rather than as inert materials with suitable physicochemical properties for plant growth, is now widely recognized as an essential component of best practices for long-term value creation from farms, greenhouses, and forests. The assemblage of soil microorganisms and microbial communities, the so-called soil microbiome, together with the plant-associated microorganisms, the plant microbiome, present points of management intervention through the addition of beneficial microorganisms with the potential for enhanced biological control, including chemical insecticides (e.g., thiamethoxam, clothianidin) and fungicides (e.g., fludioxonil, difenoconazole), stress protection including increased accessibility to otherwise unavailable nutrient pools, or as an alternative to plant growth regulation to improve crop yields.

[0003] As such, there is a growing market for microbial inoculant products. Such products can be applied directly to growing substrates such as soil, sand, peat, etc., or they can be introduced into seeds or seedlings for placement in various growing environments. The benefits and other effectiveness of such products necessarily depend on the viability of the microorganisms and their ability to survive and grow in the environments in which they are released to realize any potential benefits.

[0004] Nitrogen is an essential element required by all living organisms alike. In all cropping systems, good plant growth and therefore good yields depend on large inputs of nitrogen fertilizer to increase nitrogen availability in the soil.

[0005] Various nitrogen fertilizers have been proposed. AB) describes fertilizers that contain at least one basic L-amino acid, such as arginine or lysine, with the majority of the basic L-amino acid content being present as its monophosphate. The basic L-amino acid phosphate may be combined with a binder and / or provided with a surface layer that functions as a coating. The invention also relates to methods of enhancing plant growth by making basic L-amino acid phosphate available to plants.

[0006] In natural ecosystems, nitrogen availability to plants is regulated by a complex network of organisms, chemical reactions, and soil processes. In agricultural systems, however, deliberate addition of nitrogen through fertilization has been shown to modify soil processes, which in turn modify the plant-soil continuum in a manner that depends on the form of nitrogen. Depending on the form of nitrogen, this includes a shift from oligotrophic bacteria, sometimes capable of fixing atmospheric dinitrogen gas, to eutrophic forms, some of which are harmful to plants and can therefore negatively affect crop production.

[0007] In other words, the bacteria added to enhance plant growth are not affected by the fertilizer added. It can be affected in a negative way and the result of fertilization can be reduced growth rather than improved growth.

[0008] From the above, it is clear that there is an inherent conflict between the intentional addition of nitrogen in the form of fertilizers and the unintentional addition of nitrogen through the accumulation of nitrogen pollutants in the air, and the viability and therefore predictability of microorganisms that promote plant growth. Thus, in any cultivation system, finding a reliable and efficient simultaneous application of nitrogen and beneficial microbial inoculants is, at best, difficult to predict, and at worst, makes the management tools incompatible. This means that fertilization of the soil will necessarily limit the functionality of products containing beneficial microorganisms. Similarly, the incorporation of beneficial microorganisms into seed coatings is not always fully compatible with the incorporation of nitrogen into the same coating. Furthermore, the addition of certain nitrogen-fixing bacterial strains that form a symbiotic relationship with plants, such as Leguminosae plants, such as soybeans, peas, or legumes, is incompatible with the addition of nitrogen to enhance the growth of such plants at early growth stages before the nitrogen-fixing symbiosis is established.

[0009] The above discussed effects have been discussed and described in several research reviews, e.g., Zahran in Rhizobium-Legume Symbiosis and See Nitrogen Fixation under Severe Conditions and in an Arid Climate (Microbiology and Molecular Biology Reviews, December 1999, pp. 968-989).

[0010] Further techniques for making the field of environmentally friendly plant fertilizers are constantly being presented. For example, WO2017 / 222464 (SweTree Nutrition AB) describes a fertilizer proposed to reduce the resources required for fertilizing slow-growing plants, since it is possible to avoid repeated supply of nutrients and nitrogen leakage. More specifically, the composition of the proposed fertilizer comprises a zeolite in whose pores at least one basic L-amino acid is adsorbed.

[0011] Carlos Eduardo Flores-Tinoco et al. (in "Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation," available at https: / / www.biorxiv.org / content / 10.1101 / 741314v1?versioned=true on August 21, 2019) is an article on symbiosis between bacteria and agricultural crops, specifically, the metabolic blueprint for symbiotic nitrogen fixation. The metabolic network based on the co-catabolism of arginine and succinate provided by the plant that drives the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia is described as CATCH-N. In this article, it is concluded that the simultaneous supply of arginine and succinate stimulates nitrogenase activity.

[0012] Zhang et al. (Frontiers in Plant Science, published October 22, 2019, "Organic or Inorganic Nitrogen and Rhizobia Inoculation Provide Synergistic Growth Response of a Leguminous Plant" In "Forb and Tree" present a study on how organic and inorganic nitrogen affect plant growth and the performance of symbiotic N2-fixing rhizobia. Zhang et al. conclude that the species studied responded well to organic or inorganic N forms (or different forms of inorganic N), suggesting that nodulation responses may be plant species dependent and that N supply and nodulation may be synergistic.

[0013] US2013 / 0255338 (Agrinos) describes a composition that enhances crop production, increases plant defense processes, and / or reduces the level of plant pathogens. The composition comprises a microbial composition and a liquid fertilizer, preferably a liquid fertilizer containing at least soluble nitrogen. In one embodiment, the composition comprises a liquid fertilizer containing one or more lactic acid producing bacteria, one or more nitrogen fixing bacteria, and soluble nitrogen. In a preferred embodiment, the described composition comprises a liquid fertilizer and HYTa, a microbial consortium including Lactobacteria, nitrogen fixing bacteria, a potassium source, a phosphorus source, and a microorganism that solubilizes / mineralizes an organic carbon source, Bacillus subtilis, Bacillus thuringiensis strains HD-1 and HD-73, and Trichoderma harzianum. Alternatively, HYTd has been used, which is described as containing 12% by weight of L-amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, alanine, proline, arginine, valine, methionine, isoleucine, tryptophan, phenylalanine, lysine, and threonine), and 5% by weight of glucosamine and chitosan. HYTd also preferably contains one or more or all of soluble minerals (P, Ca, Mg, Zn, Fe, and Cu), enzymes and lactic acid from the chitin digestion process, and other polysaccharides.

[0014] Notwithstanding the above, there is a need in the field of biological control to identify means and methods that can mitigate the negative impact that nitrogen-containing fertilizers may have on the viability of growth-enhancing bacteria.

[0015] overview The present invention relates to a combination of at least one seed and at least one microorganism, the composition of which further comprises one or more macronutrients, one such macronutrient comprising nitrogen in the form of a basic L-amino acid. In particular, the present invention relates to a composition suitable for use as a liquid seed coating composition, such as a sustained release composition.

[0016] More specifically, therefore, in a first aspect, the present invention comprises a composition comprising at least one macronutrient comprising nitrogen in the form of a basic L-amino acid combined with phosphorus, and at least one microorganism.

[0017] In another embodiment, the present invention relates to seeds treated with at least one selected microorganism, macronutrients including nitrogen in the form of a basic L-amino acid, and phosphorus.

[0018] In a further aspect, the present invention relates to a method for treating seeds with a liquid composition comprising the steps of: a) providing at least one microorganism, b) providing macronutrients including nitrogen in the form of basic L-amino acids; c) applying said at least one microorganism and macronutrients to at least one seed. The present invention relates to a method comprising the steps of:

[0019] In another aspect, the present invention provides a method for producing a growing additive, such as a seed coating composition, comprising the steps of: a) providing a microbial inoculant that enhances plant growth when used as a growth additive; b) providing macronutrients including nitrogen and optionally further nutrients; and c) combining the inoculant and macronutrients with a binder. wherein the macronutrients include nitrogen in the form of basic L-amino acids.

[0020] In yet another aspect, the present invention relates to a method of cultivating plants, wherein a composition according to the first aspect, or a growth additive according to the second aspect and a seed are added to a cultivation site in an amount suitable for the specific growth requirements of said seed. [Brief description of the drawings]

[0021] [Figure 1]FIG. 1 shows agar plates testing the growth response of B. megaterium (left) and B. japonicum (right) to L-arginine phosphate. [Diagram 2] FIG. 1 shows the growth response of B. megaterium (left) and B. japonicum (right) to increasing concentrations of arGrow® Complete containing L-arginine as the only nitrogen source. [Diagram 3] FIG. 1 shows the growth response of B. megaterium (left) and B. japonicum (right) to increasing concentrations of RIKA-S containing nitrate and ammonium as nitrogen sources. [Figure 4] Figure 1 shows the growth response of B. megaterium (left) and B. japonicum (right) to different nitrogen sources (L-arginine phosphate "4.1", arGrow® Complete "4.2", and RIKA-S "4.3") with partial addition of zeolite "+". [Diagram 5] Figure 1 shows the growth response of B. megaterium (left) and B. japonicum (right) to different nitrogen sources (L-arginine phosphate "5.1", arGrow® Complete "5.2", and RIKA-S "5.3") partially supplemented with 7.4 mM succinate "+". [Figure 6] FIG. 1 shows soybean sprout dry weight 105 days after planting fertilized with different nitrogen sources (arGrow® Complete and RIKA-S) at planting. [Figure 7] Figure 7. Number (7.1) and dry weight (7.2) of soybean nodules. Plants were grown for 90 days and fertilized with different nitrogen sources (arGrow® Complete and RIKA-S) during the vegetative growth phase. [Figure 8] Figure 8. Soybean pod number (8.1) and dry weight (8.2). Plants were grown for 105 days and fertilized with different nitrogen sources (arGrow® Complete and RIKA-S) at planting. [Figure 9] Figure 9.1 shows the percentage of nodulated broad bean roots from plants grown in sand after 20 days. The color of each nodule (either pink or white) from plants grown for 57 days in soil for sowing is shown (9.2). Plants were fertilized at planting with different nitrogen sources (arGrow® Complete and RIKA-S). [Figure 10] FIG. 10. Germination rate of soybean seeds partially coated with 10 mg L-arginine phosphate per seed after 6 days on filter paper (10.1) or 10 days on sand (10.2).

[0022] Detailed Description The present invention relates to a technology that aims to reduce the incompatibility between nitrogen-containing fertilizers used to improve plant growth conditions and microorganisms.The inventors have surprisingly found that certain forms of nitrogen and their derivatives do not have a negative effect on beneficial microorganisms.Furthermore, these forms of nitrogen are also believed to stimulate the growth of such microorganisms in a wide range of concentrations or addition rates.

[0023] Thus, in a first aspect, the present invention relates to a composition comprising at least one macronutrient, comprising nitrogen in the form of a basic L-amino acid combined with phosphorus, and at least one microorganism. The microorganism is advantageously a selected microorganism, i.e. a microorganism that promotes the growth of a particular plant. The microorganism or microorganisms that are defined as being advantageous for the treatment of cancer are one or more of the microorganisms described herein. Suitable microorganisms are discussed in more detail below.

[0024] The basic L-amino acid may be linked to phosphorus via strong intermolecular interactions such as covalent or hydrogen bonds.

[0025] Some overall bond strengths are provided below for illustrative purposes. In some embodiments, the most prominent molecular interactions are selected from the group consisting of hydrogen bonds, which may be defined as the H-dipole charge of a polar bond in the range of 10-40 kJ / mol, and ion-dipole interactions, which may be defined by an ion charge-dipole charge in the range of 40-600 kJ / mol. In some embodiments, the intermolecular interactions are selected from the group consisting of overall weaker forces, such as dipole-dipole interactions, which may be defined by a dipole charge in the range of 5-25 kJ / mol, ion-induced dipole interactions, which may be defined by an ion charge-polarizable electron cloud in the range of 3-15 kJ / mol, dipole-induced dipole interactions, which may be defined by a dipole charge-polarizable electron cloud in the range of 2-10 kJ / mol, and dispersion, i.e., London interactions, which may be defined by a polarizable electron cloud in the range of 0.05-40 kJ / mol.

[0026] Thus, the present invention relates to embodiments in which the basic L-amino acid is associated with phosphorus by one or more of the interactions described above, e.g., the association is defined by about 50%, about 75%, about 80%, about 90%, or about 95% being one or more of the molecular interactions exemplified herein. Exemplary ranges are 50-60%, 50-75%, 50-95%, or 50-98% of the association is one or more of the interactions described above. In some embodiments, the association is defined by at least 50%, at least 80%, at least 90%, or at least 95% being one or more of the molecular interactions exemplified herein.

[0027] As will be appreciated by those of skill in the art, the type of interaction is affected by the pH and other conditions surrounding the compositions of the present invention.

[0028] Therefore, in this context, the expression "bound to" is understood to be different from the case where basic L-amino acids and phosphorus are added to a composition together with many other ingredients, which will dilute the combined effect of basic L-amino acids and phosphorus. Specifically, from a functional point of view, the term "bound to" is understood herein to be a link that prevents most of the degradation of basic L-amino acids by enzymes such as arginase before they are available as fertilizer to growing plants. In some embodiments, at least about 50% of the basic L-amino acids present in the composition are bound to phosphorus as defined herein. In some embodiments, at least about 60%, at least about 75%, at least about 80%, at least about 90%, or at least about 95%, or almost all, for example more than about 99%, of the basic L-amino acids present in the composition are bound to phosphorus as defined herein. Exemplary ranges are that 50-60%, 50-70%, 60-70%, 60-80%, 60-75%, 70-80%, 70-90%, 70-85%, 80-90%, 80-95%, 85-90%, 85-95%, 90-95%, 90-98%, 95-98%, 95-98%, or 98-99% of the basic L-amino acids present in the composition are bound to phosphorus, as defined herein.

[0029] Thus, the present invention utilizes the binding of phosphorus to extend the duration of the positive effect of basic L-amino acids on the growing plants derived from the seeds. Thus, the composition according to the present invention provides a long-term effect of nitrogen provided in the form of basic L-amino acids and can therefore be considered as a sustained release composition. In this context, the positive effect is enhanced plant growth. This refers to the ability of basic L-amino acids to enhance plant growth without lethal effects, i.e., toxicity, on microorganisms, which are also known to enhance plant growth.

[0030] The composition of the present invention can be a fertilizer, such as a liquid fertilizer composition. Alternatively, the composition can be formulated as a solid composition, such as a solid fertilizer, or alternatively, can be combined with water to form a liquid composition.

[0031] The liquid composition according to the invention may be a seed coating composition suitable for application to seeds by any desired means, such as coating, for example spray coating, or by impregnating the seeds with it.

[0032] Thus, if the basic L-amino acid is arginine, the basic L-amino acid associated with phosphorus can be an arginine phosphate, e.g., arginine monophosphate and / or arginine polyphosphate. Similarly, if the basic amino acid is lysine, the basic L-amino acid associated with phosphorus can be a lysine phosphate, e.g., lysine monophosphate and / or lysine polyphosphate.

[0033] However, as will be appreciated by those skilled in the art, there are other electrostatic interactions that can link basic L-amino acids to phosphorus. For example, in a covalent bond, the basic L-amino acid linked to phosphorus can be phosphoarginine or phospholysine.

[0034] In the present composition, the microorganism can be selected from any nitrogen fixing bacteria, such as mutualistic (symbiotic) bacteria, including Rhizobium associated with legumes; Frankia associated with certain dicotyledonous plant species, and certain Azospirillum genera associated with cereal plants; or free-living (non-symbiotic) bacteria, including Anabaena and Nostoc of cyanobacteria, and genera such as Azotobacter, Beijerinckia, and Clostridium.For example, the microorganism can belong to the genus Bacillus.

[0035] As mentioned above, the composition of the present invention may be a solid or liquid composition in which the microorganisms may be present in any desired form of their respective growth stages. Thus, the composition of the present invention may include, for example, one or more selected species of dormant spores or live bacteria. Advantageously, the composition includes, for example, bacteria in the spore stage of growth, preferably in an inoculum in a carrier, such as a polymer or other support structure. Such structures are well known in the field of biocontrol, including specific examples such as alginates. As will be understood by those skilled in the art, a dry form of the microorganism may be preferred, for example, when a dry composition is prepared and then subjected to dissolution in water before use.

[0036] The basic L-amino acid present in the composition according to the present invention can be any basic L-amino acid, such as arginine or lysine, and is preferably arginine.Arginine can be derivatized or reacted with another macronutrient.Therefore, the composition according to the present invention can comprise phosphate of arginine, for example arginine polyphosphate or arginine monophosphate.

[0037] Although arginine is known to be a growth-enhancing macronutrient, until now it has not been known as a substance that can protect growth-enhancing microorganisms from the toxicity commonly associated with nitrogen-containing additives.

[0038] Additional benefits are provided by combining a basic L-amino acid, such as arginine or arginine monophosphate, with a buffer, such as a microporous aluminosilicate mineral. It is believed that the composition may be used in combination with zeolite. Thus, the composition may include an organic nitrogen-containing structure, such as arginine or arginine monophosphate, in combination with zeolite. Zeolite is available as a natural material or as a synthetic structure, and the present invention is not limited to any particular form of zeolite. Advantageously, the composition according to the present invention includes zeolite that can be loaded with arginine monophosphate, for example as described in WO2017 / 222464 discussed above.

[0039] In a broader aspect, the invention also encompasses the use of zeolites in combination with nitrogen that is not present in the form of basic L-amino acids, as supported by Figure 4. This effect can therefore be exploited in compositions and granules that contain nitrogen of any chemical structure, such as ammonium nitrate, or that contain nitrogen in mixtures of basic L-amino acids with other forms of nitrogen.

[0040] Another aspect of the invention is a seed treated with at least one selected microorganism, macronutrients including nitrogen in the form of a basic L-amino acid, and phosphorus. Seeds according to the invention may be coated or otherwise treated with a composition detailed above and / or prepared as described below.

[0041] Alternatively, the seed according to the invention may comprise at least one layer of said at least one microorganism and at least one layer of said nitrogen in the form of a basic L-amino acid associated with phosphorus. All details and examples provided above regarding the chemical and beneficial effects of such association also apply to this aspect of the invention.

[0042] The seeds may, for example, be impregnated, sprayed or treated with an aqueous solution of said at least one microorganism by any suitable means. The nitrogen in the form of a basic L-amino acid in combination with phosphorus may, for example, be applied as a separate layer in an aqueous solution that is sprayed or applied by any other suitable means to the treated seeds.

[0043] As mentioned above, basic L-amino acids are advantageously associated with phosphorus via electrostatic interactions, and all details regarding the nature of such associations provided elsewhere in this application are also applicable to this aspect of the invention.

[0044] Thus, the phosphorus-linked basic L-amino acid may be arginine phosphate and / or phosphoarginine. Alternatively, the phosphorus-linked basic L-amino acid may be lysine phosphate and / or phospholysine.

[0045] Another aspect of the invention is a method for treating seeds with a liquid composition comprising the steps of: a) providing at least one microorganism, e.g., a selected microorganism; b) providing macronutrients including nitrogen in the form of basic L-amino acids; c) applying said at least one microorganism and macronutrients to at least one seed. The method includes:

[0046] Step c) may comprise combining said at least one microorganism and macronutrients with a binder to provide a liquid composition for application to said at least one seed. In an advantageous embodiment, the step of combining the microorganism with a binder comprises granulation.

[0047] The microorganism may be a nitrogen fixing bacterium as described above and the basic L-amino acid is advantageously The microorganisms may be arginine or lysine. As will be appreciated by those skilled in the art, the microorganisms are selected based on their properties as growth enhancers for the plants resulting from the treated seeds. All details regarding the microorganisms provided elsewhere in this application apply equally to this aspect of the invention.

[0048] The macronutrients of step b may advantageously further comprise phosphorus, e.g. phosphorus linked to a basic L-amino acid via electrostatic interactions. All details and examples provided above regarding the chemical and advantageous effects of such linkages also apply to this aspect of the invention.

[0049] The method may include treating the seeds with phosphorus either separately or simultaneously with the macronutrients.

[0050] The method according to the invention may be a cyclical process comprising at least one cycle of applying macronutrients, e.g. in an aqueous solution, and applying phosphorus, e.g. in an aqueous solution, and an optional step of drying the seeds between said applications.

[0051] The microorganisms may be applied to the seeds before the macronutrients and phosphorus, for example by soaking the seeds with an aqueous solution containing the microorganisms. Alternatively, the macronutrients and phosphorus may be mixed, for example in an aqueous solution, prior to their application to the seeds.

[0052] In another aspect, the present invention provides a method for producing a growth additive, such as the composition discussed above, comprising the steps of: a) providing an inoculum of a microorganism that enhances plant growth when used as a growth additive; b) providing macronutrients including nitrogen and, optionally, further macronutrients; and c) combining the inoculant and macronutrients with a binder. wherein the macronutrients include nitrogen in the form of basic L-amino acids.

[0053] The growth additive can be combined with one or more seeds during their manufacture, or alternatively at a later time, such as at the time of cultivation. As will be appreciated by those skilled in the art, all details provided elsewhere in this application regarding seeds, such as layering for the addition of ingredients, apply equally to this embodiment.

[0054] Advantageously, the step of combining the inoculant with the binder may also include granulation according to well-known methods.

[0055] The microorganism provided in the present method is selected for its advantageous growth enhancing properties and can be any bacterium, such as the nitrogen fixing bacteria discussed above. Specifically, the microorganism can belong to the genus Rhizobium or Bacillus.

[0056] The basic L-amino acid provided in the method can be in any one of the forms discussed above, for example, arginine, arginine phosphate, such as arginine monophosphate.

[0057] Monophosphates of basic L-amino acids are easily prepared by those skilled in the art according to well-known methods. Such amino acid monophosphates may be crystalline, i.e., salts. Alternatively, covalent bonds may be used to prepare the amino acid monophosphates. Furthermore, the present invention relates to, for example, the first embodiment relating to the linkage between basic L-amino acids and phosphorus. All details provided elsewhere in the application are equally applicable to this aspect of the invention.

[0058] Binders suitable for the method according to the invention are well known in the art and the skilled person can easily select a suitable material. Thus, the binder can be, for example, selected from the group consisting of polymers, such as synthetic or natural polymers, such as sugars or carbohydrates; salts; and inorganic substances.

[0059] Methods for preparing phosphates of basic L-amino acids can be, for example, as described in WO2017 / 200468 and WO2017 / 200467.

[0060] Additionally, the basic L-amino acid provided in the present method may be combined with a microporous aluminosilicate mineral, such as a zeolite, as discussed above with respect to the first aspect of the invention.

[0061] In a third aspect, the present invention relates to a method of plant cultivation, in which a composition according to the first aspect or a growth additive according to the second aspect and a seed are added to the cultivation soil in an amount suitable for the specific growth requirements of said seed. All details discussed above with respect to the first and second aspects of the present invention apply equally to this aspect.

[0062] The effect of such a method is an improvement in the overall growth of the plant due to its enhanced biological control, where the growth-stimulating nitrogen is provided in a form that does not interfere with the beneficial effects of the microorganisms. Such a method is also an environmentally friendly means of growth improvement, since the nitrogen is provided in a form that minimizes leakage from the growth environment, and chemical preparations can be avoided.

[0063] Detailed Description of the Drawings FIG. 1 shows the effect of L-arginine phosphate as the sole nitrogen source on the survival of two different bacteria. More specifically, the OD 600 10 -1 ~10 -6 Different dilutions of Bacillus megaterium (left column) and Bacillus japonicum (right column) ranging from 0.1 to 0.2 mg / ml were spotted on LB agar. The LB agar contained different concentrations of added arginine phosphate and is therefore referred to as "Treatment 1". The different added N concentrations are listed in the table below.

[0064] [Table 1]

[0065] Bacterial growth was analyzed by observing the presence of round shaped colonies after 24 hours (B. megaterium) and 3 days (B. japonicum), respectively. Treatment 1 was used as a control since no additional N supplement was added to the LB agar. -1 ~10 -3 and 10 for B. japonicum. -1 ~10 -4 The growth was observed at a dilution of 10 in B. japonicum spots, where the growth was observable by the presence of round white colonies. -6The prominent white spheres near the mark are contaminations, clearly visible due to the different shape and structure of the microorganisms. Since the experiments were performed in parallel, this control is identical in the results shown in Figures 1-4.

[0066] The maximum concentration tested, 700 mmol / l arginine phosphate (treatment 1.1), was -2 In treatment 1.2 (600 mmol N / l arginine phosphate), B. megaterium grew at a dilution of 10 -1 Growth was observed only at a dilution of 10 and no growth was observed for B. japonicum. Both bacterial strains showed growth in treatment 1.3 (500 mmol N / l arginine phosphate). B. megaterium grew at a dilution of 10 -3 While B. japonicum showed colony growth up to a dilution of 10 -1 Only in the 1.4 treatment (400 mmol / l arginine phosphate) did B. megaterium grow at 10 -3 showed colony growth up to dilution of 10 -2 The same was observed in treatment 1.5 (300 mmol / L arginine phosphate), in which B. megaterium grew for 10 -3 showed colony growth up to dilution of 10 -2 Treatment 1.6 (200 mmol / L arginine phosphate) showed colony growth up to 10 -4 Dilution attenuated the bacterial growth of both strains. The reduction in N concentration in treatment 1.7 (100 mmol N / l arginine phosphate) reduced the growth of both strains by 10 -4 The dilution was up to 100%.

[0067] In summary, the present findings indicate that arginine phosphate does not exert lethal toxicity on B. megaterium up to the maximum tested concentration of 700 mmol N / l. Consistent with this, no lethal effects were observed in the cultivation of B. japonicum at an arginine phosphate concentration of 500 mmol N / l.

[0068] Figure 1 shows the OD 600 10 -1 ~10 -6 Figure 1 shows the results of spotting different dilutions of B. megaterium (left column) and B. japonicum (right column) ranging from 0.01 to 0.01% on LB agar. The LB agar contained different concentrations of added arGrow® Complete (available from Arevo AB) and is therefore referred to as "Treatment 2". The different added N concentrations are listed in the table below.

[0069] [Table 2]

[0070] Bacterial growth was analyzed by observing the presence of round shaped colonies after 24 hours (B. megaterium) and 3 days (B. japonicum), respectively. Treatment 2 was used as a control since no further N supplement was added to the LB agar. -1 ~10 -3 and 10 for B. japonicum. -1 ~10 -4 The growth was observed at dilutions of 100% and was visible by the presence of round white colonies. 10 in -6 The prominent white spheres near the mark are contaminations, clearly visible due to the different shape and structure of the microorganisms. Since the experiments were performed in parallel, this control is identical in the results shown in Figures 1-4.

[0071] The tested N concentrations ranging from 700 mmol N / l (treatment 2.1) to 400 mmol N / l (treatment 2.4) of arGrow® Complete prevented bacterial growth in the case of both strains. Bacterial growth could be observed in treatment 2.5 (300 mmol N / l of arGrow® Complete), where B. megaterium grew at 10 -2 showed colony growth up to dilution of 10 -1 showed colony growth up to dilution of 10 -1 The prominent white colonies partially overlapping with true B. japonicum colonies present at the 10 dilution are the result of contamination. Treatment 2.6 (200 mmol N / l arGrow® Complete) -5 Stop bacterial growth of B. megaterium at dilution of 10 -3 The bacterial growth of B. japonicum was stopped by reducing the N concentration in treatment 2.7 (100 mmol N / l arGrow® Complete). -3 The dilution was up to 100%.

[0072] In summary, arGrow® Complete does not cause lethal toxicity to B. megaterium and B. japonicum up to a concentration of 300 mmol N / l.

[0073] Figure 2 shows the OD 600 10 -1 ~10 -6 Different dilutions of B. megaterium (left column) and B. japonicum (right column) ranging from 0.01 to 0.01 were spotted onto LB agar. The LB agar contained different concentrations of added RIKA-S and is therefore referred to as "Treatment 3." The different added N concentrations are listed in the table below.

[0074] [Table 3]

[0075] Bacterial growth was analyzed after 24 hours (B. megaterium) and 3 days (B. japonicum), respectively, by observing the presence of round-shaped colonies.

[0076] Treatment 3 was used as a control since no additional N supplement was added to the LB agar. -1 ~10 -3 and 10 for B. japonicum. -1 ~10 -4 The growth was observed at dilutions of 100% and was visible by the presence of round white colonies. 10 in -6 The prominent white spheres near the mark are contaminations, clearly visible due to the different shape and structure of the microorganisms. Since the experiments were performed in parallel, this control is identical in the results shown in Figures 1-4.

[0077] The tested N concentrations, ranging from 700 mmol N / l (treatment 3.1) to 200 mmol N / l (treatment 3.6) of RIKA-S, were lethally toxic to both strains and therefore inhibited bacterial growth. The visible spots in treatment 3.3 were not the result of bacterial growth but were caused by the inclusion of air in the LB agar. Bacterial growth was only visible in treatment 3.7 (RIKA-S at 100 mmol N / l), where B. megaterium grew for 10 -3 showed colony growth up to dilution of 10 -2 Colony growth was observed up to 100 μg / ml.

[0078] In summary, RIKA-S, a representative commercial fertilizer containing ammonium and nitrate as nitrogen sources, exerted lethal toxicity on B. megaterium and B. japonicum starting from concentrations already above 100 mmol N / l, thus showing to be the most lethal tested N additive with regard to the survival of B. megaterium and B. japonicum.

[0079] Figure 3 shows the OD 600 10 -1 ~10 -6 Figure 1 shows the results of spotting different dilutions of B. megaterium (left column) and B. japonicum (right column) ranging from 0.01 to 0.01 mg / ml onto LB agar. The LB agar contained different concentrations of added N forms and 200 mg of unloaded zeolite, where the zeolite was only partially added (marked with a "+"). The different additives are listed in the table below ("Treatment 4").

[0080] [Table 4]

[0081] Bacterial growth was analyzed by observing the presence of round shaped colonies after 24 hours (B. megaterium) and 3 days (B. japonicum), respectively.

[0082] Treatments 4 and 4+ were used as controls since no further N additive was added to the LB agar. Treatment 4+ further contained 200 mg of zeolite as an additive in the LB agar. In treatment 4, bacterial growth was 10 -1 ~10 -3 and 10 for B. japonicum. -1 ~10 -4 The growth was observed at a dilution of 10 in B. japonicum spots, where the growth was observable by the presence of round white colonies. -6The prominent white spheres near the mark are contaminations, clearly visible due to the different shape and structure of the microorganisms. Since the experiments were performed in parallel, this control is identical in the results shown in Figures 1-4. Upon addition of 200 mg of zeolite (treatment 4+), both strains showed similar growth responses, with B. megaterium growing at 10 -3 and B. japonicum grew up to a dilution of 10 -4 It grew until dilution.

[0083] Addition of 400 mmol / l L-arginine phosphate to LB medium was -3 Grow B. megaterium up to a dilution of 10 -2 B. japonicum was grown until a dilution of 100 mg / ml (treatment 4.1). The addition of 200 mg of zeolite (treatment 4.1+) increased the tolerance of both strains to L-arginine phosphate, increasing by 100 mg / ml each. -3 and 10 -4 The growth of bacteria was promoted up to a dilution of 400 mmol / l. When spotted on arGrow® Complete (treatment 4.2), no growth of any of the bacterial strains could be detected. Similarly, the positive effect of zeolite could be seen in LB medium supplemented with 400 mmol N / l arGrow® Complete (treatment 4.2+). With the addition of zeolite, B. megaterium grew at 10 -2 and B. japonicum grew up to a dilution of 10 -1 Both bacteria grew up to a dilution of 10 -3 (B. megaterium) and 10 -1 (B. japonicum) grew on LB agar supplemented with 100 mmol N / l RIKA-S (treatment 4.3). A positive effect of 200 mg zeolite on bacterial growth was seen in treatment 4.3+, counteracting the toxic effect of RIKA-S. B. megaterium grew at 10 -5 showed bacterial growth up to dilution of 10, while B. japonicum was 10 -2 showed bacterial growth up to dilution. In summary, it was observed that the overall bacterial lethality caused by high N concentrations was attenuated when zeolite was present in the LB agar.

[0084] Figure 5 shows the OD 600 10 -1 ~10 -6 Figure 1 shows the results of spotting different dilutions of B. megaterium (left column) and B. japonicum (right column) ranging from 0.01 to 0.01% on LB agar containing different concentrations of added N forms and 7.4 mM succinic acid, which was only partially added ("+"). The different additions are listed in the table below ("Treatment 5").

[0085] [Table 5]

[0086] Bacterial growth was analyzed after 24 hours (B. megaterium) and 4 days (B. japonicum), respectively, by observing the presence of round-shaped colonies.

[0087] Treatments 5 and 5+ were used as controls since no further N supplement was added to the LB agar. Treatment 5+ contained 7.4 mM sterile filtered succinic acid as a supplement in the LB agar. In treatment 5, bacterial growth was 10 -1 ~10 -4 and 10 for B. japonicum. -1 ~10 -5 The growth was observed at dilutions of 0.01 mg / ml and the growth was observable by the presence of round white colonies. With the addition of succinic acid (treatment 5+), neither strain grew.

[0088] Addition of 100 mmol / l L-arginine phosphate to LB medium (treatment 5.1) -2 Grow B. megaterium up to a dilution of 10 -5B. japonicum was grown until dilution of 0.01% (treatment 5.1). Addition of succinic acid (treatment 5.1+) inhibited the growth of both strains.

[0089] Addition of 100 mmol / l arGrow® Complete to LB medium (treatment 5.2) was -3 Grow B. megaterium up to a dilution of 10 -2 B. japonicum was grown in the medium until a dilution of 0.01 mg / mL. The addition of succinic acid (treatment 5.2+) inhibited the growth of both strains.

[0090] Addition of 50 mmol / l RIKA-S to LB medium (treatment 5.3) was -3 Grow B. megaterium up to a dilution of 10 -5 B. japonicum was grown until dilution of 0.01% (treatment 5.3). Addition of succinic acid (treatment 5.3+) inhibited the growth of both strains.

[0091] In summary, it was observed that the addition of succinic acid to bacterial growth media impairs bacterial growth and does not enhance bacterial viability.

[0092] Figure 6 shows the dry weight of soybean seedlings grown in "Sajord" (Hasselfors Garden), a low N potting substrate, for 105 days in greenhouse conditions. All seeds were inoculated with rhizobia. The control plants shown were not fertilized. Other plants were fertilized with either 30 kgN / ha arGrow® Complete or RIKA-S as indicated.

[0093] Treatment with arGrow® Complete at planting increased soybean sprout biomass production compared to untreated controls. Fertilization with RIKA-S did not improve biomass production compared to controls. Therefore, arGrow® Complete fertilization resulted in the highest biomass production among the treatment conditions and was the only fertilizer that increased soybean sprout biomass compared to the control.

[0094] Figure 7 shows the number of soybean nodules and dry weight of plants grown in greenhouse conditions for 90 days in "Sajord" (Hasselfors Garden), a low N potting substrate. All seeds were inoculated with rhizobia. The control plants shown were not fertilized. Other plants were fertilized at the vegetative stage with either 30 kgN / ha arGrow® Complete or RIKA-S as indicated.

[0095] Fertilization of soybean plants increased the number of nodules compared to the control (Figure 7.1). Treatment with arGrow® Complete produced 229 nodules per root, while treatment with RIKA-S produced 129 nodules per root. Also, the dry weight of nodules increased significantly after fertilization with arGrow® Complete (Figure 7.2).

[0096] Since arGrow® Complete resulted in 4 times more nodule number and 16 times more nodule dry weight compared to the control condition, it can be concluded that arGrow® Complete is the best fertilizer in terms of nodule growth. Figure 8 shows the number and dry weight of soybean pods from plants grown in greenhouse conditions for 105 days in "Sajord" (Hasselfors Garden), a low N potting substrate. All seeds were inoculated with rhizobia. The control plants shown were not fertilized. Other plants were fertilized with either 30 kgN / ha arGrow® Complete or RIKA-S as indicated at planting.

[0097] Fertilization of soybean plants increased the number of pods only when arGrow® Complete was applied (Figure 8.1). This treatment produced 45 pods per plant, while the RIKA-S treatment produced 22 pods. Pod dry weight was also significantly increased only after arGrow® Complete was applied (Figure 8.2).

[0098] Since arGrow® Complete resulted in 1.3 times more soybean pod number and dry weight compared to the control condition, it can be concluded that arGrow® Complete is the best fertilizer in terms of plant yield. Figure 9.1 shows the percentage of broad bean plants with nodulated roots. Plants were grown in sand for 20 days in greenhouse conditions. All seeds were inoculated with rhizobia. Control plants shown were not fertilized. Other plants were fertilized with either 30 kgN / ha arGrow® Complete or RIKA-S as indicated at planting.

[0099] Neither the control nor the RIKA-S treated plants showed any nodules at the early plant growth stage. Only the arGrow® Complete treatment caused nodule formation on 80% of the plant roots.

[0100] All 57-day-old plants grown in the low-N potting substrate "Sajord" (Hasselfors Garden) showed nodule formation at that time point (Figure 9.2). When the nodules were dissected, only pink nodule tissue was found in the control plants and in the plants treated with arGrow® Complete, whereas the plants treated with RIKA-S had a mixture of white and pink nodule tissue. The color of the nodule tissue (red means active, white means inactive) indicates the ability of the bacteria to actively fix dinitrogen from the atmosphere. Thus, only in the RIKA-S treatment was a mixture of active and inactive nodules found.

[0101] Therefore, it can be concluded that treatment with arGrow® Complete positively affects nodulation by promoting nodule formation at the earliest time point compared to the control. Furthermore, arGrow® Complete does not appear to have a negative effect on nodule activity, in contrast to RIKA-S. Figure 10 shows the results of a composition according to the invention. Soybean seeds were coated with 10 mg of L-arginine phosphate and rhizobacteria. Germination of the coated seeds was evaluated in comparison to uncoated but inoculated seeds. Germination of the seeds was evaluated after 6 days on filter paper (Figure 10.1) or after 10 days in sand (Figure 10.2). In both cases, coating the seeds with L-arginine phosphate increased the germination rate compared to the control.

[0102] In summary, a coating layer of L-arginine phosphate has beneficial effects on the germination of soybean seeds. EXAMPLES

[0103] experiment The following examples are provided for illustrative purposes only and should not be construed as limiting the invention, which is defined by the appended claims. All references provided below or elsewhere in this application are hereby incorporated by reference.

[0104] Example 1 Effect of different nitrogen sources on bacterial survival Commercially available bacterial strains of Bacillus megaterium MVY-011 (isolated from BACTO-K, Bioenergy) and Bradyrhizobium japonicum (isolated from RhizoFix RF-10, Feldsaaten Freudenberger) were grown overnight in 3 ml of sterile liquid LB medium (pH 7.0) at 28 °C and 200 rpm. Bacterial growth was monitored by measuring the optical density (OD) after overnight incubation.600 The bacterial culture was diluted with purified and sterilized water for 10 -1 O.D. 600 Adjusted to 10. -6 Serial dilutions were performed up to a final dilution of 100 μg / ml, resulting in a total of six dilution steps for each B. megaterium and B. japonicum culture.

[0105] 15 ml solid LB agar plates were prepared under sterile conditions. Some were supplemented with different nitrogen (N) sources (Table 1). The positive control did not contain any added nitrogen source. All other agar plates were supplemented with different N sources (Table 1).

[0106] [Table 6]

[0107] The N additives were added as sterile filtered solutions (0.2 micron pore size) to pre-autoclaved LB agar under sterile conditions in laminar flow. Different concentrations of additives were added to obtain final molar concentrations of 700 mmol N / l, 600 mmol N / l, 500 mmol N / l, 400 mmol N / l, 300 mmol N / l, 200 mmol N / l, and 100 mmol N / l in each Petri dish.

[0108] 5 μl of the bacterial dilutions were then spotted onto agar with various N concentrations under sterile conditions by using a multichannel pipette. After the plates were dried, they were sealed and incubated at 28°C. Photographs of B. megaterium were taken after 24 hours. Photographs of B. japonicum were taken after 3 days.

[0109] Example 2 Reduction of the toxic effects of nitrogen on bacterial growth via zeolites. Commercially available bacterial strains of Bacillus megaterium MVY-011 (isolated from BACTO-K, Bioenergy) and Bradyrhizobium japonicum (isolated from RhizoFix RF-10, Feldsaaten Freudenberger) were grown overnight in 3 ml of sterile liquid LB medium (pH 7.0) at 28 °C and 200 rpm. Bacterial growth was monitored by measuring the optical density (OD) after overnight incubation. 600 The bacterial culture was diluted with purified and sterilized water for 10 -1 O.D. 600 Adjusted to 10. -6 Serial dilutions up to a final dilution of 1000 mM N / l were performed, resulting in a total of six dilution steps for each B. megaterium and B. japonicum culture. 15 ml solid LB agar plates were prepared under sterile conditions. Different N sources were added separately (Table 1). N additives were added as sterile filtered solutions (0.2 micron pore size) to pre-autoclaved LB agar under sterile conditions in laminar flow. Different concentrations of additives were added to obtain a final molar concentration of 400 mmol N / l and 100 mmol N / l in each Petri dish. Some agar plates contained 200 mg of autoclaved, finely ground zeolite, which was mixed into the agar solution during the preparation of the plates.

[0110] 5 μl of the bacterial dilutions were then spotted onto agar with various N concentrations under sterile conditions by using a multichannel pipette. After the plates were dried, they were sealed and incubated at 28°C. Photographs of B. megaterium were taken after 24 hours. Photographs of B. japonicum were taken after 3 days.

[0111] Example 3 The addition of succinic acid inhibits bacterial growth on the nitrogen sources tested. Commercially available bacterial strains of Bacillus megaterium MVY-011 (isolated from BACTO-K, Bioenergy) and Bradyrhizobium japonicum (isolated from RhizoFix RF-10, Feldsaaten Freudenberger) were grown overnight in 3 ml of sterile liquid LB medium (pH 7.0) at 28 °C and 200 rpm. Bacterial growth was monitored by measuring the optical density (OD) after overnight incubation. 600 The bacterial culture was diluted with purified and sterilized water for 10 -1 O.D. 600 Adjusted to 10. -6 Serial dilutions were performed up to a final dilution of 100 μg / ml, resulting in a total of six dilution steps for each B. megaterium and B. japonicum culture. 15 ml solid LB agar plates were prepared under sterile conditions, partially supplemented with different nitrogen (N) sources (Table 1).

[0112] N additives were added as sterile filtered solutions (0.2 micron pore size) to pre-autoclaved LB agar under sterile conditions in laminar flow. Different concentrations of additives were added to obtain a final molar concentration of 100 mmol N / l or 50 mmol N / l in each Petri dish. In part, during plate preparation, sterile filtered (0.2 micron pore size) succinic acid (Sigma-Aldrich, ≧99.0%) in water for dissolution was mixed into the agar solution to a final concentration of 7.4 mM.

[0113] 5 μl of the bacterial dilutions were then spotted onto agar with various N concentrations under sterile conditions by using a multichannel pipette. After drying, the plates were sealed and incubated at 28°C. Photographs of B. megaterium were taken after 24 hours. Photographs of B. japonicum were taken after 4 days.

[0114] Example 4 Positive impact of arGrow® Complete on plant biomass, nodule number, and plant yield Soybean (Alexa variety, Skanefro AB) was treated with RhizoFix RF-10 (Feldsaaten Freudenberger) according to the manufacturer's instructions and the seeds were inoculated. One soybean seed was then planted in a pot containing 3 liters of low-N potting substrate "Sajord" (Hasselfors Garden). Vermiculite was added as a top layer and the first watering of the pot was with Nemablom (Bionema) as a one-time treatment. The plants were grown in a greenhouse and watered once a day for 90 or 105 days as indicated. The plants were fertilized once with different nitrogen sources by adding either arGrow® Complete or RIKA-S (Table 2). Two different time points were selected for the application of fertilizers: at the time of planting the seeds or at the vegetative growth stage of soybeans 33 days after planting. The control plants were inoculated but not fertilized.

[0115] [Table 7]

[0116] The different measurements were made after the plants were harvested. The plant aboveground tissues, except for the pods, were harvested after 105 days and dried at 50°C for 2 weeks. The weight of the dry plant material was then recorded. The data shown are from plants that were fertilized at the planting stage.

[0117] To assess the number and dry weight of nodules attached to the roots, the roots of the plants were washed in water after 90 days to remove the soil. The nodules along the entire root were counted and then excised from the root. The collected nodules were dried at 50°C for 2 weeks and then weighed. The data shown are from plants fertilized during the vegetative growth phase.

[0118] The number of emerged pods was counted 105 days after planting. The pods were then harvested, dried at 50° C. for 2 weeks and then weighed. The data shown are from plants that were fertilized at the time of planting.

[0119] Example 5 Positive impact of arGrow® Complete on nodule formation and activity Broad bean (Vicia faba, var. Boxer, Lantmannen) seeds were inoculated by treating them with RhizoFix RF-20 (Feldsaaten Freudenberger) according to the manufacturer's instructions. Five broad bean seeds were planted in pots containing 3 liters of sand and fertilized with different nitrogen sources at planting (Table 2). Control plants were inoculated but not fertilized. After 20 days, the number of plants bearing nodules along the roots was counted.

[0120] Broad beans, not inoculated but fertilized according to Table 2 at planting, were grown in parallel. Two seeds were added to one pot containing 3 liters of low N potting substrate "Sajord" (Hasselfors Garden). Control plants were not fertilized. After 57 days, the roots were examined and the formed nodules were cut open with a razor blade. The color of the nodule tissue (red means active, white means inactive) indicates the ability of the bacteria to actively fix dinitrogen from the atmosphere. The selected pictures are representative pictures and not a quantitative measure.

[0121] Example 6 Preparation of the composition according to the invention Soybean (Alexa variety, Skanefro AB) seeds were coated in batches using a Concept ML2000 Coating machine (Satec) with a Sp quick Pumpdrive 5206 (Heidolph) pump connected for the distribution of the binder. Fine powder of L-arginine phosphate (Table 1) was weighed out in an amount corresponding to 10 mg of L-arginine phosphate powder per soybean seed. In addition, 1% carboxymethylcellulose (CMC, Akucell AF 1505 LV, Nouryon) was added as binder. RhizoFix RF-10 (Feldsaaten Freudenberger) was used as liquid for the coating process.

[0122] The CMC powder was mixed thoroughly with the L-arginine phosphate powder. The seeds were then placed in a coating machine and liquid was added until the seeds were moistened. The powder mixture was added to the seeds until the seeds were no longer sticky. More liquid was then added until the seeds were sticky again. This cycle was repeated until all the powder was bound to the seeds. After the last powder was added, a final liquid spray was added.

[0123] Control seeds were not coated but were inoculated with RhizoFix RF-10 (Feldsaaten Freudenberger) according to the manufacturer's instructions. Seeds were thoroughly dried and germinated on filter paper. After 6 days, the germination percentage of a total of 10 seeds was calculated. In parallel, 20 seeds were planted in sand and grown in the greenhouse. After 10 days, the germination percentage was evaluated.

Claims

1. A composition comprising at least one macronutrient comprising nitrogen in the form of a basic L-amino acid combined with phosphorus, and at least one microorganism.

2. The composition of claim 1 which is a liquid seed coating composition.

3. The composition of claim 1 or 2, which is a sustained release composition.

4. 4. The composition of claim 1, wherein the at least one microorganism is a selected microorganism, such as a nitrogen fixing bacterium.

5. 2. The composition of any one of the preceding claims, wherein the at least one microorganism is present in the form of a spore.

6. 10. A composition according to any one of the preceding claims, wherein the at least one microorganism is present in an inoculum, preferably within a carrier such as a polymer or other supporting structure.

7. 2. The composition of any one of the preceding claims, wherein the basic L-amino acid is arginine or lysine.

8. 2. The composition of any one of the preceding claims, wherein the basic L-amino acid is associated with phosphorus via electrostatic interactions.

9. The composition of claim 8, wherein the basic L-amino acid associated with phosphorus is an arginine phosphate, such as arginine monophosphate and / or arginine polyphosphate.

10. The composition of claim 8, wherein the basic L-amino acid associated with phosphorus is phosphoarginine.

11. 10. A composition according to any one of the preceding claims, wherein the basic L-amino acid associated with phosphorus is present in admixture with a microporous aluminosilicate mineral such as a zeolite.

12. Seeds treated with at least one selected microorganism, macronutrients including nitrogen in the form of a basic L-amino acid, and phosphorus.

13. 13. The seed according to claim 12, wherein the treatment comprises application to the seed of at least one layer of a selected microorganism, at least one layer of the nitrogen in the form of a basic L-amino acid, and at least one layer containing phosphorus, optionally with an intermediate layer therebetween.

14. 13. Seed according to claim 12, which has been treated, e.g. coated, with a composition according to any one of claims 1 to 11.

15. 13. The seed according to claim 12, comprising at least one layer of said at least one microorganism and at least one layer of said nitrogen in the form of a basic L-amino acid combined with phosphorus.

16. The method of claim 1, wherein the treatment comprises soaking the seeds with an aqueous solution of the at least one microorganism and applying a basic L-amino acid and phosphorus in one or more layers. The seed according to claim 12.

17. 17. The seed according to claim 16, wherein the nitrogen in the form of a basic L-amino acid is applied in a layer separate from the layer containing phosphorus.

18. 17. The seed of claim 16, wherein said nitrogen in the form of a basic L-amino acid is combined with phosphorus in one or more layers.

19. 19. The seed according to claim 12, 15 to 16 or 18, wherein the basic L-amino acid associated with phosphorus is arginine phosphate and / or phosphoarginine.

20. 1. A method for treating seeds with a liquid composition comprising the steps of: a) providing at least one microorganism, e.g. a selected microorganism; b) providing macronutrients containing nitrogen in the form of basic L-amino acids; c) applying said at least one microorganism and said macronutrients to at least one seed. The method includes:

21. 21. The method of claim 20, wherein step c) comprises combining the at least one microorganism and the macronutrients with a binder to provide a liquid composition that is applied to the at least one seed.

22. 22. The method of claim 21, wherein the step of combining the microorganism with the binder comprises granulation.

23. 23. The method of any one of claims 20 to 22, wherein the at least one microorganism is a nitrogen fixing bacterium.

24. 24. The method according to any one of claims 20 to 23, wherein the basic L-amino acid is arginine or lysine.

25. 25. The method of any one of claims 20 to 24, wherein the macronutrients further comprise phosphorus.

26. 26. The method of claim 25, wherein the basic L-amino acid is linked to phosphorus, such as through electrostatic interactions.

27. 27. The method of claim 26, wherein the basic L-amino acid linked to phosphorus is an arginine phosphate, such as arginine monophosphate and / or arginine polyphosphate, and / or phosphoarginine.

28. 28. The method of any one of claims 20 to 27, comprising treating the seeds with phosphorus.

29. 30. The method of claim 28, comprising at least one cycle of applying a liquid containing macronutrients, applying a liquid containing phosphorus, and drying the seeds between said applications.

30. The at least one microorganism is applied to the seeds before the macronutrients and the phosphorus, for example by soaking the seeds with an aqueous solution containing the microorganism.

30. The method according to item 29.

31. 31. The method of claim 30, wherein the at least one microorganism, the macronutrients, and the phosphorus are mixed as a solution prior to the application thereof to seeds.

Images