Materials and methods for treating iron deficiency and iron chlorosis in plants

Abstract

The present invention provides compositions and methods for treating and / or preventing iron deficiency and iron chlorosis in plants. A composition comprising an iron trapping fraction is contacted with a plant and / or its surroundings to increase the amount of iron bioavailable to the plant.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 359,248, filed on July 8, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] In plants, iron is required for photosynthesis and chlorophyll synthesis. Iron availability in soils can determine the distribution of plant species in natural ecosystems and limit crop yields and nutritional quality. Inadequate iron absorption can lead to reduced growth, interveinal chlorosis, and decreased overall health. Adequate iron levels in food crops are important to combat iron deficiency, one of the largest nutritional disorders worldwide.

[0004] When plants are deficient in iron, the result can be a condition known as iron chlorosis, which manifests as yellowing of plant leaves. The main symptom of iron deficiency is interveinal chlorosis, or the development of leaves that appear yellow while the veins remain dark green. In severe cases, entire leaves may turn yellow or white as plant cells die. It is common for only one or half of a branch of a tree to change color while the rest remain normal.

[0005] Yellow leaves indicate a plant lack of chlorophyll. A reduction in chlorophyll during the growing season can negatively impact plant growth and vigor. If food crops experience chlorosis, they may produce smaller fruit that tastes bitter.

[0006] Iron chemistry in soil is complex, and various reactions in soil can lead to low bioavailability of iron and increased cases of iron chlorosis. Iron chlorosis typically occurs in alkaline (pH greater than 7.0) soils and soils containing lime. Although these soils may have adequate iron content, the solubility of iron is low, especially in soils with high soil pH.

[0007] Factors that further exacerbate iron deficiency and chlorosis include reduced soil temperatures and conditions that restrict air access to the soil (such as, for example, mulching, compaction, and water-saturated conditions). Chlorosis may be more severe where topsoil has been removed, exposing a lime-rich subsoil (such as, for example, soils that have been eroded or land that has been leveled).

[0008] Some plants have evolved the ability to adapt to living in high-pH soils with iron deficiency and / or low iron solubility, for example, by secreting chelated iron to mobilize iron, increasing iron stores in cells, and optimizing photosynthesis to prevent absorption of excessive light energy.

[0009] However, not all plants have these adaptations, which means growers must utilize other measures to address iron deficiency in their crops. These measures can include, for example, adding manure and fertilizer to the soil and spraying fertilizer directly on plant leaves. While these may be short-term solutions, when fertilizers are overused or applied incorrectly, they can run off into surface water, seep into groundwater, and evaporate into the air. These substances are coming under increasing scrutiny as sources of air and water pollution, making their responsible use an ecological and commercial imperative. Even when used properly, over-reliance on and long-term use of certain fertilizers can have detrimental effects on soil ecosystems.

[0010] Increasing regulatory requirements governing the availability and use of chemicals, as well as consumer demand for residue-free, sustainably grown foods are impacting the industry and sparking an evolution in thinking about how to address myriad challenges. These foods are produced with minimal environmental impact for food and fiber growth. Therefore, there is a need for effective and environmentally friendly methods and compositions to address nutrient deficiencies in plants, including treating and / or preventing iron deficiency and iron chlorosis in plants. Summary of the invention

[0011] The present invention relates to methods and compositions for treating plant nutrient deficiencies. More specifically, the present invention provides compositions and methods for increasing the amount of bioavailable iron absorbed and utilized by plants. Advantageously, the compositions and methods of the present invention are environmentally friendly, non-toxic and cost-effective agricultural solutions for improving plant health, growth and / or yield.

[0012] In a preferred embodiment, the present invention provides a method for treating and / or preventing plant nutrient deficiency, wherein a composition comprising an iron-trapping component is applied to a plant and / or the environment in which the plant grows. In a specific embodiment, the plant has been determined to suffer from iron deficiency and / or iron chlorosis.

[0013] The present invention also provides a composition comprising one or more iron-capturing components. In certain embodiments, the iron-capturing component is a beneficial microorganism, a growth byproduct of an iron-capturing microorganism, or some other compound that can bind iron and increase its solubility and / or bioavailability to plants. In certain embodiments, the composition comprises more than one iron-capturing component.

[0014] In some embodiments, the method is performed at a pH of less than 6.8, preferably less than 5.0, more preferably less than 4.8. Thus, in some embodiments, the method further comprises administering the composition in combination with an acidic pH adjusting agent.

[0015] In a preferred embodiment, the beneficial microorganisms of the present invention are non-pathogenic fungi, yeasts and / or bacteria that are able to accumulate iron naturally or through genetic modification.

[0016] In one embodiment, the beneficial microorganism is a strain of Bacillus subtilis. In a specific embodiment, the strain is Bacillus subtilis B4 (NRRL B-68031). The B4 strain is preferably applied in spore form, but grows in biofilm form when exposed to an acidic environment.

[0017] Surprisingly, it was found that B4 produces one or more compounds that can gather, chelate or otherwise capture iron. In certain embodiments, the compound is pulcherrimone and / or pulcherrimone acid. Advantageously, when the microorganism and / or the extracellular polysaccharide (EPS) of the microorganism grows in the form of a biofilm, after being exposed to an acidic pH value (e.g., less than 6.8, preferably less than 5.0), an iron trapping agent (such as pulcherrimone and / or pulcherrimone acid) is used to effectively absorb iron. In some embodiments, iron is then provided to plants as a nutrient. This can be promoted by adjusting the pH value to a pH value closer to neutrality (e.g., 6.8 to 8.0).

[0018] B4 is also particularly advantageous over other traditional probiotic microorganisms due to its ability to produce digestive enzymes including, for example, cellulases and amylases.

[0019] In certain embodiments, the composition may include other non-pathogenic microorganisms capable of producing compounds capable of sequestering, chelating or otherwise trapping iron.The microorganisms may be in biofilm form, spore form, planktonic form, or any other form.

[0020] In certain embodiments, these microorganisms are also capable of producing one or more of the following: surfactants, such as lipopeptides and / or glycolipids; bioactive substances with antimicrobial and immunomodulatory effects; polyketides; acids; peptides; anti-inflammatory compounds; enzymes, such as amylases, cellulases, proteases and / or lipases; and sources of amino acids, vitamins and other nutrients.

[0021] In certain embodiments, the iron-trapping component of the composition of the present invention is a crude or purified siderophore or plant siderophore, or other molecules with high iron affinity, for example, pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA (ethylenediaminetetraacetic acid), iron EDTA, DTPA (diethylenetriaminepentaacetic acid), EDDHA (ethylenediamine-di-o-hydroxyphenylacetic acid), N,N-dihydroxy-N,N′-diisopropyladipamide (DPH), 2,3-dihydroxybenzoic acid, azotocin, transferrin, enterocoele bacteriocin, pyocyanin, protochelatin, chelatrocin, bacteriocin, vibriocin, vibrioferrin, azotocin, aminochelatin, yersin, agarobactin, grape agglutinin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, chromatin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, ferric oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderocite, hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

[0022] In certain embodiments, the composition comprises an organic acid or an inorganic acid. Preferably, the acid is present in an amount suitable for adjusting the pH of the composition or the environment in which it is used to 6.8 or lower, preferably 5.0 or lower, more preferably 4.8 or lower.

[0023] The method of the present invention can be used to inoculate the rhizosphere of a plant with one or more beneficial microorganisms. For example, the microorganisms of the composition of the present invention can colonize the rhizosphere and provide a variety of benefits to plants whose roots grow in the rhizosphere, including protection, nutrition, and increased iron absorption.

[0024] The methods of the present invention may also be used to improve the health of the soil, for example by improving water and nutrient dispersion and / or reducing compaction.

[0025] In addition, in certain embodiments, the methods of the present invention can be used to enhance the health, growth and / or yield of plants whose health is impaired by iron deficiency. Thus, in certain embodiments, the methods of the present invention can also be used to improve the health or immune response of plants.

[0026] Advantageously, the present invention can be used without releasing large amounts of inorganic compounds into the environment. In addition, the compositions and methods utilize components that are biodegradable and toxicologically safe. Therefore, the present invention can be used as a "green" soil and plant treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1A to 1C Shown are the growth and exopolysaccharide (EPS) formation of B4 after 24 h (A) and 48 h (B) at acidic pH (top row, pH 4.8) and neutral pH (bottom row, pH 6.8).

[0028] Figure 2 The purified B4 EPS is shown to be pink.

[0029] Figure 3 Results of an amylase test of B4 are shown. Agar streaked with B4 produced an orange color around bacterial growth, indicating that starch was broken down.

[0030] Figure 4 Results of the cellulase test of B4 are shown. Agar streaked with B4 produced a yellow clearing zone around bacterial growth, indicating that cellulose was broken down.

[0031] FIG. 5A to FIG. 5C The production and activity of B4 siderophores after 6 hours in aerobic (top panel) and anaerobic (bottom panel) environments and in different growth media are shown. (A) B4 cultures grown in MRS-sucrose (left) medium and B4 cultures grown in M23-6 (right) medium are shown. (B) B4 cultures grown in minimal medium with Tween (left) and minimal medium without Tween (right) are shown. (C) Dry B4 spores grown in minimal medium are shown.

[0032] FIG. 6A to FIG. 6C The production and activity of B4 siderophores after 24 hours in aerobic (top panel) and anaerobic (bottom panel) environments and in different growth media are shown. (A) B4 cultures grown in M23-6 (left) and MRS-sucrose (right) medium are shown. (B) B4 cultures grown in minimal medium with Tween (left) and minimal medium without Tween (right) are shown. (C) Dry B4 spores grown in minimal medium are shown.

[0033] Figure 7 Shown are the results of iron assays of B4 (BSSL) cultures grown in different media. DETAILED DESCRIPTION

[0034] The present invention provides compositions and methods for treating and / or preventing iron deficiency in plants. More specifically, the present invention provides compositions that can be applied to plant leaves and / or soil in which plants grow as chelated iron to increase the amount of bioavailable iron available to the plants. Advantageously, the microbial-based products and methods of the present invention are environmentally friendly, non-toxic and cost-effective.

[0035] Selected Definitions

[0036] As used herein, "biofilm" is a complex aggregate of microorganisms (such as bacteria) in which cells adhere to each other and / or to a surface. In certain embodiments, adhesion is achieved via extracellular polysaccharide substances produced by the bacteria. Cells in a biofilm are physiologically different from planktonic cells of the same organism, which are single cells that can float or swim in a liquid culture medium.

[0037] As used herein, "preventing" or "prevention" a disease, condition, or disorder refers to delaying, arresting, inhibiting, preventing, and / or minimizing the onset or progression of specific signs or symptoms thereof. Prevention may include, but does not require, indefinite, absolute, or complete prevention over the lifetime of a subject, meaning that symptoms or signs may develop later. Prevention may include reducing the severity of the onset of such disease, condition, or disorder, and / or inhibiting the progression of a condition or disorder to a more severe condition or disorder.

[0038] As used herein, "treating" or "treatment" of a disease, condition, or disorder refers to eradication, improvement, reduction, alleviation, or reversal of at least one sign or symptom of a disease, condition, or disorder (e.g., infection). Treatment may include but does not require complete cure of the disease, condition, or disorder, meaning that treatment may also include partial eradication, improvement, reduction, alleviation, or reversal.

[0039] As used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, protein, organic compound such as a small molecule (such as those described below), or other compound is substantially free of other compounds associated with it in nature (e.g., microporous materials). For example, a purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of flanking genes or sequences in its naturally occurring state. A purified or isolated polypeptide is free of amino acids or sequences that flank it in its naturally occurring state. A purified or isolated microbial strain is removed from the environment in which it exists in nature. Thus, an isolated strain may exist, for example, as a biologically pure culture or as a spore (or other form of strain) associated with a vector.

[0040] In certain embodiments, the purified compound is at least 60% by weight of the compound of interest. Preferably, the preparation is at least 75% by weight of the compound of interest, more preferably at least 90%, and most preferably at least 99%. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100% (w / w) by weight of the desired compound. For example, purity is measured by any appropriate standard method such as column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC) analytical methods.

[0041] As used herein, "ionophores" are carboxylated polyether non-therapeutic antibiotics, wherein the carboxylated polyether non-therapeutic antibiotics disrupt the ion concentration gradient (Ca2+, K+, H+, Na+) of microorganisms, thereby causing them to enter an ineffective ion cycle. The disruption of ion concentrations makes it impossible for microorganisms to maintain normal metabolism and causes microorganisms to consume additional energy. The functions of ionophores include: selecting against Gram-positive bacteria (such as methanogens) and protists or affecting the metabolism of Gram-positive bacteria and protists.

[0042] As used herein, "siderophores" are compounds produced by various organisms to remove iron from the surrounding environment. Siderophores are generally small, low molecular weight compounds that have the ability to bind to ferric iron (Fe 3+ ) with a higher affinity, capable of forming strong iron chelate complexes that can be absorbed by organisms under certain circumstances. As used herein, "plant siderophores" are iron carriers produced by plants.

[0043] "Metabolite" refers to any substance produced by metabolism (e.g., a growth byproduct) or a substance necessary to participate in a specific metabolic process. A metabolite can be an organic compound that is a starting material, intermediate, or end product of metabolism. Examples of metabolites can include, but are not limited to, enzymes, toxins, acids, solvents, alcohols, proteins, carbohydrates, vitamins, minerals, trace elements, amino acids, polymers, polyketides, and surfactants.

[0044] As used herein, "methanogens" are microorganisms that produce methane gas as a byproduct of metabolism. Methanogens are archaea that can be found in the digestive systems and metabolic wastes of ruminants and non-ruminants (e.g., pigs, poultry, and horses, among others). Examples of methanogens include, but are not limited to, Methanobacterium (e.g., Methanobacterium formiformis), Methanobacterium Brevibacterium (e.g., Methanobacterium rumenii), Methanococcus (e.g., Methanococcus maritimus), Methanosaccharomyces (e.g., Methanosaccharomyces breisi), Methanophorone (e.g., Methanophorone staufen), Methanobacterium migratoriae, Methanothermophilus woerneri, Methanobacterium (e.g., Methanobacterium mobilis), Methanothermophilus candlerii, Methanomostatus bunei, Methanogens (e.g., Methanobacterium unitede, Methanothrix thermophilus), Methanosarcina (e.g., Methanosarcina barkeri, Methanosarcina mazei), Methanosphaeroides stuartii, Methanosphaeroides henselae, Methanothermophilus and / or Methanothrix sunnai.

[0045] As used herein, "surfactant" refers to a surface-active compound that reduces the surface tension (or interfacial tension) between two liquids, between a liquid and a gas, and / or between a liquid and a solid. Surfactants are used, for example, as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. "Biosurfactants" are surface-active molecules produced by living organisms and / or using substrates of natural origin.

[0046] As used herein, "agriculture" refers to the cultivation and propagation of plants, algae and / or fungi for food, fiber, biofuels, pharmaceuticals, cosmetics, supplements, ornamental purposes and other uses. According to the present invention, agriculture may also include gardening, landscaping, horticulture, plant protection, orchards and arboriculture. Agriculture also includes the care, monitoring and maintenance of soil.

[0047] As used herein, the term "plant" includes, but is not limited to, any kind of woody plants, ornamental or decorative plants, crops or cereals, fruit or vegetable plants, flowers or trees, macroalgae or microalgae, phytoplankton and photosynthetic algae (e.g., green algae - Chlamydomonas reinhardtii, etc.). "Plants" also include single-cell plants (e.g., microalgae, etc.) and a large number of plant cells that differentiate into colonies (such as Volvox, etc.) or structures present in any stage of plant development. Such structures include, but are not limited to, fruits, seeds, twigs, stems, leaves, roots, petals, etc. Plants can be grown alone, such as in a garden, or as part of a variety of plants (e.g., part of an orchard, crop, or pasture).

[0048] As used herein, "crop plants" refers to any plant or algae grown for profit and / or to provide food for humans, animals or aquatic life, or for human use (e.g., for textile, cosmetic and / or pharmaceutical production), or for human enjoyment (e.g., flowers or shrubs in landscaping or gardens), or any plant or algae for industrial, commercial or educational use, or parts thereof. Crop plants can be plants (including transgenic plants and plant varieties) obtained by traditional breeding and optimization methods or by biotechnology and recombinant methods, or a combination of these methods.

[0049] All plants and plant parts can benefit from the present invention. In this case, plants are understood to mean all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants).

[0050] Plant tissue and / or plant parts are understood to refer to all above-ground and underground parts and organs of plants, such as buds, leaves, flowers, roots, needles, stems, stalks, fruits, seeds, tubers and rhizomes. Plant parts also include crop materials and vegetative and reproductive propagation materials, such as cuttings, tubers, rhizomes, young branches and seeds. As used herein, "enhance" refers to improving or increasing. For example, enhancing plant health refers to improving the growth and ability of plants to thrive (including increasing seed germination, seedling emergence and / or vitality); improving the ability to withstand transplant shock; improving the ability to resist pests and / or diseases; improving the ability to compete with weeds; and improving the ability to survive under environmental stresses such as drought and / or overwatering.

[0051] Enhancing plant growth and / or enhancing plant biomass refers to, for example, increasing the size and / or mass of plants above and below ground (e.g., increasing crown / leaf volume, shoot size, height, trunk thickness, branch length, shoot length, stem length, protein content, root size / density and / or overall growth index), and / or improving the ability of a plant to achieve a desired size and / or mass.

[0052] Increasing yield refers to increasing the end product produced by plants in a crop plant, for example by increasing the amount, quantity and / or size of fruits, leaves, roots, flowers, shoots, stems, seeds, fibers, extracts and / or tubers per plant and / or improving their quality.

[0053] As used herein, "soil amendment" or "soil conditioner" refers to any compound, material, or combination of compounds or materials added to the soil to enhance the physical properties of the soil. Soil amendments can include organic and inorganic substances, and may also include fertilizers, pesticides and / or herbicides, etc. Nutrient-rich, well-drained soil is essential for plant growth and health, so soil amendments can enhance plant growth and health by changing the nutrient and moisture content of the soil. Soil amendments can also be used to improve a variety of different qualities of the soil, including but not limited to soil structure (e.g., preventing compaction); increasing nutrient concentration and storage capacity; increasing water retention in dry soils; and improving drainage in waterlogged soils.

[0054] As used herein, "abiotic stress" refers to abiotic conditions that negatively affect living organisms in a particular environment. Abiotic stress must affect the environment beyond its normal range of variation, thereby adversely affecting the population performance or individual physiology of the organism. Examples of abiotic stress include, but are not limited to, drought, extreme temperatures, floods, high winds, natural disasters, changes in soil pH, high radiation, soil compaction, pollution, etc. "Biological stress" is a biotic stress caused by living conditions, such as animal, plant, or microbial pests.

[0055] The scope provided herein is understood as a shorthand expression of all numerical values ​​in the scope. For example, the scope of 1 to 50 is understood to include any number, combination of numbers or the sub-range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and all decimal values ​​(such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9) between the above integers. About sub-range, " nested sub-range " extending from any end point of scope is particularly considered. For example, nested sub-ranges of the exemplary range 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0056] As used herein, "decrease" refers to a negative change and "increase" refers to a positive change, wherein the positive or negative change is at least 0.25%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0057] The transitional term "comprising," which is synonymous with "including" or "containing," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps "and those that do not materially affect the basic, novel characteristic." Use of the term "comprising" refers to other embodiments consisting of "consisting of" or "consisting essentially of" the referenced components.

[0058] Unless otherwise specified or obvious from the context, the term "or" as used herein should be understood to be inclusive. Unless otherwise specified or obvious from the context, as used herein, the terms "a", "and" and "the" are understood to be singular or plural.

[0059] Unless otherwise specified or apparent from the context, as used herein, the term "about" should be understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the specified value.

[0060] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0061] All references cited herein are hereby incorporated by reference in their entirety.

[0062] Composition

[0063] The present invention provides a composition for use according to the method of the present invention, wherein the composition comprises one or more iron-trapping components. In certain embodiments, the iron-trapping component is a beneficial microorganism, a growth byproduct of a microorganism, or some other compound known to bind iron. In certain embodiments, the composition comprises more than one iron-trapping component.

[0064] The total amount of iron-trapping components in the composition preferably comprises 0.0001% to 100% of the composition, 0.001 to 95%, 0.01 to 90%, 0.1% to 85%, 0.5 to 80%, 0.75% to 75%, 1.0% to 70%, 1.25% to 65%, 1.5% to 60%, 1.75% to 55%, 2.0% to 50% or 5.0% to 25% of the composition by weight or volume.

[0065] In certain embodiments, the composition is a "microbial-based composition," which means that the composition includes components produced by the growth of microorganisms or other cell cultures. Therefore, a microbial-based composition may include the microorganisms themselves and / or byproducts of microbial growth. The microorganisms may be in an asexual state, in spore form, in mycelial form, in any other form of microbial propagules, or a mixture thereof. The microorganisms may be in the form of planktonic microorganisms or biofilms, or a mixture of the two. The byproducts of growth may be metabolites, cell membrane components, expressed proteins, and / or other cell components, etc. The microorganisms may be intact microorganisms or lysed microorganisms. Cells may be completely absent, or present in an amount of, for example, at least 1×10 per milliliter of composition or per CFU / g of composition. 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×108 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or more CFU concentrations.

[0066] Advantageously, in preferred embodiments, the compositions of the present invention can concentrate iron in the soil and make it available to plants in need thereof. Thus, in some embodiments, the compositions can be used to treat iron deficiency and / or iron chlorosis in plants.

[0067] In a preferred embodiment, the beneficial microorganism of the composition of the present invention is a non-pathogenic fungus, yeast and / or bacteria that can collect iron naturally or by genetic modification. The beneficial microorganism can be in an active form, an inactive form and / or a dormant form. In a preferred embodiment, the microorganism is a microorganism described as "generally recognized as safe" by the relevant regulatory agency or GRAS.

[0068] In certain embodiments, these microorganisms are also capable of producing one or more of the following: surfactants, such as lipopeptides and / or glycolipids; bioactive substances with antimicrobial and immunomodulatory effects; polyketides; acids; peptides; anti-inflammatory compounds; enzymes, such as amylases, cellulases, proteases and / or lipases; and sources of amino acids, vitamins and other nutrients.

[0069] The microorganism of the present invention can be a natural microorganism or a genetically modified microorganism. For example, the microorganism can be transformed with a specific gene to show a specific characteristic. The microorganism can be a mutant of a desired strain. As used herein, "mutant" refers to a strain, genetic variant or subtype of a standard bacterium, wherein the mutant has one or more genetic variations (e.g., point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation or amplification mutation) compared to the standard bacterium. The process of preparing mutants is well known in the field of microbiology. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.

[0070] In some embodiments, beneficial microorganisms are selected based on natural or acquired resistance to certain antibiotics applied to an environment containing iron-trapping pathogens, for example, to control pathogenic and / or harmful microorganisms in a living organism or elsewhere in the environment.

[0071] In one embodiment, the composition includes about 1×10 6 About 1×10 13 , about 1×10 7 About 1×10 12 , about 1×10 8About 1×10 11 , or about 1×10 9 About 1×10 10 CFU / g of various types of microorganisms.

[0072] In one embodiment, the composition includes a total of about 0.001% to 100%, about 1% to 90%, or about 10% to 75% total microorganisms by volume.

[0073] In certain embodiments, the composition comprises growth byproducts of microorganisms, but does not comprise live microorganisms. For example, in certain embodiments, pathogenic microorganisms are only used in the production of growth byproducts of the composition according to the present invention, rather than being directly applied to the environment.

[0074] The microorganisms may include yeast, bacteria and / or fungi, such as Acaulospora, Acidithiobacillus spp. (e.g., A.ferooxidans, A.albertensis, A.caldus, A.cuprithermicus, A.ferrianus, A.ferridurans, A.ferriphilus, A.ferrivorans, A.ferrooxidans, A.sulfuriphilus, and A.thiooxidans), Acremonium chrysogenum, Agrobacterium (e.g., A. radiobacter), Aspergillus, Aureobasidium (e.g., A. pullulans), Azospirillum (e.g., A. brasiliensis), Azotobacter (A. vinelandii, A. chroococcum), Bacillus (e.g., B. amyloliquefaciens, B. coagulans, B. firmus, B. laterosporus), orus, B. licheniformis, B. megaterium, B. mucilaginosus, B. subtilis), Blakeslea, Candida (e.g., C. albicans, C. apicola, C. batistae, C. bombicola, C. floricola, C. kuoi, C. riodocensis, C. nodaensis, C. stellate), Cryptococcus, Debaryomyces (e.g., D. hansenii, D.hansenii), Dipodascopsism, Entomophthora, Escherichia coli, Frateuria (e.g., F. aurantia), Hanseniaspora (e.g., H. uvarum), Hansenula, Issatchenkia, Kluyveromyces (e.g., K. phaffii), Lentinula spp. (e.g., L. edodes), Legionella pneumophila, Lipomyces, Magnetospirillummagneticum, Magnetococcus marinus, methanogens, Metschnikowia sp. (M. pulcherrimia), Meyerozyma (e.g., M. guilliermondii, M. caribbica), Monascus purpureus, Mortierella, Mucor (e.g., M. piriformis), Neisseria meningitidis, Pantoea (e.g., P. agglomerans, P. allii), Penicillium, Phythium, Phycomyces, Pichia (e.g., P. anomala, P. guilliermondii, P. occidentalis, P. kudriavzevii), Pleurotus erythrocytes, otus (e.g., P. ostreatus, P. ostreatus, P. sajorcaju, P. cystidiosus, P. cornucopiae, P. pulmonarius, P. tuberregium, P. citrinopileatus, and P. flabellatus), Pseudomonas (e.g., P. chlororaphis, P. aeruginosa, P.aeruginosa, Pseudomonas koreensis), Pseudozyma (e.g., P. aphidis, P. antarctica), Rhizobium radiobacter, Rhizopus, Rhodospirillum (e.g., R. rubrum), Rhodotorula (e.g., R. bogoriensis), Saccharomyces (e.g., Saccharomyces cerevisiae, S. boulardii, S. torula), Sphingomonas (e.g., S. pauc imobilis), Starmerella (e.g., S. bombicola), Streptomyces, Torulopsis, Thraustochytrium, Trichoderma (e.g., T. reesei, T. harzianum, T. viridae), Ustilago (e.g., U. maydis), Vibrio cholerae. cholerae, Wickerhamiella (e.g., W. domericqiae), Wickerhamomyces (e.g., W. anomalus), Williopsis (e.g., W. mrakii), Zygosaccharomyces (e.g., Z. bailii), and others (including those listed as pathogens elsewhere in this disclosure).

[0075] If present, the fungus may be in the form of living or non-living cells, mycelium, spores and / or fruiting bodies. The fruiting bodies (if present) may be, for example, chopped and / or mixed into granular and / or powdered form.

[0076] If present, the yeast may be in the form of live or inactivated cells or spores, and may be in the form of dry cells and / or dormant cells (eg, yeast hydrolysate).

[0077] If present, the bacteria may be in the form of vegetative or planktonic cells, biofilms, spores, and / or dried cell or spore masses.

[0078] In some embodiments, dried microorganisms (eg, spores) may be mixed with fillers known in the art, such as, for example, microcrystalline cellulose (MCC).

[0079] In one embodiment, the composition comprises one or more Bacillus bacteria and / or growth byproducts thereof. In certain embodiments, the Bacillus is Bacillus amyloliquefaciens, Bacillus subtilis, Spore lactic acid bacteria and / or Bacillus licheniformis.

[0080] In one embodiment, the composition comprises Bacillus amyloliquefaciens NRRL B-67928 "B.amy" and / or its growth byproducts. Cultures of the Bacillus amyloliquefaciens "B.amy" microorganism have been deposited at the Northern Regional Research Laboratory (NRRL) Culture Collection of the Agricultural Research Service at 1815 N. University St., Peoria, IL, USA. The deposit has been assigned the accession number NRRL B-67928 and was deposited on February 26, 2020.

[0081] In one embodiment, the composition comprises a strain of Bacillus subtilis and / or its growth byproducts. In a specific embodiment, the strain is Bacillus subtilis B4 (NRRL B-68031). Cultures of the B4 microorganism have been deposited at the Northern Regional Research Laboratory (NRRL) Culture Collection of the Agricultural Research Service at 1815 North University St., Peoria, IL, USA. The deposit has been assigned a deposit number, NRRL B-68031, and was deposited on May 6, 2021.

[0082] B4 is a Gram-positive spore-forming strain of Bacillus subtilis capable of anaerobic growth (obligate anaerobe). The B4 strain is preferably applied in spore form, but germinates in an acidic environment where it can grow in a biofilm. Surprisingly, it was found that B4, when grown in a biofilm, can produce one or more compounds that can gather, chelate or otherwise trap iron. In certain embodiments, the compound is pulcherrimone and / or pulcherrimone acid.

[0083] Advantageously, the microorganisms and / or exopolysaccharides (EPS) of the biofilm effectively absorb iron using an iron capture agent such as pulcherrimone and / or pulcherrimone acid when passing through low pH values ​​(e.g., less than 6.8, less than 5.0, or less than 4.8). Once, for example, the pH value rises to somewhat near neutrality (e.g., 6.8 to 8.0), the microorganisms can make the iron more bioavailable to the plant.

[0084] B4 has a particular advantage over other agricultural microorganisms because it produces relatively large amounts of the lipopeptide surfactin (e.g., higher than wild-type Bacillus subtilis), as well as digestive enzymes including, for example, cellulases and amylases. These enzymes help break down nutrients into smaller units, such as volatile fatty acids (e.g., propionic acid, acetic acid, butyric acid), glucose, and amino acids.

[0085] The proprietary culture described herein has been deposited under conditions that ensure that it is available during the pendency of this patent application to persons whom the Director of the Patent and Trademark Office deems entitled to access it pursuant to 37 CFR 1.14 and 35 U.S.C. 122. The deposit may be made available in accordance with the requirements of foreign patent laws in countries in which the counterpart of this application or its progeny has been filed. It should be understood, however, that the provision of the deposit does not constitute a derogation from patent rights granted by governmental action, but rather constitutes a license to practice the invention.

[0086] Furthermore, each of the deposited cultures in this deposit is maintained and made available to the public, in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms, by all necessary means to preserve its viability and protect it from contamination, for a period of at least five years after the last request for samples of the deposited culture and in any case for at least 30 (thirty) years after the date of deposit or for the enforceability of any patent that may issue disclosing the culture. The depositor acknowledges that it is responsible for replacing the deposited culture if the depositor is unable to provide samples upon request due to the state of the deposit. Upon the grant of a patent disclosing the culture deposit of the present invention, all restrictions on making the culture available to the public are irrevocably lifted.

[0087] In certain embodiments, the composition may include other microorganisms that are capable of naturally producing or genetically engineering pulcherrimin and / or pulcherriminic acid, or other compounds that are capable of sequestering, chelating or otherwise trapping iron. In specific embodiments, the microorganisms are capable of growing as a biofilm.

[0088] In certain embodiments, the microorganism is a naturally occurring microorganism or a transgenic microorganism capable of regulating genes involved in iron capture and transport, for example, HFE, GDF15, TWSG1, ERFE, matrix enzyme 2, TF, TFR1, TFR2, HAMP, and HJV.

[0089] In certain embodiments, the composition may comprise a crude or purified siderophore or plant siderophore, or other molecules with high iron affinity, for example, pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA (ethylenediaminetetraacetic acid), iron EDTA, DTPA (diethylenetriaminepentaacetic acid), EDDHA (ethylenediamine-di-o-hydroxyphenylacetic acid), N,N-dihydroxy-N,N′-diisopropyladipamide (DPH), 2,3-dihydroxybenzoic acid, azotocin, transferrin, enterobactin, purulent azotocin, protochelatin, chelatin, bacteriocin, vibriocin, vibrioferrin, azotocin, aminochelatin, yersin, agarobactin, grape agglutinin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, achromatin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, ferric oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderocite, hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

[0090] The composition may also include other microbial growth byproducts. The microbial growth byproducts may be produced by the microorganisms of the composition, and / or they may be produced separately, such as by the microorganisms listed herein and added to the composition.

[0091] In certain embodiments, the composition may include residual substrates from the culture process and / or purified or unpurified growth byproducts, such as biosurfactants, killer factors, enzymes, polyketides, and / or other metabolites. The microorganism may be active or inactive, although in preferred embodiments, if the microorganism is considered a pathogen, the microorganism is inactivated and / or removed from the composition.

[0092] In one embodiment, the growth by-products are purified from the substratum producing the by-products. In addition, in one embodiment, the growth by-products are used in a crude form. The crude form may include, for example, a liquid supernatant (comprising residual cells and / or nutrients) generated by the microorganisms that culture the relevant growth by-products.

[0093] Growth byproducts may include metabolites or other biochemical substances produced by cell growth, including amino acids, peptides, polyketides, antibiotics, proteins, enzymes, biosurfactants, solvents, vitamins and / or other metabolites.

[0094] Additional components

[0095] Other components may be added to the composition, such as carriers, other microbial-based compositions, biosurfactants, enzymes, catalysts, solvents, buffers, emulsifiers, lubricants, solubility regulators, preservatives, stabilizers, UV inhibitors, viscosity regulators, preservatives, tracers, antimicrobial agents, surfactants, pH adjusters, essential oils, plant extracts, cross-linking agents, chelating agents, fatty acids, alcohols, reducing agents, synthetic agents, dyes, colorants, fragrances, antimicrobial compounds, antibiotics, foaming agents, foam reducers, polymers, thickeners, chelates and other components specific to the intended use.

[0096] In certain embodiments, the composition includes a chelating agent, including but not limited to ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), phosphonates, succinic acid (DMSA), diethylenetriaminepentaacetic acid (DTPA), N-acetylcysteine, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), organic acids with multiple coordinating groups (e.g., erythrine), STPP (sodium tripolyphosphate, Na5P3O 10 ), trisodium phosphate (TSP), water, carbohydrates, organic acids with more than one coordinating group (e.g., citric acid), lipids, steroids, amino acids or related compounds of the above (e.g., glutathione), peptides, phosphates, nucleotides, tetrapyrroles, ferroamines, ionophores, or phenols, sodium citrate, sodium gluconate, ethylenediamine disuccinic acid (EDDS), iminodisuccinic acid (IDS), L-glutamic acid diacetic acid (GLDA), GLDA Na4, methylglycine diacetic acid (MGDA), polyaspartic acid (PASA), hemoglobin, chlorophyll, lipophilic β-diketone, and (14,16)-heteronedione, ethylenediamine-N,N'-diglutaric acid (EDDG), ethylenediamine-N,N'-malonic acid (EDDM), 3-hydroxy-2,2-iminodisuccinic acid (HIDS), 2-hydroxyethyliminodiacetic acid (HEIDA), pyridine-2,6-dicarboxylic acid (PDA), trimethylglycine (TMG), titanium reagent, or any combination thereof.

[0097] In certain embodiments, the composition includes a germination promoter for enhancing the germination of spore-shaped microorganisms in a microbial-based composition. In a specific embodiment, the germination promoter is an amino acid, such as, for example, L-alanine and / or L-leucine. In one embodiment, the germination promoter is manganese.

[0098] In certain embodiments, the composition comprises an organic acid selected from the group consisting of, for example, acetic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid, tartaric acid, uric acid, propionic acid, butyric acid, sorbic acid, fumaric acid, benzoic acid, hydrofluoric acid, caproic acid, salicylic acid, gluconic acid, pyruvic acid, adipic acid, trichloroacetic acid, glycolic acid, cinnamic acid, carboxylic acid, succinic acid, carbonic acid, glutaric acid, capric acid, and ascorbic acid. In some embodiments, the composition comprises an inorganic acid selected from the group consisting of, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, perchloric acid, hydrofluoric acid, hydrobromic acid, and sulfonic acid. Preferably, the acid is present in an amount suitable for adjusting the pH value of the composition or the environment in which it is used to 6.8 or less, preferably 5.0 or less, more preferably 4.8 or less.

[0099] In one embodiment, the composition includes one or more fatty acids. The fatty acids can be produced by the microorganisms of the composition, and / or produced separately and included as additional components. In certain preferred embodiments, the fatty acids are saturated long-chain fatty acids having a 14 to 20 carbon backbone, such as, for example, myristic acid, palmitic acid, or stearic acid. In some embodiments, a combination of two or more saturated long-chain fatty acids is included in the composition. In some embodiments, saturated long-chain fatty acids can inhibit methane production and / or increase cell membrane permeability of methanogens.

[0100] In certain embodiments, the composition comprises one or more enzymes that help break down food sources into smaller units, such as volatile fatty acids (e.g., propionic acid, acetic acid, butyric acid), glucose, and amino acids. These enzymes can be produced by the microorganisms of the composition, and / or produced separately and included as additional components. Non-limiting examples of digestive enzymes include amylase, maltase, lactase, lipase, protease, sucrase, and cellulase.

[0101] In some embodiments, the composition may include additional components that are known to reduce methane production in methanogens, such as, for example, nitrates (e.g., calcium nitrate, ammonium nitrate, sodium nitrate, potassium nitrate, and magnesium nitrate); seaweed (e.g., Asparagus spp. and / or Asparagus spinosa, etc.); macroalgae; nitrooxypropanols (e.g., 3-nitrooxypropanol and / or ethyl 3-nitrooxypropanol, etc.); anthraquinones; ionophores (e.g., monensin and / or lasalocid); etc.); polyphenols (e.g., saponins, tannins, etc.); yucca extracts (e.g., steroidal saponin-producing plant species, etc.); soap bark extracts (triterpenoid saponin-producing plant species, etc.); organosulfur (e.g., garlic extract, etc.); flavonoids (e.g., quercetin, rutin, kaempferol, naringin, and anthocyanins, etc.); bioflavonoids from green citrus fruits, rose hips, and / or red currants; carboxylic acids; and / or terpenes (e.g., d-limonene, pinene, and citrus extract, etc.).

[0102] In one embodiment, the composition includes one or more biosurfactants. Biosurfactants are a group of structurally diverse surfactants produced by microorganisms that are biodegradable and can be efficiently produced using selected organisms on renewable substrates. All biosurfactants are amphiphilic molecules. Amphiphilic molecules consist of two parts: a polar (hydrophilic) group and a non-polar (hydrophobic) group. The common lipophilic part of the biosurfactant molecule is the hydrocarbon chain of a fatty acid, while the hydrophilic part is formed by an ester or alcohol group of a neutral lipid, a carboxylate group of a fatty acid or an amino acid (or peptide), an organic acid in a flavonoid or glycolipid, or a carbohydrate.

[0103] Biosurfactants, due to their amphiphilic structure, increase the surface area of ​​hydrophobic insoluble substances, improve the water bioavailability of these substances, and change the properties of bacterial cell surfaces. Biosurfactants accumulate at interfaces, thereby reducing interfacial tension, resulting in the formation of agglomerated micellar structures in solution. Safe and effective microbial biosurfactants reduce surface tension and interfacial tension between liquid, solid and gas molecules. The ability of biosurfactants to form pores and destabilize biofilms allows biosurfactants to be used as antibacterial, antifungal and hemolytic agents.

[0104] Advantageously, in certain embodiments, biosurfactants can help disrupt and / or penetrate biofilms to increase the effectiveness of antimicrobial compounds. Additionally, in certain embodiments, biosurfactants can promote increased transport and / or bioavailability of nutrients through and into cells, including into plant roots.

[0105] The biosurfactant according to the present invention may include, for example, glycolipids, lipopeptides, flavonoids, phospholipids, fatty acid esters, and high molecular weight polymers (such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes, etc.).

[0106] In one embodiment, the biosurfactant is a glycolipid. Glycolipids may include sophorolipids, rhamnolipids, cellobiose lipids, mannoerythritol lipids and trehalose lipids, etc. In one embodiment, the biosurfactant is a lipopeptide. Lipopeptides may include, for example, surfactin, iturin, sphingomyelin, mucin, fengycin and lichenin. In certain embodiments, a mixture of biosurfactants is used.

[0107] In one embodiment, the biosurfactant has been purified from a fermentation medium in which the biosurfactant was produced. In addition, in one embodiment, the biosurfactant is used in a crude form (including a fermentation broth produced by culturing a microorganism that produces the biosurfactant). The crude biosurfactant solution may include about 0.001% to 99%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, or about 50% pure biosurfactant, as well as residual cells and / or nutrients.

[0108] In one embodiment, the composition includes 1 ml / L to 10 ml / L of saponin, or 2 ml / L to 6 ml / L of rumen fluid. Saponin is a natural surfactant present in many plants and has properties similar to microbial surfactants (e.g., self-binding and interacting with biofilms). Saponin has three basic categories: triterpenoid saponins, steroidal saponins, and steroidal glycoalkaloids.

[0109] Some well-known triterpenoid saponin plant families include Leguminosae, Amaranthaceae, Apiaceae, Caryophyllaceae, Aquifoliaceae, Araliaceae, Cucurbitaceae, Berberidaceae, Chenopodiaceae, Myrsinaceae and Tribulaceae, etc. Quillaja sapodilla and beans (such as soybean, kidney bean and pea) are rich sources of triterpenoid saponins. Steroidal saponins are usually found in Agavaceae, Alliumceae, Asparagaceae, Dioscoreaceae, Liliaceae, Amaryllidaceae, Bromeliaceae, Palmaceae and Scrophulariaceae, and are accumulated in large quantities in crop plants such as Dioscorea, Allium, Asparagus, Fenugreek, Yucca and Ginseng. Steroidal glycoalkaloids are usually found in Solanaceae plants such as tomato, potato, eggplant and pepper.

[0110] In one embodiment, the composition of the present invention may include one or more additional substances and / or nutrients to supplement the needs of the beneficial microorganisms in the composition and / or to supplement the needs of the soil, water or plants in the application environment. These may include, for example, sources of amino acids (including essential amino acids), peptides, proteins, vitamins, trace elements, fats, fatty acids, lipids, carbohydrates, sterols, enzymes and minerals (such as calcium, magnesium, phosphorus, potassium, sodium, chlorine, sulfur, chromium, cobalt, copper, iodine, iron, manganese, molybdenum, nickel, selenium and zinc). In some embodiments, the microorganisms of the composition generate and / or provide these substances.

[0111] In certain embodiments, the composition may further comprise one or more carriers and / or excipients suitable for delivering the composition to soil, water, or plants.

[0112] Carriers and / or excipients according to the present invention may include any and all solvents, diluents, buffers (such as, for example, neutral buffered saline, phosphate buffered saline or optionally Tris-HCl, acetate or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents, solubilizers (such as, for example, Tween 80, polysorbate 80), colloids, dispersion media, carriers, fillers (e.g., MCC), chelating agents (such as, for example, EDTA or glutathione), amino acids (such as, for example, glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, fragrances, thickeners, coatings, preservatives (such as, for example, thimerosal, benzyl alcohol), antioxidants (such as, for example, ascorbic acid, sodium metabisulfite), tonicity control agents, absorption delaying agents, adjuvants, bulking agents (such as, for example, lactose, mannitol), etc. In certain embodiments, the composition comprises a filler, such as microcrystalline cellulose (MCC).

[0113] In certain embodiments, the composition may include glucose (eg, in the form of molasses), glycerol, and / or glycerol as an osmotic pressure substance or as a supplement to an osmotic pressure substance to increase the osmotic pressure during storage and transportation of the dry product.

[0114] In order to improve or stabilize the effect of the composition, it can be mixed with a suitable adjuvant and then used directly or diluted (if necessary). In a preferred embodiment, the composition is formulated into a liquid, a concentrated liquid or a dry powder or granules, which can be mixed with water and other components to form a liquid product.

[0115] The composition may also include other compounds and / or methods for effectively enhancing plant health, growth and / or yield, and / or supplementing the growth of beneficial microorganisms. For example, in one embodiment, the composition may include and / or may be applied simultaneously with nutrients and / or micronutrients to promote the growth of plants and / or microorganisms, such as magnesium, phosphate, nitrogen, potassium, selenium, calcium, sulfur, iron, copper and zinc; and / or one or more prebiotics, such as kelp extract, fulvic acid, chitin, humate and / or humic acid. A grower or agricultural scientist who benefits from the present disclosure can determine the exact materials and their amounts.

[0116] The composition may also contain and / or be used in conjunction with other agricultural compounds and / or crop management systems. In one embodiment, the composition may optionally contain or be applied with, for example, natural and / or chemical pesticides, insect repellents, herbicides, fertilizers, water treatment agents, nonionic surfactants and / or soil conditioners. If the composition is mixed with compatible chemical additives, the chemical is preferably diluted with water before being added to the composition of the present invention.

[0117] The composition can be formulated into, for example, solid, semi-solid, liquid or gaseous forms such as tablets, capsules, compressed granules, powders, granules, gels, solutions, steaks / spikes (for soil), drops, aerosols, suspensions, concentrates and other formulations suitable for a particular application. Liquids, dusts, granules, microgranules, granules, wettable powders, flowable powders, emulsions, microcapsules, oils or aerosols.

[0118] Exemplary embodiments

[0119] In some embodiments, the compositions of the present invention comprise the following:

[0120] A) one or more microorganisms (yeast, fungi and / or bacteria) capable of capturing iron and / or producing iron capturing growth byproducts, wherein preferably at least one of the one or more microorganisms is of the genus Bacillus, and wherein even more preferably the Bacillus is Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928;

[0121] B) Iron-capturing substances selected from the group consisting of pulcherrimone, pulcherrimone acid, citric acid, citrate, EDTA (ethylenediaminetetraacetic acid), iron EDTA, DTPA (diethylenetriaminepentaacetic acid), EDDHA (ethylenediamine-di(o-hydroxyphenylacetic acid)), N,N-dihydroxy-N,N′-diisopropylhexanediamine (DPH), 2,3-dihydroxybenzoic acid, azotocin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, chromothioate, ornithine bacteriocin, rhodotorulic acid, lysine, glutamic acid, gluconic acid, iron oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderite, transferrin, enterobactin, bacteriocin, vibriocin, azotocin, aminochelatin, pyocyanin, yersinic acid, agarobactin, grape agglutinin, hydroxamic acid, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid, enterobactin, pyocyanin, protochelatin, chelatin, vibrioferrin and / or nicotinamide.

[0122] C) carriers / excipients, which include solvents, diluents, buffers (such as, for example, neutral buffered saline, phosphate buffered saline or optionally Tris-HCl, acetate or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents, solubilizers (such as, for example, Tween 80, polysorbate 80), colloids, dispersion media, carriers and / or fillers (e.g., MCC);

[0123] D) one or more acids selected from the group consisting of, for example, acetic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid, tartaric acid, uric acid, propionic acid, butyric acid, sorbic acid, fumaric acid, benzoic acid, hydrofluoric acid, caproic acid, salicylic acid, gluconic acid, pyruvic acid, adipic acid, trichloroacetic acid, glycolic acid, cinnamic acid, carboxylic acid, succinic acid, carbonic acid, glutaric acid, capric acid, ascorbic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, perchloric acid, hydrofluoric acid, hydrobromic acid and sulfonic acid in an amount suitable for adjusting the pH of the composition or its application environment to 6.8 or less, preferably 5.0 or less, more preferably 4.8 or less; and / or

[0124] E) A biosurfactant selected from the group consisting of sophorolipids, rhamnolipids, cellobiose lipids, mannoerythritol lipids, trehalose lipids, surfactin, iturin, actin, mucin, fengycin and lichenin.

[0125] In some embodiments, the composition comprises each of component A to component E. In some embodiments, the composition comprises any combination of component A to component E, or comprises any one of component A to component E alone.

[0126] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; and pulcherrimin and / or pulcherriminic acid.

[0127] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; and a carrier / excipient.

[0128] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form and / or biofilm form; and one or more acids listed in point D).

[0129] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; pulcherrimin and / or pulcherriminic acid; and a carrier / excipient.

[0130] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form and / or biofilm form; pulcherrimin and / or pulcherriminic acid; a carrier; and one or more acids listed in point D).

[0131] In some embodiments, the composition comprises Bacillus subtilis NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928 in spore form, plant form, and / or biofilm form; pulcherrimin and / or pulcherriminic acid; a carrier; and a biosurfactant.

[0132] Production of microorganisms and / or microbial growth by-products

[0133] The present invention utilizes methods for the cultivation of microorganisms and the generation of microbial metabolites and / or other byproducts of microbial growth. The present invention further utilizes culture methods suitable for cultivating microorganisms and generating microbial metabolites at a desired scale. These culture processes include, but are not limited to, liquid culture / fermentation, solid state fermentation (SSF) and modifications, hybrids and / or combinations thereof.

[0134] As used herein, "fermentation" refers to the cultivation or growth of cells under controlled conditions. Growth can be aerobic or anaerobic. In a preferred embodiment, the microorganisms are grown using SSF and / or a modified form thereof.

[0135] In one embodiment, the invention provides materials and methods for generating biomass (eg, living cellular material), extracellular metabolites, residual nutrients, and / or intracellular components.

[0136] The microbial growth vessel used according to the present invention can be any fermenter or culture reactor for industrial use. In one embodiment, the vessel can have functional controls / sensors or can be connected to functional controls / sensors to measure important factors in the culture process, such as pH, oxygen, pressure, temperature, humidity, microbial density and / or metabolite concentration, etc.

[0137] In another embodiment, the vessel can also monitor the growth of microorganisms in the vessel (e.g., measuring cell number and growth stage). In addition, daily samples can be taken from the vessel and counted by techniques known in the art (such as dilution plate techniques).

[0138] In one embodiment, the method comprises supplementing the culture with a nitrogen source. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea and / or ammonium chloride, etc. These nitrogen sources can be used alone or in a combination of two or more.

[0139] This method can provide oxygenation for the growing culture. One embodiment utilizes the slow movement of air to remove air with low oxygen content and introduce oxygenated air. In the case of submerged fermentation, the oxygenated air can be ambient air supplemented daily by mechanical means including an impeller for mechanically agitating the liquid and an air distributor for supplying gas bubbles to the liquid to dissolve oxygen into the liquid.

[0140] The method may further include supplementing the culture with a carbon source. The carbon source is typically a carbohydrate such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol and / or maltose; an organic acid such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid and / or pyruvic acid; an alcohol such as ethanol, propanol, butanol, amyl alcohol, hexanol, isobutanol and / or glycerol; and fats and oils such as soybean oil, rapeseed oil, rice bran oil, olive oil, corn oil, sesame oil and / or linseed oil. These carbon sources may be used alone or in combination with two or more forms.

[0141] In one embodiment, the growth factor and micronutrient of microorganism are included in the culture medium. When the growth microorganism can not generate all the vitamins required for them, this is particularly preferred. Inorganic nutrients can also be included in the culture medium, and the inorganic nutrients include trace elements such as iron, zinc, copper, manganese, molybdenum and / or cobalt. In addition, the source of vitamins, essential amino acids and trace elements can be included in the culture medium in the form of, for example, fine powder or coarse meal (such as corn flour) or in the form of extracts (such as yeast extract, potato extract, beef extract, soybean extract, banana peel extract, etc.) or in purified form. Amino acid (such as, for example, amino acids that can be used for protein biosynthesis) can also be included.

[0142] In one embodiment, the culture medium may also include inorganic salts. Available inorganic salts may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, sodium chloride, calcium carbonate and / or sodium carbonate. These inorganic salts may be used alone or in a combination of two or more.

[0143] In one embodiment, one or more biostimulants, meaning substances that enhance the growth rate of microorganisms, may also be included. Biostimulants may be species specific and may enhance the growth rate of various species.

[0144] In some embodiments, the method for culturing may further include adding an antimicrobial agent to the culture medium before and / or during the culturing process.

[0145] In certain embodiments, antibiotics can be added to the culture at low concentrations to generate microorganisms that are resistant to antibiotics. Microorganisms that survive contact with the antibiotics are selected and repeatedly re-cultured with increasing antibiotic concentrations to obtain cultures that are resistant to antibiotics. This can be done in a laboratory setting or on an industrial scale using methods known in the art of microbiology. In certain embodiments, for example, the amount of antibiotic in the culture starts at 0.0001 ppm and increases by about 0.001 ppm to 0.1 ppm each iteration until the concentration in the culture is equal to or approximately equal to the dose typically applied to iron-trapped pathogens.

[0146] The pH of the mixture should be appropriate for the microorganism of interest. Buffers and pH adjusters (such as carbonates and phosphates) can be used to stabilize the pH near the preferred value. When metal ions are present in high concentrations, it may be necessary to use chelating agents in the culture medium.

[0147] The microorganisms can grow in a planktonic form or in a biofilm. If a biofilm is present, the vessel can contain a substrate on which the microorganisms can grow in a biofilm state. The system can also have the ability to apply stimuli (such as shear stress) that stimulate and / or enhance the growth characteristics of the biofilm.

[0148] In one embodiment, the culture method of the microorganism is carried out at about 5° to about 100° C., preferably 15° C. to 60° C., more preferably 25° C. to 50° C. In another embodiment, the culture can be carried out continuously at a constant temperature. In another embodiment, the culture is affected by temperature changes.

[0149] In one embodiment, the equipment used in the method and the culture process is a sterile device. Culture equipment (such as a reactor / vessel) can be separated from a sterilizing device (for example, an autoclave), but is connected thereto. Culture equipment can also have a sterilizing device that sterilizes on the spot before starting inoculation. Air can be sterilized by methods known in the art. For example, ambient air can pass through at least one filter before being introduced into the vessel. In other embodiments, the culture medium can be pasteurized, or alternatively not heated at all, but can utilize low water activity and low pH value to control the growth of undesirable bacteria.

[0150] In one embodiment, the present invention further provides a method for producing microbial metabolites by culturing the microbial strain of the present invention under conditions suitable for growth and metabolite production, and optionally purifying the metabolites, such as, for example, biosurfactants, enzymes, proteins, ethanol, lactic acid, β-glucan, peptides, metabolic intermediates, polyunsaturated fatty acids and lipids, etc. The content of the metabolites produced by the method can be at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, etc.

[0151] The biomass content of the fermentation medium may be, for example, 5 g / l to 180 g / l or more, or 10 g / l to 150 g / l. The cell concentration may be, for example, at least 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 or 1×10 13 cells.

[0152] Microbial growth byproducts produced by the microorganism of interest can be retained in the microorganism or secreted into the growth medium. The medium can contain compounds that stabilize the activity of the microbial growth byproducts.

[0153] The methods and apparatus for culturing microorganisms and producing microbial byproducts can be performed in a batch process, a quasi-continuous process, or a continuous process.

[0154] In one embodiment, all of the microbial culture composition is removed when the culture is complete (eg, when a desired cell density or density of a particular metabolite is reached). In this batch process, a completely new batch is started when the first batch is harvested.

[0155] In another embodiment, only a portion of the fermentation product is removed at any one time. In this embodiment, the biomass with living cells, spores, conidia, hyphae and / or mycelium remains in the vessel as an inoculant for a new culture batch. The removed composition can be a cell-free culture medium or contain cells, spores or other reproductive propagules and / or combinations thereof. A quasi-continuous system is created in this way.

[0156] Advantageously, the method does not require complex equipment or high energy consumption. The microorganisms of interest can be cultured and utilized on site on a small or large scale, even while still mixed with their culture medium.

[0157] Preparation of microbial-based products

[0158] "Microbial-based products" are products that are applied in practice to achieve a desired effect. Microbial-based products may be microbial-based compositions obtained only from microbial culture processes. In addition, microbial-based products may include further components that have been added. These additional components may include, for example, stabilizers, buffers, carriers (e.g., water or saline), nutrients that support further microbial growth addition, non-nutrient growth promoters and / or agents that help track microorganisms and / or compositions in their application environments. Microbial-based products may also include mixtures based on microbial compositions. Microbial-based products may also include one or more components based on microbial compositions that have been treated in some manner, such as but not limited to filtration, centrifugation, lysis, drying, purification, etc.

[0159] A microbial-based product of the present invention is simply a fermentation medium containing the microorganism and / or microbial metabolites and / or any residual nutrients produced by the microorganism. The fermented product can be used directly without extraction or purification. If necessary, extraction and purification can be easily achieved using standard extraction and / or purification methods or techniques described in the literature.

[0160] The microorganisms in the microbial products can be in active or inactivated form. In addition, the microorganisms and the residual culture used can be removed from the composition. The microbial products can be used without further stabilization, preservation and storage. Advantageously, the direct use of these microbial products maintains the high vitality of the microorganisms, reduces the possibility of contamination by foreign agents and undesirable microorganisms, and maintains the activity of the by-products of microbial growth.

[0161] The microorganisms and / or culture medium (eg, fermentation broth or solid matrix) produced by the growth of the microorganisms can be removed from the growth vessel and transferred via tubing or the like for immediate use.

[0162] In one embodiment, the microbial-based product is the only microbial growth byproduct.For example, a biosurfactant produced by a microorganism can be collected from a liquid fermentation tank in a crude form that includes about 50% pure biosurfactant in the liquid fermentation broth.

[0163] In other embodiments, the microbial-based product (microorganism, culture medium, or microorganism and culture medium) can be placed in a container of appropriate size, taking into account, for example, the intended use, the intended method of use, the size of the fermenter, and any mode of transportation from the microbial growth facility to the point of use. Thus, the container in which the microbial-based composition is placed can be, for example, 1 gallon to 1,000 gallons or larger. In other embodiments, the container is 2 gallons, 5 gallons, 25 gallons or larger.

[0164] For example, when the yeast fermentation product is harvested from the growth vessel, when the obtained product is placed in a container and / or piped (or otherwise transported for use), other components may be added. Additives may be buffers, carriers, other microbial-based compositions produced in the same or different facilities, viscosity modifiers, preservatives, nutrients for microbial growth, tracers, solvents, antimicrobial agents, other microorganisms, and other ingredients specific to the intended use, etc.

[0165] Other suitable additives that may be included in the formulations according to the invention include substances commonly used in such preparations. Examples of such additives include surfactants, emulsifiers, lubricants, buffers, solubility regulators, pH regulators, preservatives, stabilizers and UV inhibitors.

[0166] In one embodiment, the product may also include a buffer comprising an organic acid and an amino acid or its salt. Suitable buffers include citrate, gluconate, tartrate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactate, gluconate, propanol glycolate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine, and mixtures thereof. Phosphoric acid and phosphorous acid, or their salts may also be used. Synthetic buffers may be used, but preferably natural buffers may be used, such as the organic acids and amino acids listed above, or their salts.

[0167] In another embodiment, the pH adjuster comprises potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid, or a mixture.

[0168] In one embodiment, additional components may be included in the formulation, for example, an aqueous solution formulation of a salt (such as sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate, sodium hydrogen phosphate).

[0169] Advantageously, according to the present invention, microbial-based products may include fermentation broth in which the microorganisms are grown. The product may be, for example, at least 1%, 5%, 10%, 25%, 50%, 75% or 100% fermentation broth by weight. The biomass in the product may be, for example, 0% to 100% (including all percentages between 0% and 100%) by weight.

[0170] Alternatively, the product may be stored prior to use. The storage time is preferably short. Thus, the storage time may be less than 60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day or 12 hours. In a preferred embodiment, if living cells are present in the product, the product is stored at a cool temperature, such as, for example, below 20° C., 15° C., 10° C. or 5° C. On the other hand, the biosurfactant composition can generally be stored at ambient temperature.

[0171] Methods for treating and / or preventing iron deficiency in plants

[0172] The present invention provides methods for treating plant iron deficiency and iron chlorosis, wherein a composition according to the present invention is applied to a plant and / or its surroundings. In certain embodiments, the methods work by increasing the bioavailability of iron and other nutrients to the plant. Thus, these methods can also be used to increase the absorption of nutrients by plants.

[0173] As used herein, "applying" a composition or product refers to contacting the composition or product with a target or site, causing the composition or product to have an effect on the target or site. This effect can be due to, for example, microbial growth and / or interaction with the plant, as well as the effects of metabolites, enzymes, biosurfactants, or other microbial growth byproducts. Applying can also include "treating" a target or site with a composition. In some embodiments, a variety of plants and / or their surroundings are treated according to the methods of the present invention.

[0174] The "surrounding environment" of a plant refers to the soil and / or other culture medium, which may include the rhizosphere, that the plant grows in. In certain embodiments, the surrounding environment does not extend beyond a radius of, for example, at least 5 miles, 1 mile, 1000 feet, 500 feet, 300 feet, 100 feet, 10 feet, 8 feet, or 6 feet from the plant.

[0175] In some embodiments, the method comprises applying a composition according to the invention together with an acid, wherein the acid adjusts the pH of the composition or environment to 6.8 or less, preferably 5.0 or less, more preferably 4.8 or less. The acid may be an organic acid selected from the group consisting of, for example, acetic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid, tartaric acid, uric acid, propionic acid, butyric acid, sorbic acid, fumaric acid, benzoic acid, hydrofluoric acid, caproic acid, salicylic acid, gluconic acid, pyruvic acid, adipic acid, trichloroacetic acid, glycolic acid, cinnamic acid, carboxylic acid, succinic acid, carbonic acid, glutaric acid, capric acid, and ascorbic acid. The acid may also be an inorganic acid selected from the group consisting of, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, perchloric acid, hydrofluoric acid, hydrobromic acid, and sulfonic acid.

[0176] In some embodiments, the method comprises applying a composition according to the invention together with chelated iron and / or ferric sulfate.

[0177] If a microorganism is present in the composition, the microorganism may be alive (or viable) or inactive at the time of application. In some embodiments, the microorganism is in the form of a yeast extract and / or another microbial hydrolysate.

[0178] Microbial growth by-products may be used in addition to the microbial growth by-products of the composition, or may be used alone without the microorganisms.

[0179] The method may also include adding materials to promote microbial growth (e.g., adding nutrients and / or prebiotics) during the application process. Thus, live microorganisms can grow in situ and produce active compounds in situ. Thus, high concentrations of microorganisms and their growth byproducts can be easily and continuously achieved in the environment.

[0180] In some embodiments, the method comprises applying the whole microbial culture including inactivated cells in a submerged or solid-state fermentation medium. Advantageously, this reduces the amount of waste products generated during the production of the composition of the invention while increasing production efficiency by eliminating extraction and / or purification steps of microbial metabolites. In addition, the inclusion of inactive cells and residual fermentation medium provides a rich source of organic and inorganic nutrients that are essential for supporting soil and / or plant health.

[0181] In one embodiment, the composition is dispersed in the environment while being supported on a carrier. The carrier can be made of a material that can relatively gently retain the microorganism, thereby allowing the microorganisms that are thus proliferated to be easily released. The carrier is preferably cheap, and in some embodiments can serve as a nutrient source for the microorganisms thus applied, particularly a nutrient source that can be gradually released. Biodegradable carrier materials include corn husks, sugar industry waste or any agricultural waste. The water content of the carrier is, by weight, typically 1% to 99%, preferably 5% to 90%, more preferably 10% to 85%.

[0182] Application can include contacting the microbial-based product directly with the plant, plant part and / or the plant's surrounding environment (e.g., soil or rhizosphere). The microbial product can be applied as a seed treatment or applied to the soil surface, or applied to the surface of the plant or plant part (e.g., applied to the surface of the root, tuber, stem, flower, leaf, fruit or flower). It can be sprayed, poured, sprinkled, injected or diffused in the form of a liquid, dry powder, dust, granule, microparticle, pellet, wettable powder, flowable powder, emulsion, microcapsule, oil, gel, paste or aerosol.

[0183] In some embodiments, the composition is contacted with one or more roots of the plant. The composition can be applied directly to the roots by spraying or soaking the roots, etc., and / or indirectly to the roots by applying the composition to the soil (or rhizosphere) in which the plant grows. The composition can be applied to the seeds of the plant before or at the time of planting, or to any other part of the plant and / or its surroundings.

[0184] In one embodiment, mechanical action may be used, such as by tilling, to work the composition into the soil. In another embodiment, after application of the composition, a liquid (such as water) may be subsequently applied. Water may be applied as a spray using standard methods known to those of ordinary skill in the art. Other liquid wetting agents and wetting formulations may also be used.

[0185] In some embodiments, the composition is applied to the soil surface without mechanical bonding. The beneficial effects of soil application can be activated by rainfall, sprinkler irrigation, flooding or drip irrigation and subsequently delivered to, for example, the roots of plants.

[0186] In some embodiments, the composition is applied as a seed treatment in the form of a dry or liquid formulation or applied to the surface of a plant or plant part (eg, applied to the surface of a plant leaf or root).

[0187] In some embodiments, the composition is contacted with the leaves of the plant, for example by spraying. In some embodiments, the composition is injected or otherwise implanted into the stem or trunk of the plant, making the composition available to the interior and vascular tissues of the plant.

[0188] Plants and / or their environment can be treated at any point in the process of growing plants. For example, soil treatment compositions can be applied to the soil before, at the same time as, or after seeds are planted therein. Applications can also be made at any time during the development and growth of the plant thereafter, including when the plant is flowering, fruiting, and during and / or after leaf shedding.

[0189] In one embodiment, the method can be used in large-scale agricultural environments. The method can include applying the composition to a tank connected to an irrigation system that is used to supply water, fertilizer, or other liquid compositions to crops, orchards, or fields. Therefore, the soil around the plants and / or plants can be treated with the composition in the following ways: for example, soil injection, soil irrigation, or using a center pivot irrigation system, or spraying in the seed furrow, or using a sprinkler or dripper. Advantageously, the method is suitable for treating hundreds of acres of crops, orchards, or fields at one time.

[0190] In one embodiment, the method can be used in a smaller scale environment, such as a home garden or greenhouse. In such cases, the method can include spraying the composition onto the plants and / or their surroundings using a handheld lawn and garden sprayer. The composition can be mixed with water and, optionally, with other lawn and garden treatments, such as fertilizers and pesticides. The composition can also be mixed into a standard handheld waterer and poured onto the soil.

[0191] The compositions and methods of the present invention can be used alone or in combination with other methods and compounds to effectively enhance the health, growth and / or yield of plants; and can also be used in combination with other compounds to effectively treat and prevent plant pathogenic pests. For example, the methods can be used in conjunction with the following sources of nutrients and / or micronutrients for promoting plant and / or microbial growth: such as magnesium, phosphate, nitrogen, potassium, selenium, calcium, sulfur, iron, copper and zinc; and / or can be used in conjunction with one or more of the following prebiotics: such as kelp extract, fulvic acid, chitin, humate and / or humic acid. The exact materials and their amounts can be determined by a grower or agricultural scientist who benefits from the present disclosure.

[0192] The composition can also be used in combination with other agricultural compounds and / or crop management systems. In one embodiment, the composition can optionally include and / or be used with, for example, natural and / or chemical pesticides, insect repellents, herbicides, fertilizers, water treatment agents, nonionic surfactants and / or soil conditioners.

[0193] In one embodiment, the method can be used to inoculate the rhizosphere with one or more beneficial microorganisms. For example, in a preferred embodiment, the microorganisms of the composition can colonize the rhizosphere and provide a variety of benefits (including protection and nutrition) to the plant whose roots grow therein in addition to increasing the amount of bioavailable iron to the plant.

[0194] Advantageously, the methods of the present invention can be used to enhance the health, growth and / or yield of plants whose immune health is compromised due to infection by pathogens or environmental stressors (such as, for example, drought). Therefore, in certain embodiments, the methods of the present invention can also be used to improve the immune health or immune response of a plant.

[0195] These methods can help increase agricultural production even in poor or damaged soils; restore depleted green areas such as pastures, forests, wetlands and grasslands; and restore non-cultivable land to make it available for agriculture, reforestation and / or natural regeneration of plant ecosystems. In addition, through improved agricultural practices, these methods can help reduce pollution caused by greenhouse gas emissions.

[0196] In certain embodiments, enhancing soil health includes improving one or more properties of the soil. This may include, for example, removing and / or reducing pollutants in the soil, increasing the nutrient content and nutrient availability of the soil, improving the drainage and / or moisture retention properties of the soil, increasing soil salinity, increasing soil microbiome diversity, and / or controlling soil-borne pests and diseases. Other improvements may include adding volume and / or structure to soil that is eroded by wind and / or water, and preventing and / or slowing wind and / or water erosion of the soil.

[0197] In certain embodiments, the method includes a step of characterizing the soil type and / or soil health prior to treating the soil according to the methods of the invention. Thus, the method may also include tailoring the composition to meet specific soil type and / or soil health requirements. Soil characterization methods are known in the agronomic art.

[0198] In some embodiments, the methods are used to restore soil health, where the soil being treated was once healthy but has deteriorated over a period of time. Remediation can restore the soil to a previous state of health and / or an enhanced state of health.

[0199] In certain embodiments, the methods result in the removal and / or reduction of contaminants from soil, including remediation of soil contaminated with iron. In some embodiments, the contaminants are directly degraded by the microorganisms employed in the composition.

[0200] In certain embodiments, the methods can also help improve the diversity of the soil microbiome by promoting colonization of beneficial soil microorganisms in the soil and the roots of plants growing therein. The growth of nutrient-fixing microorganisms (such as rhizobia and / or mycorrhizae) and other endogenous and applied microorganisms can be promoted, thereby increasing the number of different species in the soil microbiome.

[0201] In one embodiment, the present invention can also be used to improve one or more qualities of soil, thereby enhancing the performance of the soil for agricultural, domestic and horticultural purposes.

[0202] In certain embodiments, soil treatment compositions may also be applied to promote colonization of the plant's roots and / or rhizosphere and vascular system, thereby enhancing plant health and vigor. Thus, nutrient-fixing microorganisms, as well as other beneficial endogenous and exogenous microorganisms and / or their byproducts that promote crop growth, health and / or yield may be promoted.

[0203] In one embodiment, the methods can be used to enhance the penetration of beneficial molecules through the outer layer of root cells.

[0204] The present invention can be used to improve any quantity in any type of soil (e.g., clay, sandy, silty, peaty, chalky, loamy, and / or combinations thereof. In addition, the methods and compositions can be used to improve the quality of dry, waterlogged, porous, depleted, compacted soils, and / or combinations thereof.

[0205] In one embodiment, the method can be used to improve drainage and / or diffusion of water in waterlogged soil. In one embodiment, the method can be used to improve water retention in dry soil.

[0206] In one embodiment, the methods can be used to increase nutrient retention in porous and / or poor soils.

[0207] All plants and plant parts can be treated according to the invention. In this case, plants are understood to mean all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants (including transgenic plants and plant varieties) obtained by traditional breeding and optimization methods or by biotechnology and recombinant methods, or a combination of these methods.

[0208] Plant parts are understood to be all above-ground and below-ground parts and organs of plants (such as buds, leaves, flowers and roots etc.), the following examples being mentioned: leaves, needles, stems, flowers, fruit bodies, fruits and seeds, but also roots, tubers and rhizomes. Plant parts also include crop material and vegetative and sexual propagation material, for example cuttings, tubers, rhizomes, young shoots and seeds.

[0209] Example

[0210] The present invention and its many advantages can be better understood by the following examples given by way of illustration. The following examples illustrate some methods, applications, embodiments and variations of the present invention. None of them should be considered as limitations of the present invention. For the present invention, a large number of changes and modifications may be made.

[0211] Example 1 - pH growth test of B4 strain

[0212] The B4 strain was spread on tryptic soy agar (TSA) at neutral (6.8) and acidic (4.8) pH levels to look for growth differences, in an effort to determine how it performs in the various pH environments found within the dairy cow digestive system.

[0213] Growth was faster on neutral pH (6.8) compared to acidic plates within 24 hours, but after 48 hours, growth on pH 4.8 agar was equal or faster. Figure 1A to Figure 1B In addition, when grown on acid agar, it produces large amounts of exopolysaccharides (EPS). Figure 1C . This is the result of environmental pressure.

[0214] Dried B4 spores were also added to sterile PBS adjusted to pH 2.8 and left for 24 hours. They were then plated on neutral (6.8) TSA plates. A lawn of growth appeared, but no EPS was produced (similar to the pH 6.8 plates in Figure 1). This shows that the dried spores remained intact after being exposed to an overall harsh environment. Overall, the pH of the growth medium / environment can affect EPS production.

[0215] Example 2 - EPS

[0216] B4 was grown in liquid medium at pH 4.8 to specifically produce EPS. Putative EPS was isolated from the culture and purified.

[0217] FTIR analysis confirmed that the purified sample was EPS, and HPLC analysis confirmed the presence of a large peak, indicative of sugar oligomers. C=O bonds were also observed via UV absorption.

[0218] Furthermore, surprisingly, when the cultures were processed for EPS extraction, purple streaks appeared in the cell pellets. When EPS purification was complete, the samples appeared pink, indicating the presence of pulcherrimone or pulcherrimone acid. Figure 2 .

[0219] Example 3 - Enzyme Assay Test - Amylase

[0220] Amylase is an enzyme that hydrolyzes the glycosidic bonds in starch molecules by converting complex carbohydrates into monosaccharides. Agar (starch) is inoculated with B4 and cultured for growth. Figure 3 After running the plate assay, an orange color is observed around the bacterial growth, indicating starch breakdown. This is a positive amylase test.

[0221] Example 4 - Enzyme Assay Test - Cellulase

[0222] Cellulase is an enzyme that converts cellulose into glucose. Carboxymethylcellulose agar (CMCA) medium plates were used to test the cellulase activity of B4.

[0223] B4 was grown in tryptone soy broth with and without cellobiose. After 48 hours of growth, the liquid culture was streaked onto CMCA plates. After inoculation, once growth appeared on the agar, an iodine solution was introduced into the plates. The yellow transparent area around the bacterial growth indicated the decomposition of cellulose and the presence of cellulase. Figure 4 .

[0224] Example 5 - Detection of siderophores

[0225] The chrome azurol S (CAS) assay was used to detect siderophores from B4 cultures and dried B4 spores. These tests were performed in aerobic and anaerobic environments in different growth media: MRS-sucrose, M23-6, minimal medium with Tween, and minimal medium without Tween. FIG. 5A to FIG. 5C ) and 24 hours ( FIG. 6A to FIG. 6C )Observe multiple plates.

[0226] Siderophores scavenge iron from the Fe-CAS-cetyltrimethylammonium chloride complex and the release of the CAS dye results in a color change from blue to orange. Any observable color change on the CAS agar plate indicates qualitative detection of siderophores.

[0227] All B4 medium cultures tested showed positive responses for siderophore production and activity. MRS-sucrose (the richest medium) produced the strongest activity. There were no differences in siderophore activity between the four medium cultures under aerobic and anaerobic conditions.

[0228] The siderophore activity produced by dried B4 spores was lower compared to that of B4 cultures, which may be due to the dormancy of the spores. The dried spores performed better under aerobic conditions.

[0229] Example 6 - Iron activity determination

[0230] To understand how B4 interacts with iron and to determine differences in iron activity between cultures, an iron assay kit (Sigma-Aldrich) was used to measure the amount of ferrous iron (Fe) present in different B4 cultures. 2+ ) concentration, iron (Fe 3+ ) concentration and total iron concentration.

[0231] Iron is released from the sample by the addition of an acidic buffer. The released iron reacts with the chromogen to produce a colorimetric (593 nm) product proportional to the iron present.

[0232] Figure 7 The results in Figures 5 and 6 correlate with the siderophore results reported in Figures 5 to 6, meaning that higher siderophore activity correlates with lower total iron levels. For example, MRS-sucrose medium is the richest medium with the highest siderophore activity and the lowest total iron content. In addition, the minimal medium used is designed to increase the production of pulchermin, which is chelated iron. Higher levels of pulchermin in the culture increase both the iron concentration and the total iron concentration.

Claims

1. A method for treating and / or preventing iron deficiency and / or iron chlorosis in plants, wherein the method comprises applying a composition comprising one or more iron trapping ingredients to the plants and / or their surroundings.

2. The method of claim 1, further comprising applying an acid to the environment to achieve a pH of 6.8 or less.

3. The method of claim 1, wherein the composition is applied to the soil in which the plant is growing.

4. The method of claim 3, wherein the composition is applied to the soil via an irrigation system or via a sprayer applicator.

5. The method of claim 1, wherein the composition is applied directly to the plant.

6. The method of claim 5, wherein the composition is injected or implanted into the stem or trunk of the plant.

7. The method of claim 5, wherein the composition is applied as a seed treatment.

8. The method of claim 5, wherein the composition is applied to the roots and / or leaves of the plant.

9. The method of claim 1, wherein the iron-trapping component is a non-pathogenic microorganism that is capable of trapping iron or produces a growth byproduct that is capable of trapping iron.

10. The method according to claim 9, wherein the microorganism is a bacterium of the genus Bacillus.

11. The method of claim 9, wherein the microorganism is Bacillus subtilis B4 NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928.

12. The method of claim 9, wherein the microorganism is applied in the form of spores but grows in the form of a biofilm when in contact with an acidic environment having a pH of 6.8 or less.

13. The method according to claim 1, wherein the iron capture component is selected from the following: pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA ethylenediaminetetraacetic acid, iron EDTA, DTPA diethylenetriaminepentaacetic acid, EDDHA ethylenediamine-di-o-hydroxyphenylacetic acid, N,N-dihydroxy-N,N′-diisopropyladipamide DPH, 2,3-dihydroxybenzoic acid, nitrogen carrying bacteria, transferrin, enterobactin, pyocyanin, protochelatin, ferritin, Bacteriocins, vibriocins, vibrioferrin, azotocin, aminochelatins, yersin, agarobactin, grape agglutinin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, chromobactin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, iron oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderite hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

14. The method of claim 1, wherein the composition further comprises one or more microbial growth byproducts selected from the group consisting of biosurfactants, enzymes, organic acids, fatty acids, amino acids, proteins, peptides, alcohols, polyketides, natural antibiotics, aldehydes, amines, sterols, and vitamins.

15. The method of claim 1, wherein the composition is applied to the plant and / or its surroundings along with a source of one or more nutrients and / or prebiotics.

16. A composition for treating and / or preventing iron deficiency and / or iron chlorosis, said composition comprising one or more iron-trapping components and, optionally, a carrier.

17. The composition according to claim 16, further comprising an amount of an acid to adjust the pH of the composition to 6.8 or less.

18. The composition of claim 16, wherein the iron-trapping component is a non-pathogenic microorganism that is capable of trapping iron or produces a growth byproduct that is capable of trapping iron.

19. The composition according to claim 18, wherein the microorganism is a bacterium of the genus Bacillus.

20. The composition of claim 19, wherein the microorganism is Bacillus subtilis B4 NRRL B-68031 or Bacillus amyloliquefaciens NRRL B-67928.

21. The composition of claim 18, wherein the microorganism is in spore form or biofilm form.

22. The composition of claim 18, wherein the iron-trapping component is selected from the group consisting of pulcherrimin, pulcherriminic acid, citrate, citric acid, EDTA ethylenediaminetetraacetic acid, iron EDTA, DTPA diethylenetriaminepentaacetic acid, EDDHA ethylenediamine-di-o-hydroxyphenylacetic acid, N,N-dihydroxy-N,N′-diisopropyladipamide DPH, 2,3-dihydroxybenzoic acid, nitrogen-carrying bacteria, transferrin, enterobactin, pyocyanin, protochelatin The invention can be used to treat schizolin, chelatin, chelatin, bacteriocin, vibriocin, vibrioferrin, azotocin, aminochelatin, yersin, ferrochrome, deferasirox, deferiprone, deferoxamine, fusarin, chrysin, chromatin, ornithine, rhodotorulic acid, lysine, glutamic acid, gluconic acid, ferric oxyhydroxide minerals, ferrihydrite, magnetite, hematite, goethite, siderite, hydroxamate, catecholase, salicylate, carboxylate, mugineic acid, ferulic acid, caffeic acid and / or nicotinamide.

23. The composition of claim 16, further comprising a germination promoter, wherein the germination promoter is L-alanine, L-leucine or manganese.

24. The composition of claim 16, further comprising one or more microbial growth byproducts selected from the group consisting of biosurfactants, enzymes, organic acids, fatty acids, amino acids, proteins, peptides, alcohols, polyketides, natural antibiotics, aldehydes, amines, sterols, and vitamins.

25. The composition of claim 16, further comprising a nutrient or a prebiotic.

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