Compositions and methods for nitrogen fixation cluster modulation

Abstract

The present disclosure relates to genetically modified microorganisms for improving the phenotype of plants, such as nitrogen utilization in non-leguminous plants. Comprising novel strains of microbial bodies, microbial consortia and agricultural compositions comprising the same. In addition, the present disclosure teaches methods of utilizing the microorganisms, microbial consortia, and agricultural compositions comprising the same in methods of conferring beneficial properties to target plant species. In particular aspects, the disclosure provides methods of enhancing desired plant traits in agronomically important species, such as fixation, utilization, regulation, absorption, acquisition, tolerance and / or processing of nitrogen in plants.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 477,306, filed on December 27, 2022, the entire disclosure of which is incorporated herein by reference.

[0003] Reference to a Sequence Listing Submitted Electronically

[0004] The official copy of the sequence listing is submitted electronically as a WIPOST26 - compliant XML sequence listing and is filed concurrently with this specification. The file name of the sequence listing is 22038_SeqListing.xml, created on December 06, 2023, and is 479,270 bytes in size. The sequence listing contained in this file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to isolated and genetically modified microorganisms having applications especially in agriculture. The disclosed microorganisms can be used in their isolated and biologically pure state or formulated into agriculturally acceptable compositions. Methods of using the isolated microorganisms or agriculturally acceptable compositions in agricultural applications are also disclosed. Background Art

[0006] Scientists estimate that if the global agricultural "yield gap" (i.e., the difference between the observed best yields and those achieved elsewhere) could be closed, global crop production would increase by 45% to 70%. That is, if all farmers (regardless of their location in the world) could achieve the highest attainable yields expected for their respective regions, most of the shortfalls in global food production could be addressed. However, it is difficult to solve the problem of how to achieve higher yields in the heterogeneous global landscape. Generally, the yield gap can be explained by water shortages, sub - standard farming practices, insufficient fertilizers, and the inability to use herbicides and pesticides. However, significantly increasing the use of water, fertilizers, herbicides, and pesticides globally is not only economically infeasible for most regions of the world but would also have adverse environmental consequences.

[0007] Therefore, it is completely infeasible to meet global agricultural yield expectations by simply scaling up the current high - input agricultural systems used in most developed countries.

[0008] Accordingly, there is a pressing need in the art for improved methods of enhancing crop performance and conferring beneficial traits to desired plant species. The techniques described herein include modifying the intergenic regions in nitrogen - fixation clusters to alter the amount of atmospheric nitrogen fixed by bacteria.

[0009] Nitrogen is the most abundant gas in the Earth's atmosphere; however, N2 is not bioavailable to plants that require nitrogen for proper growth and production. Synthetic nitrogen fertilizers in the form of ammonia or ammonium (NH3 / NH4 + ) are widely used in agriculture and are most commonly industrially prepared by the Haber-Bosch process, which requires high energy input along with natural gas and produces greenhouse gases as by-products.

[0010] Biological nitrogen fixation can occur in bacteria that utilize nitrogenase to convert N2 into bioavailable ammonia. Organisms expressing this enzyme complex may be able to reduce the dependence of modern agriculture on the Haber-Bosch process and the extensive application of synthetic nitrogen fertilizers. Despite this possibility, methods for regulating the fixation of atmospheric nitrogen are limited to using the natural nitrogen fixation genes of bacteria, which are thought to be under the control of a single regulatory element, thus limiting the regulation of nitrogen fixation to meet the needs of specific applications. SUMMARY OF THE INVENTION

[0011] Modifications to the nitrogen fixation gene cluster ("nif" cluster) in bacteria are described herein to improve nitrogen fixation ability. Regions containing regulatory elements that have hitherto been unknown within the cluster are described and modified.

[0012] The present disclosure includes microbial organisms having applications in various fields including agriculture. The disclosed microbial organisms can be used in their isolated and biologically pure state or formulated into agriculturally acceptable compositions. Also provided are agriculturally beneficial microbial consortia (which comprise at least two members of the disclosed microbial organisms) and methods of utilizing such consortia in agricultural applications. In some aspects, genomic modification of the microorganisms (individuals, consortia, and / or communities) is envisioned to improve microbial traits and the plants associated with the microorganisms.

[0013] The present disclosure addresses the important problem of how to improve plant performance, thereby narrowing the yield gap worldwide while providing ways to endow other beneficial traits to plant species. The modified strains described herein improve plant performance by enabling the plant to increase and / or improve nitrogen utilization, fixation, uptake, acquisition, tolerance, distribution, regulation, processing, and / or any combination of any of the foregoing and / or any item.

[0014] In some embodiments, the plant is a non-leguminous crop plant.

[0015] In some embodiments, the plant is a dicotyledonous plant. In some embodiments, the plant is a vegetable, herbaceous plant, ornamental plant, or fruit plant. In some embodiments, the plant is selected from the group consisting of: kale, spinach, lettuce, carrot, potato, beet, radish, tomato, broccoli, cauliflower, squash, mustard, berry, pepper, leafy green, cowpea, melon, cucumber, basil, grape, and okra.

[0016] In some embodiments, the plant is a monocotyledonous plant. In some embodiments, the plant is a C3 monocotyledonous plant. In some embodiments, the plant is a C4 monocotyledonous plant. In some embodiments, the plant is selected from the group consisting of: corn, wheat, rice, sorghum, sugarcane, onion, bamboo, palm, garlic, ginger, lily, daffodil, iris, orchid, bluebell, tulip, amaryllis, banana, plantain, ginger, turmeric, cardamom, asparagus, pineapple, sedge, rush, chive, forage grass, turf grass, buckwheat, quinoa, chia, and millet.

[0017] The solutions for improving crop performance and increasing yields provided by the present disclosure are harmless to the earth's resources because they do not rely on increasing water consumption or the input of synthetic chemicals into the system. Instead, the present disclosure utilizes microorganisms to confer beneficial traits to the desired plants, including increased yields.

[0018] Accordingly, the present disclosure provides environmentally sustainable solutions that allow farmers to increase the yields of important crops and that do not rely on increasing the use of synthetic herbicides and insecticides.

[0019] In an embodiment, the present disclosure provides an effective and widely applicable agricultural platform that utilizes microorganisms and microbial aggregates (multiple microorganisms, in some aspects, multiple microorganisms that modify the health or desired phenotype (e.g., agronomic traits) of the associated plants) that promote one or more desired plant traits.

[0020] The microorganisms disclosed herein modify the performance of plants (e.g., crop plants) through direct and indirect mechanisms. In some aspects, the microorganisms form a symbiotic relationship with the plant. In some aspects, the microorganisms produce compounds (e.g., metabolites, toxins, proteins, lipopeptides, or other compositions) that confer benefits to the plant or that the plant can utilize for modification. In some aspects, the microorganisms increase the solubility of one or more compositions (e.g., nutrients), thereby benefiting the plant. In some aspects, the microorganisms confer tolerance to exogenous substances (e.g., herbicides or insecticides) to the plant. In some aspects, the microorganisms produce compositions that are harmful to plant pests (e.g., insects). In some aspects, the microorganisms fix nitrogen, thereby modifying the nutritional status of the plant. Other aspects are envisioned in addition to the above-listed exemplary and non-limiting aspects.

[0021] In some embodiments, a single microorganism is utilized. In some aspects, the single microorganism is isolated and purified. In some aspects, the single microorganism is a taxonomic species of bacteria. In some aspects, the single microorganism is a distinguishable strain of a taxonomic species of bacteria. In some aspects, the single microorganism is a novel, recently discovered strain of a taxonomic species of bacteria.

[0022] In some aspects, the single microorganism (whether a taxonomically distinguishable species or strain) is combined with one or more other microorganisms of a different species or strain. In certain aspects, the combination of two or more microorganisms forms consortia or a consortium. The terms consortia and consortium are used interchangeably.

[0023] In certain aspects, the present disclosure provides for the development of highly functional microbial consortia that contribute to the promotion of the development and expression of desired phenotypic or genotypic plant traits. In some embodiments, the consortia of the present disclosure possess functional attributes that do not exist in nature when the individual microorganisms live alone. That is, in various embodiments, specific microbial species are combined into a consortium such that the microbial combination possesses functional attributes that none of the individual members of the consortium possess when considered alone.

[0024] In some embodiments, the functional attribute possessed by the microbial consortia is the ability to confer one or more beneficial characteristics to a plant species, such as: increased growth, increased yield, increased nutrient utilization (e.g., nitrogen, phosphate, etc.), increased nitrogen use efficiency, enhanced stress tolerance, enhanced drought tolerance, increased photosynthetic rate, enhanced water use efficiency, enhanced pathogen resistance, modification of plant architecture (which does not necessarily affect plant yield but addresses plant function), etc. Beneficial characteristics of pest resistance and / or tolerance are also contemplated, including adverse effects on nematodes, insects, or other pests.

[0025] In some embodiments, the individual microorganisms do not possess the ability to confer these beneficial characteristics to plants when present in nature. Instead, in some embodiments, these microorganisms are combined into a consortium by human handiwork to form a functional composition that possesses attributes and functional characteristics that do not exist in nature. In some embodiments, the consortium may include microorganisms that have been genetically edited, altered, or modified by techniques known to those of ordinary skill in the art to modify the cellular composition (including DNA, RNA, proteins, and / or combinations thereof).

[0026] However, in other embodiments, the present disclosure provides isolated and biologically pure individual microorganisms that are capable of conferring beneficial traits to a desired plant species without the need to combine the microorganisms into a consortium.

[0027] In some embodiments, the microorganism is a strain of Paenibacillus that has been genetically modified to enhance nitrogen fixation ability. The one or more genetic modifications are characterized as providing improved nitrogen fixation activity to the genetically modified microorganism as compared to a non-genetically modified strain of the microorganism.

[0028] Accordingly, the present disclosure provides environmentally sustainable solutions that allow farmers to increase yields of important crops without relying on increased use of synthetic fertilizers, herbicides, and / or pesticides. For example, in one aspect, the present disclosure describes isolated microorganisms that have been genetically modified to enhance nitrogen fixation ability. In some embodiments, the endogenous nif genes of the isolated microorganism, particularly an intergenic region (an "intergenic spacer") between known genes, are genetically modified to alter the nitrogen fixation ability of the microorganism.

[0029] The present disclosure also relates to agricultural compositions that include one or more strains of the isolated genetically modified microorganisms disclosed herein and an agriculturally acceptable carrier. In some embodiments, the agricultural composition includes one or more additional agriculturally beneficial agents (such as fertilizers, biofertilizers, biocidal nematocides, biostimulants, synthetic insecticides, and / or synthetic herbicides).

[0030] Also disclosed herein are methods of conferring one or more beneficial traits to a plant, the methods including applying an agriculturally effective amount of one or more of the isolated genetically modified microorganisms or agricultural compositions disclosed herein.

[0031] Any strain disclosed herein can be further combined with one or more additional microorganisms, thereby forming a microbial consortium. A microbial consortium can be any combination of one or more individual microorganisms. In certain embodiments, the microbial consortium comprises two microorganisms or three microorganisms or four microorganisms or five microorganisms or six microorganisms or seven microorganisms or eight microorganisms or nine microorganisms or 10 microorganisms or more than 10 microorganisms.

[0032] Another object of the present disclosure is to design microbial consortia that are capable of performing a common multi-dimensional activity. In certain aspects, the microorganisms that make up the consortium act synergistically. In various aspects, the effect of the microbial consortium on a particular plant trait is greater than the effect that would be observed when using any one individual microbial member of the consortium alone. That is, in some aspects, the consortium exhibits a greater-than-additive effect on the desired plant trait as compared to the effect that would be found when using any individual member of the consortium alone.

[0033] In some aspects, the consortiums initiate the establishment of other plant-microbe interactions, such as by serving as primary colonizers or founder populations that set the trajectory for future microbiome development.

[0034] In embodiments, the present disclosure relates to synergistic combinations (or mixtures) of microbial isolates.

[0035] In some aspects, the consortiums taught herein provide a wide range of agricultural applications, including: increasing the yields of grains, fruits, and flowers; enhancing the growth of plant parts; improving the ability to utilize nutrients (e.g., nitrogen, phosphate, etc.); increasing disease resistance; biopesticidal effects, including increasing resistance to fungi, insects, and nematodes; increasing survival rates in extreme climates; and improving other desired plant phenotypic characteristics. Clearly, these benefits to plants and / or adverse effects on target pests and / or pathogens can be obtained without any harmful side effects on the environment.

[0036] In some aspects, the individual microorganisms of the present disclosure or the consortiums containing them can be combined into agriculturally acceptable compositions.

[0037] In some embodiments, the agricultural compositions of the present disclosure include, but are not limited to: wetting agents, solubilizers, defoamers, detergents, chelating agents, drift reducers, neutralizing agents, buffers, corrosion inhibitors, dyes, odorants, extenders, penetration aids, adhesives, binders, dispersants, thickeners, stabilizers, emulsifiers, freeze point depressants, antimicrobials, fertilizers, pesticides, nematicides, insecticides, herbicides, inert carriers, polymers, etc.

[0038] In one embodiment of the present disclosure, the microorganisms (including isolated single species or strains, consortia, or combinations thereof, such as metabolites) are provided in the form of a seed coating or other seed application. In an embodiment, the seed coating can be applied to bare and untreated seeds. In other embodiments, the seed coating can be applied to previously treated seeds. Thus, in some embodiments, the present disclosure teaches a method of treating seeds that includes applying an isolated bacterial strain or microbial consortium to the seeds. In certain embodiments, the isolated bacterial strain or microbial consortium is applied in the form of an agricultural composition that includes an agriculturally acceptable carrier. In some embodiments, the agricultural composition can be formulated as: a soil drench, a foliar spray, a soak treatment, an in-furrow treatment, a soil amendment, a granule, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural composition can be applied alone or in a rotational spray program with other agricultural products. In some embodiments, the agricultural composition can be tank-mix compatible. In some embodiments, the agricultural composition can be tank-mix compatible with other agricultural products. In some embodiments, the agricultural composition can be compatible with equipment for ground, aerial, and irrigation applications.

[0039] In some embodiments, the applied microorganisms can become endophytic and thus can be present in the treated growing plants and their progeny. In other embodiments, the microorganisms may be applied in a co-treatment form simultaneously with the seed treatment.

[0040] In one embodiment of the present disclosure, the microorganisms are provided in the form of granules or plugs applied to the plant growth medium or as a soil drench. In other embodiments, the microorganisms are provided in the form of a foliar application, such as a foliar spray or a liquid composition. The foliar spray or liquid application can be applied to the growing plants or the plant growth medium, such as soil, a hydroponic system, a gel.

[0041] In other embodiments, the microorganisms (including isolated single species or strains or consortia) and / or their combinations (such as metabolites) are provided in the form of a fertilizer, pesticide, or other amendment that can be applied to the soil. In some embodiments, the microorganisms are provided in the form of a fertilizer, pesticide, or other amendment applied to the soil before planting. In some embodiments, the microorganisms are provided in the form of a fertilizer, pesticide, or other amendment applied to the soil at the time of planting. In some embodiments, the microorganisms are provided in the form of a fertilizer, pesticide, or other amendment applied to the soil after planting.

[0042] In other embodiments of the present disclosure, the microorganisms (including isolated single species or strains or consortia) and / or their combinations (such as metabolites) are provided in the form of a post-harvest disease control application.

[0043] In embodiments, the agricultural compositions of the present disclosure can be formulated as: (1) solutions; (2) wettable powders; (3) dusts; (4) soluble powders; (5) emulsion or suspension concentrates; (6) seed dressings, (7) tablets; (8) water-dispersible granules; (9) water-soluble granules (slow or quick release); (10) microencapsulated granules or suspensions; (11) as an irrigation component, and (12) components of fertilizers, pesticides, and other compatible amendments, etc. In some aspects, the composition can be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure can be applied to soil, plants, seeds, rhizospheres, root sheaths, or other areas where application of the microbial composition will be beneficial.

[0044] Another object of the present disclosure relates to agricultural compositions that are formulated to provide a high colony-forming unit (CFU) bacterial population or aggregate. In some aspects, the agricultural composition has an adjuvant that provides a relevant shelf life. In embodiments, the CFU concentration of the taught agricultural composition is higher than the concentration at which the microorganisms would naturally occur outside of the disclosed method. In another embodiment, the agricultural composition contains microbial cells at a concentration of 10^2 - 10^12 CFU / gram of carrier or 10^5 - 10^9 CFU / gram of carrier. In one aspect, the microbial cells are applied directly to the seeds as a seed coating at a concentration of 10^5 - 10^9 CFU. In other aspects, the microbial cells are applied as an overcoat on top of another seed coating as a seed overcoat at a concentration of 10^5 - 10^9 CFU. In other aspects, the microbial cells are applied as a co-treatment agent with another seed treatment agent at a ratio of 10^5 - 10^9 CFU.

[0045] In various aspects, the present disclosure relates to agricultural microbial preparations for promoting plant growth. In various aspects, the present disclosure provides the taught isolated microorganisms and aggregates containing them formulated as agricultural inoculants. The taught inoculants can be applied to plants, seeds, or soil, or in combination with fertilizers, pesticides, and other compatible amendments. Suitable examples of formulating inoculants containing isolated microorganisms can be found in U.S. Patent No. 7,097,830.

[0046] The disclosed microbial preparations can: reduce the need for nitrogen-containing fertilizers, solubilize minerals, provide biological insecticidal protection for plants, protect plants from pathogens (e.g., fungi, insects, and nematodes), and make valuable nutrients, such as nitrogen and / or phosphate, available to plants, thereby reducing and eliminating the need to use chemical insecticides and chemical fertilizers.

[0047] In some embodiments, the isolated and biologically pure microorganisms of the present disclosure can be utilized in a method of conferring one or more beneficial properties or traits to a desired plant species.

[0048] In some embodiments, in a method of conferring one or more beneficial properties or traits to a desired plant species, an agriculturally acceptable composition comprising the isolated and biologically pure microorganisms of the present disclosure can be utilized.

[0049] In some embodiments, in a method of conferring one or more beneficial properties or traits to a desired plant species, the consortiums of the present disclosure can be utilized.

[0050] In some embodiments, in a method of conferring one or more beneficial properties or traits to a desired plant species, an agriculturally acceptable composition comprising the consortiums of the present disclosure can be utilized.

[0051] The present disclosure provides a method for effectively enhancing a plant component or a plant part by coating the plant component or the plant part with an isolated microorganism or a microbial consortium in an amount that is not normally found on the plant component or the plant part.

[0052] Some embodiments described herein are methods for preparing an agricultural seed composition or a seed coating, the method comprising: contacting the surface of a seed with a formulation comprising a purified microbial population comprising at least one isolated microorganism that is heterologous to the seed or is rarely present on the seed. Further embodiments require preparing an agricultural plant composition, which comprises: contacting the surface of a plant with a formulation comprising a purified microbial population comprising at least one isolated microorganism that is heterologous to the plant. In other aspects, the formulation or the microorganism is introduced into the interior of the seed, such as into the cotyledon or the germ or other seed tissues.

[0053] In some aspects, applying the isolated microorganisms, microbial aggregates, exudates, metabolites, and / or agricultural compositions of the present disclosure to seeds or plants can modulate agronomically important traits. Agronomically important traits can be, for example, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, increased water use efficiency, increased nitrogen use efficiency, increased nitrogen stress resistance, increased nitrogen fixation, increased utilization of nutrients (e.g., phosphate, potassium, etc.), insect resistance, herbivore resistance, pathogen resistance, reduced pathogen levels (e.g., via secretion of metabolites that affect pathogen survival), increased yield, increased yield under water-limited conditions, enhanced health, increased vigor, improved growth, increased photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, increased seed weight, faster seed germination, altered seed carbohydrate composition, altered seed oil composition, pod number, delayed senescence, stay-green, and altered seed protein composition. In some aspects, at least 2, 3, 4, or more agronomically important traits are modulated. In some aspects, the modulation is a positive effect on one of the aforementioned agronomic traits.

[0054] In some aspects, the isolated microorganisms, aggregates, and / or agricultural compositions of the present disclosure can be applied to plants in order to modulate or alter plant characteristics, such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, spike weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, increased nitrogen use efficiency, improved root architecture, increased water use efficiency, increased biomass, decreased biomass, increased root length, decreased root length, increased seed weight, increased shoot length, decreased shoot length, increased yield, increased yield under water-limited conditions, grain quality, grain water content, metal tolerance, spike number, number of grains per spike, pod number, enhanced nutrition, pathogen resistance, insect resistance, increased photosynthetic capacity, salt tolerance, stay-green, increased vigor, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, decreased number of wilted leaves per plant, decreased number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, detectable modulation of metabolite levels, detectable modulation of transcript levels, and detectable modulation of the proteome.

[0055] In some embodiments, the agricultural formulations taught herein comprise at least one member selected from the group consisting of agriculturally compatible carriers, thickeners, microbial stabilizers, fungicides, antibacterial agents, herbicides, nematicides, insecticides, plant growth regulators, rodenticides, and nutrients.

[0056] The methods described herein can include contacting a seed or a plant with at least 100 CFU or spores, at least 300 CFU or spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000 CFU or spores, at least 1,000,000 CFU or spores or more of the microorganisms taught herein.

[0057] The methods described herein can include contacting a seed or a plant with a composition comprising metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising at least 1 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising at least 10 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising at least 100 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising at least 1 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising at least 10 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising at least 100 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising at least 1 kg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or a plant with a composition comprising more than 1 kg of metabolites produced by a single microorganism or microbial consortium disclosed herein.

[0058] In some embodiments of the methods described herein, the isolated microorganisms of the present disclosure are present in the formulation in an amount that is effectively detectable within and / or on the target tissue of an agricultural plant. For example, at least 100 CFU or spores, at least 300 CFU or spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000 CFU or spores, at least 1,000,000 CFU or spores or more of the microorganisms are detected among and / or on the target tissue of the plant. Alternatively or additionally, the microorganisms of the present disclosure may be present in the formulation in an amount that effectively increases the biomass and / or yield of the plants to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied. Alternatively or additionally, the microorganisms of the present disclosure may be present in the formulation in an amount that effectively and detectably modulates an agronomic trait of interest of the plants to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied.

[0059] In some embodiments of the methods described herein, one or more metabolites isolated from the microorganisms or consortia of the present disclosure are present in the formulation in an amount that is effectively detectable within and / or on the target tissues of an agricultural plant. For example, at least 1 mg, at least 10 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 400 mg, at least 600 mg, at least 800 mg, at least 1 g or more of the metabolite is detected within and / or on the target tissues of the plant. Alternatively or additionally, the metabolites isolated from the microorganisms and consortia of the present disclosure may be present in the formulation in an amount that effectively increases the biomass and / or yield of the plants to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more when compared to a reference agricultural plant that has not been applied the formulation of the present disclosure. Alternatively or additionally, the metabolites isolated from the microorganisms and consortia of the present disclosure may be present in the formulation in an amount that effectively and detectably modulates the agronomic trait of interest of the plants to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more when compared to a reference agricultural plant that has not been applied the formulation of the present disclosure.

[0060] In some embodiments, the agricultural compositions taught herein are shelf-stable. In some aspects, the microorganisms taught herein are freeze-dried. In some aspects, the microorganisms taught herein are spray-dried. In some aspects, the microorganisms taught herein are placed in a liquid formulation. In some aspects, the microorganisms taught herein are present on granules.

[0061] The present disclosure also describes a plurality of isolated microorganisms enclosed within an article selected from the group consisting of a bottle, a flask, an ampoule, a package, a vessel, a bag, a box, a storage bin, an envelope, a cardboard box, a container, a silo, a shipping container, a railroad car, and a crate.

[0062] In some aspects, combining selected plant species with the disclosed microorganisms (operational taxonomic units (OTUs), strains, or compositions comprising any of the foregoing) results in increased crop yields and the production of their products. Thus, in one aspect, the present disclosure provides a synthetic combination of seeds of a first plant and a microbial preparation coated onto the surface of the seeds of the first plant such that the microorganisms are present on the surface of the seeds at a higher level compared to what is present on the surface of uncoated reference seeds. In another aspect, the present disclosure provides a synthetic combination of a part of a first plant and a microbial preparation coated onto the surface of that part of the first plant such that the microorganisms are present on the surface of that part of the first plant at a higher level compared to what is present on the surface of an uncoated reference plant part. The above methods can be used alone or in conjunction with plant breeding and transgenic technologies.

[0063] In some embodiments, the bacterial strain has morphological and physiological characteristics that are substantially similar to the isolated bacterial strains of the present disclosure. In some embodiments, the isolated bacterial strain has genetic characteristics that are substantially similar to the isolated bacterial strains of the present disclosure. In some embodiments, the isolated bacterial strain is a naturally occurring or artificially created mutant of the isolated bacterial strains of the present disclosure. In some embodiments, the isolated bacterial strain is a genetically edited, altered, or modified bacterial strain. In some embodiments, the isolated bacterial strains of the present disclosure are in a substantially pure culture. In some embodiments, the isolated bacterial strains of the present disclosure are in a pure culture. In some embodiments, the isolated bacterial strains of the present disclosure are in a cell fraction, extract, or supernatant.

[0064] In some embodiments, the progeny and / or mutants of the isolated bacterial strains of the present disclosure are contemplated. In some embodiments, the progeny, mutants, and / or genetically modified forms of the isolated bacterial strains of the present disclosure are contemplated.

[0065] In some embodiments, cell-free or inactivated preparations of the isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains, are contemplated. In some embodiments, cell-free or inactivated preparations of the isolated bacterial strains of the present disclosure, or mutants or genetically edited, altered, or modified variants of the isolated bacterial strains, are contemplated. In some embodiments, metabolites produced by the isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains, are contemplated. In some embodiments, metabolites produced by the isolated bacterial strains of the present disclosure, or mutants or genetically modified variants of the isolated bacterial strains, are contemplated.

[0066] In some embodiments, the agricultural composition comprises a isolated bacterial strain and an agriculturally acceptable carrier. The isolated bacterial strain can be present in the composition at 1×10^2 to 1×10^12 CFU / gram. The agricultural composition can be formulated as a seed coating.

[0067] In some embodiments, a method of conferring at least one beneficial trait to a plant species comprises applying an isolated bacterial strain to the plant or the growth medium in which the plant is located. In some embodiments, a method of conferring at least one beneficial trait to a plant species comprises applying the agricultural composition of the present disclosure to the plant or the growth medium in which the plant is located.

[0068] In some embodiments, the plant is a non-leguminous crop plant. In some embodiments, the plant is a monocotyledonous plant. In some embodiments, the plant is a C3 monocotyledonous plant. In some embodiments, the plant is a C4 monocotyledonous plant. In some embodiments, the plant is selected from the group consisting of: maize, wheat, rice, sorghum, sugarcane, onion, bamboo, palm, garlic, ginger, lily, daffodil, iris, orchid, bluebell, tulip, amaryllis, banana, plantain, ginger, turmeric, cardamom, asparagus, pineapple, sedge, rush, leek, forage, buckwheat, quinoa, chia, and millet.

[0069] In some embodiments, the present disclosure teaches a method of growing a plant having at least one beneficial trait. In some embodiments, the method comprises applying an isolated bacterial strain or a microbial consortium to a seed of the plant; sowing or planting the seed; and growing the plant. In certain embodiments, the isolated bacterial strain or the microbial consortium is applied in the form of an agricultural composition further comprising an agriculturally acceptable carrier.

[0070] In some embodiments, the microbial consortium has morphological and physiological properties that are substantially similar to the microbial consortium of the present disclosure. In some embodiments, the microbial consortium has genetic properties that are substantially similar to the microbial consortium of the present disclosure. In some embodiments, the microbial consortium is in a substantially pure culture. In some embodiments, subsequent generations of any of the microorganisms of the microbial consortium are contemplated. In some embodiments, mutants of any of the microorganisms of the microbial consortium are contemplated. In some embodiments, genetically edited, altered, or modified variants of any of the microorganisms of the microbial consortium are contemplated. In some embodiments, cell-free or inactivated preparations of the microbial consortium or mutants or genetically edited, altered, or modified variants of any of the microorganisms in the microbial consortium are contemplated. In some embodiments, metabolites produced by the microbial consortium or mutants or genetically edited, altered, or modified variants of any of the microorganisms in the microbial consortium are contemplated.

[0071] In some embodiments, the agricultural composition comprises a microbial consortium and an agriculturally acceptable carrier. The microbial consortium of the agricultural composition can be present in the composition at 1×10^3 to 1×10^12 bacterial cells / gram. In some embodiments, the agricultural composition is formulated as a seed coating. In some embodiments, a method of conferring at least one beneficial trait to a plant species comprises applying the microbial consortium to the plant or the growth medium in which the plant is located. In some embodiments, a method of conferring at least one beneficial trait to a plant species comprises applying the agricultural composition to the plant or the growth medium in which the plant is located. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0073] The present disclosure can be more fully understood by the following detailed description and the accompanying drawings that form a part of this application.

[0074] Figure 1A and 1B depicts a typical monocistronic (single transcriptional unit) model of the nitrogen fixation operon (nfix operon or nfix cluster) that responds to the binding of GlnR to site I under the control of the nifB promoter. Figure 1A depicts a model of the Paenibacillus subgroup I cluster. Figure 1B depicts a model of the Paenibacillus subgroup II cluster.

[0075] Figure 1C depicts the intergenic region named "Int_1" between nifH and nifD. Figure 1D depicts the intergenic region named "Int_2" between nifK and nifE. Figure 1E depicts the intergenic region named "Int_3" between nifX (in some species) or orf1 (in some species) and hesA.

[0076] Figure 2 shows the typical model presented in the literature where GlnR binds to site I upstream of the nif cluster to induce transcription of the cluster under the control of the nifB promoter (the first gene of the cluster).

[0077] Figure 3 depicts the distances (number of nucleotides) between different components of the nif cluster among different species and strains of Paenibacillus.

[0078] Figure 4A shows the model of nif cluster transcription described in the literature. For example, Figure 4A in the original typical model, oxidative stress conditions each time require the synthesis of a complete complex, which is energetically costly for bacteria.Figure 4B Displays an alternative model developed by the inventors of the present case, showing different transcriptional units within the nif cluster, which can confer a more efficient regulatable nitrogen fixation mechanism in bacteria that have promoters and / or other non-coding regulatory elements in some or all of the intergenic regions of the nfix cluster genes.

[0079] Figure 4C Shows the phylogenetic relationship between the "Int_1" regions of different strains (labeled). Int_1 is the polynucleotide sequence between nifH and nifD in Paenibacillus.

[0080] Figure 4D Shows the phylogenetic relationship between the "Int_2" regions of different strains (labeled). Int_2 is the polynucleotide sequence between nifK and nifE in Paenibacillus.

[0081] Figure 4E Shows the phylogenetic relationship between the "Int_3" regions of different strains (labeled). Int_3 is the polynucleotide sequence immediately upstream of hesA in Paenibacillus.

[0082] Figure 5A Depicts the expression levels of the nifB':GFP fusions of different strains when expressed in strain 77155 under aerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars). Figure 5B Depicts the expression levels of the nifB':GFP fusions of different strains when expressed in strain 77155 under anaerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars).

[0083] Figure 6A Depicts the expression levels of the nifB':GFP fusions of different strains when expressed in a mutant strain of strain 77155 under aerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars). Figure 6B Depicts the expression levels of the nifB':GFP fusions of different strains when expressed in strain 77155 under anaerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars).

[0084] Figure 7A Depicts the expression levels of the nifD':GFP fusions of different strains when expressed in strain 77155 under aerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars). Figure 7BDepicts the expression levels of nifD' promoter:GFP fusions of different strains when expressed in strain 77155 under anaerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars).

[0085] Figure 8A Depicts the expression levels of nifE' promoter:GFP fusions of different strains when expressed in strain 77155 under aerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars). Figure 8B Depicts the expression levels of nifE' promoter:GFP fusions of different strains when expressed in strain 77155 under anaerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars).

[0086] Figure 9A Depicts the expression levels of hesA' promoter:GFP fusions of different strains when expressed in strain 77155 under aerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars). Figure 9B Depicts the expression levels of hesA' promoter:GFP fusions of different strains when expressed in strain 77155 under anaerobic conditions (absence of nitrogen represented by square bars, presence of nitrogen represented by dot bars).

[0087] Figure 10A Depicts the percentage of ethylene conversion in the acetylene reduction assay (ARA) for exchange between different neutral genes in different strains under low nitrogen conditions. Figure 10B Depicts the percentage of ethylene conversion in the acetylene reduction assay (ARA) for exchange between different neutral genes in different strains under high nitrogen conditions. In both figures, the edits (G#) are as follows: Individual edits: G76 - G78 = 77155 nifD' neutral exchange; G81 - G83 = 77155 nifE' neutral exchange; G103 and G104 = 77155 hesA' neutral exchange. Combined edits: G109 and G110 = 77155 nifD' and nifE' neutral exchange; G113 - G115 = 77155 nifD' and hesA' neutral exchange; G118 - G120 = 77155 nifE' and hesA' neutral exchange.

[0088] The present disclosure can be more fully understood by the following detailed description and sequence listing that form a part of this application. The sequence description and the accompanying sequence listing comply with the rules for disclosure of nucleotide and amino acid sequences in regulatory patent applications as set forth in 37 C.F.R. §§ 1.821 and 1.825.

[0089] The description of the sequences is given in Table 1 below.

[0090] Table 1: Description of the Sequences

[0091] The description of the sequences in the Sequence Listing is given in the table below. *Note: The strain identifier may further include an optional prefix (e.g., “CM” or “PM”). For example, strain 8619 may optionally be synonymously referred to as CM8619. “Int_#” refers to one of the intergenic regions described in the Examples. Int_1 (or Int 1, int_1, etc.) is the region between nifH and nifD. Int_2 is the region between nifK and nifE. Int_3 is the region between nifX and hesA or between orf1 and hesA, depending on the species and strain.

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[0107] Detailed Description of the Invention

[0108] Although the following terms are considered well understood by one of ordinary skill in the art, the following terms are set forth to facilitate explanation of the subject matter disclosed herein.

[0109] The term "a" or "an" means one or more of that entity, i.e., it can refer to multiple referents. Thus, the terms "a" or "an", "one or more", and "at least one" may be used interchangeably herein. In addition, the reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility of there being more than one element, unless the context clearly requires that there be exactly one element.

[0110] As used herein, the terms "microbiome" or "microbe" should be interpreted broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains (i.e., bacteria and archaea), as well as eukaryotic fungi and protists. In some embodiments, the present disclosure relates to the "microbes" of the passages present in the present disclosure. Such characterization can refer not only to the classified bacterial genera identified in the tables, but also to the classified species identified, as well as to various novel and newly identified bacterial strains in the tables.

[0111] As used herein, the term "microbe" or "microbiome" refers to any species or taxonomic unit of a microbiome, including, but not limited to, archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microsporidia, nanobacteria, oomycetes, and protozoa. In some embodiments, a microbe or microbiome encompasses a single cell (e.g., a unicellular microbiome) or more than one cell (e.g., a multicellular microbiome). Thus, a "population of microbiomes" can refer to multiple cells of a single microbiome, where these cells share a common genetic derivation.

[0112] As used herein, the term "bacteria" generally refers to any prokaryotic organism and can refer to organisms from the domain Bacteria (eubacteria), the domain Archaea (archaea), or both. In some cases, a bacterial genus or other taxonomic classification may be in flux, having been re-designated for other reasons (e.g., but not limited to, the evolutionary field of whole genome sequencing), and / or may be variable based on methodology, and it should be understood that such naming variability is within the scope of any claimed taxonomy. For example, certain species of the genus Erwinia have been described in the literature as belonging to the genus Pantoea (Zhang, Y., Qiu, S. Examining phylogenetic relationships of Erwinia and Pantoea species using whole genome sequence data. Antonie van Leeuwenhoek 108, 1037-1046 (2015)).

[0113] The term "16S" refers to the DNA sequence of the 16S ribosomal RNA (rRNA) of bacteria. 16S rRNA gene sequencing is an established method for studying the phylogeny and taxonomy of bacteria.

[00166] As used herein, the term "fungus" generally refers to any organism from the fungal kingdom. Historically, fungi have been taxonomically classified based on morphological manifestations. Since the mid-nineteenth century, it has been recognized that some fungi have polymorphic life cycles, and different nomenclatural names have been used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association established rules for naming fungi as anamorphs, teleomorphs, or holomorphs based on their fungal status (Taylor, J.W. One Fungus = One Name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2, 113-120 (2011)). With the development of genome sequencing, it has become clear that taxonomic classification based on molecular phylogenetics does not conform to morphology-based nomenclature (Shenoy, B.D., Jeewon, R., and Hyde, K.D. (2007). Impact of DNA sequence-data on the taxonomy of anamorphic fungi. Fungal Diversity 26:1-54). Thus, in 2011, the International Botanical Congress adopted a resolution approving the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) (2012), the result of which was the designation "one fungus = one name" (Hawksworth, D.L. Managing and coping with names of pleomorphic fungi in a period of transition. IMA Fungus 3, 15-24 (2012)).

[0114] The term "Internal Transcribed Spacer" ("ITS") refers to the spacer DNA (non-coding DNA) between the small subunit ribosomal RNA (rRNA) and the large subunit (LSU) rRNA genes in the corresponding transcribed regions located in chromosomes or in polycistronic rRNA precursor transcripts. ITS gene sequencing is an established method for studying fungal phylogeny and taxonomy. In some cases, the "Large Subunit" ("LSU") sequence is used to identify fungi. LSU gene sequencing is an established method for studying fungal phylogeny and taxonomy. Some fungal microorganisms of the present invention can be described by ITS sequences, and some fungal microorganisms can be described by LSU sequences. It should be understood that both are equally descriptive and accurate for determining taxonomy.

[0115] The term "microbial consortia" or "microbial consortium" refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as performing a common function, or which can be described as participating in or causing or being associated with an identifiable parameter or plant phenotypic trait. The community can comprise one or more species of microorganisms, or strains of a species. In some cases, these microorganisms coexist symbiotically within the community.

[0116] The term "microbial community" means a group of microorganisms comprising two or more species or strains. Unlike microbial consortia, a microbial community does not necessarily perform a common function, or participate in or cause or be associated with an identifiable parameter or plant phenotypic trait.

[0117] The term "Accelerated Microbial Selection" or "AMS" can be used interchangeably with the term "Directed Microbial Selection" or "DMS" and refers to an iterative selection method which, in some embodiments of the present disclosure, is used to obtain the claimed microbial species or a consortium of said species.

[0118] As used herein, the terms "isolate", "isolated", "isolated microorganism" and similar terms are intended to mean that one or more microbial bodies have been separated from at least one material with which they were associated in a particular environment (e.g., soil, water, plant tissue).

[0119] Thus, an "isolated microorganism" does not exist in its natural environment; rather, the microorganism has been removed from its natural environment and placed in a state of non-natural existence by various techniques described herein. Thus, an isolated strain can exist, for example, in a biologically pure culture or in spore form (or other form of the strain) in combination with an agricultural carrier.

[0120] In certain aspects of the present disclosure, the isolated microorganism exists in the form of an isolated and biologically pure culture. Those skilled in the art will understand that an isolated and biologically pure culture of a particular microorganism means that the culture is substantially free (to the extent scientifically reasonable) of other living organisms and contains only the single microorganism under discussion. The culture may contain different concentrations of the microorganism. The present disclosure states that isolated and biologically pure microorganisms are generally "necessarily different from less pure or impure materials". See, for example, In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), see also In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microorganisms), see also Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand, discussing purified adrenaline), affirmed in part, reversed in part, 196 F. 496 (2d Cir. 1912), each of these documents is incorporated herein by reference. In addition, in some aspects, the present disclosure provides certain quantitative measures of the concentration or purity limits that will necessarily be found within an isolated and biologically pure microorganism culture. In certain embodiments, the presence of these purity values is another property that distinguishes the microorganisms disclosed by the present invention from those that exist in their natural state. See, for example, Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing the purity limits of vitamin B12 produced by microorganisms).

[0121] As used herein, "single isolate" shall be taken to mean a composition or culture that, after separation from one or more other microorganisms, consists primarily of a single genus, species, or strain of microorganism. The phrase should not be taken to indicate the degree of isolation or purification of the microorganism. However, a "single isolate" may consist essentially of only one genus, species, or strain of microorganism.

[0122] In the context of microorganisms, the term "modified" means that the microorganism has been changed in some way compared to its natural state as found. In this context, "modified" is synonymous with "engineered" and indicates human involvement in creating the modification. In some cases, the modification includes a change in a polynucleotide within the microorganism (e.g., in its genome). The modification can include a deletion, insertion, substitution, and / or chemical alteration of at least one nucleotide and can result in a change in the phenotype of the microorganism (e.g., upregulation of a particular pathway, downregulation of a particular pathway, knockout of gene or protein function) and / or a change in the phenotype of another heterologous organism associated with or becoming associated with the microorganism.

[0123] The term "modulate" (or "modulated", "modulating", etc.) means that a particular property has changed from a previously existing property. For example, a modulated nitrogen fixation ability (or capability) means that an organism has an increase in nitrogen fixation, a decrease in nitrogen fixation, a different temporal or spatial expression of nitrogen fixation, or other changes different from the previous nitrogen fixation ability.

[0124] As used herein, the term "growth medium" is any medium suitable for supporting plant growth. By way of example, the medium can be natural or artificial and includes, but is not limited to: soil, potting mix, bark, vermiculite, hydroponic solutions applied alone and to solid plant support systems, and tissue culture gels. It should be understood that the medium can be used alone or in combination with one or more other media. It can also be used with or without the addition of exogenous nutrients for roots and leaves and a physical support system.

[0125] In one embodiment, the growth medium is a naturally occurring medium such as soil, sand, mud, clay, humus, topsoil, rock, or water. In another embodiment, the growth medium is artificial. Such an artificial growth medium can be constructed to mimic the conditions of a naturally occurring medium; however, this is not required. The artificial growth medium can be made of one or more of any number and combination of materials including sand, minerals, glass, rock, water, metal, salts, nutrients, water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0126] The medium can be amended or fertilized with additional compounds or components, such as components that can contribute to the interaction and / or selection of specific populations of microorganisms with plants and with each other. For example, antibiotics (such as penicillin) or sterilants (such as quaternary ammonium salts and oxidants) can be present, and / or the physical conditions (such as salinity, plant nutrients (such as organic and inorganic minerals (such as phosphorus, nitrogen salts, ammonia, potassium, and micronutrients such as cobalt and magnesium)), pH, and / or temperature) can be modified.

[0127] The term "plant" generally includes the whole plant, plant organs, plant tissues, seeds, plant cells, seeds and their progeny. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. As used herein, the term "plant part" refers to plant cells, plant protoplasts, plant cell tissue cultures from which a plant can be regenerated, intact plant callus, plant clumps, and plant cells in a plant or plant part such as an embryo, pollen, ovule, seed, leaf, flower, shoot, fruit, kernel, ear, ear axis, husk, stalk, root, root tip, anther, etc. and parts thereof themselves. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the present invention, provided that these parts contain the introduced polynucleotide.

[0128] "Plant part" is intended to refer to the whole plant or a plant component, which may contain differentiated and / or undifferentiated tissues, such as, but not limited to, plant tissues, parts, and cell types. In one embodiment, the plant part is one of the following: whole plant, seedling, meristem, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, offset, bud, bract, tumor tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, callus). The term "plant organ" refers to a plant tissue or group of tissues that make up a morphologically and functionally distinct part of a plant. As used herein, "plant part" is synonymous with "part" of a plant and refers to any part of a plant, and may include different tissues and / or organs, and may be used interchangeably throughout with the term "tissue".

[0129] Similarly, "plant propagation part" is intended to generally refer to any part of a plant that is capable of initiating other plants via sexual or asexual reproduction of the plant, such as, but not limited to: seed, seedling, root, shoot, cutting, scion, graftling, stolon, bulb, tuber, corm, offset, or bract. A plant part can be located in a plant or in a plant organ, tissue culture, or cell culture.

[0130] "Progeny" includes any subsequent generation of an organism produced via sexual or asexual reproduction.

[0131] "Grain" is intended to mean mature seeds produced by commercial growers for purposes other than growing or propagating the species.

[0132] The term "monocotyledonous" or "monocot" refers to a subclass of angiosperms, also known as "Monocotyledoneae", whose seeds generally contain only one cotyledon or seed leaf. The term includes references to the whole plant, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny.

[0133] The term "dicotyledonous" or "dicotyledon" refers to a subclass of angiosperms, also known as the "Dicotyledoneae", whose seeds typically contain two embryonic leaves or cotyledons. The term includes references to whole plants, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny.

[0134] As used herein, the term "cultivar" refers to a plant variety, strain, or race that has been produced by horticultural or agronomic techniques and that is not normally present in wild-type populations.

[0135] As used herein, "improved" should be construed broadly to encompass the improvement of a plant's characteristics as compared to a control plant or as compared to the known average amount associated with the characteristic under discussion. For example, "improved" plant biomass associated with the application of the beneficial microorganisms or aggregates of the present disclosure can be demonstrated by comparing the biomass of plants treated with the microorganisms taught herein with the biomass of untreated control plants. Alternatively, the biomass of plants treated with the microorganisms taught herein can be compared to the average biomass typically achieved by a given plant (as represented in scientific or agricultural publications known to those of skill in the art). In the present disclosure, "improved" does not necessarily require that the data be statistically significant (e.g., p < 0.05); rather, any quantifiable difference indicating that one value (e.g., an average treatment value) is different from another value (e.g., an average control value) can rise to the level of "improved".

[0136] As used herein, "inhibit and repress" and similar terms should not be construed as requiring complete inhibition or repression, although in some embodiments this may be required.

[0137] As used herein, the term "genotype" refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or population of organisms.

[0138] The compositions and methods herein can provide plants with improved "agronomic traits" or "agronomically important traits" or "traits of agronomic interest", which can include, but are not limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, increased water use efficiency, increased nitrogen use efficiency, increased nitrogen fixation, insect resistance, herbivore resistance, pathogen resistance, increased yield, enhanced health, increased vigor, improved growth, increased photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, metabolite regulation, proteome regulation, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, and altered seed nutrient composition as compared to isoline plants that do not contain the modifications from the methods or compositions herein.

[0139] "Agronomic trait potential" is intended to mean the ability of a plant part to exhibit a phenotype (preferably, an improved agronomic trait) at some point in its life cycle or to transmit that phenotype to another plant part in the same plant with which it is associated.

[0140] As used herein, the terms "molecular marker", "marker", or "genetic marker" refer to an indicator used in methods for observing differences in nucleic acid sequence characteristics. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers, or isozyme markers or combinations of markers described herein (which define specific gene and chromosomal locations). The mapping of molecular markers near an allele is a procedure that can be performed by an ordinary person with experience in molecular biotechnology.

[0141] As used herein, the term "trait" refers to a characteristic or phenotype. For example, in the context of some embodiments of the present disclosure, crop yield relates to the amount of marketable biomass (e.g., fruit, fiber, grain) produced by a plant. Desirable traits may also include other plant characteristics, including but not limited to: water use efficiency, nutrient use efficiency, productivity, mechanical harvestability, fruit maturity, shelf life, insect / disease resistance, early plant maturity, stress tolerance, etc. Traits can be inherited in a dominant or recessive manner, or in a partial or incomplete dominant manner. Traits can be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by more than one locus) or can also be produced by the interaction of one or more genes with the environment.

[0142] As used herein, the term "phenotype" refers to the observable characteristics of an individual cell, cell culture, organism (e.g., a plant), or population of organisms, which are produced by the interaction between the genetic makeup (i.e., genotype) of the individual and the environment.

[0143] As used herein, a "synthetic nucleotide sequence" or "synthetic polynucleotide sequence" is a nucleotide sequence that is known not to exist in nature or is not naturally occurring. Generally speaking, when compared to any other naturally occurring nucleotide sequence, such a synthetic nucleotide sequence will contain at least one nucleotide difference.

[0144] As used herein, the term "nucleic acid" refers to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length or their analogs. The term refers to the primary structure of the molecule and thus includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. It also includes modified nucleic acids, such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone-modified nucleic acids, etc. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0145] As used herein, the term "gene" refers to any DNA fragment associated with a biological function. Thus, a gene includes, but is not limited to, coding sequences and / or regulatory sequences required for its expression. A gene may also include non-expressed DNA fragments, which form, for example, recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from sources of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0146] As used herein, the terms "homologous" or "homolog" or "ortholog" are known in the art and refer to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms "homology", "homologous", "substantially similar", and "substantially corresponding" are used interchangeably herein. They refer to nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the present disclosure, such as deletions or insertions of one or more nucleotides that do not substantially change the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. Thus, it should be understood that, as will be appreciated by those skilled in the art, the present disclosure encompasses more than specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, cultivar, or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar, or strain. For the purposes of the present disclosure, homologous sequences are compared. "Homologous sequences" or "homologs" or "orthologs" are considered, regarded as, or known to be functionally related. The functional relationship can be indicated in any of a variety of ways, including but not limited to: (a) the degree of sequence identity and / or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., 1987) Supplement 30, Section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania), and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), which uses default parameters.

[0147] As used herein, the term "nucleotide change" refers to, for example, nucleotide substitution, deletion, insertion, chemical modification, or any of the foregoing, as well understood in the art.

[0148] As used herein, the term "protein modification" refers to, for example, amino acid substitution, amino acid modification, deletion, and / or insertion, as well understood in the art.

[0149] As used herein, the term "at least a portion" or "fragment" of a nucleic acid or polypeptide means a portion having the smallest size characteristic of such a sequence, or any larger fragment of the full-length molecule (up to and including the full-length molecule). Fragments of the polynucleotides of the present disclosure can encode biologically active portions of gene regulatory elements. Biologically active portions of gene regulatory elements can be prepared by isolating the portion of one of the polynucleotides of the present disclosure that contains the gene regulatory element and assessing activity as described herein. Similarly, a portion of a polypeptide can be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, etc., up to the full-length polypeptide. The length of the portion to be used will depend on the particular application. A portion of a nucleic acid that can be used as a hybridization probe can be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide that can be used as an epitope can be as short as 4 amino acids. A portion of a polypeptide that functions as the full-length polypeptide will generally be longer than 4 amino acids.

[0150] As used herein, the term "primer" refers to an oligonucleotide that is capable of annealing to an amplification target to permit DNA polymerase attachment, thereby serving as a starting point for DNA synthesis under conditions that induce synthesis of a primer extension product (i.e., in the presence of nucleotides and reagents for polymerization such as DNA polymerase and at a suitable temperature and pH). (Amplification) primers are preferably single-stranded to obtain maximum amplification efficiency. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of an extension product in the presence of reagents for polymerization. The exact length of the primer will depend on many factors, including temperature and the composition of the primer (A / T vs. G / C content). A pair of bidirectional primers consists of a forward primer and a reverse primer as commonly used in the art of DNA amplification (e.g., PCR amplification).

[0151] The term "stringency" or "stringent hybridization conditions" refers to hybridization conditions that affect the stability of a hybrid, such as temperature, salt concentration, pH, formamide concentration, etc. These conditions are optimized empirically to maximize the specific binding of a primer or probe to its target nucleic acid sequence and to minimize non-specific binding. The terms used include reference to conditions under which a probe or primer will hybridize to its target sequence to a detectably greater extent (e.g., at least 2-fold greater than background) compared to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm is the temperature at which 50% of the complementary target sequence hybridizes to a perfectly matched probe or primer (at a defined ionic strength and pH). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ions at pH 7.0 to 8.3, usually about 0.01 to 1.0 M Na+ ion concentration (or other salts), and the temperature is at least about 30°C (for short probes or primers (e.g., 10 to 50 nucleotides)) and at least about 60°C (for long probes or primers (e.g., greater than 50 nucleotides)). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions or "reduced stringency conditions" include hybridization in a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and washing in 2× SSC at 40°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and washing in 0.1× SSC at 60°C. Hybridization procedures are well known in the art and are described, for example, by Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, the stringent conditions are hybridization at 45°C in 0.25 M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5% to 20% sodium dodecyl sulfate (e.g., 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%) followed by washing at 55°C to 65°C in 5× SSC containing 0.1% (w / v) sodium dodecyl sulfate.

[0152] In some embodiments, a cell or organism has at least one heterologous trait. As used herein, the term "heterologous trait" refers to a phenotype conferred on a cell or organism by an exogenous molecule or other organism (e.g., a microorganism), a DNA fragment, a heterologous polynucleotide, or a heterologous nucleic acid.

[0153] A variety of phenotypic changes are of interest in the present disclosure, including but not limited to modifying the fatty acid composition in plants, altering the amino acid content of plants, changing the pathogen defense mechanisms of plants, increasing the yield of plants having economically important traits (e.g., grain yield, forage yield, etc.), and the like. These results can be achieved by using the methods and compositions of the present disclosure to provide the expression of heterologous products or increased expression of endogenous products in plants.

[0154] A "synthetic consortium" can include a combination of plants and microorganisms of the present disclosure. The combination can be achieved, for example, by coating the surface of the seeds of a plant (e.g., an agricultural plant) or a host plant tissue (roots, stems, leaves, etc.) with a microorganism of the present disclosure. Additionally, a "synthetic consortium" can include a combination of microorganisms of various strains or species. The synthetic consortium has at least one variable that can distinguish the combination from any combination that exists in nature. The variable can particularly be the concentration of the microorganism on the seed or plant tissue that does not occur naturally, or a combination of a microorganism and a plant that does not occur naturally, or a combination of microorganisms or strains that do not naturally coexist. In each of these cases, the synthetic consortium shows signs of being artificial and has structural and / or functional properties that do not exist when considering the individual elements of the combination separately.

[0155] In some embodiments, the microorganism can be "endogenous" to the seed or plant. As used herein, a microorganism is considered to be "endogenous" to a plant or seed if the microorganism is derived from a plant sample that is its source. That is, the microorganism is found to be associated with the plant in nature. In embodiments in which an endogenous microorganism is applied to a plant, the endogenous microorganism is applied in an amount different from the level found on the plant in nature. Thus, if a microorganism that is endogenous to a given plant is present on the plant at a level that does not exist in nature, the microorganism can still form a synthetic consortium with the plant.

[0156] In some embodiments, a composition (e.g., a microorganism) can be “heterologous” (also referred to as “exogenous”) to another composition (e.g., a seed or a plant), and in some aspects, is referred to herein as a “heterologous composition”. As used herein, a microorganism is considered to be “heterologous” to a plant or a seed if the microorganism does not originate from the plant sample from which it is sourced. That is, the microorganism is found in a situation where it is not associated with the plant in nature. For example, a microorganism that is typically associated with the leaf tissue of a maize plant is considered exogenous to the leaf tissue of another maize plant that does not contain the microorganism in nature. In another example, a microorganism that is typically associated with a maize plant is considered exogenous to a wheat plant that does not contain the microorganism in nature. Similarly, a nucleotide or amino acid sequence can be considered “heterologous” to a locus in an organism or a different organism from which it was initially obtained. Broadly speaking, “heterologous” means not naturally present at the particular location, or at the particular time, or with a particular additional component.

[0157] When a composition is applied, artificially inoculated, associated, or placed mechanically or manually onto or into a plant component, seedling, plant, or in or onto a plant growth medium or treatment formulation such that the treatment agent is present in or on the plant component, seedling, plant, plant growth medium, or formulation in a manner not found in nature prior to the application of the treatment agent, e.g., the composition not found in nature in that plant variety, at that stage of plant development, in that plant tissue, at that abundance, or in that growth environment (e.g., drought). In some embodiments, such a manner is contemplated to be selected from the group consisting of: the presence of a microorganism; the presence of a microorganism in a different cell number, concentration, or amount; the presence of a microorganism in a different physical location in a different plant component, tissue, cell type, or in or on a plant; the presence of a microorganism at different time periods such as during the development period of a plant or plant component, time of day, time of season, and combinations thereof. In some embodiments, "being heterologously placed" means that the microorganism is applied to a tissue or cell type of a plant component different from the location where the microorganism naturally occurs. In some embodiments, "being heterologously placed" means that the microorganism is applied to a certain developmental stage of a plant component, seedling, or plant, and the microorganism is not associated in nature at that developmental stage but may be associated at other stages. For example, if a microorganism is normally found during the flowering stage of a plant and not found at other stages, the microorganism applied during the seedling stage can be considered to be heterologously placed. In some embodiments, if a microorganism is normally found in the root tissue of a plant component and not found in the leaf tissue, and the microorganism is applied to the leaf, the microorganism is heterologously placed. In another non-limiting example, if a microorganism is present in the mesophyll layer of leaf tissue in nature but is applied to the epidermal layer, the microorganism will be considered to be heterologously placed. In some embodiments, "being heterologously placed" means that a natural plant component, seedling, or plant does not contain a detectable level of the microorganism in the same plant component, seedling, or plant. In some embodiments, "being heterologously placed" means that the microorganism is applied to the plant component, seedling, or plant in a greater concentration, number, or amount compared to the concentration, number, or amount present in nature in the plant component, seedling, or plant. For example, the microorganism is heterologously placed when present in a number, amount, or concentration that is at least 1.5 times greater, between 1.5 and 2 times greater, 2 times greater, between 2 and 3 times greater, 3 times greater, between 3 and 5 times greater, 5 times greater, between 5 and 7 times greater, 7 times greater, between 7 and 10 times greater, 10 times greater, or even more than 10 times greater compared to the concentration present prior to the placement of the microorganism. In another non-limiting example, a microorganism present in cupressaceous tree tissue in nature will be considered heterologous to the tissues of maize, wheat, cotton, and soybean plants.In another instance, a microorganism that is present in the leaf tissue of maize, spring wheat, cotton, or soybean plants in nature is considered heterologous to the leaf tissue of another maize, spring wheat, cotton, or soybean plant that does not contain the microorganism or contains a different amount of the microorganism in nature.

[0158] A microorganism can also be "heterologously placed" onto a given plant tissue. This means that the microorganism is placed on a plant tissue on which it is not found in nature. For example, if a given microorganism is only naturally present on the roots of a given plant, the microorganism can be exogenously applied to the above-ground tissue of the plant and will thus be "heterologously placed" onto the plant tissue. Accordingly, when applied to a plant that does not naturally contain the microorganism or does not naturally have the microorganism present in the amount applied, the microorganism is considered to be heterologously placed.

[0159] The compositions and methods herein can provide a "modulated", "agronomic trait", or "agronomically important trait" to a host plant, which can include but are not limited to the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, increased nitrogen use efficiency, improved root architecture, increased water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel quality, kernel water content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutritional enhancement, pathogen resistance, insect resistance, increased photosynthetic capacity, salt tolerance, stay-green, increased vigor, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, decreased number of wilted leaves per plant, decreased number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, detectable modulation of metabolite levels, detectable modulation of transcript levels, and detectable modulation of the proteome. The term "modulated" is intended to mean a change in an agronomic trait that is altered by the presence of a microorganism, exudate, culture broth, metabolite, etc. In various aspects, the modulation provides the conferral of a beneficial trait.

[0160] Microorganisms and Microbiomes

[0161] As used herein, the term "microbiome" should be interpreted broadly. It includes but is not limited to prokaryotic bacteria and archaea, as well as eukaryotic fungi and protists.

[0162] In certain embodiments, the microbiota is an endophyte, or an epiphyte, or a microbiota that inhabits the rhizosphere or rhizoplane of a plant. That is, the microbiota can be found in the soil material adhering to the roots of a plant or in the region immediately adjacent to the roots of the plant.

[0163] In one embodiment, the microbiota is an endophyte. Endophytes can be beneficial to the host plant by preventing pathogenic organisms from colonizing it. The extensive colonization of plant tissues by endophytes creates a "barrier effect" in which the local endophytes prevail and prevent pathogenic organisms from persisting. Endophytes can also produce chemicals that inhibit the growth of competitors, including pathogenic organisms.

[0164] In some embodiments, the microbiota is non-culturable. This is to be taken to mean that it is not known whether the microbe is culturable or is difficult to culture using methods known to those skilled in the art.

[0165] The microbiota of the present disclosure can be collected or obtained from any source, or be contained within and / or associated with materials collected from any source.

[0166] In one embodiment, the microbiota or a combination of microbiota can provide possible or predicted benefits to a plant. For example, it is predictable that the microbiota: enhances nitrogen fixation; releases phosphate from soil organic matter; releases phosphate from inorganic forms of phosphate (e.g., rock phosphate); "fixed carbon" in root microspheres; lives in the rhizosphere of a plant, thereby helping the plant absorb nutrients from the surrounding soil and then making these nutrients more readily available to the plant; increases the number of nodules on plant roots, thereby increasing the number of symbiotic nitrogen-fixing bacteria (e.g., Rhizobium species) per plant and the amount of nitrogen fixed by the plant; triggers plant defense responses, such as ISR (induced systemic resistance) or SAR (systemic acquired resistance), which helps the plant resist the invasion and spread of pathogenic microorganisms; competes with microorganisms harmful to plant growth or health through antagonism or competitive utilization of resources (e.g., nutrients or space); changes the color of one or more parts of the plant, or changes the chemical condition, its odor, taste, or one or more other characteristics of the plant.

[0167] The microbial bodies of the present disclosure can be isolated in a substantially pure or mixed culture. They can be concentrated, diluted, or provided at their natural concentration as present in the source material. For example, microbial bodies from salt deposits can be isolated for the present disclosure by suspending the deposit in fresh water and allowing the deposit to settle to the bottom. After an appropriate settling period, the water containing most of the microbial bodies can be removed by decantation and applied directly to the plant growth medium, or concentrated by filtration or centrifugation, diluted to an appropriate concentration, and applied to the plant growth medium together with most of the salts removed. As another example, microbial bodies from mineralized or toxic sources can be treated similarly to recover the microbes for application to plant growth materials, thereby minimizing the likelihood of damaging the plants.

[0168] In some embodiments, a mixed population of microbial bodies is used in the methods of the present disclosure.

[0169] Nitrogen fixation in bacteria

[0170] Glutamine (Gln) is a universal nitrogen signal in all free-living nitrogen-fixing organisms (see, e.g., Wang et al., PLOS Genetics, 2018). Gram-negative bacteria, such as Klebsiella and Pseudomonas, have well-defined nitrogen pathways and are more amenable to easier and more predictable gene delivery and expression for genomically modified strains. In the Gram-negative organism Klebsiella, NifL is a negative regulator of the nif operon. When intracellular glutamine is high (nitrogen in excess), NifL forms a repressor complex that inactivates nif operon expression.

[0171] In the Gram-negative organism Azospirillum, NifA activates the transcription of the nif operon. Glutamine regulates the expression of nifA through the phosphorylation of NtrB by NtrC. Nitrogenase is inactivated post-transcriptionally.

[0172] In contrast, Gram-positive bacteria, such as Paenibacillus herein, are more difficult to transform and have less studied nitrogen fixation pathways.

[0173] In Gram-positive bacteria, the nif cluster encodes nitrogen fixation genes regulated by GlnR. The binding of GlnR to site I activates Nif expression, while the binding of GlnR to site II represses Nif expression. The nitrogenase complex in Gram-positive bacteria is encoded by 9 to 10 genes (e.g., in Paenibacillus azotofixans species) and is activated / inhibited respectively by the binding of GlnR to site I / II ( Figure 1A and 1B ).

[0174] Therefore, for Gram-positive bacteria such as Paenibacillus, successful genomic modifications leading to greater nitrogen fixation ability are not only surprising but highly desirable in agricultural biotechnology. Due to the sporulation ability of Paenibacillus, the commercial potential of products containing gene-edited Paenibacillus strains that improve nitrogen fixation in crop plants is increased.

[0175] Therefore, the gene targets for improving nitrogen fixation in Paenibacillus are the Nif activator / repressor GlnR and its binding sites.

[0176] In the genus Paenibacillus, the nif operon (cluster) contains multiple genes, including: nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, and nifV. Although the DNA sequences of the nif cluster are highly conserved among Paenibacillus strains that fix N2, there are some variations, and these Paenibacillus can be divided into two subgroups: subgroup I and subgroup II. The nine genes nifBHDKENXhesAnifV of the nif gene cluster are contiguous within subgroup I, while there is an ORF of 261 to 561 bp between nifX and hesA within subgroup II, and the predicted product is unknown. Paenibacillus species Paenibacillus polymyxa and Paenibacillus triticisoli are examples of subgroup I. Paenibacillus species Paenibacillus albus, Paenibacillus anaericanus, Paenibacillus azotifigens, Paenibacillus borealis, Paenibacillus donghaensis, Paenibacillus ehimensis, Paenibacillus graminis, Paenibacillus jilunlii, Paenibacillus odorifer, Paenibacillus panacisoli, Paenibacillus phoenicis, Paenibacillus pocheonensis, Paenibacillus rhizoplanae, Paenibacillus silage, Paenibacillus taohuashanense, Paenibacillus thermophilus, Paenibacillus typhae, and Paenibacillus wynnii are examples of subgroup II. In the genus Paenibacillus, there are two different subgroups, subgroup I and subgroup II, and each subgroup contains a different operon composition. Subgroup I Paenibacillus, such as Paenibacillus polymyxa, contains, in this order: nifB, nifH, nifD, nifK, nifE, nifN, nifZ, hesA, nifV. Subgroup II Paenibacillus, such as Paenibacillus graminis, contains, in this order: nifB, nifH, nifD, nifK, nifE, nifN, nifX, orf1, hesA, nifV.

[0177] Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, which is distributed in bacteria and archaea. This enzyme consists of two component proteins, namely MoFe protein and Fe protein. The MoFe protein component is an α2β2 heterotetramer containing two metal clusters (encoded by nifD and nifK); FeMo-co is a [Mo-7Fe-9S-C-homocitrate] cluster that serves as the active site for substrate binding and reduction; while the P cluster is an [8Fe-7S] cluster that transfers electrons to FeMo-co. The Fe protein (encoded by nifH) is a homodimer bridged by [4Fe-4S] clusters between subunits and serves as the obligate electron donor for the MoFe protein. The assembly pathway for the biosynthesis of nitrogenase is complex. In addition to the structural subunits encoded by nifH, nifD, and nifK, the biosynthesis of metal clusters requires several genes, as well as other gene products necessary for the production of a fully functional enzyme. Currently, it has been well established through genetic and biochemical analyses that nifE, nifN, nifX, nifB, nifQ, nifV, nifY, and nifH contribute to the synthesis of FeMo-co and its insertion into nitrogenase, nifU, nifS, and nifZ play important roles in the synthesis of metal clusters, and nifM is essential for the correct folding of the Fe protein of nitrogenase. (Wang et al., PLoS Genet. October 2013; 9(10):e1003865).

[0178] Although most current biological N2 reduction is catalyzed by Mo-nitrogenase, in Mo-limited environments, two homologous alternative nitrogenases: V-nitrogenase and Fe-nitrogenase are important biological sources for fixing nitrogen. V-nitrogenase and Fe-nitrogenase are encoded by vnf genes and anf genes. The distribution of Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase in nature is not uniform. Most nitrogen-fixing organisms, such as Klebsiella pneumoniae (K. pneumoniae), only have Mo-nitrogenase, while some organisms, such as Azotobacter vinelandii (A. vinelandii), have all three types of nitrogenase, and other organisms, such as Rhodobacter capsulatus and Rhodospirillum rubrum, carry Mo-nitrogenase and Fe-nitrogenase (Xie et al., 2014, PLoS Genetics 10(3):e1004231).

[0179] The nitrogenase complex consists of two conserved proteins: the MoFe protein, which is composed of subunits encoded by the nifD and nifK genes; and the Fe protein encoded by the nifH gene. The nitrogenase ferredoxin gene nifH is one of the oldest functional genes extant in the evolutionary history of genes. The nucleotide sequences of the coding regions of the nifHDK genes are highly conserved among all nitrogen-fixing organisms. However, the copy numbers and arrangements of nifH, nifD, and nifK are different among different nitrogen-fixing bacteria.

[0180] According to the literature, the nif operon is a component cluster of genes that are transcribed as a single unit (monocistron) due to GlnR binding to binding site I upstream of the nif operon ( Figure 2 ). However, the inventors of the present case found that intergenic regions exist between some of the larger initial coding sequences ( Figure 3 ). Assuming that other regulatory elements are located between some or all of the individual genes of the operon, this may lead to differential regulation of the individual components as well as of the operon itself.

[0181] Therefore, the inventors of the present case envision that a novel method of controlling nitrogen fixation in bacteria, as exemplified in the genus Paenibacillus, is feasible. The control of the nif cluster is crucial for understanding the gene regulation of nitrogen fixation in bacteria and ultimately for developing better products for various applications, such as agriculture.

[0182] Edit targets of various polynucleotides in the bacterial genome are selected to increase nitrogen fixation in the absence of exogenously applied nitrogen, in the presence of a minimal amount of added nitrogen (e.g., ammonium), and in the presence of added nitrogen.

[0183] Genomic modification of microorganisms

[0184] In some embodiments, a microorganism can alter its genome in such a way as to provide a desirable modified trait, such as improved nitrogen fixation in non-leguminous crops.

[0185] Various methods are known in the art for modifying polynucleotides in cells (including but not limited to any polynucleotide sequences contained within the cell, including genomic, chromosomal, and plasmid DNA). Briefly, a single-strand break or a double-strand break is introduced into the target polynucleotide (the object to be modified) according to the needs of the practitioner, and the result can be the insertion of at least one nucleotide, the deletion of at least one nucleotide, the substitution of at least one nucleotide, or any combination of the foregoing.

[0186] A single-strand break or a double-strand break (SSB or DSB) can be accomplished by any of a variety of methods, including the use of chemicals or radiation, the result of a homologous recombination process, by introducing a specific or non-specific nuclease, or by any combination of the foregoing methods.

[0187] Enzymes that affect polynucleotide cleavage are known in the art and can include, without limitation: restriction endonucleases, meganucleases, TALENs, zinc fingers, or Cas endonucleases.

[0188] Alternatively, homologous recombination can be utilized to insert or remove using the same or nearly the same sequences flanking the polynucleotide of interest.

[0189] In some aspects, the present disclosure relates to isolated genetically modified microorganisms that have improved nitrogen fixation activity compared to non-genetically modified variants of the same microbial species or strain.

[0190] Microbial consortium

[0191] In various aspects, the present disclosure provides microbial consortia comprising a combination of at least any two microorganisms, wherein one microorganism is a strain having a modified nif cluster. In some embodiments, the microorganism is a Paenibacillus strain, and in some cases, a species selected from the group consisting of: Paenibacillus polymyxa, Paenibacillus triticisoli, Paenibacillus albus, Paenibacillus anaerobius, Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus donghaensis, Paenibacillus ehimensis, Paenibacillus cerealis, Paenibacillus lijunlii, Paenibacillus putidus, Paenibacillus ginsengiterrae, Paenibacillus phoenixensis, Paenibacillus sichuanensis, Paenibacillus rhizosphaerae, Paenibacillus silage, Paenibacillus taohuashanensis, Paenibacillus thermophilus, Paenibacillus typha and Paenibacillus wynnii. In some embodiments, the Paenibacillus strain belongs to Subgroup I. In some embodiments, the Paenibacillus strain belongs to Subgroup II.

[0192] In certain embodiments, the consortia of the present disclosure comprise two or three or four or five or six or seven or eight or nine or ten or more microorganisms. The microorganisms of the consortia are different microbial species, or different strains of microbial species.

[0193] Compositions produced by microorganisms

[0194] In some cases, the microorganisms of the present disclosure can produce one or more compounds and / or have one or more activities, such as one or more of the following: produce metabolites, produce phytohormones (such as auxin), produce acetoin, produce antimicrobial compounds, produce siderophores, produce polyketides, produce phenazines, produce cellulases, produce pectinases, produce chitinases, produce glucanases, produce xylanases or proteases or organic acids or lipopeptides or polynucleotides or polypeptides, nitrogen fixation, mineral phosphate solubilization, or any combination and / or multiple of the foregoing.

[0195] For example, the microorganisms of the present disclosure can produce phytohormones selected from the group consisting of auxin, cytokinin, gibberellin, ethylene, brassinolide, and abscisic acid.

[0196] Accordingly, "metabolites produced by the microorganisms of the present disclosure" is intended to encompass any molecule (small molecule, vitamin, mineral, protein, nucleic acid, lipid, fat, carbohydrate, etc.) produced by a microorganism. Generally, the precise mechanism by which the microorganisms of the present disclosure confer beneficial traits to a given plant species is unknown. It is hypothesized that, in some cases, the microorganisms produce metabolites that are beneficial to plants. Thus, in some aspects, cell-free or inactivated preparations of the microorganisms are beneficial to plants because the microorganisms do not have to be alive to confer beneficial traits to a given plant species, provided that the preparation includes metabolites produced by the microorganisms that are beneficial to plants.

[0197] In one embodiment, the microorganisms of the present disclosure can produce auxin (e.g., indole-3-acetic acid (IAA)). The production of auxin can be analyzed. Many of the microorganisms described herein are capable of producing the phytohormone auxin indole-3-acetic acid (IAA) when grown in culture. Auxin plays a key role in altering the physiology of plants, including the degree of root growth.

[0198] Thus, in one embodiment, the microorganisms of the present disclosure are present in a population disposed on the surface or within the tissue of a given plant species. The microorganisms can produce a composition, such as a metabolite, in an amount that effectively causes a detectable increase in the amount of the composition found on or within the plant as compared to a reference plant not treated with the microorganisms of the present disclosure or a cell-free or non-active preparation. The composition produced by the population of microorganisms can be beneficial to the plant species.

[0199] The composition produced by such microorganisms can be present in the cell culture broth or medium in which the microorganisms are grown, or can encompass exudates produced by the microorganisms. As used herein, "exudate" refers to one or more compositions secreted or extracted from one or more microbial cells. As used herein, "culture broth" refers to the common composition of the cell culture medium after the microbial cells are placed in the medium. The composition of the culture broth can change over time, during different stages of microbial growth and / or development. The culture broth and / or exudate can improve the traits of the plant with which it becomes associated.

[0200] Microorganism-Induced Traits in Plants

[0201] The present disclosure utilizes microorganisms to confer beneficial properties (or beneficial traits) to desired plant species (e.g., agronomic species of interest). In the present disclosure, the terms "beneficial property", "beneficial trait", or "trait of interest" are used interchangeably and refer to the modulation of a desired plant phenotype or genetic property of interest by the application of a microorganism or microbial consortium as described herein. As previously mentioned, in some aspects, it may be highly desirable that metabolites produced by a given microorganism ultimately be responsible for modulating or conferring beneficial traits to a given plant.

[0202] There are many beneficial traits that can be modulated by the application of the microorganisms of the present disclosure. For example, the microorganisms may have the ability to confer one or more beneficial properties to a plant species, such as: increased growth, increased yield, increased nitrogen use efficiency, enhanced stress tolerance, enhanced drought tolerance, increased photosynthetic rate, enhanced water use efficiency, enhanced pathogen resistance, modification of plant architecture (which does not necessarily affect plant yield but addresses plant function and causes the plant to increase the production of metabolites of interest), etc.

[0203] In various aspects, the microorganisms taught herein provide a wide range of agricultural applications, including: increasing cereal, fruit, and flower yields; increasing the growth of plant parts; increasing the ability to utilize nutrients (e.g., nitrogen, phosphate, etc.); increasing disease resistance; biocidal effects (including increasing resistance to fungi, insects, and / or nematodes); increasing survival rates in extreme climates; and improving other desired plant phenotypic properties.

[0204] In some aspects, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to modulate or alter plant characteristics, such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, increased nitrogen use efficiency, increased utilization of nutrients (e.g., phosphate, potassium, etc.), improved root architecture, increased water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel quality, kernel water content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutritional enhancement, pathogen resistance, reduced pathogen levels (e.g., via secretion of metabolites that affect pathogen survival), insect resistance, increased photosynthetic capacity, salt tolerance, stay-green, increased vigor, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, detectable modulation of metabolite levels, detectable modulation of transcript levels, and detectable modulation of the proteome, relative to a reference plant.

[0205] In some aspects, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to negatively modulate specific plant characteristics. For example, in some aspects, the microorganisms of the present disclosure are capable of reducing a phenotypic trait of interest, as this functionality may be desirable in some applications. For example, the microorganisms of the present disclosure may possess the ability to reduce root growth or root length. Or the microorganisms may possess the ability to reduce shoot growth or the plant growth rate, as these modulations of plant traits may be desirable in certain applications.

[0206] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to confer nematode stress tolerance to the plants.

[0207] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to provide biostimulation (biostimulant effect) to the plants. In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to provide disease tolerance to the plants.

[0208] Agricultural composition

[0209] In some embodiments, the microorganisms of the present disclosure are combined with agricultural compositions. Agricultural compositions generally refer to such organic and inorganic compounds, which may include compositions that promote the cultivation of microorganisms and / or plant components; compositions for formulating microorganisms to be applied to plant components (e.g., but not limited to: wetting agents, solubilizers (also known as "compatibilizers"), defoamers, detergents, chelating agents, drift reducers, neutralizers and buffers, corrosion inhibitors, dyes, odorants, extenders (also known as "spreaders"), penetration aids (also known as "penetrants"), adhesives (also known as "binders" or "bonding agents"), dispersants, thickeners (also known as "thickeners"), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, etc.); compositions for providing protection to plant components or plants (e.g., but not limited to: insecticides, nematicides, fungicides, bactericides, herbicides, etc.); and other compositions that may be of interest for specific applications.

[0210] In some embodiments, the agricultural compositions of the present disclosure are solids. When using solid compositions, it may be desirable to include one or more carrier materials with the isolated active microorganisms or aggregates. In some embodiments, the present disclosure teaches the use of carriers including but not limited to: mineral soils such as silica, silica gel, silicates, talc, kaolin, activated clay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea, and urea, plant-derived products such as cereal flour, bark powder, wood powder, and fruit shell powder, cellulose powder, palygorskite, montmorillonite, mica, vermiculite, synthetic silica, and synthetic calcium silicate, or combinations of these materials.

[0211] Growth composition

[0212] In some embodiments, compositions that promote growth and development are provided to the microorganisms and / or plant components. Exemplary compositions include liquids (e.g., culture broths, media) and / or solids (e.g., soil, nutrients). Various organic or inorganic compounds can be added to the growth compositions either alone or in combination with the plant components to benefit the health of the microorganisms, such as but not limited to: amino acids, vitamins, minerals, carbohydrates, monosaccharides, lipids.

[0213] Formulation composition

[0214] One or more compositions other than the microorganisms or microorganism-produced compositions may be combined for various application, stability, activity, and / or storage reasons. The additional compositions may be referred to as "formulation components".

[0215] In some embodiments, the agricultural compositions disclosed herein can be formulated as liquids, solids, gases, or gels.

[0216] Accordingly, in some embodiments, the present disclosure teaches that the agricultural compositions disclosed herein can include compounds or salts such as monoethanolamine salts, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium bisulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium bicarbonate, ammonium thiosulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate.

[0217] In some embodiments, the present disclosure teaches that the agricultural compositions can include binders such as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, vinylpyrrolidone / vinyl acetate copolymers, and polyvinyl acetate, or combinations thereof; lubricants such as magnesium stearate, sodium stearate, talc, or polyethylene glycol, or combinations thereof; antifoaming agents such as silicone emulsions, long-chain alcohols, phosphate esters, acetylenic glycols, fatty acids, or organofluorine compounds; and complexing agents such as salts of ethylenediaminetetraacetic acid (EDTA), salts of nitrilotriacetic acid, or salts of polyphosphoric acid, or combinations thereof.

[0218] In some embodiments, the agricultural composition comprises a surfactant. In some embodiments, the surfactant is added to a liquid agricultural composition. In other embodiments, the surfactant is added to solid formulations, particularly those designed to be diluted with a carrier prior to application. Thus, in some embodiments, the agricultural composition comprises a surfactant. Surfactants are sometimes used alone or in combination with other additives (such as minerals or vegetable oils) as adjuvants in spray tank mixes to improve the biological performance of the microorganism against the target. The type of surfactant used for the bioenhancer generally depends on the nature of the microorganism and the mode of action. Surfactants can be characterized as anionic, cationic or non-ionic and can be used as emulsifiers, wetting agents, suspending agents or for other purposes. In some embodiments, the surfactant is a non-ionic surfactant such as: alkyl ethoxylates, linear alcohol ethoxylates and fatty amine ethoxylates. Surfactants commonly used in the formulation field and which can also be used in the formulations of the present invention are described in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, N.J., 1998 and Encyclopedia of Surfactants, Volumes I - III, Chemical Publishing Co., New York, 1980 - 81. In some embodiments, the present disclosure teaches the use of surfactants including aromatic sulfonic acids (such as, lignosulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid) and alkali metal, alkaline earth metal or ammonium salts of fatty acids of aryl sulfonates, alkyl ethers, dodecyl ethers, fatty alcohol sulfates and fatty alcohol diol ether sulfates; condensates of sulfonated naphthalene and its derivatives with formaldehyde; condensates of naphthalene or naphthalenesulfonic acid with phenol and formaldehyde; condensates of phenol or phenolsulfonic acid with formaldehyde; condensates of phenol with formaldehyde and sodium sulfite; polyoxyethylene octylphenyl ether; ethoxylated isooctylphenol, ethoxylated octylphenol or ethoxylated nonylphenol; tributylphenyl polyglycol ether; alkylaryl polyether alcohol; isotridecanol; ethoxylated castor oil; ethoxylated triaryl phenol; salts of phosphorylated triaryl phenol ethoxylates; dodecanol polyglycol ether acetate; sorbitan esters; lignin-sulfite waste liquor or methyl cellulose, or combinations of these substances.

[0219] In some embodiments, the present disclosure teaches other suitable surfactants, including salts of alkyl sulfates such as diethanolammonium lauryl sulfate; alkyl aryl sulfonates such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide adducts such as nonylphenol-C18 ethoxylate; ethanol-alkylene oxide adducts such as tridecyl alcohol-C16 ethoxylate; soaps such as sodium stearate; alkylnaphthalene-sulfonates such as sodium dibutylnaphthalenesulfonate; dialkyl esters of sulfosuccinates such as sodium di(2-ethylhexyl) sulfosuccinate; sorbitan esters such as sorbitan oleate; quaternary amines such as dodecyltrimethylammonium chloride; polyethylene glycol esters of fatty acids such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of monoalkyl and dialkyl phosphates; vegetable oils such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, etc.; and esters of the above vegetable oils, particularly methyl esters.

[0220] In some embodiments, the agricultural composition comprises a wetting agent. A wetting agent is a substance that, when added to a liquid, increases the spreading or penetrating ability of the liquid by reducing the interfacial tension between the liquid and the surface on which it spreads. Wetting agents are used in agrochemical formulations for two main functions: increasing the wetting rate of powders in water during processing and manufacturing to prepare soluble liquid concentrates or suspension concentrates; and shortening the wetting time of wettable powders and increasing the penetration rate of water into water-dispersible granules during mixing of the product with water in a spray tank or other container. In some embodiments, examples of wetting agents used in the agricultural compositions of the present disclosure (including wettable powders, suspension concentrates, and water-dispersible granule formulations) are: sodium dodecyl sulfate; sodium dioctyl sulfosuccinate; alkylphenol ethoxylates; and fatty alcohol ethoxylates.

[0221] In some embodiments, the agricultural composition of the present disclosure comprises a dispersant. A dispersant is a substance that adsorbs on the surface of particles and helps to keep the particles in a dispersed state and prevent their re-aggregation. In some embodiments, a dispersant is added to the agricultural composition of the present disclosure to promote dispersion and suspension during manufacturing and to ensure re-dispersion of the particles in water in a spray tank. In some embodiments, dispersants are used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants used as dispersants have the ability to strongly adsorb onto the particle surface and provide an electrostatic or steric barrier against particle re-aggregation. In some embodiments, the most commonly used surfactants are anionic, nonionic, or a mixture of these two types.

[0222] In some embodiments, for wettable powder formulations, the most commonly used dispersant is sodium lignosulfonate. In some embodiments, the suspension concentrate uses polyelectrolytes (such as naphthalene sulfonate formaldehyde condensate) to provide excellent adsorption and stabilization. In some embodiments, triphenyl ethylene phenol ethoxylate phosphate is also used. In some embodiments, for example, alkyl aryl ethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionic surfactants as dispersants for suspension concentrates.

[0223] In some embodiments, the agricultural compositions of the present disclosure include polymeric surfactants. In some embodiments, the polymeric surfactants have extremely long hydrophobic "backbones" and a large number of ethylene oxide chains, which form the "teeth" of the "comb-like" surfactants. In some embodiments, these high molecular weight polymers can give the suspension concentrate excellent long-term stability because the hydrophobic backbone has many points that anchor to the particle surface. In some embodiments, examples of dispersants used in the agricultural compositions of the present disclosure are: sodium lignosulfonate; naphthalene sulfonate formaldehyde condensate; triphenyl ethylene phenol ethoxylate phosphate; fatty alcohol ethoxylate; alkyl ethoxylate; EO-PO block copolymer; and graft copolymer.

[0224] In some embodiments, the agricultural compositions of the present disclosure include emulsifiers. An emulsifier is a substance that stabilizes a suspension of droplets of one liquid phase in another liquid phase. In the absence of an emulsifier, the two liquids will separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkyl phenols or fatty alcohols having 12 or more ethylene oxide units and oil-soluble calcium salts of dodecyl benzene sulfonic acid. Hydrophilic-lipophilic balance ("HLB") values in the range of 8 to 18 will generally provide a well-stabilized emulsion. In some embodiments, the emulsion stability can sometimes be improved by adding a small amount of an EO-PO block copolymer surfactant.

[0225] In some embodiments, the agricultural compositions of the present disclosure include solubilizers. A solubilizer is a surfactant that forms micelles in water at a concentration above the critical micelle concentration. The micelles can then solubilize or dissolve water-insoluble materials within the hydrophobic portion of the micelles. The types of surfactants commonly used for solubilization are nonionic surfactants: sorbitan monooleate; sorbitan monooleate ethoxylate; and methyl oleate.

[0226] In some embodiments, the agricultural compositions of the present disclosure include an organic solvent. The organic solvent is mainly used in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent in granular formulations. Sometimes mixtures of solvents are used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffin oils such as kerosene or refined paraffin. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons can be used as co-solvents to prevent crystallization of the pesticide when the formulation is emulsified in water. Sometimes alcohols are used as co-solvents to increase solubility.

[0227] In some embodiments, the agricultural composition includes a gelling agent. Thickeners or gelling agents are mainly used in the formulation of suspension concentrates, emulsions, and suspo-emulsions to modify the rheology or fluidity of the liquid and prevent separation and sedimentation of the dispersed particles or droplets. Thickeners, gelling agents, and anti-settling agents generally fall into two categories, namely water-insoluble particles and water-soluble polymers. It is possible to use clays and silica to produce suspension concentrate formulations. In some embodiments, the agricultural composition includes one or more thickeners including, but not limited to: montmorillonite such as bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickeners. The most commonly used types of polysaccharides are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Some embodiments utilize xanthan gum and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickeners including, but not limited to: guar gum; locust bean gum; carrageenan; alginate; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starch, polyacrylate, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0228] In some embodiments, the presence of surfactants (which reduce the interfacial tension) can cause foaming during mixing operations in the preparation of water-based formulations and during application through a spray tank. Therefore, in some embodiments, to reduce the foaming tendency, an anti-foaming agent is usually added during the preparation stage or before filling into bottles / spray tanks. Generally speaking, there are two types of anti-foaming agents, namely silicone and non-silicone. Silicone is usually an aqueous emulsion of dimethylpolysiloxane, and non-silicone anti-foaming agents are water-insoluble oils such as octanol and nonanol, or silica. In both cases, the function of the anti-foaming agent is to transfer the surfactant from the air-water interface.

[0229] In some embodiments, the agricultural composition includes a preservative.

[0230] In some embodiments, the agricultural composition can be formulated as: a soil drench, a foliar spray, an immersion treatment, an in-furrow treatment, a soil amendment, a granule, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural composition can be applied alone or in a rotational spray program with other agricultural products.

[0231] In some embodiments, the agricultural composition can be tank mix compatible. In some embodiments, the agricultural composition can be tank mix compatible with other agricultural products. In some embodiments, the agricultural composition can be compatible with equipment for ground, aerial, and irrigation applications.

[0232] In some embodiments, the agricultural composition can be applied to genetically modified seeds or plants.

[0233] Protection composition

[0234] In addition, individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with known active agents available in the agricultural field, such as: insecticides, herbicides, fungicides, fungicides, insecticides, virucides, acaricides, nematicides, scabicides, plant growth regulators, rodenticides, algicides, biocontrols, or beneficial agents. In addition, the microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with known fertilizers. Such combinations can exhibit synergistic properties. In addition, individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with inert ingredients. Additionally, in some aspects, the disclosed microorganisms are combined with bioactive agents.

[0235] In some embodiments, individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with biopesticides that act as herbicides, fungicides, fungicides, insecticides, virucides, acaricides, nematicides, scabicides, rodenticides, and / or algicides. Such biopesticides can be, but are not limited to, large biological organisms (e.g., beneficial nematodes, etc.), microbial organisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemicals (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola rapeseed oil).

[0236] Insecticides and biopesticides

[0237] In some embodiments, the agricultural composition of the present disclosure contains an insecticide for use in combination with the taught microorganisms. In some embodiments, the agricultural composition of the present disclosure contains a biopesticide for use in combination with the taught microorganisms.

[0238] In some embodiments, a single microorganism or microbial aggregate or microbial community developed according to the disclosed methods can be combined with known pesticides in the agricultural field, such as: pesticides that act as herbicides, fungicides, fungistats, insecticides, virucides, acaricides, nematicides, scabicides, rodenticides, and / or algicides.

[0239] In some embodiments, a single microorganism or microbial aggregate or microbial community developed according to the disclosed methods can be combined with known biopesticides in the agricultural field, such as: biopesticides that act as herbicides, fungicides, fungistats, insecticides, virucides, acaricides, nematicides, scabicides, rodenticides, and / or algicides.

[0240] For example, in some embodiments, the present disclosure teaches agricultural compositions that comprise one or more of the following active ingredients, including: macroorganisms (e.g., beneficial nematodes, etc.), microbial organisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemicals (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola oil).

[0241] In some embodiments, a single microorganism or microbial aggregate or microbial community developed according to the disclosed methods can be combined with herbicides selected from the group consisting of: acetamides selected from the group consisting of: acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, pretilachlor, metolachlor, pyrazolate, napropamide, clethodim, propachlor, propisochlor, and thenylchlor; amino acid derivatives selected from the group consisting of: bilanafos, glufosinate, and bialaphos; aryloxyphenoxypropionates selected from the group consisting of: clodinafop-propargyl, cyhalofop-butyl, fenoxaprop-p-ethyl, fluazifop-p-butyl, haloxyfop-methyl, pinoxaden, clodinafop, quizalofop-p-ethyl, and quizalofop-tefuryl; diquat and paraquat; (thio)carbamates selected from the group consisting of: asulam, butylate, carbetamide, desmedipham, piperophos, EPTC, pebulate, molinate, dimepiperate, phenmedipham, pyributicarb, pyrazolate, and triallate; cyclohexanediones selected from the group consisting of: butroxydim, clethodim, thiazopyr, bicyclopyrone, sethoxydim, pyraclonil, and tralkoxydim; dinitroanilines selected from the group consisting of: benefin, ethalfluralin, oryzalin, pendimethalin, trifluralin, and fluralin; diphenyl ethers selected from the group consisting of: acifluorfen, bifenox, chlomethoxyfen, diclofop-methyl, fluroglycofen-ethyl, fomesafen, lactofen, and oxyfluorfen; hydroxybenzonitriles selected from the group consisting of: bromoxynil, dichlobenil, and ioxynil; imidazolinones selected from the group consisting of: imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin, and imazethapyr; phenoxyacetic acids selected from the group consisting of: carfentrazone-ethyl, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dichlorprop, MCPA, MCPA-thioethyl, MCPB, and mecoprop; pyrazines selected from the group consisting of: chloridazon, flufenpyr-ethyl, pyrazoxyfen, norflurazon, and pyridate; pyridines selected from the group consisting of: aminopyralid, clopyralid, picolinafen, fluthiacet-methyl, fluridone, fluroxypyr, triclopyr, picoxystrobin, and thiazopyr; sulfonylureas selected from the group consisting of: amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, ethoxysulfuron, cyclosulfamuron, ethametsulfuron-methyl, flazasulfuron, flucarbazone-sodium, foramsulfuron, halosulfuron-methyl, halosulfuron, imazosulfuron, iodosulfuron-methyl-sodium, mesosulfuron-methyl, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron-methyl, prosulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, tribenuron-methyl, tritosulfuron, trifloxysulfuron-sodium, florasulam, tritosulfuron, and 14(2-chloro-6-propyl-imidazo[1,2]pyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxypyrimidin-2-yl)urea;Triazines selected from the group consisting of: ametryn, atrazine, cyanazine, dimethametryn, ethalfluralin, hexazinone, metobenzuron, metamitron, prometryn, simazine, terbutryn, desmetryn, and flufenetulam; urea compounds selected from the group consisting of: chlorotoluron, chloroxuron, diuron, fluometuron, isoproturon, linuron, methabenzthiazuron, and buthiuron; acetolactate synthase inhibitors selected from the group consisting of: bispyribac-sodium, cloransulam-methyl, diclosulam, florasulam, flucarbazone, flumetsulam, metosulam, orthosulfamuron, penoxsulam, propoxycarbazone, pyribenzoxim, pyribambenz-propyl, pyriminobac-methyl, pyrithiobac-sodium, pyraflufen-ethyl, pyrazolynate, and sulfosulfuron; and compounds selected from the group consisting of: amicarbazone, aminotriazole, anilofos, butafenacil, benazolin, benzfendizone, benfluresate, benzofenap, bentazone, bicyclopyrone, bromacil, bromobutide, butafenacil-isopropyl, butamifos, cafenstrole, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, clomazone, clopyralid, cumyluron, cyclosulfamuron, 2,4-D, diflufenican, dimefuron, dithiopyr, ethalfluralin, fenoxaprop-ethyl, flamprop-M, flamprop-isopropyl, flazasulfuron, florasulam, flucarbazone-sodium, flufenacet, flumetsulam, flumiclorac-pentyl, flumioxazin, flupoxam, flupropacil, flurtamone, fluthiacet-methyl, fomesafen, glufosinate-ammonium, glyphosate, halosafen, haloxyfop-methyl, imazamethabenz-methyl, imazapic, imazapyr, imazaquin, imazethapyr, iodosulfuron-methyl-sodium, isoxaben, isoxachlortole, isoxaflutole, lactofen, mesotrione, metamitron, metazachlor, metobenzuron, metosulam, metribuzin, nicosulfuron, norflurazon, oryzalin, oxadiargyl, oxadiazon, oxasulfuron, penoxsulam, pendimethalin, propachlor, propanil, prosulfuron, pyraflufen-ethyl, pyrazolynate, pyrazosulfuron-ethyl, pyribenzoxim, pyribambenz-propyl, pyriminobac-methyl, pyrithiobac-sodium, pyriftalid, quinclorac, quinmerac, rimsulfuron, sulfentrazone, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, topramezone, triafamone, 4-hydroxy-3-[2-(2-methoxy-ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-bicyclo[3.2.1]oct-3-ene-2-one, ethyl (3-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydro-2H-pyrimidin-1-yl)-phenoxy]-pyridin-2-yloxy)-acetate, methyl 6-amino-5-chloro-2-cyclopropyl-pyrimidine-4-carboxylate, 6-chloro-3-(2-cyclopropyl-6-methyl-phenoxy)-pyridazin-4-ol, 4-amino-3-chloro-6-(4-chloro-phenyl)-5-fluoro-pyridine-2-carboxylic acid, methyl 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-phenyl)-pyridine-2-carboxylate, and methyl 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridine-2-carboxylate.;

[0242] In some embodiments, a separate microorganism or microbial aggregate or microbial community developed according to the disclosed methods can be combined with an insecticide selected from the group consisting of: organo(thio)phosphates selected from the group consisting of acephate, isocarbophos,azinphos-methyl, chlorpyrifos, chlorpyrifos-methyl, dichlorvos, diazinon, dichlorvos, dimefox, dimethoate, disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion, methamidophos, methidathion, methyl parathion, mevinphos, monocrotophos, oxydemeton-methyl, paraoxon, parathion, phenthoate, phosalone, phosmet, phosphamidon, phorate, phoxim, pirimiphos-methyl, profenofos, prothiofos, tetrachlorvinphos, terbufos, triazophos, and trichlorfon; carbamates selected from the group consisting of alanycarb, aldicarb, bendiocarb, benfuracarb, carbaryl, carbofuran, carbosulfan, fenoxycarb, furathiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb, and triazamate; pyrethroids selected from the group consisting of allethrin, bifenthrin, cyfluthrin, cyhalothrin, cyphenothrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, imiprothrin, lambda-cyhalothrin, permethrin, prallethrin, pyrethrins I and II, resmethrin, silafluofen, tau-fluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin, and profluthrin; insect growth regulators selected from the group consisting of: a) chitin synthesis inhibitors, wherein the chitin synthesis inhibitor is a benzoylurea selected from the group consisting of chlorfluazuron, cyramazin, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, and teflubenzuron; buprofezin, phenoxycarb, hexythiazox, etoxazole, and clofentezine; b) ecdysone antagonists selected from the group consisting of halofenozide, methoxyfenozide, tebufenozide, and azadirachtin; c) juvenile hormone analogs selected from the group consisting of pyriproxyfen, hydroprene, and fenoxycarb; or d) lipid biosynthesis inhibitors selected from the group consisting of spirodiclofen, spiromesifen, and spirotetramat; nicotinic receptor agonist / antagonist compounds selected from the group consisting of clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, acetamiprid, thiacloprid, and 1-(2-chloro-thiazol-5-ylmethyl)-2-nitroimino-3,5-dimethyl-[1,3,5]triazinan; GABA antagonist compounds selected from the group consisting of endosulfan, ethiprole, fipronil, pyrafluprole, pyrafluprole, pyrafluprole, pyrafluprole, and 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfinamoyl-1H-pyrazole-3-thiocarboxamide;A macrolide insecticide selected from the group consisting of avermectin, emamectin, mibemycin, lepidomectin, spinosad and ethyl spinosad; a mitochondrial electron transport inhibitor (METI) I scabicide selected from the group consisting of fenazaquin, pyraclostrobin, tebufenpyrad, tolfenpyrad and pyrimidine; a METI selected from the group consisting of II and III compounds: acequinoxaline, fluacyprim and hydrazone; chlorfenapyr; oxidative phosphorylation inhibitors selected from the group consisting of tricyclam, diafenthiuron, fenbutatin and propargite; cryomazine; piperonyl butoxide; sodium channel blockers selected from the group consisting of indoxacarb and metaflumizone; and compounds selected from the group consisting of benclothiaz, bifenazate, batan, flonicamid, pyridalyl, pymetrozine, sulfur, cypermethrin, flubendiamide, chlorfenapyr, cyantraniliprole (HGW86), cypermethrin, flufenapyr, fluazifop, sulfamethoxam, cyanamide, imicyafos, bistrifluan and pyrifos. ;

[0243] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial consortia disclosed herein with known pesticides in the agricultural field, such as: pesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0244] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial consortia disclosed in the present invention with known biopesticides in the agricultural field, such as: biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0245] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a pesticide, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a pesticide, a synergistic effect on a plant phenotypic trait of interest is observed.

[0246] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, a synergistic effect on a plant phenotypic trait of interest is observed.

[0247] The synergistic effect obtained by the taught method can be quantified according to the Colby formula (i.e., (E) = X + Y - (X * Y / 100)). See Colby, R.S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, Vol. 15, pp. 20 - 22. Thus, the so - called “synergy” is intended to be the component that increases the desired effect beyond the additive amount when it is present.

[0248] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agricultural active pesticidal compounds and agricultural adjuvant pesticidal compounds in a synergistic manner.

[0249] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agricultural active biopesticidal compounds and agricultural adjuvant biopesticidal compounds in a synergistic manner.

[0250] Plant growth regulators and biostimulants

[0251] In some embodiments, the agricultural compositions of the present disclosure comprise plant growth regulators and / or biostimulants used in combination with the taught microorganisms.

[0252] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed method can be combined with known plant growth regulators in the agricultural field, such as: auxins, gibberellins, cytokinins, ethylene producers, growth inhibitors, and growth retardants.

[0253] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients, including: ancymidol, butralin, alcohols, chlormequat chloride, cytokinins, daminozide, ethephon, fenfuram, gibberellic acid, gibberellin mixtures, indole - 3 - butyric acid (IBA), maleic hydrazide, mefludide, mepiquat chloride, mepiquat pentaborate, naphthaleneacetic acid (NAA), 1 - naphthylacetamide (NAD), n - decanol, paclobutrazol, calcium cyclamate, trinexapac - ethyl, uniconazole, salicylic acid, abscisic acid, ethylene, brassinolide, jasmonates, polyamines, nitric oxide, strigolactones, or karrikins, etc.

[0254] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed method can be combined with known seed inoculants in the agricultural field, such as: RHIZO - etc. In some embodiments, Bradyrhizobium inoculants are used in combination with any single microorganism or microbial consortium disclosed herein. In certain aspects, when one of the foregoing inoculants (e.g., or Bradyrhizobium) is combined with a microorganism or microbial consortium as taught herein, a synergistic effect is observed.

[0255] In some embodiments, the agricultural compositions of the present disclosure comprise a plant growth regulator that includes: kinetin, gibberellic acid, and indolebutyric acid, as well as copper, manganese, and zinc.

[0256] In some embodiments, the present disclosure teaches agricultural compositions that comprise one or more commercially available plant growth regulators, including but not limited to: A- Royaltac Sucker- Off- Contact- E- Early Foli- X- B- Boll Cotton Quali- SA- Super Early Foli- Green N- PGR Pro- Rite- Sucker Sta- DipN Hi- FST- Turf Armor T- T- and

[0257] In some embodiments, the present invention teaches the synergistic use of the disclosed microorganisms or microbial aggregates with plant growth regulators and / or stimulants (such as phytohormones or chemicals that affect the production or destruction of plant growth regulators).

[0258] In some embodiments, the present invention teaches phytohormones, which may include: auxins (e.g., indole-3-acetic acid IAA), gibberellins, cytokinins (e.g., kinetin), abscisic acid, ethylene (and its production, such as regulated by ACC synthase and destroyed by ACC deaminase).

[0259] In some embodiments, individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with biostimulants. Such biostimulants can be, but are not limited to, microbial organisms, plant extracts, seaweeds, acids, biochar, etc.

[0260] In some embodiments, individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with fertilizers, which can be organic (e.g., manure, blood meal, fish meal, etc.), nitrogen-based (e.g., nitrates, ammonium, urea, etc.), phosphate, and potash fertilizers. Such fertilizers can also contain micronutrients, including but not limited to sulfur, iron, zinc, etc.

[0261] In some embodiments, the present invention teaches additional chemicals that promote plant growth and can act synergistically with the microorganisms and microbial aggregates disclosed herein, such as: humic acid, fulvic acid, amino acids, polyphenols, and protein hydrolysates.

[0262] Thus, in some embodiments, the present disclosure provides for the combined application of the taught microorganisms with to any crop. Additionally, the present disclosure provides for the combined application of the taught microorganisms with to any crop and using any method or application rate.

[0263] In some embodiments, the present disclosure teaches agricultural compositions having biostimulants.

[0264] As used herein, the term "biostimulant" refers to any substance used to stimulate the growth of microbial organisms that may be present in soil or other plant growth media.

[0265] The level of microbiota in the soil or growth medium is directly related to plant health. Microbiota feed on biodegradable carbon sources, and thus plant health is also related to the amount of organic matter in the soil. While fertilizers provide nutrients for plant nourishment and growth, in some embodiments, biostimulants provide biodegradable carbon (e.g., molasses, carbohydrates (e.g., sugars)) for the nourishment and growth of microbiota. Unless otherwise expressly stated, a biostimulant may comprise a single component, or a combination of several different components, which, due to the effects of one or more of said components (acting alone or in combination), are capable of enhancing microbiota activity or plant growth and development.

[0266] In some embodiments, a biostimulant is a compound that produces a non-nutritional plant growth response. In some embodiments, many of the important benefits of biostimulants are based on their ability to affect hormone activity. Hormones in plants (phytohormones) are chemical messengers that regulate normal plant development as well as responses to the environment. Root and shoot growth, among other growth responses, are regulated by phytohormones. In some embodiments, the compounds in a biostimulant can alter the hormonal status of a plant and have a significant impact on its growth and health. Thus, in some embodiments, the present disclosure teaches kelp, humic acid, fulvic acid, and vitamin B as common components of biostimulants. In some embodiments, the biostimulants of the present disclosure enhance antioxidant activity, thereby enhancing the plant's defense system. In some embodiments, vitamin C, vitamin E, and amino acids (e.g., glycine) are antioxidants contained in biostimulants.

[0267] In other embodiments, biostimulants can be used to stimulate the growth of microbiota present in the soil or other plant growth media. Previous studies have shown that when certain biostimulants containing specific organic seed extracts (e.g., soybeans) are used in combination with a microbial inoculum, the biostimulant is capable of stimulating the growth of the microorganisms included in the microbial inoculum. Thus, in some embodiments, the present disclosure teaches one or more biostimulants that, when used in conjunction with a microbial inoculum, are capable of enhancing the populations of indigenous and inoculated microorganisms. For a review of some of the popular uses of biostimulants, see Calvo et al., 2014, Plant Soil 383:3-41.

[0268] Combinations of plant components, microorganisms, and agricultural compositions

[0269] In some embodiments, the present disclosure teaches that individual microorganisms or microbial aggregates or microbial communities or any combination of the foregoing (e.g., microbiota comprising any one or more of the genome-edited Paenibacillus strains described herein) can be applied to plant components (optionally in combination with any agricultural composition) to modify the plant phenotype.

[0270] Isolated microorganisms or communities or aggregates (which are generally interchangeably referred to as “microbes” or “microbe”) can be applied to heterologous plant components to form synthetic combinations. A microorganism is considered heterologous to a plant component if it is not normally associated with the plant component in nature or is applied in an amount different from that present in nature when present. In some embodiments, a microorganism may be present in one part of a plant in nature and not in another part, and the introduction of the microorganism into the other part of the plant is considered a heterologous association.

[0271] It is further contemplated that isolated or plant- or plant-component-associated microorganisms can be further associated with one or more agricultural compositions (such as those agricultural compositions described above).

[0272] Synthetic combinations of microorganisms with plant components, microorganisms with agricultural compositions, and microorganisms with plant components and agricultural compositions are contemplated (commonly referred to as “synthetic compositions,” i.e., compositions that contain components that are not normally found associated in nature).

[0273] Treatment of plant parts

[0274] In some embodiments, the present disclosure also relates to the discovery that treating plant components with a combination of one or more of the microorganisms or agricultural compositions of the present disclosure before sowing or planting can enhance desired plant traits, such as plant growth, plant health, and / or plant insect resistance.

[0275] Thus, in some embodiments, the present disclosure teaches the use of one or more of the microorganisms or microbial aggregates as plant component treatment agents. A plant component treatment agent can be a plant component coating that is directly applied to an untreated and “naked” plant component. However, a plant component treatment agent can be an overcoat of a plant component that is applied to a plant component that has been coated with one or more previous plant component coatings or plant component treatment agents. The previous plant component treatment agent can include one or more active compounds (chemical or biological) and one or more inert ingredients.

[0276] The term “plant component treatment agent” generally refers to the application of a material to a plant component before or during planting in soil. A plant component treatment agent having the microorganisms and other agricultural compositions of the present disclosure has the advantage of delivering the treatment agent to the area where the plant component is planted shortly before germination and emergence of the plant component.

[0277] In other embodiments, the present disclosure also teaches that the use of a plant component treatment agent can minimize the amount of microorganisms or agricultural compositions required to successfully treat a plant and further limit the amount of contact of workers with the microorganisms and compositions compared to application techniques such as spraying above the soil or above the emerged plant components.

[0278] In addition, in some embodiments, the present disclosure teaches that the microorganisms disclosed herein are important for enhancing the early stages of plant life (e.g., within the first thirty days after emergence of the plant component). Thus, in some embodiments, delivering the microorganisms and / or compositions of the present disclosure in the form of a plant component treatment agent can place them in the area of action at a time that is important for microbial activity.

[0279] In some embodiments, the microbial compositions of the present disclosure are formulated as plant component treatment agents. In some embodiments, it is contemplated that one or more layers of the microorganisms and / or agricultural compositions disclosed herein can be substantially uniformly coated on the plant components by using treatment agent application equipment that is specifically designed and manufactured to accurately, safely, and effectively apply plant component treatment products to the plant components, using conventional mixing, spraying methods, or combinations thereof. Such equipment uses various types of coating techniques, such as spin coaters, drum coaters, fluidized bed techniques, spouted beds, rotary spraying, or combinations thereof. Liquid plant component treatment agents (such as the liquid plant component treatment agents of the present disclosure) can be applied through a spin "atomizer" disk or spray nozzles that evenly distribute the plant component treatment agent on the plant components as they move in a spray pattern. In various aspects, the plant components are then mixed or tumbled for a period of time to achieve additional treatment agent distribution and drying.

[0280] Prior to coating with the microbial composition, the plant components can be primed or unprimed to improve the uniformity of germination and emergence. In an alternative embodiment, a dry powder formulation can be metered onto the moving plant components and allowed to mix until fully distributed.

[0281] In some embodiments, at least a portion of the surface area of the plant component is coated with the microbial composition according to the present disclosure. In some embodiments, the plant component coating comprising the microbial composition is applied directly to the bare plant component. In some embodiments, the overcoat of the plant component comprising the microbial composition is applied to the plant component to which the plant component coating has been previously applied. In some aspects, the plant component may have a plant component coating that comprises, for example, clothianidin and / or Bacillus firmus - I-1582, on top of which the composition of the present invention will be applied in the form of an overcoat of the plant component. In some aspects, the taught microbial composition is applied in the form of an overcoat of the plant component to the plant component that has been treated with PONCHO TM VOTiVO TM In some aspects, the plant component may have a plant component coating that comprises, for example, metalaxyl and / or clothianidin and / or Bacillus firmus - I-1582, on top of which the composition of the present invention will be applied in the form of an overcoat of the plant component. In some aspects, the taught microbial composition is applied in the form of an overcoat of the plant component to the plant component that has been treated with ACCELERON TM treatment.

[0282] In some embodiments, the plant component treated with the microbe has a microbial spore concentration or microbial cell concentration as follows: about 10^2 to 10^12, 10^2 to 10^11, 10^2 to 10^10, 10^2 to 10^9, 1^02 to 10^8, 10^2 to 10^7, 10^2 to 10^6, 10^2 to 10^5, 10^2 to 10^4, or 10^2 to 10^3 microbial spores or microbial cells per plant component.

[0283] In some embodiments, the plant component treated with the microbe has a microbial spore concentration or microbial cell concentration as follows: about 10^3 to 10^12, 10^3 to 10^11, 10^3 to 10^10, 10^3 to 10^9, 10^3 to 10^8, 10^3 to 10^7, 10^3 to 10^6, 10^3 to 10^5, or 10^3 to 10^4 microbial spores or microbial cells per plant component.

[0284] In some embodiments, the plant component treated with the microbe has a microbial spore concentration or microbial cell concentration as follows: about 10^4 to 10^12, 10^4 to 10^11, 10^4 to 10^10, 10^4 to 10^9, 10^4 to 10^8, 10^4 to 10^7, 10^4 to 10^6, or 10^4 to 10^5 microbial spores or microbial cells per plant component.

[0285] In some embodiments, the microbially treated plant part has a microbial spore concentration or microbial cell concentration of about 10^5 to 10^12, 10^5 to 10^11, 10^5 to 10^10, 10^5 to 10^9, 10^5 to 10^8, 10^5 to 10^7, or 10^5 to 10^6 per plant part.

[0286] In some embodiments, the microbially treated plant part has a microbial spore concentration or microbial cell concentration of about 10^5 to 10^9 per plant part.

[0287] In some embodiments, the microbially treated plant part has a microbial spore concentration or microbial cell concentration of at least about 1×10^3 or 1×10^4 or 1×10^5 or 1×10^6 or 1×10^7 or 1×10^8 or 1×10^9 per plant part.

[0288] In some embodiments, the amount of one or more of the microorganisms and / or agricultural compositions applied to the plant part depends on the final formulation and the size or type of the plant or plant part utilized. In some embodiments, one or more of the microorganisms are present at about 2% w / w to about 80% w / w of the total formulation. In some embodiments, one or more of the microorganisms employed in the composition are about 5% w / w to about 65% w / w, or 10% w / w to about 60% w / w of the total formulation by weight.

[0289] In some embodiments, the plant part may also have more spores or microbial cells per plant part, such as about 10^2, 10^3, 10^4, 10^5, 10^6, 10^7, 10^8, 10^9, 10^10, 10^11, 10^12, 10^13, 10^14, 10^15, 10^16, or 10^17 spores or cells per plant part.

[0290] In some embodiments, the thickness of the coating of the plant component of the present disclosure can be up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm,1550 μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430 μm, 2440 μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510 μm, 2520 μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590 μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm2980 μm, 2990 μm, or 3000 μm.

[0291] In some embodiments, the thickness of the coating of the plant component of the present disclosure can be 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0292] In some embodiments, the coating of the plant component of the present disclosure can be at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, or 50% of the weight of the uncoated plant component.

[0293] In some embodiments, the microbial spores and / or cells can be freely coated onto the plant component or they can be formulated in a liquid or solid composition before being coated onto the plant component. For example, a solid composition containing the microbial bodies can be prepared by mixing a solid carrier with a suspension of spores until the solid carrier is impregnated with the spores or cell suspension. The mixture can then be dried to obtain the desired granules.

[0294] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents such as activated carbon, nutrients (fertilizers), and other reagents capable of improving the germination and quality of the product or combinations thereof.

[0295] Known plant component coating methods and compositions in the art can be particularly useful when modified by adding one of the embodiments of the present disclosure. Such coating methods and their application devices are disclosed, for example, in U.S. Patent Nos. 5,916,029, 5,918,413, 5,554,445, 5,389,399, 4,759,945, 4,465,017 and U.S. Patent Application No. 13 / 260,310.

[0296] Plant component coating compositions are disclosed, for example, in U.S. Patent Nos. 5,939,356, 5,876,739, 5,849,320, 5,791,084, 5,661,103, 5,580,544, 5,328,942, 4,735,015, 4,634,587, 4,372,080, 4,339,456 and 4,245,432.

[0297] In some embodiments, a variety of additives can be added to the plant component treatment preparation comprising the composition of the present invention. A binder can be added, and the binder includes a binder composed of a natural or synthetic adhesive polymer that has no phytotoxic effect on the coated plant component. The binder can be selected from polyvinyl acetate; polyvinyl acetate copolymers; ethylene-vinyl acetate (EVA) copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; celluloses, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose; polyvinylpyrrolidone; polysaccharides, including starch, modified starch, dextrin, maltodextrin, alginate and chitosan; fats; oils; proteins, including gelatin and zein; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonate; acrylic copolymers; polyvinyl acrylate; polyethylene oxide; acrylamide polymers and copolymers; hydroxyethyl acrylate, methacrylamide monomers; and polychloroprene.

[0298] Any of a variety of colorants can be employed, including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methylene, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be added include micronutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

[0299] A polymer or other dust control agent can be applied to keep the treatment agent on the surface of the plant component.

[0300] In some specific embodiments, in addition to microbial cells or spores, the coating may further comprise an adhesive layer. The adhesive should be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetate; polyvinyl acetate copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; cellulose, such as methyl cellulose, hydroxy methyl cellulose, and hydroxy methyl propyl cellulose; dextrin; alginate; sugars; molasses; polyvinyl pyrrolidone; polysaccharides; proteins; fats; oils; gum arabic; gelatin; syrups; and starches. More examples can be found, for example, in U.S. Patent No. 7,213,367.

[0301] The plant component treatment preparation may also include various additives, such as adhesives, dispersants, surfactants, and nutrients, as well as buffering components. Other conventional plant component treatment additives include, but are not limited to: coating agents, wetting agents, buffering agents, and polysaccharides. At least one agriculturally acceptable carrier, such as water, solid, or dry powder, can be added to the plant component treatment preparation. The dry powder can be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon, and various phosphorus compounds.

[0302] In some embodiments, the plant component coating composition may comprise at least one filler, which is an organic or inorganic, natural or synthetic component, where the active components are combined to facilitate their application on the plant component. In various aspects, the filler is an inert solid, such as clay, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (such as ammonium salts), natural soil minerals (such as kaolin, clay, talc, lime, quartz, attapulgite, montmorillonite, bentonite, or diatomaceous earth), or synthetic minerals (such as silica, alumina, or silicates, especially aluminum silicate or magnesium silicate).

[0303] In some embodiments, the plant component treatment formulation may further include one or more of the following ingredients: other pesticides, including compounds that act only below the ground; fungicides such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of these materials; herbicides, including compounds selected from glyphosate, carbamate, thiocarbamate, acetamide, triazine, dinitroaniline, glycerol ether, pyridazinone, uracil, phenoxy, urea, and benzoic acid; herbicide safeners such as benzoxazine, diphenylmethyl derivatives, N,N-diallyldichloroacetamide, various dihaloacetyl groups, oxazolidinyl and thiazolidinyl compounds, acetone, phthalic anhydride compounds, and oxime derivatives; chemical fertilizers; biofertilizers; and biocontrol agents such as other naturally occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium, and mycorrhizal fungi. These ingredients may be added as separate layers on the plant component, or alternatively may be added as part of the plant component coating composition of the present disclosure.

[0304] In some embodiments, the formulation for treating plant components in the present disclosure may be in the form of: a suspension; an emulsion; a slurry of particles in an aqueous medium (e.g., water); a wettable powder; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation may be from about 0.5 wt% to about 99 wt% (w / w), or 5 wt% to 40 wt%, or otherwise formulated by those skilled in the art.

[0305] As mentioned above, other conventional inactive or inert ingredients may be incorporated into the formulation. Such inert ingredients include, but are not limited to: conventional adhesives; dispersants such as methylcellulose, e.g., acting as a combined dispersant / adhesive for the plant component treatment agent; polyvinyl alcohol; lecithin, polymeric dispersants (e.g., polyvinylpyrrolidone / vinyl acetate); thickeners (e.g., clay thickeners for increasing viscosity and reducing sedimentation of particle suspensions); emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea), dyes, colorants, etc. Additional inert ingredients useful in the present disclosure can be found in McCutcheon's, Volume 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.

[0306] The coated preparation of the plant component of the present disclosure can be applied to the plant component by various methods, including but not limited to: mixing in a container (such as a bottle or a bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert materials can be used to bring the plant component into contact with the microbial composition according to the present disclosure.

[0307] In some embodiments, the amount of the microbial or agricultural composition used to treat the plant component will vary depending on the type of the plant component and the type of the active ingredient, but the treatment will involve bringing the plant component into contact with an agriculturally effective amount of the composition of the present invention.

[0308] As discussed above, the effective amount means the amount of the composition of the present invention sufficient to affect a beneficial or desired result. The effective amount can be applied during one or more applications.

[0309] In some embodiments, in addition to the coating layer, the plant component can be treated with one or more of the following components: other pesticides, including fungicides and herbicides; herbicide safeners; fertilizers and / or biocontrol agents. These components can be added as separate layers or alternatively can be added in the coating layer.

[0310] In some embodiments, the coated preparation of the plant component of the present disclosure can be applied to the plant component using a variety of techniques and machines, such as fluidized bed technology, roll mill method, drum electrostatic plant component processor, and drum coater. Other methods (such as fountain bed) can also be useful. The plant component can be pre-sized before coating. After coating, the plant component is usually dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.

[0311] In some embodiments, the plant component treated with the microorganism can also be wrapped with an outer film coating to protect the coating. Such outer coatings are known in the art and can be applied using fluidized bed and cylinder film coating techniques.

[0312] In other embodiments of the present disclosure, the compositions according to the present disclosure can be introduced onto plant components by using solid matrix priming. For example, an amount of the composition of the present invention can be mixed with a solid matrix material, and then a plant component can be placed in contact with the solid matrix material for a period of time to allow the composition to be introduced onto the plant component. Then, the plant component can optionally be separated from the solid matrix material and stored or used, or the mixture of the solid matrix material plus the plant component can be directly stored or planted. Solid matrix materials that can be used in the present disclosure include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyureas, polyacrylates, or any other material capable of absorbing or adsorbing the composition of the present invention for a period of time and releasing the composition into or onto the plant component. It is useful to ensure that the composition of the present invention and the solid matrix material are compatible with each other. For example, the solid matrix material should be selected such that it can release the composition at a reasonable rate (e.g., over a period of minutes, hours, or days).

[0313] In some embodiments, the present disclosure teaches that individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with any plant biostimulant.

[0314] In some embodiments, the present disclosure teaches agricultural compositions that comprise one or more commercially available biostimulants, including but not limited to: Diehard TM Diehard TM Fe, Diehard TM Soluble Kelp, Diehard TM Humate SP, Foliar Plus TM , Plant Plus TM , Accomplish Soil Builder TM , Nutri Life, SoilSolution TM , Seed Coat TM , PercPlus TM , Plant Thrust TM , and etc.

[0315] In some embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with an active chemical agent, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with an active chemical agent, a synergistic effect on a plant phenotypic trait of interest is observed.

[0316] In some embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with a fertilizer, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with a fertilizer, a synergistic effect on a plant phenotypic trait of interest is observed.

[0317] In some embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with a plant growth regulator, an additive effect on a plant phenotypic trait of interest is observed. In some embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with a plant growth regulator, a synergistic effect is observed. In some aspects, the microorganisms of the present disclosure are combined with and a synergistic effect on one or more phenotypic traits of interest is observed.

[0318] In some embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with a biostimulant, an additive effect on a plant phenotypic trait of interest is observed. In some embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with a biostimulant, a synergistic effect is observed.

[0319] The synergistic effect obtained by the taught methods can be quantified according to the Colby formula (i.e., (E)=X+Y-(X*Y / 100)). See Colby, R.S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, Vol. 15, pp. 20-22. Thus, “synergistic” is intended to be a component that, when present, increases the desired effect beyond the additive amount.

[0320] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agricultural active compounds as well as agricultural adjuvant compounds in a synergistic manner.

[0321] In other embodiments, when a microorganism or microbial consortium identified according to the taught methods is combined with a fertilizer, a synergistic effect is observed.

[0322] In addition, in certain embodiments, the present disclosure utilizes synergistic interactions to define microbial consortia. That is, in certain aspects, the present disclosure combines certain isolated microbial species that exhibit synergistic effects into consortia that confer beneficial traits to plants or that are associated with enhancing beneficial plant traits.

[0323] Agricultural compositions developed according to the present disclosure can be formulated with certain adjuvants to enhance the activity of known active agricultural compounds. This has the advantage of reducing the amount of active ingredient in the formulation while maintaining the efficacy of the active compound, thus keeping costs as low as possible and complying with any official regulations. In individual cases, it is also possible to broaden the scope of action of the active compound, since plants (which were not successfully treated with a specific active ingredient without addition) can actually be successfully treated by adding certain adjuvants as well as the disclosed microbial isolates and consortia. In addition, when environmental conditions are unfavorable, the performance of the active substance can be increased in individual cases by means of a suitable formulation.

[0324] Such adjuvants that can be used in agricultural compositions can be adjuvants. Generally, adjuvants are in the form of surface-active or salt compounds. Depending on their mode of action, they can be roughly classified as regulators, activators, fertilizers, pH buffers, etc. Regulators affect the wetting, sticking, and spreading properties of the formulation. Activators disrupt the waxy surface layer of plants and enhance the penetration of the active ingredient into the surface layer (short-term (a few minutes) and long-term (a few hours)). Fertilizers (such as ammonium sulfate, ammonium nitrate, or urea) enhance the absorption and solubility of the active ingredient and can reduce the antagonistic behavior of the active ingredient. pH buffers are routinely used to adjust the formulation to the optimal pH.

[0325] In some embodiments, the plant component is a plant reproductive component (e.g., seeds, tubers, bulbs, and / or buds). In some embodiments, the plant component is other than a plant reproductive component (e.g., leaves, stems, and / or roots). In some embodiments, multiple plant components are associated with the microorganisms described herein.

[0326] In some embodiments, a plant or plant component is associated with one or more microorganisms described herein via an indirect method, such as but not limited to treatment of the growth medium in which the plant or plant component is placed.

[0327] For additional embodiments of the agricultural compositions of the present disclosure, see “Chemistry and Technology of Agrochemical Formulations”, edited by D.A. Knowles, copyright 1998, Kluwer Academic Publishers.

[0328] Plant and Agronomic Benefits

[0329] A variety of plants (including those cultivated agriculturally) can benefit from the application of microorganisms (such as those described herein, including single microorganisms, consortia, and / or compositions produced therefrom or comprising any of the foregoing). Many different plants can be used in the methods of the present disclosure, including mosses, lichens, and algae. In embodiments, the plants have economic, social, or environmental value. For example, the plants can include those used as food crops, fiber crops, oil crops, for forestry, for the pulp and paper industry, as raw materials for biofuel production, and as ornamental plants.

[0330] The genetically modified microorganisms disclosed herein can be applied to improve nitrogen fixation in plants. In some embodiments, such plants include those lacking natural nitrogen-fixing symbionts (such as non-leguminous crops), such as, but not limited to: wheat, maize (corn), rice, and vegetables. In some embodiments, such plants include those that would benefit from additional nitrogen fixation.

[0331] Method of application

[0332] Any suitable technique known in the art can be used to apply the microbial bodies to plants, seedlings, cuttings, propagules, etc. and / or to the growth medium containing the plants.

[0333] However, by way of example, the microorganisms, consortia, or compositions comprising them and / or compositions produced therefrom can be applied to plants, seedlings, cuttings, propagules, etc. by spraying, coating, broadcasting, or any other method known in the art.

[0334] In another embodiment, the isolated microorganisms, consortia, or compositions comprising them can be applied directly to plant seeds prior to sowing.

[0335] In another embodiment, the isolated microorganisms, consortia, or compositions comprising them can be applied directly to plant seeds in the form of a seed coating.

[0336] In one embodiment of the present disclosure, the isolated microorganisms, consortia, or compositions comprising them are supplied in the form of granules or plugs or soil drenches for application to the plant growth medium.

[0337] In other embodiments, the isolated microorganisms, consortia, or compositions comprising them are supplied in the form of a foliar application, such as a foliar spray or a liquid composition. The foliar spray or liquid application can be applied to growing plants or to the growth medium, such as soil.

[0338] In some embodiments, the isolated microorganism, consortium, or composition comprising the same is supplied in a form selected from: soil drench, foliar spray, dip treatment, in-furrow treatment, soil amendment, granule, broadcast treatment, post-harvest disease control treatment, or seed treatment. In some embodiments, the agricultural composition can be applied alone or according to a rotary spray program.

[0339] In some embodiments, the isolated microorganism, consortium, or composition comprising the same can be tank-mix compatible. In some embodiments, the agricultural composition can be tank-mix compatible with other agricultural products. In some embodiments, the agricultural composition can be compatible with equipment for ground, aerial, and irrigation applications.

[0340] In another embodiment, the isolated microorganism, consortium, or composition comprising the same can be formulated into granules and applied beside the seeds during planting. Or the granules can be applied after planting. Or the granules can be applied before planting.

[0341] In some embodiments, the isolated microorganism, consortium, or composition comprising the same is applied to the plant or growth medium in the form of a topical application and / or drench to improve crop growth, yield, and quality. The topical application can be carried out by using a dry mixture or powder or dusting composition or can be a liquid-based formulation.

[0342] In embodiments, the isolated microorganism, consortium, or composition comprising the same can be formulated as: (1) a solution; (2) a wettable powder; (3) a dusting powder; (4) a soluble powder; (5) an emulsion or suspension concentrate; (6) a seed dresser or seed coating, (7) a tablet; (8) a water-dispersible granule; (9) a water-soluble granule (slow release or fast release); (10) a microencapsulated granule or suspension; (11) as an irrigation component, and (12) a component of fertilizers, pesticides, and other compatible amendments, etc. In certain aspects, the composition can be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure can be applied to soil, plants, seeds, rhizosphere, root sheath, or other areas where the application of the microbial composition will be beneficial. Additionally, the shock method can be utilized as a means for introducing endophytic microorganisms.

[0343] In various aspects, the composition is applied to the leaves of the plant. The composition can be applied to the leaves of the plant in the form of an emulsion or suspension concentrate, a liquid solution, or a foliar spray. The application of the composition can be carried out in a laboratory, growth chamber, greenhouse, or field.

[0344] In another embodiment, the microorganism can be inoculated into the plant by: pruning the roots or stems and exposing the plant surface to the microorganism by spraying, dipping, or otherwise applying a liquid microbial suspension or gel or powder.

[0345] In another embodiment, the microbial bodies can be directly injected into the leaf surface or root tissue, or otherwise directly inoculated into or onto the leaf or root pruning sites, or into the excised embryo or radicle or coleoptile. These inoculated plants can then be further exposed to a growth medium containing additional microbial bodies; however, this is not necessary.

[0346] In other embodiments, particularly where the microorganism is non-culturable, the microbial bodies can be transferred to the plant by any one or combination of the following: grafting, inserting an explant, aspiration, electroporation, wounding, root pruning, inducing stomatal opening, or any physical, chemical, or biological treatment that provides an opportunity for the microorganism to enter the plant cells or intercellular spaces. Those skilled in the art can readily appreciate many alternative techniques that can be used.

[0347] In one embodiment, the microbial bodies infiltrate parts of the plant (such as roots, stems, leaves, and / or reproductive plant parts (become endophytes)), and / or grow on the surfaces of roots, stems, leaves, and / or reproductive plant parts (become epiphytes) and / or grow in the plant rhizosphere. In one embodiment, the microbial bodies form a symbiotic relationship with the plant.

[0348] Although the present invention has been particularly shown and described in connection with preferred embodiments and various alternative embodiments, those skilled in the relevant art should understand that various changes can be made to the forms and details thereof without departing from the spirit and scope of the present invention. Various changes, modifications, and improvements to the present disclosure (including certain changes, modifications, substitutions, and improvements) that are readily conceivable by those skilled in the art are also part of the present disclosure. For example, although the following specific examples may illustrate the methods and embodiments described herein using specific plants, the principles in these examples can be applied to any plant. Therefore, it should be understood that the scope of the present invention is covered by the embodiments described herein, rather than being covered only by the specific examples illustrated below.

[0349] All cited publications, patents, patent applications, patent application publications, and non-patent literature mentioned in this application are incorporated herein by reference in their entirety for all purposes to the extent as if each cited patent and publication were individually and specifically incorporated by reference.

[0350] Examples

[0351] The methods and compositions presented herein (based on the use of the disclosed isolated microorganisms, communities, aggregates, and / or compositions comprising microorganisms or aggregates or communities and / or produced by microorganisms or aggregates or communities) improve one or more characteristics of plants (such as nitrogen fixation in agricultural crops). The abbreviation "uL" means "microliter", and "ug" means "microgram".

[0352] As the results in these examples show, the control of the nfix cluster by GlnR has been well studied and tested. Surprisingly, the inventors of the present case have found a more complex regulatory scheme than has already been described in the literature.

[0353] Example 1: Microbial Cultivation, Sequencing, and Target Selection

[0354] A Paenibacillus strain was grown in a medium to allow the cells to grow to full growth.

[0355] Then, a subsample of each strain was aseptically transferred to a growth medium and incubated under microaerobic conditions for 72 hours.

[0356] The isolates of interest were grown to mid-logarithmic phase in R2A medium. DNA was extracted using the Qiagen Powersoil DNA extraction kit, and a sequencing library was constructed using the Twist 96-Plex Library Prep kit (formerly known as the iGenomix RipTide kit) according to the manufacturer's instructions. Sequencing was performed on an Illumina HiSeq with PE150. The raw Illumina reads were trimmed, assembled, and annotated using the Bactopia nextflow pipeline, where phiX was removed, the reads were trimmed to Q20, and the minimum assembly was 3 Mb. All assemblies passed the quality control of checkM. Only assemblies with an integrity greater than 95% and a contamination less than 5% were used for this analysis. Taxonomic assignment was performed using GTDB-tk with default parameters against the April 2023 database (Pierre-Alain Chaumeil, Aaron J Mussig, Philip Hugenholtz, Donovan HParks, GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database, Bioinformatics, Volume 36, Issue 6, 15 March 2020, Pages 1925-1927).

[0357] Bioinformatics analysis was performed on the nif gene cluster (components of the typical nif operon) and upstream and downstream sequences to evaluate putative novel regulatory elements, including the presence of promoters. Phylogenetic analysis was performed to determine the relative relationships between strains, species, and genera across Gram-positive and Gram-negative bacteria that putatively possess promoters and those that do not have putative intergenic promoters. Hypotheses were established regarding the acquisition, transfer, and loss of putative intergenic promoters.

[0358] Table 2a lists the nif cluster genes identified in different species and strains of the genus Paenibacillus. In some cases, different variants of the same gene were identified, such as nifH and nifH1, hesA1 and hesA2. Table 2b lists the intergenic spacings of Int_1 (the region between nifH and nifD), Int_2 (the region between nifK and nifE), and Int-3 (the region between (nifX or orf1) and hesA) in those strains. There are conserved motif regions present in the sequences of each intergenic region (Int_1, Int_2, and Int_3), and the sequences are given as SEQ ID NO. 1-501.

[0359] The phylogenetic relationships of each intergenic region (Int_1, Int_2, and Int_3) of different Paenibacillus strains in humans are depicted respectively in Figure 4C , 4D and 4E.

[0360] Multiple sets of conserved and near-conserved regions were identified in each intergenic region. These and other regions and motifs can be observed on the sequences disclosed herein using methods known in the art.

[0361] A canonical Shine-Dalgarno sequence (AGGAGG), which is the ribosome binding site (RBS) in prokaryotes, was found in the following intergenic regions (listed as StrainID_int_#; for example, 8619_int_3 would be the intergenic region 3 of strain 8619, which is the polynucleotide sequence immediately upstream of the hesA gene): 100087_int_1, 100101_int_1, 100102_int_1, 101117_int_1, 101545_int_1, 102018_int_1, 102020_int_1, 102088_int_1, 102091_int_1, 102547_int_1, 102548_int_1, 102550_int_1, 102554_int_1, 102555_int_1, 102561_int_1, 102565_int_1, 102566_int_1, 102571_int_1, 102577_int_1, 102579_int_1, 102585_int_1, 102586_int_1, 102587_int_1, 102603_int_1, 102702_int_1, 102705_int_1, 103143_int_1, 103256_int_1, 103282_int_1, 103327_int_1, 103408_int_1, 103412_int_1, 104080_int_1, 104107_int_1, 104114_int_1, 104115_int_1, 104182_int_1, 104208_int_1, 104492_int_1, 104495_int_1, 105487_int_1, 105578_int_1, 105667_int_1, 106158_int_1, 106159_int_1, 106172_int_1, 106213_int_1, 106221_int_1, 106222_int_1, 106226_int_1, 106236_int_1, 106250_int_1, 106276_int_1, 106697_int_1, 106818_int_1, 106839_int_1, 106840_int_1, 106939_int_1, 107135_int_1, 1118_int_1, 1400_int_1, 1401_int_1, 17414_int_1, 17895_int_1, 17896_int_1, 17897_int_1,17899_int_1, 17907_int_1, 17908_int_1, 17910_int_1, 17911_int_1, 17912_int_1, 17918_int_1, 17921_int_1, 17924_int_1, 17925_int_1, 17947_int_1, 17948_int_1, 17960_int_1, 17973_int_1, 17974_int_1, 17975_int_1, 17978_int_1, 17982_int_1, 17983_int_1, 17985_int_1, 17987_int_1, 17988_int_1, 18005_int_1, 18018_int_1, 18022_int_1, 18023_int_1, 18026_int_1, 18027_int_1, 18030_int_1, 18033_int_1, 18035_int_1, 18037_int_1, 1923_int_1, 2026_int_1, 2649_int_1, 3393_int_1, 4328_int_1, 4345_int_1, 5263_int_1, 6050_int_1, 6053_int_1, 6055_int_1, 6128_int_1, 6219_int_1, 6242_int_1, 7037_int_1, 7681_int_1, 8051_int_1, 8619_int_1, 38431_int_1, 48304_int_1, 48309_int_1, 53072_int_1, 53107_int_1, 53144_int_1, 53145_int_1, 53146_int_1, 53150_int_1, 53378_int_1, 53953_int_1, 54701_int_1, 54805_int_1, 54911_int_1, 54997_int_1, 55026_int_1, 55083_int_1, 55136_int_1, 55146_int_1, 55470_int_1, 55965_int_1, 56089_int_1, 57529_int_1, 62529_int_1, 63764_int_1, 66545_int_1, 67533_int_1, 68870_int_1, 68890_int_1, 69168_int_1, 69170_int_1, 70947_int_1, 70952_int_1, 70971_int_1, 70995_int_171264_int_1, 72994_int_1, 77155_int_1, 77357_int_1, 77359_int_1, 77370_int_1, 77457_int_1, 77458_int_1, 77969_int_1, 102590_int_2, 104107_int_2, 104182_int_2, 104492_int_2, 104495_int_2, 105667_int_2, 106221_int_2, 106222_int_2, 4328_int_2, 102565_int_3, 102587_int_3, 102603_int_3, 103412_int_3, 106192_int_3, 106839_int_3, 106840_int_3, 1401_int_3, 17907_int_3, 17918_int_3, 17924_int_3, 17925_int_3, 17947_int_3, 17948_int_3, 17960_int_3, 17978_int_3, 17988_int_3, 18018_int_3, 3393_int_3, 4345_int_3, 6219_int_3. Other RBS motifs and other different motifs may exist.,

[0362] Table 2a: nif cluster genes in species and strains of Paenibacillus

[0363]

[0364]

[0365]

[0366]

[0367] Table 2b: Intergenic distances (number of nucleotides) in species and strains of Paenibacillus

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] Example 2: Promoter fusions

[0374] Transcriptional reporter gene fusions were constructed to evaluate the potential promoter activity of the intergenic regions of nif cluster genes in Paenibacillus species. Transcriptional reporter gene fusions with green fluorescent protein (GFP) were operably linked to promoters of different Paenibacillus strains and tested in different strains to evaluate the potential promoter activity of the intergenic regions of nif cluster genes. Culture fluorescence was measured with a plate reader and cell fluorescence was evaluated with a microscope.

[0375] Putative promoters from different strains were each operably linked to GFP to generate promoter fusions and tested in different strains, including Paenibacillus strains with known nitrogen fixation, closely related species of Paenibacillus with unknown nitrogen fixation, Gram-positive Firmicutes, and nitrogen-fixing Gram-negative bacteria (Klebsiella variicola).

[0376] These results (shown in Table 3 below) demonstrate that different regions within the typical nif cluster can be activated in both Gram-positive and Gram-negative nitrogen-fixing bacteria

[0377] nifB regulatory element

[0378] Figure 5A (in aerobic conditions with and without nitrogen) and Figure 5B (in anaerobic conditions with and without nitrogen) confirm that the nifB promoter is expressed (activated under low nitrogen conditions) as expected in each strain. Figure 6A and 6B show the activities of different mutant strains of 77155.

[0379] nifD regulatory element

[0380] Fusions of the nifH-nifD intergenic region with GFP for each strain showed activity, indicating the presence of a putative nifD' promoter. The results are shown in Figure 7A (in aerobic conditions with and without nitrogen) and Figure 7B (in anaerobic conditions with and without nitrogen). These results suggest that there may be a promoter between nifH and nifD in at least Paenibacillus borealis and Paenibacillus odorifer.

[0381] nifE regulatory element

[0382] Fusions of the nifK-nifE intergenic region with GFP showed a range of constitutive activities from low to very high. The results are in Figure 8A (in aerobic conditions with and without nitrogen) and Figure 8B(Shown in (anaerobic conditions with and without nitrogen). These results suggest that there may be promoters between nifK and nifE in at least Paenibacillus borealis, Paenibacillus odorifer, and Paenibacillus durus.)

[0383] hesA regulatory element

[0384] Fusions of the intergenic region upstream of hesA with GFP also showed activity. In Paenibacillus strains of subgroup I, the intergenic region is between nifX and hesA. In Paenibacillus strains of subgroup II, the intergenic region is between orf1 and hesA. The results were shown in Figure 9A (aerobic conditions with and without nitrogen) and Figure 9B (anaerobic conditions with and without nitrogen). These results suggest that there may be a promoter upstream of hesA.)

[0385] Table 3: Reporter gene activities in Gram-positive and Gram-negative bacteria

[0386] A "putative promoter" is the region immediately upstream of a designated gene from a designated strain (e.g., nifB' would be the putative promoter of nifB) (e.g., "17911nifB'" is the putative nifB promoter in strain 17911). Blank cells indicate not tested.)

[0387]

[0388]

[0389] These data suggest that multiple novel promoters within the nif cluster generate multiple transcripts, and the individual transcript structures of the nif operon units are not necessarily conserved among species. These novel putative promoters could be new editing targets to improve the nitrogen fixation ability of bacteria. The nitrogenase activity could be increased further than previously achieved.)

[0390] Variations in promoters and differences in flanking cluster genes among species suggest multiple acquisitions of nif by Paenibacillus.)

[0391] Using the methods described herein, additional experiments on intergenic regions of nif cluster genes were conducted on other strains of Paenibacillus (both nitrogen-fixing and non-nitrogen-fixing Paenibacillus), as well as other nitrogen-fixing bacteria of the same and different genera across Gram-positive bacteria (including Firmicutes and Actinobacteria) and Gram-negative bacteria, and cyanobacteria and Archaebacteria.)

[0392] Example 3: Intergenic region manipulation

[0393] Neutral intergenic exchange

[0394] The intergenic region upstream of nifD, nifE, and hesA was replaced with a neutral 6-bp sequence (GAAAAT) between its nifB and nifH genes obtained from strain 77155 to force the open reading frames of the nif cluster to be transcribed together as a polycistronic operon. The resulting mutant strains were analyzed by the acetylene reduction assay (see Example 8) to evaluate the effect of replacing the intergenic region.

[0395] The results are shown in Table 4 and Figure 10A (low nitrogen condition) and 10B (high nitrogen condition). These data indicate that removing the intergenic region is not favorable for nitrogenase activity.

[0396] Table 4: Ethylene conversion rate % of neutral intergenic exchange

[0397] Six base pairs within the "edited position" described in the table were replaced with the neutral six-base pair sequence (GAAAAT), and each edited strain was retested under low ammonia (0 mM) and high ammonia (5 mM) conditions 48 hours after inoculation according to the protocol described herein. The results are given as % ethylene conversion rate / OD600 to normalize for cell density.

[0398]

[0399]

[0400] Intergenic truncation

[0401] The regions upstream of the nifD, nifE, and hesA genes were removed, except for the corresponding putative ribosome binding site (RBS). The consensus sequence of the RBS (or Shine-Dalgarno sequence) is typically AGGAGG or a similar sequence. It is speculated that the RBS will be important for translation based on the data from the neutral exchange that removed the above-mentioned intergenic region. The results are shown in Table 5.

[0402] Table 5: Ethylene conversion rate % of intergenic truncation

[0403] Each edited strain was retested under low ammonia (0 mM) and high ammonia (5 mM) conditions 48 hours after inoculation according to the protocol described herein. The results are given as % ethylene conversion rate / OD600 to normalize for cell density.

[0404]

[0405] Inter-species exchange

[0406] The intergenic regions of different wild-type and mutant strains were replaced with intergenic regions from the same positions of the Bacillus firmus strain 103408. In some cases, the parental strain for in situ editing was itself a previously edited strain, as described in PCT patent application publication number WO2022204062A1 published on September 29, 2022, and PCT / US2023 / 074808 filed on September 21, 2023. The edited strains for interspecies exchange included: 8619-G50: inactivation and duplication of the GlnR binding site II; 17899-G13: GlnR frameshift truncation + GlnA SNP; 17899-G43: GlnR frameshift truncation + GlnA SNP + ORF1 knockout; 17899-G113: GlnR C25 truncation; 77155-G3: inactivation of the GlnR binding site II; 77155-G9: inactivation and duplication of the GlnR binding site II; 77155-G46: GlnR C25 truncation. The results are shown in Table 6.

[0407] Table 6: Ethylene conversion rate % for interspecies exchange

[0408] According to the protocol described herein, each edited strain was retested at 48 hours post-inoculation under low ammonia (0 mM) and high ammonia (5 mM) conditions. The results are given as % ethylene conversion rate / OD600 and are normalized against the cell density.

[0409]

[0410] Example 4: Transcript assessment

[0411] For example, transcript production from individual cluster components was assessed by RNA-Seq. 5'-RACE mapping of the 5'-ends of potential transcripts was performed to confirm the presence of additional promoters within each nif cluster.

[0412] Example 5: Intergenic region assessment and testing

[0413] The intergenic region sequences were evaluated, and experiments were conducted to assess the minimum essential and sufficient length of the optimal nif promoter. The composition was evaluated to identify the key nucleotides necessary for promoter activity. As described above, experiments were conducted to test each of the minimum promoter and potential universal promoters.

[0414] The intergenic regions in the nif cluster can be exchanged or altered to increase or decrease the expression of nitrogenase subunits.

[0415] The disruption of the native nif cluster can be carried out to place each gene under the control of regulatory elements such as, but not limited to: inducible regulatory elements, highly active intragenic regulatory elements, and / or binding sites. In some cases, the regulatory element is a promoter.

[0416] The nif cluster can be modified such that each component gene is under the control of the same promoter (e.g., a GlnR-regulated promoter), or different promoters, or a combination thereof.

[0417] Constructs are generated and integrated into different bacterial cells to generate strains with modified nitrogenase activity. In some aspects, an increase in nitrogenase activity is desired. In some aspects, a decrease in nitrogenase activity is desired. Insertions, replacements, modifications, and / or deletions can be made within the NIF cluster and in the different intergenic regions flanking the cluster to effect any desired modulation of the nitrogen fixation ability of the microorganism.

[0418] Example 6: Genome Editing in Paenibacillus

[0419] Scarless homologous recombination

[0420] This is a general protocol for genome editing in Paenibacillus using a temperature-sensitive scarless homologous recombination plasmid and is used for the editing described herein. This protocol was developed for editing and is widely applicable to Paenibacillus isolates. This protocol requires the pre-assembly of one or more editing vectors designed to perform the desired editing using the pMMmob backbone in an E. coli donor strain and one or more Paenibacillus recipient strains, with the recipient strains having confirmed susceptibility to the relevant antibiotic resistance marker.

[0421] Conjugation

[0422] The desired recipient strain is grown overnight in an appropriate growth medium. The donor strain is grown overnight in an appropriate growth medium supplemented with the relevant antibiotic marker to maintain the mobilizable plasmid. Aliquots of the overnight cultures are washed, combined, and plated on an agar medium suitable for the growth of both strains. The plates are incubated overnight at the permissive temperature for plasmid replication in the recipient strain.

[0423] The mating mixture is recovered, washed, and re-plated on an agar plate supplemented with the appropriate antibiotic marker for selection of the transconjugant recipient strain. The plates are incubated overnight at the permissive temperature for plasmid replication until transconjugant colonies appear.

[0424] Integration

[0425] In the presence of the selectable marker, the transconjugant colonies are grown overnight in a liquid culture at the permissive temperature for plasmid replication. Dilutions of the liquid culture are plated on an agar plate supplemented with the selective antibiotic and incubated overnight at the restrictive temperature for plasmid replication. It is assumed that the colonies recovered under these conditions have integrated the editing plasmid by homologous recombination.

[0426] Excision

[0427] The integrated colonies were inoculated into liquid cultures and grown to turbidity at the permissive temperature for plasmid replication, then subcultured into fresh medium lacking antibiotics and grown overnight again at the permissive temperature. This serial subculture was repeated 2 - 3 times, and dilutions of the final subculture were plated on agar plates lacking antibiotics.

[0428] Plasmid loss in the recovered colonies was determined by re - plating onto media with and without antibiotics. Colonies that grew in the absence of antibiotics but not in the presence of antibiotics were confirmed to have excised and lost the plasmid and were identified as putative edited strains.

[0429] Confirmation

[0430] Putative edited strains were screened to determine whether the editing was successfully delivered or whether the strain had reverted to wild - type by amplifying the edited region via polymerase chain reaction (PCR). The PCR products were analyzed by gel electrophoresis and Sanger sequencing to confirm the product size and sequence suitable for editing. Colonies with successfully delivered editing were confirmed by Sanger sequencing to check that no off - target mutations were added to the edited region and that there was no growth on media containing antibiotics to confirm the absence of the plasmid backbone.

[0431] Other methods

[0432] Alternatively, any other method known in the art can be employed to achieve any one or more of the polynucleotide edits described herein, such as but not limited to: targeted and / or homing nucleases, restriction endonucleases, zinc - finger nucleases, meganucleases, Cas endonucleases, TAL effector nucleases, guide nucleases, random site mutagenesis, blind editing, chemical mutagenesis, or radiation mutagenesis. Generally, a double - strand break is generated at or near the target site to be edited, and the double - strand break is repaired by intracellular processes such as non - homologous end joining, homologous recombination, or homology - directed repair. The net effect can be any one or more of the following: insertion of at least one nucleotide, deletion of at least one nucleotide, substitution of at least one nucleotide, chemical alteration of at least one nucleotide. For the editing of the Bacillus - like strains described herein, any technique desired by the practitioner can be used to obtain the final result.

[0433] Example 7: Identification and Storage of Microorganisms

[0434] Sequencing preparation and long - term storage for microorganism identification were performed by the following method:

[0435] Day 1:

[0436] Transfer the colonies from the plate to a flask containing the appropriate liquid growth medium using a 10 uL sterile pipette tip. Place the isolates on a shaker at room temperature and incubate for 2 days.

[0437] Day 3:

[0438] After 2 days on the shaker, the tubes may be turbid. Analyze all samples by PCR. Vortex each tube, collect 50 uL of sample from each vortexed tube, and dispense into a 96-well plate. Using a multi-channel pipette, dispense 15 uL of the 50 uL sample into a new 96-well plate. The 96-well plate containing 35 uL of each sample will be used for phenotyping, and the 96-well plate containing 15 uL of each sample will be used for PCR analysis. The 27F / 1492R primers are commonly used for 16S PCR analysis because they give better results than PB36 / 38. The plate should include appropriate negative controls and these negative controls should be analyzed by PCR. Analyze the plate by PCR using an Eppendorf thermal cycler. Once PCR is complete, run the gel using standard gel electrophoresis techniques. This is important because most of the isolates have grown to a sufficient extent and ideally they should be cryopreserved on day 3. Use PCR and gel electrophoresis analysis to confirm that these isolates contain bacteria and not other microorganisms. For isolates that do not pass PCR or have a clarified culture broth, vortex the tube and streak on a Petri dish using an inoculation loop. Check after a few days to see if anything has grown or if the tube has become contaminated. For isolates that pass PCR, dispense 600 μL of 50% glycerol into a 2 ml screw-cap tube and add 1200 μL of the bacterial culture such that the culture broth is stored in 20% glycerol. Store the glycerol stock at -80 °C and record an image of the gel of the PCR samples.

[0439] Day 4:

[0440] Check the growth of the Petri dish of the streaked isolates that did not pass PCR. (During this time, the 2 ml culture broth tubes will remain on the shaker.) Once there is growth on the plate and the colonies appear to have been successfully isolated, dispense 600 μL of the culture broth-glycerol mixture into small tubes and place both tubes into their respective -80 boxes. Isolates may fail the PCR test for any of the following reasons: the primers may not be effective for all bacteria, the isolate is actually a fungus, the isolate has strong adherence and is therefore not homogenized in the culture broth, the isolate produces too much EPS and therefore needs to be diluted before PCR setup, or the isolate grows very slowly. Over the next few days, continue to check the plate to confirm that only a single bacterial species has been isolated. If contamination is observed, prepare new isolates. The viability of the prepared glycerol stocks should be verified.

[0441] Example 8: Formulation of Microorganisms

[0442] The microorganisms identified according to the previous examples can be formulated with additional components for application via methods such as, but not limited to, the following: seed treatment, root watering, root washing, seedling soaking, foliar application, soil inoculum, in-furrow application, side application, soil pretreatment, wound inoculation, drip tape irrigation, vector-mediated via pollinators, injection, osmopriming, hydroponics, aquaponics, aeroponics. Formulations containing the microorganisms are prepared in the form of liquid, solid, or gaseous preparations for agricultural applications. Application to plants is achieved, for example, in the form of powders for surface deposition onto plant leaves, sprays onto the whole plant or selected plant parts, a portion of the drip solution to the soil or roots, or coatings on plant parts before planting. Such examples are intended to be illustrative and do not limit the scope of the invention.

[0443] The culture medium components for the exemplary microorganism preparation are shown in Tables 7a to 7d. Add all the contents and require a 50% final water volume, and stir the solution at high temperature until dissolved. After all the contents have dissolved, use sterile RO water to make up the solution to the final required volume. Usually, 4x preparations are used for field trial preparation.

[0444] Table 7a: Exemplary culture medium components and concentrations of the microbial preparation

[0445]

[0446] Table 7b: Exemplary culture medium components of the microbial preparation

[0447] Component CAS# Sucrose 57-50-1 Proflo 68308-87-2 Yeast extract 8013-01-2 Tryptone 91079-40-2 MgSO4.7H2O 10034-99-8 KH2PO4 7778-77-0 K2HPO4 7758-11-04 NaNH4HPO4.4H2O 7783-13-3 CaCl2 10043-52-4 MnSO4.1H2O 10034-96-5 ZnSO4.7H2O 7446-20-0 CuSO4.5H2O 7758-99-8 Na2MoO4*2H20 10102-40-6 FeSO4*7H20 7782-63-0

[0448] Table 7c: Exemplary culture medium components of the microbial preparation

[0449] Component CAS# Tryptone 91079-40-2 Soy peptone 91079-46-8 NaCl 7647-14-5 K2HPO4 7758-11-4 Glucose 50-99-7

[0450] Table 7d: Exemplary culture medium components of the microbial preparation

[0451] Component CAS# Trehalose 6138-23-4 Isomaltulose 64519-82-0 Xanthan gum 11138-66-2

[0452] The procedure for the mixed TIX preparation is as follows: Measure all the dry ingredients into a 50 ml tube. Vortex these ingredients well to ensure that xanthan gum is "separated" by other carbon sources. Add approximately half of the total sterile RO water to this mixture and vortex. Use the long end of an L-shaped spreader to break up large chunks as much as possible. Heat some sterile RO water in a microwave to the warm water bath temperature (45 - 50 °C). Add the remaining sterile RO water to the mixture and vortex. Repeat step 4 as needed and vortex until a clear solution without lumps is obtained. Briefly spin down the foam formed during the mixing process by using a centrifuge tube for "quick centrifugation" for 5 to 10 seconds. Remember to balance to counteract the preparation (TIX) tube. Let the preparation cool to room temperature. 1. Mix in a microbial consortium. Vortex to ensure homogeneity. Ideally, add microorganisms at a concentration of 10^9 CFU / ml to this preparation.

[0453] Apply this preparation to a plant or plant part for testing in a field trial.

[0454] Example 9: Applying microorganisms to plant parts and culturing them

[0455] Prepare a microbial composition (comprising one or more isolated microorganisms, consortia, communities, combinations, or any combination of the foregoing of a single strain) according to the previous examples. The microbial composition comprises one or more microorganisms, optionally in combination with one or more additional microorganisms disclosed herein.

[0456] Microbial composition for application

[0457] In some methods, the microbial composition is dried and directly applied to a plant part.

[0458] In some methods, the microbial composition is suspended in a liquid formulation for application to a plant part.

[0459] In some methods, the microbial composition is combined with another composition, such as but not limited to: a carrier, a wetting agent, a stabilizer, a salt. In some methods, this another composition comprises molecules that bring additional agricultural beneficial results to the plants to which the microbial composition is applied. This another composition includes, for example but not limited to: herbicides, fungicides, bactericides, insecticides, insect repellents, nematicides, biostimulants.

[0460] Type of application

[0461] The microbial composition is applied to a plant component at a time during development appropriate for the desired result, such as: in a formulation applied by soil irrigation / furrow application before planting; as a treatment of seeds or other propagation components; as an application to propagation components after planting; as an in-furrow, drip irrigation or irrigation application after planting; as a direct application to a plant component (e.g., roots, leaves, stems); as an application to a harvested plant component (e.g., fruits or grains). Combinations of application types were also tested.

[0462] Method of application

[0463] The microbial composition is applied to (inoculated onto) a plant or plant component or plant product (before planting, after planting, before harvest or after harvest). This can be done, for example, by applying an agricultural composition to a hopper or spreader or tank containing the microbial composition and configured to spread the microbial composition.

[0464] A seed coating of the microbial composition is applied to one or more seeds of a crop plant. After applying the isolated microbe in the form of a seed coating, the seeds are planted and cultivated according to the customary practice established for that crop.

[0465] Alternatively, the microbial composition is applied to the soil to benefit plants present in the soil. Methods of soil application include in-furrow treatment, irrigation and drip irrigation application.

[0466] Alternatively, the microbial composition is applied to the surface of a plant or plant part after germination.

[0467] Alternatively, the microbial composition is applied to materials obtained from plants after harvest.

[0468] Control plots of plants were also planted that were not inoculated with the isolated microbe. Plants associated with the microbial composition exhibited improved properties of interest.

[0469] The application method can be carried out according to any protocol known in the art.

[0470] Diseases or pests can be further inoculated onto plant components, plants or growth media (e.g., soil) according to the purpose of the test.

[0471] An exemplary non-limiting protocol for irrigating tomato plants is given below:

[0472] 1. Ten days after planting, carefully separate the plants so that each treatment group has 6 replicates. The plants are delicate and the leaves can be easily torn off. Ensure that the size and overall appearance of the plants are as uniform as possible (the purpose of thinning is to continue to maintain a uniform plant population). If there are not enough plants in each replicate, transplant. See Step 3 for guidelines on transplanting.

[0473] 2. Start thinning the flowerpots so that each pot has one plant. Remove the smaller plants, i.e., the unhealthy or malformed plants in some aspects. If there are 2 or more healthy plants in each pot, the extra plants can be transplanted into another pot. Use the soil pre-treated by the initial planting or the remaining soil from the flowerpots where the seeds did not germinate.

[0474] 3. Transplanting: If some flowerpots did not germinate, fill them with plants from another container. For this, simply use a scoopula to dig out the extra plants (try to dig out as much of the root mass as possible without disturbing other plants) and place them in the holes dug in the empty flowerpots. Gently press with your fingers to compact the soil around the plants.

[0475] 4. Separate the flowerpots into 6 rows of flowerpots (1 row of flowerpots for each treatment group), and 4 RL98 trays are needed. Once completed, look at all the treatment groups and consider making some flowerpot exchanges to ensure that some treatment groups do not have all the tall plants and other treatment groups have all the tall plants.

[0476] 5. If necessary, change gloves. Label each flowerpot with the Avery labels you prepared. The treatment groups should be labeled as rows of 6 replicates, i.e., 1-1, 1-2, 1-3 to 1-6, etc. This makes it easier to find all the replicates of each treatment group

[0477] 6. Two weeks after planting (about 4 days after thinning and labeling), obtain the treatment agent from the microbiology team; place the tray of prepared plants on the table. Collect combitips, repeater pipettes, and RO water. (Note: A little water should be watered to the plants on the day of treatment)

[0478] 7. Mix the microbial solution by inverting the test tube / container (microbial treatment agent) 2 to 3 times or gently shaking. Set the combitip to dispense 2 ml. Collect the treatment fluid into the combitip and dispense the first step back into the test tube. Ensure that the treatment agent you have corresponds to the row of plants to be treated. Once confirmed, gently dispense 2 ml of the treatment agent onto the surface soil of each flowerpot, close to the stem but avoiding direct contact with the stem and leaves.

[0479] 8. Dispose of the pipette tips and repeat step 6 for all treatments. For the inoculated controls (IC or InoCon) and the untreated controls (UTC), apply RO water instead of the treatment agent. Once all treatment agents have been applied, return the plants to the growth chamber for (optional inoculation), growth, and assessment.

[0480] Visualization of microorganisms associated with plant parts

[0481] Individual microorganisms can be labeled with fluorescent proteins according to methods known in the art. Microscopic image analysis confirmed that the microorganisms disclosed herein are associated with various plant tissues.

[0482] Example 10: In Vitro Testing

[0483] Evaluate root colonization, acetylene reduction activity, biofilm formation, turbidity (OD at 600 nm), oxygen tolerance, and gene expression of wild-type strains and genome-edited strains. Unless otherwise noted, the protocols are performed using methods known in the art.

[0484] ARA and GC-FID of Gram-positive strains

[0485] Ensure that all equipment and materials are sterilized. Before autoclaving, wrap and seal the equipment containers with foil so that they can be opened in the anaerobic chamber transfer box and enter the anaerobic chamber aseptically. Seal the bottle necks with foil before sterilization. A loose "seal" is required to allow gas exchange in the transfer box.

[0486] 1. Streak the isolates from -80°C and incubate at 30°C or 25°C until colonies are observed.

[0487] 2. Spread one plate per isolate and incubate at 25°C or 30°C until a lawn is observed.

[0488] 3. Harvest the plates and balance the OD600 of each isolate to approximately 0.3 to normalize the inoculum.

[0489] 4. Prepare the anaerobic chamber by cleaning the surfaces and sealing the equipment - ensure that the containers allow gas exchange.

[0490] 5. Add 30 mL of NF11 per vial - 3 replicates per isolate.

[0491] 6. Add 150 μL of inoculum per vial, using sterile water to balance to an OD600 of 0.3.

[0492] 7. Pass the vials through the anaerobic chamber and seal under anaerobic conditions with and without nitrogen - include one empty vial (with a foil "cap") to add an anaerobic indicator for QC purposes.

[0493] 8. Place the vial at 30 °C and 200 rpm for 5 hours.

[0494] 9. After working in the fume hood for 5 hours, remove 10% (4 mL) from the headspace of each vial and replace it with the same volume of acetylene gas. Note: Acetylene gas is highly reactive and explosive, so the bag must be kept in the fume hood while working.

[0495] 10. Incubate at 30 °C and 200 rpm for 48 hours.

[0496] 11. After 48 hours (or other known time points), take 1 mL of the headspace sample and place it in a GC collection tube.

[0497] 12. Run the sample in the GC using the instrumental method for ethylene analysis "Split 4" to measure the acetylene peak and ethylene content

[0498] 13. Quantify the gas amount by peak area.

[0499] 14. Take the OD600 reading and the TVC of the 200 μL culture.

[0500] 15. Analyze the ethylene gas as a percentage of acetylene converted to ethylene. This gives an estimate of the total conversion rate.

[0501] The volume of the gas produced (ethylene) can be quantified using the calibration points in Chromeleon or by calculation from the % peak area. The % peak area of acetylene + ethylene must = 100%. Based on the known amount of acetylene added, the ethylene produced can be determined in mL. 1 M of gas = 24 dm3 or 24,000 mL. So 1 mM of gas = 24 mL.

[0502] To calculate how many mM of ethylene are produced, divide the amount by 24: mM ET = mL / 24

[0503] To calculate the rate: mM / hour / CFU, the mM needs to be calculated as described above, and it is necessary to know how much of the headspace was sampled (if using calibration calculations, for example, 1 mL of headspace was sampled with x mM of gas, but there was a total of 6 mL of headspace, so the total ethylene produced = 6x mM). If only using the % peak area calculation, the above steps are not necessary, only the amount of acetylene added needs to be known. The number of hours of incubation with acetylene needs to be known. A TVC needs to be performed to calculate CFU / mL, and then multiply your CFU value by the mL of culture, for example, 4 mL (g negative) or 30 mL (g positive).

[0504] Rate = total ethylene mM / (time (hours) x total CFU)

[0505] ARA and oxygen tolerance test protocol

[0506] Ensure that all equipment and materials are sterilized. Before autoclaving, wrap and seal the equipment containers with foil so that they can be opened in the transfer box of the anaerobic chamber and enter the anaerobic chamber aseptically. Seal the bottle mouths with foil before sterilization. A loose "seal" is required to allow gas exchange in the transfer box.

[0507] 1. Streak the isolate from -80°C and incubate at 30°C or 25°C until colonies are observed.

[0508] 2. Spread one plate / isolate and incubate at 25°C or 30°C until a lawn is observed.

[0509] 3. Harvest the plate and balance the OD600 of each isolate to approximately 0.3 to normalize the inoculum.

[0510] 4. Prepare the anaerobic chamber by cleaning the surface and sealing the equipment - ensure that the containers allow gas exchange.

[0511] 5. After cleaning, place the NF11 medium into the anaerobic chamber. Add 20 g / L of agar to the NF11 and place it on a hot plate. Briefly boil to melt the agar. After the agar has melted, pour 30 mL of warm agar from the side into 70 mL to maximize its surface area to form a slant.

[0512] 6. Add 150 μL of inoculum / vial and balance to an OD600 of 0.3 using sterile water. Try to do this to maximize the surface area exposed to the inoculum.

[0513] 7. Pass the vials through the anaerobic chamber and seal them under anaerobic conditions with and without nitrogen - including an empty vial (with a foil "cap") to add an anaerobic indicator for QC purposes.

[0514] 8. To adjust the oxygen level, after sealing the vials, withdraw some anaerobic air from them using a fine needle syringe and replace it with 100% pure medical - grade oxygen.

[0515] 9. The assay has been run under different oxygen conditions from 0% oxygen to 22% oxygen and can be increased to higher oxygen conditions due to the manual addition of oxygen. For example, to obtain 5% oxygen, manually remove 2.2 mL of anaerobic gas and add 2 mL of 100% pure oxygen under this condition.

[0516] 10. Place the vials in a 30°C incubator for 5 hours.

[0517] 11. After working in the fume hood for 5 hours, 10% (4 mL) was removed from the headspace of each vial and replaced with the same volume of acetylene gas. Note: Acetylene gas is highly reactive and explosive, so the bag must be kept in the fume hood while working.

[0518] 12. Incubate at 30°C and 200 rpm for 48 hours.

[0519] 13. After 48 hours (or other known time points), take 1 mL of the headspace sample and place it in a GC collection tube.

[0520] 14. Run the sample in the GC using the instrumental method for ethylene analysis "Split 4" to measure the acetylene peak and ethylene content

[0521] 15. Quantify the gas amount by peak area.

[0522] 16. Analyze the ethylene gas as a percentage of acetylene converted to ethylene. This gives an estimate of the total conversion rate.

[0523] The volume of the gas produced (ethylene) can be quantified using the calibration points in Chromeleon or by calculation from the % peak area. The % peak area of acetylene + ethylene must = 100%. Based on the known acetylene addition, the ethylene produced can be determined in mL. 1 M gas = 24 dm3 or 24,000 mL. So 1 mM gas = 24 mL. To calculate how many mM of ethylene are produced, divide the amount by 24: mM ET = mL / 24.

[0524] To calculate the rate: mM / hour / CFU, mM needs to be calculated as described above. It is necessary to know how much of the headspace was sampled (if using calibration calculation, for example, 1 mL of headspace was sampled with x mM of gas, but there was a total of 6 mL of headspace, so the total ethylene produced = 6x mM). If only using the % peak area calculation, the above steps are not necessary - only need to know how much acetylene you added. It is necessary to know the number of hours incubated with acetylene. It is necessary to perform a TVC to calculate CFU / mL, and then multiply your CFU value by the mL of culture, for example, 4 mL (g negative) or 30 mL (g positive).

[0525] Rate = total ethylene mM / (time (hours) x total CFU)

[0526] Root colonization

[0527] Using techniques known in the art, a bacterial strain with the GFP gene integrated into its genome was prepared. Seeds were treated with the strain, and using aseptic techniques, the inoculated seeds were dropped into tubes of phytagel. The tubes were placed in a suitable growth chamber and covered for 5 days to allow germination. Root tissues were separated from the seeds and sprouts using EtOH and flame-sterilized forceps and scalpels. The root tissues were cut such that they were all in the same focal plane and pressed at the same level onto 0.8% water-agar in a square plate for imaging. The same was done for the shoot tissues. Fluorescence microscopy was used to image bacterial colonization in the plant tissues.

[0528] Biofilm assay protocol

[0529] The protocol is based on the literature: “Effects of an EPS Biosynthesis Gene Cluster of Paenibacillus polymyxa WLY78 on Biofilm Formation and Nitrogen Fixation under Aerobic Conditions” (Chen 2021). Materials: 3 mL sterile glass tubes, “Biofilm Broth (BFB)” medium, 0.1% crystal violet (aqueous) solution, 40% acetic acid. Optimal overall biofilm results are obtained at 7 days; some isolates may give better results at 5 days and start to degrade after this time point. Prepare using aseptic techniques.

[0530] The formulation of BFB includes: 5 g / L KH2PO4, 5 g / L K2HPO4, 0.86 g / L monosodium glutamate, 0.1 g / L yeast extract, 1 g / L NH4Cl pH 7. After autoclaving, filter sterilize: 36 g / L glucose, 0.03 g / L MgSO4·7H2O, 0.02 g / L CaCl2·2H2O, 1 mL / L trace element solution.

[0531] The steps of the method are:

[0532] 1. Streak the isolates from -80°C.

[0533] 2. Make spread plates for each isolate.

[0534] 3. Autoclave 3 mL glass tubes in a tube rack (x3 / isolate), using foil as the lid

[0535] 4. Harvest the spread plates and balance the OD600 to ~0.3

[0536] 5. Fill each tube with 1 mL of BFB.

[0537] 6. Inoculate with 10 μl / tube of the spread plate harvest.

[0538] 7. Replace the foil lid on the tube and incubate at 30 °C for 7 days, standing still.

[0539] 8. After 7 days, start to take out the culture from the tube using a long (1250 μL) pipette tip - collect the culture in a 2 mL snap-cap tube.

[0540] 9. Add water to the culture to reach a final volume of 1 mL - read the OD600 reading.

[0541] 10. Wash the glass tube with RO water; fill it about half, hold the tube, seal the top and shake to remove the excess cell material. Rinse several times.

[0542] 11. Use a long pipette tip to remove the excess water.

[0543] 12. Add 1 mL / tube of 0.1% crystal violet solution and incubate at room temperature for 10 minutes.

[0544] 13. Pipette the crystal violet solution into a waste container (e.g., 50 mL falcon tube) and place it in the incineration bin.

[0545] 14. Rinse the glass tube until the water runs clear.

[0546] 15. Dry the glass tube (usually overnight).

[0547] 16. Add 1 mL of 40% acetic acid solution to dissolve the stained biofilm ring.

[0548] 17. Take the OD570 reading.

[0549] 18. Normalize OD570 with OD600 (if appropriate).

[0550] Example 11: In-plant testing

[0551] The edited microorganisms described above are tested in at least one plant species in a repeated manner.

[0552] The plants are associated with the wild-type and / or edited microorganisms described above and tested in the greenhouse and large-scale field trials. The association can be achieved by any one or more of the following methods: seed treatment, foliar treatment, in-furrow application, irrigation, side dressing.

[0553] In one example, multiple replicate samples of corn (maize; Zea mays) plants are treated with the microorganisms described herein and allowed to grow for at least 19 days (range 19 - 34 days). Data collected includes biomass, leaf area, plant height, root area, shoot nitrogen, greenness, NDVI (captures how much near-infrared light is reflected compared to visible red light; a measure of plant health based on how a plant reflects specific frequencies of light), NPCI (normalized pigment chlorophyll ratio index), PSRI (plant senescence reflectance index), and CCI (chlorophyll content index), and is compared to untreated controls.

Claims

1. A method for regulating nitrogen fixation in bacteria, the method comprising: a. obtaining bacteria, b. identifying the nif cluster region within the bacteria, c. identifying at least one intergenic region within the nif cluster, d. modifying at least one intergenic region downstream of the nifB gene in the cluster, and e. assessing the regulated nitrogen fixation activity of the bacteria.

2. The method according to claim 1, wherein the modification in step (d) is modifying the intergenic region between nifH and nifD.

3. The method according to claim 1, wherein the modification in step (d) is modifying the intergenic region between nifK and nifE.

4. The method according to claim 1, wherein the modification in step (d) is modifying the intergenic region upstream of hesA.

5. The method according to any one of claims 2 to 4, wherein the modification is inserting at least one heterologous promoter.

6. The method according to any one of claims 2 to 4, wherein the modification is inserting at least one heterologous promoter, wherein the heterologous promoter is inducible.

7. The method according to any one of claims 2 to 4, wherein the modification is inserting a GlnR binding site sequence.

8. The method according to any one of claims 2 to 4, wherein the modification is replacing at least one native sequence.

9. The method according to any one of claims 2 to 4, wherein the modification is deleting at least one native sequence.

10. The method according to any one of claims 2 to 4, wherein the modification is selected from the group consisting of modification, insertion, replacement, molecular alteration, chemical alteration, deletion, any combination of the foregoing, and any plurality of the foregoing.

11. The method according to any one of the preceding claims, wherein the regulation is an increase in the nitrogen fixation ability of the bacteria.

12. The method according to any one of the preceding claims, wherein the regulation is a decrease in the nitrogen fixation ability of the bacteria.

13. The method according to any one of the preceding claims, wherein the bacteria is a Gram-positive bacterium.

14. The method according to any one of the preceding claims, wherein the bacteria is a Gram-negative bacterium.

15. An artificial nif cluster sequence, wherein, compared to the wild-type sequence, the nif cluster sequence comprises a modification in at least one intergenic region downstream of the nifB gene.

16. A bacterium comprising the artificial nif cluster according to claim 15.

17. The bacterium according to claim 16, wherein the bacterium does not contain a nif cluster in its natural state.

18. The bacterium according to claim 16, wherein the bacterium contains an unmodified nif cluster in its natural state.

19. The bacterium according to claim 18, wherein the unmodified nif cluster is replaced by a modified nif cluster.

20. A synthetic combination of a plant or a plant component and a bacterium, wherein the bacterium comprises a modification in at least one intergenic region of the nif cluster located downstream of the nifB gene.

21. An artificial polynucleotide sequence comprising a polynucleotide sequence from at least one intergenic region of a nif cluster operably linked to a heterologous sequence.

22. A method of a composition according to any one of the preceding claims, wherein the nif cluster is identified from, obtained from, and / or derived from a Paenibacillus bacterium.

23. A synthetic composition comprising a plant component and a bacterium heterologously disposed with respect to the plant component, wherein the bacterium comprises an edit in one or more loci of its genome; wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide, and / or a substitution of at least one nucleotide in an intergenic region between two nif cluster components; or any combination of edits or multiple edits at any one or more of the genomic loci; wherein the bacterium exhibits an improved phenotype as compared to a bacterium not comprising the edit, wherein the improved phenotype is selected from the group consisting of: increased acetylene reduction ability, improved nitrogen fixation ability, improved biofilm formation, increased culture turbidity, stronger nitrogen fixation tolerance to oxygen levels, improved nitrogen fixation under higher nitrogen conditions, improved nitrogen fixation under lower nitrogen conditions, and any plurality and / or combination of the foregoing.

24. The synthetic composition according to claim 23, wherein the edit is made at a locus selected from the group consisting of: between nifH and nifD, between nifK and nifE, between orf1 and hesA, between nifX and hesA, and any plurality and / or combination of the foregoing.

25. The synthetic composition according to claim 23, wherein the bacterium belongs to the genus Paenibacillus.

26. The synthetic composition according to claim 23, wherein the bacterium comprises a sequence selected from the group consisting of SEQ ID NO: 1-501.

27. The synthetic composition according to claim 23, further comprising formulation components and / or agricultural compositions.

28. The synthetic composition according to claim 23, wherein the Paenibacillus bacterium is present in a liquid formulation at a concentration of at least about 10^2 CFU / mL, or in a non-liquid formulation at a concentration of at least about 10^2 CFU / gram.

29. The synthetic composition according to claim 23, further comprising at least one additional microorganism.

30. The synthetic composition according to claim 23, wherein the plant component is a seed.

31. The synthetic composition according to claim 23, wherein the plant component is a seed comprising a transgene.

32. The synthetic composition according to claim 23, wherein the plant component is obtained from vegetative tissue.

33. The synthetic composition according to claim 23, wherein the plant component is a plant propagation component.

34. The synthetic composition according to claim 23, wherein the plant component is a whole plant.

35. The synthetic composition according to claim 23, wherein the formulation components are selected from the group consisting of: compounds that enhance the stability of the microorganism, preservatives, carriers, surfactants, anti-compound agents, and any plurality and / or combination thereof.

36. The synthetic composition according to claim 23, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, an insecticide, a herbicide, micronutrients, macronutrients, nitrogen, phosphorus, potassium, or any plurality and / or combination of the foregoing.

37. A plurality of synthetic compositions according to claim 23, wherein the synthetic compositions are substantially enclosed within an item selected from the group consisting of: tubes, bottles, jars, ampoules, packages, vessels, bags, boxes, storage tanks, envelopes, cartons, containers, silos, shipping containers, carriages, and cases.

38. A plurality of synthetic compositions according to claim 37, wherein the synthetic compositions are at a temperature below zero degrees Celsius.

39. The synthetic composition according to claim 23, wherein the plant part is obtained from a monocotyledonous plant.

40. The synthetic composition according to claim 39, wherein the monocotyledonous plant is a C3 monocotyledonous plant.

41. The synthetic composition according to claim 39, wherein the monocotyledonous plant is a C4 monocotyledonous plant.

42. The synthetic composition according to claim 23, wherein the plant part is obtained from a dicotyledonous plant.

43. The synthetic composition according to claim 23, wherein the agricultural composition comprises a growth medium.

44. The synthetic composition according to claim 43, wherein the growth medium comprises soil.

45. A plurality of synthetic compositions according to claim 23, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between the respective synthetic compositions.

46. A plant obtained, grown, or derived from the synthetic composition according to claim 23, wherein the plant comprises the bacterium.

47. The plant according to claim 46, wherein the plant exhibits improved agronomically important traits compared to a plant that is not obtained, grown, or derived from the synthetic composition according to claim 23.

48. The plant according to claim 47, wherein the improved agronomically important traits are yield, plant health, improved nitrogen utilization, and / or plant vigor.

49. A method of improving the health, yield, and / or vigor of a plant, the method comprising: a. associating a plant part with a bacterium having an edit in one or more loci of its genome; wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide, and / or a substitution of at least one nucleotide at or near one or more of the following genomic loci: between nifH and nifD, between nifK and nifE, between orf1 and hesA, between nifX and hesA, or any combination of edits or multiple edits at any one or more of the genomic loci; b. placing the plant part of the crop plant in a culture medium that supports plant growth; c. growing a plant from the plant part of the crop plant; d. evaluating one or more characteristics of the plant, wherein at least one of the characteristics is improved compared to the same characteristic of a plant obtained from a plant part not associated with the bacterium of (a).

50. The method according to claim 49, wherein the one or more characteristics of (d) include an improvement in nitrogen fixation, an increase in biomass, an increase in leaf area, an increase in plant height, an increase in root area, an increase in shoot nitrogen composition, an increase in greenness, an increase in NDVI, an increase in NPCI, an increase in PSRI, an increase in CCI, an increase in yield, and any combination of the foregoing.

51. The method according to claim 49, further comprising at least one additional microorganism.

52. The method according to claim 49, wherein associating the plant part of the crop plant with a bacterium having an edit in one or more loci of its genome is achieved by a method selected from the group consisting of in-furrow application, soil drench application, side-dress application, and any combination of the foregoing.

53. The method according to claim 49, wherein associating the plant part of the crop plant with a bacterium having an edit in one or more loci of its genome is achieved by coating the plant part with a liquid formulation of the bacterium.

54. The method according to claim 49, wherein associating the plant part of the crop plant with a bacterium having an edit in one or more loci of its genome is achieved by coating the plant part with a substantially non-liquid formulation of the bacterium.

55. The method according to claim 49, wherein the plant part is a plant reproductive part.

56. The method according to claim 49, wherein the plant part is a leaf.

57. The method according to claim 49, wherein the plant part is a whole plant.

58. A modified Paenibacillus bacterium, wherein the Paenibacillus bacterium contains edits in one or more loci of its genome, and wherein the edits are deletions of at least one nucleotide, insertions of at least one nucleotide, and / or substitutions of at least one nucleotide at or near one or more of the following genomic loci: between nifH and nifD, between nifK and nifE, between orf1 and hesA, between nifX and hesA, or any combination of edits at any one or more of the genomic loci or multiple edits.

59. The modified Paenibacillus bacterium according to claim 58, wherein the Paenibacillus bacterium exhibits an improved phenotype compared to a Paenibacillus bacterium that does not contain the edits, and wherein the improved phenotype is selected from the group consisting of: increased acetylene reduction ability, improved biofilm formation, increased culture turbidity, stronger nitrogen fixation tolerance to oxygen levels, and any combination of the foregoing.

60. The modified Paenibacillus bacterium according to claim 58, wherein the Paenibacillus bacterium contains a sequence selected from the group consisting of SEQ ID NO: 1-123.

61. A substantially pure composition comprising the modified Paenibacillus bacterium according to claim 58.

62. A bacterial culture comprising the modified Paenibacillus bacterium according to claim 58.

63. A fermentation culture comprising the modified Paenibacillus bacterium according to claim 58.

64. An agricultural composition comprising the modified Paenibacillus bacterium according to claim 58 and an agriculturally acceptable carrier.

65. The agricultural composition according to claim 64, further comprising a plant or a plant part, wherein the modified Paenibacillus bacterium is present in the agricultural composition in an amount effective to produce an improved phenotype in the plant.

66. The agricultural composition according to claim 65, wherein the improved phenotype is an increase in the health, yield, and / or vigor of the plant.

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