Improved diazotrophic microorganisms for use in agriculture.

Abstract

The present disclosure relates to genetically modified microorganisms for improving plant phenotypes, such as nitrogen availability in legume plants. Novel microbial strains, microbial consortia, and agricultural compositions containing the same are included. Additionally, the present disclosure teaches methods for utilizing the described microorganisms, microbial consortia, and agricultural compositions containing the same in methods for imparting beneficial characteristics to target plant species. In certain aspects, the present disclosure provides methods for increasing desirable plant traits in agriculturally important species, such as plant nitrogen fixation, utilization, regulation, uptake, acquisition, tolerance, and / or treatment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 489,771, filed March 12, 2023, which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing An official copy of the Sequence Listing has been submitted electronically as a WIPO ST26 compliant XML Sequence Listing with the filename 23031_SequenceListing.xml, created on February 12, 2024, and having a size of 51,695 bytes, and is being filed herewith. The Sequence Listing contained in this document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to isolated, biologically pure microorganisms, particularly for agricultural applications. The disclosed microorganisms can be utilized in their isolated, biologically pure state or can be formulated into agriculturally acceptable compositions. Methods of using the isolated microorganisms or agriculturally acceptable compositions in agricultural applications are also disclosed. [Background technology]

[0004] According to the United Nations World Food Programme, there are nearly 900 million malnourished people in the world. The prevalence of malnutrition is particularly pronounced in the world's developing countries, where one in six children is underweight. The lack of available food can be attributed to many socioeconomic factors. However, whatever the ultimate cause, the fact remains that there is insufficient food to feed the growing world population, which is expected to reach 9 billion by 2035. The United Nations estimates that agricultural yields will have to increase by 70 to 100 percent to feed the projected world population in 2050.

[0005] These staggering global population and malnutrition figures highlight the importance of agricultural efficiency and productivity to sustain a growing world population. The technological advances achieved by modern row-crop agriculture are impressive, resulting in crop yields never before seen. However, despite advances made through technological innovations such as genetically modified crops and new novel insecticidal and herbicidal compounds, there remains a need for improved crop yields to meet the demands of an exponentially growing world population.

[0006] Scientists estimate that global crop production could increase by 45–70% if the world's agricultural "yield gap"—the difference between the most commonly observed yields and results elsewhere—could be closed. In other words, if all farmers, regardless of location, could achieve the highest yields expected in their region, a large portion of the world's food production shortfall could be addressed. However, the problem of how to achieve higher yields in different parts of the world is difficult.

[0007] In many cases, yield gaps can be explained by insufficient water, substandard agricultural practices, inadequate fertilizer, or the failure to use herbicides or pesticides. However, significantly increasing global use of water, fertilizer, herbicides, and pesticides would not only be economically unfeasible for most of the world, but would also have adverse environmental effects.

[0008] Therefore, it is simply not feasible to meet agricultural production expectations by simply scaling up the current high-input agricultural systems used in most developed countries.

[0009] Thus, there is an urgent need in the art for improved methods for increasing crop yields and imparting beneficial traits to desired plant species.

[0010] The technology described herein is, for example, environmental nitrogen-fixing bacteria that are gene-edited using scarless homologous recombination techniques to increase the amount of fixed atmospheric nitrogen.

[0011] These gene-edited bacterial strains confer improved phenotypes to plants, such as legume crops, increasing the amount of nitrogen available to the plant and increasing final yield. Summary of the Invention

[0012] Included are isolated, biologically pure microorganisms, particularly those applicable to agriculture. The disclosed microorganisms can be utilized in their isolated, biologically pure state or formulated into agriculturally acceptable compositions. Additionally, agriculturally beneficial microbial consortia comprising at least two of the disclosed microorganisms, as well as methods for utilizing the consortia in agricultural applications, are provided. In some embodiments, genomic modification of microorganisms (individuals, consortia, and / or communities) is contemplated for the improvement of microbial traits and the improvement of microorganism-associated plants.

[0013] Herein, we present successful genome-edited strains of the Gram-positive spore-forming bacterium Paenibacillus across several different species. Several different edits were evaluated in several different strains.

[0014] The present disclosure addresses this important problem of how to improve plant performance, thereby closing the global yield gap and providing methods for imparting other beneficial traits to plant species. The novel genome-edited strains of Paenibacillus described herein improve plant performance by enabling increases and / or improvements in plant nitrogen availability, fixation, uptake, acquisition, tolerance, distribution, regulation, processing, and / or any multiple and / or combination of any of the foregoing.

[0015] In some embodiments, the plant is a dicotyledonous plant. In some embodiments, the plant is a legume crop plant. In some embodiments, the plant is soybean.

[0016] The solutions provided by the present disclosure to increasing crop yields and productivity are not detrimental to global resources because they do not rely on increased water consumption or increased synthetic chemical inputs to production systems. Rather, the present disclosure utilizes microorganisms to impart beneficial traits, including increased yield, to desirable plants.

[0017] Thus, the present disclosure provides an environmentally sustainable solution that enables farmers to increase yields of important crops and does not rely on increased use of synthetic herbicides and pesticides.

[0018] In embodiments, the present disclosure provides an efficient and broadly applicable agricultural platform that utilizes microorganisms and microbial consortia (multiple microorganisms, in some aspects multiple microorganisms that improve the health or desirable phenotype of the plant, such as an agronomic trait, with which it is associated) that promote one or more desirable plant characteristics.

[0019] The microorganisms disclosed herein improve plant yield, such as in crop plants, through both direct and indirect mechanisms. In some embodiments, the microorganisms are symbiotic with the plant. In some embodiments, the microorganisms produce compounds (e.g., metabolites, toxins, proteins, lipopeptides, or other compositions) that confer a benefit to the plant or that the plant can use for improved characteristics. In some embodiments, the microorganisms improve the solubility of one or more compositions, such as nutrients, thereby benefiting the plant. In some embodiments, the microorganisms confer resistance to exogenous substances, such as herbicides or insecticides, on the plant. In some embodiments, the microorganisms produce compositions that are harmful to plant pests, such as insects. In some embodiments, the microorganisms fix nitrogen, thereby improving the nutritional status of the plant. Other embodiments beyond the exemplary, non-limiting embodiments listed above are also contemplated.

[0020] 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 bacterial species. In some aspects, the single microorganism is an identifiable strain of a taxonomic bacterial species. In some aspects, the single microorganism is a newly discovered novel strain of a taxonomic bacterial species.

[0021] In some embodiments, the single microorganism, whether a taxonomically identifiable species or a taxonomically identifiable strain, is mixed with one or more other microorganisms of different species or strains. In certain embodiments, the combination of two or more microorganisms forms a consortia or consortium. The terms consortia and consortium are used interchangeably.

[0022] In certain aspects, the present disclosure provides for the development of highly functional microbial consortia that are useful for promoting the development and expression of desirable phenotypic or genotypic plant traits. In some embodiments, the consortia of the present disclosure possess functional properties not found in nature when individual microorganisms live alone. That is, in various embodiments, mixing specific microbial species into a consortium results in a mixture of microorganisms that possess functional properties not possessed by any single member of the consortium when viewed individually.

[0023] In some embodiments, the functional property possessed by the microbial consortium is the ability to confer one or more beneficial properties to a plant species, such as increased growth, increased yield, increased nutrient utilization (e.g., nitrogen, phosphate, etc.), improved nitrogen use efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, enhanced water use efficiency, increased pathogen resistance, modifications to plant architecture that do not necessarily affect plant yield, but rather address plant functionality, etc. Additionally, beneficial properties of pest resistance and / or tolerance, including adverse effects on nematodes, insects, or other pests, are also contemplated.

[0024] In some embodiments, these individual microorganisms do not possess the ability to confer these beneficial properties to plants when they exist in nature. Rather, in some embodiments, humans combine these microorganisms into consortia to develop functional compositions that possess attributes and functional properties not found in nature. In some embodiments, the consortia may include microorganisms that have been gene-edited, engineered, or modified through modifications of cellular compositions, including DNA, RNA, proteins, and / or combinations thereof, via techniques known to those of skill in the art.

[0025] However, in other embodiments, the present disclosure provides isolated and biologically pure individual microorganisms that can impart beneficial traits to desired plant species and do not require mixing into a consortium.

[0026] In some embodiments, the microorganism is a strain of the genus Paenibacillus that has been genetically modified to improve its nitrogen fixation capacity.

[0027] Thus, the present disclosure provides environmentally sustainable solutions that enable 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 improve the microorganism's ability to fix nitrogen. In some embodiments, the endogenous nif gene of the isolated microorganism is genetically modified to improve the microorganism's ability to fix nitrogen. In some embodiments, the endogenous glnR gene encoding the protein GlnR is genetically modified to improve the microorganism's ability to fix nitrogen.

[0028] In some embodiments, the present disclosure relates to an isolated genetically modified microorganism comprising one or more genetic modifications selected from a genetic modification to an endogenous glnR gene encoding GlnR and a genetic modification to a regulatory sequence of an endogenous nif gene. The genetic modification to the endogenous glnR gene encoding GlnR in the genetically modified microorganism is characterized by providing a mutant glnR gene that produces a GlnR protein variant. Further, the genetic modification to the regulatory sequence within the endogenous nif gene is characterized by providing improved binding affinity for GlnR compared to the regulatory sequence that is not genetically modified. The one or more genetic modifications are characterized by providing the genetically modified microorganism with improved nitrogen fixation activity compared to a microbial strain that is not genetically modified.

[0029] In some embodiments, the isolated genetically modified microorganisms described herein are characterized by constitutive expression of nif genes, regardless of the local nitrogen concentration in the environment surrounding the microorganism. For example, in some embodiments, the isolated genetically modified microorganisms described herein are characterized by constitutive expression of nif genes under nitrogen-limiting conditions. In some embodiments, the isolated genetically modified microorganisms described herein are characterized by constitutive expression of nif genes under nitrogen-replete conditions.

[0030] The present disclosure also relates to a process for preparing an isolated genetically modified microorganism, the process comprising genetically modifying an endogenous glnR gene encoding GlnR, genetically modifying a regulatory sequence within an endogenous nif gene, or a combination thereof, and isolating the microorganism. Genetically modifying the endogenous glnR gene encoding GlnR comprises editing the endogenous glnR gene to produce a mutant gene encoding a GlnR protein variant. Genetically modifying the regulatory sequence within the endogenous nif gene comprises replacing the regulatory sequence with a DNA sequence characterized by providing improved binding affinity for GlnR compared to the native regulatory sequence. Additionally, the microorganism is characterized by having nitrogen fixation activity, and genetic modification of the endogenous glnR gene encoding GlnR and / or genetic modification of the regulatory sequence within the endogenous nif gene provides improved nitrogen fixation activity compared to an unmodified microbial strain.

[0031] The present disclosure further relates to agricultural compositions comprising one or more strains of the isolated genetically modified microorganisms disclosed herein and an agriculturally acceptable carrier. In some embodiments, the agricultural compositions comprise one or more additional agriculturally beneficial agents (e.g., fertilizers, biofertilizers, bionematicides, biostimulants, synthetic insecticides, and / or synthetic herbicides).

[0032] Also disclosed herein are methods for imparting one or more beneficial traits to a plant, the methods comprising applying an agriculturally effective amount of one or more of the isolated genetically modified microorganisms or agricultural compositions disclosed herein.

[0033] In some embodiments, the Paenibacillus strain is listed in Table 1a, Table 1b, or Table 1c. In some embodiments, the Paenibacillus strain comprises a polynucleotide sequence sharing at least 90% identity with any one or more of SEQ ID NOs: 1-12. In some embodiments, the Paenibacillus strain is a species selected from the group consisting of polymyxa, tritici, albidus, anaericanus, azotifigens, borealis, donghaensis, ehimensis, graminis, jilunlii, odorifer, panacisoli, phoenicis, pocheonensis, rhizoplanae, silage, taohuashanense, thermophilus, typhae, and wynnii. In some embodiments, the Paenibacillus strain is of subgroup I. In some embodiments, the Paenibacillus strain is of subgroup II.

[0034] Any strain disclosed herein can be further combined with one or more additional microorganisms to form a microbial consortium. The microbial consortium can be any combination of one or more individual microorganisms. In certain embodiments, the microbial consortium comprises two, three, four, five, six, seven, eight, nine, ten, or more than ten microorganisms.

[0035] Another objective of the present disclosure is to design a microbial consortium that can perform multidimensional activities in the same way. In certain embodiments, the microorganisms that make up the consortium act synergistically. In certain embodiments, the effect that the microbial consortium has on a particular plant trait is greater than the effect observed when any one specific microbial member of the consortium is used alone. That is, in some embodiments, the consortium exhibits an effect on a desired plant trait that is greater than the sum of the effects seen when any one specific member of the consortium is used alone.

[0036] In some embodiments, the consortia establish other plant-microbe interactions, for example, by serving as primary colonizers or founder populations that set the trajectory for future microbiome development.

[0037] In embodiments, the present disclosure is directed to synergistic combinations (or mixtures) of microbial isolates.

[0038] In some aspects, the wild-type microorganisms, gene-edited microorganisms, and / or consortia taught herein provide a wide range of agricultural uses, including improved grain, fruit, and flower yields, improved growth of plant parts, and improved ability to utilize nutrients (e.g., nitrogen, phosphate, and the like). Significantly, these benefits to the plant can be obtained without any adverse side effects to the environment.

[0039] In some aspects, individual microorganisms or consortia comprising microorganisms of the present disclosure can be combined into agriculturally acceptable compositions.

[0040] In some embodiments, agricultural compositions of the present disclosure include, but are not limited to, wetting agents, compatibilizers, defoamers, detergents, sequestering agents, drift reducing agents, neutralizing agents, buffers, corrosion inhibitors, dyes, odorants, spreading agents, penetration aids, tackifiers, binders, dispersants, thickeners, stabilizers, emulsifiers, freezing point depressants, antimicrobials, fertilizers, pesticides, nematicides, insecticides, herbicides, inert carriers, polymers, and the like.

[0041] In one embodiment of the present disclosure, the microorganisms (including isolated single species, or strains, consortia, or compositions thereof, such as metabolites) are provided to seeds in the form of a seed coating or other application. In embodiments, the seed coating may be applied to bare, untreated seeds. In other embodiments, the seed coating may be applied to previously treated seeds. Thus, in some embodiments, the present disclosure teaches a method of treating seeds comprising applying an isolated bacterial strain or microbial consortium to seeds. In certain embodiments, the isolated bacterial strain or microbial consortium is applied as an agricultural composition comprising an agriculturally acceptable carrier. In some embodiments, the agricultural composition may be formulated as a soil drench, foliar spray, dip treatment, in-furrow treatment, soil amendment, granule, broad-spectrum treatment, post-harvest disease control treatment, or seed treatment. In some embodiments, the agricultural composition may be applied alone or in a rotational application program with other agricultural products. In some embodiments, the agricultural composition may be compatible with tank-mixing. In some embodiments, the agricultural composition may be compatible with tank-mixing with other agricultural products. In some embodiments, the agricultural compositions may be compatible with equipment used in ground, aerial, and irrigation applications.

[0042] In some embodiments, the applied microorganisms may be endophytic, resulting in their presence on the treated growing plants and their progeny, while in other embodiments, the microorganisms may be applied simultaneously as a co-treatment with the seed treatment.

[0043] In one embodiment of the present disclosure, the microorganisms are provided in the form of granules, plugs, or soil drench applied to the plant growth medium. In other embodiments, the microorganisms are provided in the form of a foliar treatment, such as a foliar spray composition or a foliar liquid composition. The foliar spray or liquid treatment may be applied to the growing plant or to the growth medium, such as soil.

[0044] In other embodiments, the microorganisms (including isolated single species, or strains, or consortia, or compositions thereof, such as metabolites) are provided as fertilizers, pesticides, or other amendments that can be applied to the soil. In some embodiments, the microorganisms are provided as fertilizers, pesticides, or other amendments that are applied to the soil prior to planting. In some embodiments, the microorganisms are provided as fertilizers, pesticides, or other amendments that are applied to the soil simultaneously with planting. In some embodiments, the microorganisms are provided as fertilizers, pesticides, or other amendments that are applied to the soil after planting.

[0045] In other embodiments of the present disclosure, the microorganisms (including isolated single species or strains, or consortia) and / or compositions thereof (e.g., metabolites) are provided in the form of a post-harvest disease control agent.

[0046] In embodiments, the agricultural compositions of the present disclosure may be formulated as, inter alia, (1) solutions, (2) wettable powders, (3) dusts, (4) soluble powders, (5) emulsifiable concentrates or suspensions, (6) seed dressings, (7) tablets, (8) water-dispersible granules, (9) water-soluble granules (slow-release or fast-release), (10) microencapsulated granules or suspensions, (11) irrigation components, and (12) components of fertilizers, pesticides, and other compatibility improvers. In certain aspects, the compositions may be diluted in an aqueous medium before conventional spray applications. The compositions of the present disclosure may be applied to soil, plants, seeds, the rhizosphere, rhizosheath, or other areas where applying a microbial composition is beneficial.

[0047] Yet another object of the present disclosure relates to agricultural compositions formulated to provide a bacterial population or consortium with a high colony forming unit (CFU). In some aspects, these agricultural compositions have an adjuvant that provides adequate shelf life. In embodiments, the CFU concentration of the agricultural compositions of the present teachings is higher than the concentration of the microorganisms present in nature, i.e., outside of the methods of the present disclosure. In another embodiment, the agricultural composition includes microbial cells at a concentration of 10^2 to 10^12 CFU per gram of carrier or 10^5 to 10^9 CFU per gram of carrier. In one aspect, the microbial cells are applied directly to seeds as a seed coating at a concentration of 10^5 to 10^9 CFU. In another aspect, the microbial cells are applied over another seed coating as a seed overcoating at a concentration of 10^5 to 10^9 CFU. In another aspect, the microbial cells are applied as a co-treatment with another seed treatment at a rate of 10^5 to 10^9 CFU.

[0048] In an aspect, the present disclosure is directed to agricultural microbial formulations that promote plant growth. In an aspect, the present disclosure provides isolated microorganisms of the present teachings and consortia comprising the same, formulated as agricultural bioinoculants. The bioinoculants of the teachings can be applied to plants, seeds, or soil, or can be combined with fertilizers, pesticides, and other compatibility improvers. Suitable examples of bioinoculant formulations comprising isolated microorganisms can be found in U.S. Patent No. 7,097,830, incorporated herein by reference.

[0049] The disclosed microbial formulations can reduce the need for nitrogen-containing fertilizers, solubilize minerals, provide bio-pesticide 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, thus reducing and eliminating the need to use chemical pesticides and fertilizers.

[0050] In some embodiments, the isolated, biologically pure microorganisms of the present disclosure may be utilized in methods to impart one or more beneficial properties or traits to a desired plant species.

[0051] In some embodiments, agriculturally acceptable compositions containing isolated biologically pure microorganisms of the present disclosure can be utilized in methods to impart one or more beneficial properties or traits to desired plant species.

[0052] In some embodiments, the consortia of the present disclosure may be utilized in methods to impart one or more beneficial properties or traits to a desired plant species.

[0053] In some embodiments, agriculturally acceptable compositions containing the consortia of the present disclosure may be utilized in methods to impart one or more beneficial properties or traits to desired plant species.

[0054] In some embodiments, the isolated, biologically pure microorganisms of the present disclosure and / or consortia of the present disclosure are derived from an accelerated microbial selection process ("AMS" process). The AMS process utilized in some embodiments of the present disclosure is described, for example, in (1) International Patent Application No. PCT / NZ2012 / 000041, published on September 20, 2012 as WO 2012125050A1, and (2) International Patent Application No. PCT / NZ2013 / 000171, published on March 27, 2014 as WO 2014046553A1, each of which is incorporated herein by reference in its entirety for all purposes.

[0055] However, in other embodiments, the microorganisms of the present disclosure are not derived from an accelerated microbial selection process. In some aspects, the microorganisms utilized in embodiments of the present disclosure are selected from among members of the microorganisms present in the database. In certain aspects, the microorganisms utilized in embodiments of the present disclosure are selected from microorganisms present in the database based on specific characteristics of the microorganism.

[0056] The present disclosure provides that plant elements or plant parts can be effectively enhanced by coating the plant elements or plant parts with an amount of isolated microorganisms or microbial consortia not normally found in the plant elements or plant parts.

[0057] Some embodiments described herein are methods for preparing agricultural seed compositions or seed coatings, comprising contacting the surface of a seed with a formulation comprising a purified population of microorganisms, the purified population comprising at least one isolated microorganism that is heterologous to the seed or that is rarely present on the seed. Further embodiments involve preparing agricultural plant compositions, comprising contacting the surface of a plant with a formulation comprising a purified population of microorganisms, the purified population comprising at least one isolated microorganism that is heterologous to the plant. In other aspects, the formulation or microorganism is introduced into the interior of the seed, for example, into other seed tissues such as cotyledons or embryos.

[0058] In some aspects, applying the isolated microorganisms, microbial consortia, exudates, metabolites, and / or agricultural compositions of the present disclosure to seeds or plants modulates 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, improved water use efficiency, improved nitrogen utilization, improved tolerance to nitrogen stress, improved nitrogen fixation, improved nutrient utilization (e.g., phosphate, potassium, etc.), pest and disease resistance, herbivore resistance, pathogen resistance, reduced pathogen levels (e.g., via excretion of metabolites that impair pathogen survival), increased yield, increased yield under water-limited conditions, improved health, improved vigor, improved growth, improved photosynthetic capacity, nutritional enhancement, modified protein content, modified oil content, increased biomass, increased shoot length, increased root length, improved root architecture, increased seed weight, promoted seed germination, altered seed carbohydrate composition, altered seed oil composition, pod number, delayed senescence, greening, and altered seed protein composition. In some embodiments, at least two, three, four, or more agronomically important traits are modulated. In some embodiments, the modulation is a positive effect on one of the aforementioned agronomic traits.

[0059] In some embodiments, relative to a reference plant, the genetically modified plant exhibits an increase in oil content, an increase in protein content, an increase in seed carbohydrate composition, an increase in seed oil composition, an increase in seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, panicle weight, improved growth, improved health, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved root architecture, improved 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 mass, grain moisture content, metal tolerance, number of panicles, number of kernels per panicle, number of pods, nutritional enhancement, pathogen The isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to regulate or modify plant characteristics such as resistance, pest and disease resistance, improved photosynthetic capacity, salt tolerance, greening, improved plant vigor, increased mature seed dry weight, increased mature seed fresh weight, 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.

[0060] In some embodiments, the agricultural formulations taught herein comprise at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antimicrobial, a herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

[0061] The methods described herein can include contacting a seed or 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 a microorganism taught herein.

[0062] The methods described herein can include contacting a seed or plant with a composition comprising a metabolite produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method includes contacting a seed or plant with a composition comprising at least 1 mg of a metabolite produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method includes contacting a seed or plant with a composition comprising at least 10 mg of a metabolite produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method includes contacting a seed or plant with a composition comprising at least 100 mg of a metabolite produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method includes contacting a seed or plant with a composition comprising at least 1 g of a metabolite produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method includes contacting a seed or plant with a composition comprising at least 10 g of a metabolite produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method comprises contacting the seed or plant with a composition comprising at least 100 g of metabolites produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method comprises contacting the seed or plant with a composition comprising at least 1 kg of metabolites produced by a single microorganism or a microbial consortium disclosed herein. In some embodiments, the method comprises contacting the seed or plant with a composition comprising more than 1 kg of metabolites produced by a single microorganism or a microbial consortium disclosed herein.

[0063] In some embodiments of the methods described herein, the isolated microorganism of the present disclosure is present in the formulation in an amount effective to be detectable in and / or on a target tissue of a crop plant. For example, the microorganism is detected in and / or on a target tissue of a plant in an amount of at least 100 CFUs or spores, at least 300 CFUs or spores, at least 1,000 CFUs or spores, at least 3,000 CFUs or spores, at least 10,000 CFUs or spores, at least 30,000 CFUs or spores, at least 100,000 CFUs or spores, at least 300,000 CFUs or spores, at least 1,000,000 CFUs or spores, or more. Alternatively, or additionally, the microorganisms of the present disclosure may be present in the formulation in an amount effective to increase the biomass and / or yield of a plant to which a formulation of the present disclosure has been 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, as compared to a reference crop plant to which a 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 effective to detectably modulate a desired agronomic trait of a plant to which the formulation of the present disclosure has been applied by at least 1% or more, 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, relative to a reference crop plant to which the formulation of the present disclosure has not been applied.

[0064] In some embodiments of the methods described herein, one or more metabolites isolated from a microorganism or consortium of the present disclosure are present in the formulation in an amount effective to be detectable in and / or on a target tissue of a crop plant, e.g., the metabolite is detected in and / or on a target tissue of a plant in an amount of 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. Alternatively, or additionally, metabolites isolated from the microorganisms and consortia of the present disclosure may be present in the formulation in an amount effective to increase the biomass and / or yield of a plant to which a formulation of the present disclosure has been 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, as compared to a reference crop plant to which a formulation of the present disclosure has not been applied. Alternatively, or additionally, metabolites isolated from the microorganisms and consortia of the present disclosure may be present in the formulation in an amount effective to detectably modulate an agronomic trait of interest in a plant to which the formulation of the present disclosure has been applied by at least 1% or more, 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, as compared to a reference crop plant to which the formulation of the present disclosure has not been applied.

[0065] 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 in a liquid formulation. In some aspects, the microorganisms taught herein are present on a granule.

[0066] Also described herein are a plurality of isolated microorganisms contained within an object selected from the group consisting of a bottle, a jar, an ampoule, a package, a container, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

[0067] In some aspects, combining selected plant species with the disclosed microorganisms, i.e., operational taxonomic units (OTUs), strains, or compositions comprising any of the foregoing, improves crop yield and improves production of the products of those crops. Thus, in one aspect, the disclosure provides a synthetic combination comprising a seed of a first plant and a preparation of a microorganism coated on the surface of the seed of the first plant, such that the microorganism is present on the surface of the seed at a level higher than that present on the surface of an uncoated, reference seed. In another aspect, the disclosure provides a synthetic combination comprising a portion of a first plant and a preparation of a microorganism coated on the surface of the portion of the first plant, such that the microorganism is present on the surface of the portion of the first plant at a level higher than that present on the surface of an uncoated, reference plant. The foregoing methods can be used alone or in conjunction with plant breeding and plant gene transfer techniques.

[0068] In some embodiments, the Paenibacillus strain is listed in Table 1a, Table 1b, or Table 1c. In some embodiments, the Paenibacillus strain comprises a polynucleotide sequence sharing at least 90% identity with any one or more of SEQ ID NOs: 1-12. In some embodiments, the Paenibacillus strain is a species selected from the group consisting of polymyxa, tritici, albidus, anaericanus, azotifigens, borealis, donghaensis, ehimensis, graminis, jilunlii, odorifer, panacisoli, phoenicis, pocheonensis, rhizoplanae, silage, taohuashanense, thermophilus, typhae, and wynnii. In some embodiments, the Paenibacillus strain is of subgroup I. In some embodiments, the Paenibacillus strain is of subgroup II.

[0069] In some embodiments, the isolated bacterial strain has substantially similar morphological and physiological characteristics to the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain has substantially similar genetic characteristics to the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a naturally occurring or artificial variant of the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a gene-edited, altered, or modified bacterial strain. In some embodiments, the isolated bacterial strain of the present disclosure is in a substantially pure culture. In some embodiments, the isolated bacterial strain of the present disclosure is in a pure culture. In some embodiments, the isolated bacterial strain of the present disclosure is in a cell fraction, extract, or supernatant.

[0070] In some embodiments, progeny and / or mutants of the isolated bacterial strains of the present disclosure are contemplated. In some embodiments, progeny, mutants, and / or genetically modified versions of the isolated bacterial strains of the present disclosure are contemplated.

[0071] In some embodiments, cell-free or inactivated preparations of the isolated bacterial strains of the present disclosure, or mutants of said 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 gene-edited, engineered, or modified variants of said isolated bacterial strains, are contemplated. In some embodiments, metabolites produced by the isolated bacterial strains of the present disclosure, or mutants of said 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 said isolated bacterial strains, are contemplated.

[0072] In some embodiments, agricultural compositions include an isolated bacterial strain and an agriculturally acceptable carrier. The isolated bacterial strain may be present in the composition at 1 x 10^2 to 1 x 10^12 CFU per gram. The agricultural composition may be formulated as a seed coating.

[0073] In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying an isolated bacterial strain to the plant or to a growth medium in which the plant is located. In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying an agricultural composition of the present disclosure to the plant or to a growth medium in which the plant is located.

[0074] In some embodiments, the present disclosure teaches methods of cultivating plants having at least one beneficial trait. In some embodiments, the methods include applying an isolated bacterial strain or microbial consortium to plant seeds, sowing or planting the seeds, and growing the plants. In certain embodiments, the isolated bacterial strain or microbial consortium is applied as an agricultural composition further comprising an agriculturally acceptable carrier.

[0075] In some embodiments, the microbial consortium has morphological and physiological characteristics substantially similar to the microbial consortium of the present disclosure. In some embodiments, the microbial consortium has genetic characteristics 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, progeny of any microorganism of the microbial consortium are contemplated. In some embodiments, mutants of any microorganism of the microbial consortium are contemplated. In some embodiments, gene-edited, engineered, or modified variants of any microorganism of the microbial consortium are contemplated. In some embodiments, cell-free or inactivated preparations of the microbial consortium, or mutants or gene-edited, engineered, or modified variants of any microorganism in the microbial consortium are contemplated. In some embodiments, metabolites produced by the microbial consortium, or mutants or gene-edited, engineered, or modified variants of any microorganism in the microbial consortium are contemplated.

[0076] In some embodiments, the agricultural composition comprises a microbial consortium and an agriculturally acceptable carrier. The microbial consortium of the agricultural composition may be present in the composition at 1 x 10^3 to 1 x 10^12 bacterial cells per gram. In some embodiments, the agricultural composition is formulated as a seed coating. In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying a microbial consortium to a plant or a growth medium in which the plant is located. In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying an agricultural composition to a plant or a growth medium in which the plant is located.

[0077] In any of the methods, the microorganism can comprise a 16S rRNA nucleic acid sequence having at least 97% sequence identity to a 16S rRNA nucleic acid sequence of a bacterium selected from the organisms provided in Table 1a, Table 1b, and / or Table 1C. DETAILED DESCRIPTION OF THE INVENTION

[0078] The present disclosure can be more fully understood from the following detailed description and sequence listing, which form a part of this specification.

[0079] The sequence descriptions and sequence listing accompanying this application comply with the rules governing the disclosure of nucleotide and amino acid sequences in patent applications as set forth in 37 CFR §§ 1.821 and 1.825.

[0080] Descriptions of the parent strains, edited strains, and sequences disclosed herein are provided in Table 1a, Table 1b, and Table 1c.

[0081] Table 1a: Paenibacillus parent strains, taxa, and sources Species designations are provided by 16S rRNA determinations as well as whole genome sequencing (WGS) determinations. Variation can be attributed to factors such as sequencing quality, reference database content, bioinformatics algorithms, and taxonomic flow. *Note: As indicated in the table, strain identifiers may further include optional prefixes. For example, strain 17899 may optionally be referred to synonymously as CM17899. [Table 1]

[0082] Table 1b: Paenibacillus edited strains As shown in the table, strain identifiers may include optional prefixes. For example, parent strain (PM)55083 with edit ID 14 is "(PE)55083-G14," with the prefixes "PM" (see Table 1a) and "PE" for the parent and edited strain, respectively, which are optional additional designations. [Table 2]

[0083] Table 1c: Sequences [Table 3-1] [Table 3-2]

[0084] The microorganisms described in this application are deposited with the Agricultural Research Service Culture Collection (NRRL), an international depository institution located at 1815 North University Street, Peoria, IL 61604, USA. The deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The deposits are made in accordance with and to satisfy the standards set forth in 37 CFR §§ 1.801-1.809 and U.S. Manual of Patent Examining Procedures §§ 2402-2411.05.

[0085] Paenibacillus odorifer strain 17899 was deposited with the NRRL on August 18, 2022, under accession number B-68192.

[0086] Paenibacillus polymyxa strain 77155 was deposited with the NRRL on August 18, 2022, under accession number B-68191.

[0087] Paenibacillus polymyxa strain 8619 was deposited with the NRRL on August 18, 2022, under accession number B-68193.

[0088] Paenibacillus polymyxa strain ID 55083-G14 was deposited with the NRRL on March 11, 2022, under accession number B-68106.

[0089] Paenibacillus polymyxa strain ID 8619-G50 was deposited with the NRRL on March 11, 2022, under accession number B-68108.

[0090] Paenibacillus polymyxa strain ID 68890-G12 was deposited with the NRRL on March 11, 2022, under accession number B-68103.

[0091] Paenibacillus polymyxa strain ID 77155-G3 was deposited with the NRRL on March 11, 2022, under accession number B-68105.

[0092] Paenibacillus odorifer strain ID 17899-G13A was deposited with the NRRL on March 11, 2022, under accession number B-68110.

[0093] Although the following terms are believed to be well understood by those of ordinary skill in the art, the following are set forth to facilitate explanation of the subject matter of the present disclosure.

[0094] The term "a" or "an" refers to one or more of that entity, i.e., can refer to a plurality of referents. Thus, the terms "a" or "an," "one or more," and "at least one" are used interchangeably herein. In addition, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements are present, unless the context clearly requires that there be only one of that element.

[0095] As used herein, the terms "microorganism" or "microbe" should be interpreted broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic classes, namely, bacteria and archaea, and eukaryotic fungi and protists. In some embodiments, the present disclosure refers to the "microorganisms" of Table 1A or various other tables or paragraphs present in this disclosure. This characterization can refer not only to the identified taxonomic bacterial genera of a table, but also to the identified taxonomic bacterial species, and to various novel and newly identified bacterial strains of the table.

[0096] As used herein, the terms "microbe" or "microorganism" refer to any microbial species or taxon, including, but not limited to, archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, a microbe or microorganism encompasses an individual cell (e.g., a unicellular microorganism) or multiple cells (e.g., a multicellular microorganism). Thus, a "population of microorganisms" can refer to multiple cells of a single microorganism that share a common genetic origin.

[0097] As used herein, the term "bacterium" generally refers to any prokaryotic organism and may refer to organisms of either the kingdom Eubacteria (bacteria), the kingdom Archaea (archaea), or both. In some cases, bacterial genera or other taxonomic classifications may be in taxonomic flux, reassigned for various reasons (such as, but not limited to, the evolving field of whole genome sequencing), and / or may be variable based on methodology, and it is understood that such nomenclature reassignments are within the scope of any claimed taxonomy. For example, certain species of the genus Erwinia are 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)).

[0098] The term "16S" refers to the DNA sequence of a bacterial 16S ribosomal RNA (rRNA) sequence. 16S rRNA gene sequencing is an established method for studying bacterial phylogeny and taxonomy.

[0099] As used herein, the term "fungus" or "fungi" generally refers to any organism from the kingdom Fungi. Historically, fungi have been classified by morphological appearance. Beginning in the mid-1800s, it was recognized that some fungi have a pleomorphic life cycle, with different nomenclature designations being used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association established rules for naming fungi according to their status as anamorph, teleomorph, or holomorph (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 became clear that taxonomic classification based on molecular phylogeny was incompatible with 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.). As a result, in 2011, the International Botanical Congress adopted a resolution approving the International Code of Nomenclature for Algae, Fungi, and Plants (Melbourne Code) (2012), which described the consequence of designating "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)).

[0100] The term "internal transcribed spacer" (ITS) refers to the spacer DNA (non-coding DNA) located between the small subunit ribosomal RNA (rRNA) gene and the large subunit (LSU) rRNA gene in a chromosome, or the corresponding transcribed region in a polycistronic rRNA precursor transcript. 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 their ITS sequence, and some by their LSU sequence. It is understood that both are equally descriptive and accurate for determining taxonomy.

[0101] The term "microbial consortia" or "microbial consortium" refers to a subset of a microbial community consisting of individual microbial species or strains of species, which may be described as performing a common function or may be described as relating to, causing, or correlating with a recognizable parameter or plant phenotypic trait. A community may include one or more species or strains of species of microorganisms. In some cases, microorganisms coexist symbiotically within a community.

[0102] The term "microbial community" refers to a group of microorganisms comprising two or more species or strains. Unlike a microbial consortium, a microbial community does not necessarily perform a common function or relate to, cause, or correlate with a discernible parameter or plant phenotypic trait.

[0103] The term "rapid microbial sorting" or "AMS" is used interchangeably with the term "directional microbial sorting" or "DMS" and refers, in some embodiments of the present disclosure, to an iterative sorting method utilized to obtain the claimed microbial species or consortia of such species.

[0104] As used herein, "isolate," "isolated," "isolated microorganism," and similar terms are intended to mean that one or more microorganisms are separated from at least one of the substances with which they are associated in a particular environment (e.g., soil, water, plant tissue).

[0105] Thus, an "isolated microorganism" is not present in its naturally occurring environment. Rather, through various techniques described herein, the microorganism has been removed from its natural environment and placed in a non-naturally occurring state. Thus, the isolated strain may exist, for example, as a biologically pure culture or as spores (or other form of the strain) associated with an agricultural carrier.

[0106] In certain embodiments of the present disclosure, the isolated microorganism exists as an isolated, biologically pure culture. An isolated, biologically pure culture of a particular microorganism is understood by those skilled in the art to indicate that other organisms are substantially absent from the culture (for scientific reasons), and that only the individual microorganism in question is present in the culture. The culture may contain various concentrations of the microorganism. The present disclosure points out that isolated, biologically pure microorganisms are often "necessarily distinct from less pure or impure material." See, e.g., re. Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins); and re. Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microorganisms); and Parke-Davis & Co. v. HK Mulford & Co., 189 F.95 (SDNY 1911) (Learned Hand discussing purified adrenaline), partially affirmed and partially overturned, 196 F.496 (2d Cir. 1912), each of which is incorporated herein by reference. Furthermore, in some aspects, the present disclosure prescribes certain quantitative measures of concentration or purity limits that must be found in isolated, biologically pure microbial cultures. In certain embodiments, the presence of these purity values ​​is an additional attribute that distinguishes the microorganisms of the present disclosure from those present in nature. See, for example, Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958), which is incorporated herein by reference (discussing purity limits for microbially produced vitamin B12).

[0107] As used herein, "individual isolate" should be understood to mean a composition or culture that predominantly contains microorganisms of a single genus, species, or strain after separation from one or more other microorganisms. The phrase should not be understood to refer to the extent to which the microorganism has been isolated or purified. However, an "individual isolate" can contain microorganisms of substantially only one genus, species, or strain.

[0108] With respect to a microorganism, the term "modified" means that the microorganism has been changed in some way compared to the natural state in which it is found. In this context, "modified" is synonymous with "engineered" and indicates that the hand of man was involved in creating the modification. In some cases, the modification involves a change in a polynucleotide within the microorganism, for example, in its genome. The modification may include a deletion, insertion, substitution, and / or chemical alteration of at least one nucleotide, which may result in a change in the phenotype of the microorganism (e.g., upregulation of a particular pathway, downregulation of a particular pathway, knockout of a gene or protein function), and / or a change in the phenotype of another, heterologous organism with which the microorganism is or becomes associated.

[0109] As used herein, the term "growth medium" refers to any medium suitable for supporting plant growth. By way of example, the medium may be a natural or artificial medium, including, but not limited to, soil, potting soil, bark, vermiculite, hydroponic solutions alone and applied to solid plant support systems, and tissue culture gels. It should be understood that these media may be used alone or in combination with one or more other media. These media may also be used with or without the addition of exogenous nutrients and physical support systems for the roots and leaves.

[0110] In one embodiment, the growth medium is a naturally occurring medium such as soil, sand, mud, clay, humus, topsoil, stone, or water. In another embodiment, the growth medium is artificial. Such artificial growth media may be constructed to mimic the conditions of naturally occurring media; however, this is not necessary. Artificial growth media can be made from one or more of any number and combination of materials, including sand, minerals, glass, rock, water, metals, salts, nutrients, and water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0111] The media may be amended or enriched with additional compounds or components, such as components that may aid in the interaction and / or selection of specific groups of microorganisms with plants and with each other. For example, antibiotics (such as penicillin) or sterilizing agents (e.g., quaternary ammonium salts and oxidizing agents) may be present, and / or physical conditions (salt, plant nutrients (e.g., organic and inorganic minerals, such as phosphorus, nitrogen-containing salts, ammonia, potassium, and micronutrients such as cobalt and magnesium), pH, and / or temperature) may be modified.

[0112] The term "plant" generally includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and their progeny. Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. As used herein, the term "plant element" refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps, and intact plant cells in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, husks, stems, roots, root tips, anthers, and the like, as well as plants themselves. Progeny, variants, and mutants of regenerated plants are also within the scope of the present invention, provided that these parts contain the introduced polynucleotide.

[0113] "Plant element" is intended to refer to either a whole plant or plant components, which may include differentiated and / or undifferentiated tissues, such as, but not limited to, plant tissues, parts, and cell types. In one embodiment, the plant element is one of the following: a whole plant, seedling, meristem, ground tissue, vascular tissue, skin tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keiki, shoot, bud, tumor tissue, and various forms of cells and cultures (e.g., single cell, protoplast, embryo, callus tissue). The term "plant organ" refers to a plant tissue or group of tissues that constitutes a morphologically and functionally distinct part of a plant. As used herein, "plant part" is synonymous with "part" of a plant, refers to any part of a plant, can include distinct tissues and / or organs, and may be used interchangeably with the term "tissue" throughout.

[0114] Similarly, "plant reproductive element" is generally intended to refer to any part of a plant that is capable of initiating another plant through either sexual or asexual reproduction of that plant, such as, but not limited to, a seed, seedling, root, shoot, cutting, scion, explant, stolon, bulb, tuber, corm, keiki, or sprout. A plant element can be in a plant, or in a plant organ, tissue culture, or cell culture.

[0115] "Progeny" includes any descendant of an organism produced through sexual or asexual reproduction.

[0116] "Grain" is intended to mean mature seed produced by commercial growers for purposes other than seed growth or reproduction.

[0117] The terms "monocotyledonous" or "monocotyledonous plant" refer to the subclass of angiosperms, also known as "monocotyledons," whose seeds typically contain only one germ leaf or cotyledon. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny.

[0118] The terms "dicotyledonous" or "dicot" refer to the subclass of angiosperms, also known as "dicots," whose seeds typically contain two germ leaves or cotyledons. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny.

[0119] As used herein, the term "cultivar" refers to a variety, strain, or variety of a plant created by horticultural or agronomic techniques and not normally found in natural populations.

[0120] As used herein, "improved" should be broadly understood to encompass an improvement in a plant characteristic compared to a control plant or compared to a known average amount associated with the characteristic in question. For example, "improved" plant biomass associated with the application of beneficial microorganisms or consortia of the present disclosure can be demonstrated by comparing the biomass of a plant treated with a microorganism taught herein to the biomass of an untreated control plant. Alternatively, it is possible to compare the biomass of a plant treated with a microorganism taught herein to the average biomass normally achieved by a given plant as shown in scientific or agricultural publications known to those of skill in the art. In the present disclosure, "improvement" does not necessarily require that the data be statistically significant (e.g., p<0.05). Rather, any quantifiable difference demonstrating that one value (e.g., the average treatment value) is different from another value (e.g., the average control value) can rise to the level of "improvement."

[0121] As used herein, "inhibiting and suppressing" and similar terms should not be construed as requiring complete inhibition or suppression, although this may be desirable in some embodiments.

[0122] As used herein, the term "genotype" refers to the genetic constitution of an individual cell, cell culture, tissue, organism (e.g., plant), or group of organisms.

[0123] The compositions and methods herein may provide improved "agronomic traits" or "agronomically important characteristics" or "traits of agronomic interest" in plants, which may include, but are not limited to, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest and disease resistance, herbivore resistance, pathogen resistance, improved yield, improved health, improved vigor, improved growth, improved photosynthetic capacity, nutritional enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of metabolites, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, compared to an isogenic plant that does not include the modification resulting from the methods or compositions herein.

[0124] "Agronomic trait potential" is intended to mean the ability of a plant element, at a certain point in its life cycle, to exhibit a phenotype, preferably an improved agronomic trait, or to transmit said phenotype to another plant element with which it is associated in the same plant.

[0125] As used herein, the terms "molecular marker," "marker," or "genetic marker" refer to indicators used in methods for visualizing 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-specific amplified regions (SCARs), cleaved amplified polymorphic sequences (CAPS) markers, or isozyme markers, or combinations of markers described herein, which define specific genetic and chromosomal locations. Mapping molecular markers near an allele is a technique that can be performed by those skilled in the art of molecular biology.

[0126] 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 characteristics of a plant, including, but not limited to, water use efficiency, nutrient use efficiency, yield, feasibility of mechanical harvesting, fruit maturity, storage life, pest / disease resistance, early plant maturity, stress tolerance, etc. Traits may be dominantly or recessively inherited, or may be partially or incompletely dominantly inherited. Traits may be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by multiple loci), or may result from the interaction of one or more genes with the environment.

[0127] As used herein, the term "phenotype" refers to the observable characteristics of an individual cell, cell culture, organism (e.g., plant), or group of organisms that result from the interaction between the genetic constitution (i.e., genotype) of the individual and the environment.

[0128] As used herein, a "synthetic nucleotide sequence" or "synthetic polynucleotide sequence" is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will contain at least one nucleotide difference compared to any other naturally occurring nucleotide sequence.

[0129] As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or their analogs. The term refers to the primary structure of the molecule and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. The term also includes modified nucleic acids, such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, nucleic acids containing backbone modifications, etc. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0130] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Thus, a gene includes, but is not limited to, a coding sequence and / or regulatory sequences required for expression of the coding sequence. Genes can also include non-expressed DNA segments that form, for example, recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and can include sequences designed to have desired parameters.

[0131] As used herein, the terms "homologous," "homologue," "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 "corresponding substantially" are used interchangeably herein. These terms refer to nucleic acid fragments whose ability to mediate gene expression or generate a particular phenotype has not been affected by a change in one or more nucleotide bases. These terms also refer to modifications of the nucleic acid fragments of the present disclosure, such as the deletion or insertion of one or more nucleotides, that do not substantially alter the functional characteristics of the resulting nucleic acid fragment compared to the original, unmodified fragment. Therefore, it is understood that the present disclosure encompasses sequences other than the specific exemplary sequences, as will be understood by one of skill in the art. 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 purposes of this disclosure, homologous sequences are compared. "Homologous sequences" or "homologs" or "orthologs" are considered, believed, or known to be functionally related. Functional relationship can be indicated in any one of a number of ways, including, but not limited to, (a) degree of sequence identity and / or (b) identical 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 (F.M. Ausubel et al., eds., 1987), Supplement 30, section 7.718, Table 7.71.Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK), ALIGN Plus (Scientific and Educational Software, Pennsylvania), and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.

[0132] As used herein, the term "nucleotide change" refers to, for example, a nucleotide substitution, deletion, insertion, chemical modification, or any of the foregoing, as is well understood in the art.

[0133] As used herein, the term "protein modification" refers to, for example, amino acid substitutions, amino acid modifications, deletions, and / or insertions, as is well understood in the art.

[0134] As used herein, the term "at least a portion" or "fragment" of a nucleic acid or polypeptide refers to a minimum size characteristic of such a sequence, or any larger fragment of a full-length molecule, up to and including the full-length molecule. A fragment of a polynucleotide of the present disclosure may encode a biologically active portion of a gene regulatory element. A biologically active portion of a gene regulatory element can be prepared by isolating a 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 four amino acids, five amino acids, six amino acids, seven amino acids, etc., up to the full-length polypeptide. The length of the portion used will depend on the particular application. A portion of a nucleic acid useful as a hybridization probe can be as short as 12 nucleotides. In some embodiments, this is 20 nucleotides. A portion of a polypeptide useful as an epitope can be as long as four amino acids. A portion of a polypeptide that performs the function of the full-length polypeptide will generally be longer than four amino acids.

[0135] As used herein, the term "primer" refers to an oligonucleotide capable of annealing to an amplification target when placed under conditions conducive to the synthesis of a primer extension product, i.e., in the presence of nucleotides and a polymerization agent, such as a DNA polymerase, and a suitable temperature and pH, allowing the DNA polymerase to attach and thereby serving as a starting point for DNA synthesis. (Amplification) primers are preferably single-stranded to maximize amplification efficiency. The primer is preferably an oligodeoxyribonucleotide. The primer must be sufficiently long to initiate the synthesis of an extension product in the presence of a polymerization agent. The exact length of the primers depends on many factors, including temperature and primer composition (A / T vs. G / C content). A bidirectional primer pair consists of one forward and one reverse primer, as commonly used in the art of DNA amplification, such as primers in PCR amplification.

[0136] The terms "stringency" or "stringent hybridization conditions" refer to hybridization conditions, such as temperature, salt concentration, pH, and formamide concentration, that affect hybrid stability. These conditions are empirically optimized to maximize specific binding of a primer or probe to its target nucleic acid sequence and minimize nonspecific binding. As used herein, the terms include reference to conditions under which a probe or primer hybridizes to its target sequence at a detectable level (e.g., at least 2-fold over background) relative to other sequences. Stringent conditions are sequence-dependent and will vary under various circumstances. Larger sequences hybridize specifically at higher temperatures. Stringent conditions are generally selected to be approximately 5°C lower than the melting temperature (Tm) of a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Stringent conditions typically involve a pH of 7.0-8.3, a salt concentration of less than about 1.0 M Na+ ions, typically about 0.01-1.0 M Na+ ions (or other salts), and a temperature of at least about 30°C for short probes or primers (e.g., 10-50 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 the addition of destabilizing agents such as formamide. Exemplary low stringency or "reduced stringency" conditions include hybridization with a buffer of 30% formamide, 1 M NaCl, and 1% SDS at 37°C, followed by a wash in 2×SSC at 40°C. Exemplary "high stringency" conditions include hybridization in 50% formamide, 1 M NaCl, and 1% SDS at 37°C, followed by a wash in 0.1×SSC at 60°C. Hybridization procedures are well known in the art and are described, for example, in Ausubel et al., 1998 and Sambrook et al., 2001.In some embodiments, stringent conditions are hybridization in 0.25 M NaHPO buffer (pH 7.2) containing 1 mM NaEDTA, 0.5-20%, 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% sodium dodecyl sulfate at 45°C, followed by washing in 5x SSC containing 0.1% (weight / volume) sodium dodecyl sulfate at 55°C to 65°C.

[0137] In some embodiments, the 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 another organism (e.g., a microorganism), DNA segment, heterologous polynucleotide, or heterologous nucleic acid.

[0138] A variety of phenotypic changes are of interest to the present disclosure, including, but not limited to, modifying the fatty acid composition in plants, altering the amino acid content of plants, altering the pathogen defense mechanisms of plants, increasing plant yield for economically important traits (e.g., grain yield, forage yield, etc.), etc. These results can be achieved by expressing heterologous products or by increasing the expression of endogenous products in plants using the methods and compositions of the present disclosure.

[0139] An "artificial mixture" can include a mixture of a plant and a microorganism of the present disclosure. The mixture can be achieved, for example, by coating the surface of a plant seed, such as an agricultural plant, or host plant tissue (roots, stems, leaves, etc.) with a microorganism of the present disclosure. Furthermore, an "artificial mixture" can include a mixture of various strains or species of microorganisms. An artificial mixture has at least one variable that distinguishes it from any mixture occurring in nature. This variable may be, in particular, a concentration of microorganisms on the seed or plant tissue that does not occur in nature, or a combination of microorganisms and plants that does not occur in nature, or a combination of microorganisms or strains that does not occur in nature. In each of these examples, the artificial mixture exhibits artificiality and possesses structural and / or functional properties that are not present when the individual components of the mixture are considered in isolation.

[0140] In some embodiments, a microorganism may be "endogenous" to a seed or plant. As used herein, a microorganism is considered "endogenous" to a plant or seed if it originates from the herbarium specimen from which it is procured; that is, if the microorganism is found naturally in association with the plant. In embodiments in which an endogenous microorganism is applied to a plant, the endogenous microorganism is applied at a level that differs from that 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 occur naturally, the microorganism may still form an artificial mixture with the plant.

[0141] In some embodiments, a composition (such as a microorganism) can be "heterologous" (also referred to as "exogenous") to another composition (such as a seed or plant), and in some aspects is referred to herein as a "heterologous composition." As used herein, a microorganism is considered "heterologous" to a plant or seed if it is not native to the plant specimen from which it is obtained; that is, if the microorganism is not naturally found in association with that plant. For example, a microorganism normally associated with the leaf tissue of one corn plant would be considered foreign to the leaf tissue of another corn plant that does not naturally harbor that microorganism. In another example, a microorganism normally associated with a corn plant would be considered foreign to a wheat plant that does not naturally harbor that microorganism.

[0142] A composition is "heterologously disposed" when it is mechanically or manually applied, artificially inoculated, associated, or disposed on or in a plant element, seedling, plant, plant growth medium, or treatment formulation in a manner not found in nature prior to application of the treatment, e.g., such that the treatment is present on or in a plant element, seedling, plant, plant growth medium, or treatment formulation in a manner not found in nature prior to application of the treatment, e.g., such that the combination is not found in nature in that plant variety, at that plant developmental stage, in that plant tissue, in 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 the microorganism; the presence of the microorganism in different numbers of cells, concentrations, or amounts; the presence of the microorganism in different plant elements, tissues, cell types, or other physical locations within or on the plant; the presence of the microorganism over different periods of time, e.g., developmental stages, days, or seasons of the plant or plant element; and combinations thereof. In some embodiments, "heterologously disposed" means that the microorganism is applied to a tissue or cell type of a plant element that is different from that in which the microorganism is found in nature. In some embodiments, "heterologously located" means that a microorganism is applied to a plant element, seedling, or developmental stage of a plant with which the microorganism is not naturally associated, but may be associated at other stages. For example, if a microorganism is normally found at the flowering stage of a plant but not at other stages, the microorganism applied at the seedling stage would be considered heterologously located. In some embodiments, a microorganism is heterologously located where the microorganism is normally found in the root tissue of a plant element but not in the leaf tissue, and the microorganism is applied to the leaf. In another non-limiting example, if a microorganism is naturally found in the mesophyll layer of leaf tissue but is applied to the epidermis layer, the microorganism would be considered heterologously located. In some embodiments, "heterologously located" means that a native plant element, seedling, or plant does not contain detectable levels of the microorganism in that same plant element, seedling, or plant. In some embodiments, "heterologously located" means that the microorganism being applied is in a concentration, number, or amount in the plant element, seedling, or plant that exceeds the concentration, number, or amount naturally found in the plant element, seedling, or plant.For example, a microorganism is heterologously disposed if it is present in a number, amount, or concentration that is at least 1.5-fold higher, 1.5-2-fold higher, 2-fold higher, 2-3-fold higher, 3-fold higher, 3-5-fold higher, 5-fold higher, 5-7-fold higher, 7-fold higher, 7-10-fold higher, 10-fold higher, or even greater than 10-fold higher than the concentration present prior to disposition of the microorganism. In another non-limiting example, a microorganism naturally found in the tissue of a tree of the Cupressaceae family would be considered heterologous to the tissue of a corn, wheat, cotton, or soybean plant. In another example, a microorganism naturally found in the leaf tissue of a corn, spring wheat, cotton, or soybean plant would be considered heterologous to the leaf tissue of another corn, spring wheat, cotton, or soybean plant that naturally lacks the microorganism or contains the microorganism in a different amount.

[0143] A microorganism can also be "heterologously distributed" on a given plant tissue. This means disposing the microorganism on plant tissue where it is not naturally found. For example, if a given microorganism naturally occurs only in the roots of a given plant, the microorganism can be exogenously applied to the above-ground tissue of the plant, thereby "heterologously disposing" the microorganism on the plant tissue. Thus, a microorganism is considered to be heterologously distributed when it does not naturally have any other microorganisms present, or when it does not naturally have any other microorganisms present in the numbers being applied.

[0144] The compositions and methods herein may provide a host plant with "modulated" "agronomic traits" or "agronomically important traits," including, but 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, improved health, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, compared to an isogenic plant grown from seed without the use of the seed treatment formulation. , increased yield, increased yield under limited water conditions, grain mass, grain moisture content, metal tolerance, panicle number, grain number per panicle, pod number, nutritional enhancement, pathogen resistance, pest and disease resistance, improved photosynthetic capacity, salt tolerance, greening, improved plant vigor, increased mature seed dry weight, increased mature seed fresh weight, increased number of mature seeds per plant, 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 unwilted leaves per plant, a detectable change in the level of a metabolite, a detectable change in the level of a transcript, and a detectable change in the proteome. By the term "modulated" it is intended to refer to a change in an agronomic trait that is modulated by the presence of a microorganism, exudate, broth, metabolite, etc. In an aspect, the modulation provides for the impartation of a beneficial trait.

[0145] Microbe and microorganism As used herein, the term "microorganism" should be interpreted broadly and includes, but is not limited to, prokaryotic bacteria and archaea, as well as eukaryotic bacteria and protists.

[0146] In certain embodiments, the microorganism is an endophyte or epiphyte, or a microorganism that resides in the plant rhizosphere or rhizocteth, i.e., the microorganism may be found present in soil material attached to the roots of the plant or in the area immediately adjacent to the roots of the plant.

[0147] In embodiments, the microorganism is an endophyte. Endophytes may benefit the host plant by preventing pathogenic organisms from infecting the host plant. The widespread infection of plant tissues by endophytes creates a "barrier effect" in which the endophytes in that area outcompete pathogenic organisms and prevent pathogenic organisms from establishing themselves. Endophytes may also produce chemicals that inhibit the growth of competitors, including pathogenic organisms.

[0148] In certain embodiments, the microorganism is non-culturable, which should be understood to mean that the microorganism is not known to be culturable or that it is difficult to cultivate the microorganism using methods known to those skilled in the art.

[0149] The microorganisms of the present disclosure may be collected or obtained from any source, or may be contained within and / or associated with material collected from any source.

[0150] In one embodiment, a microorganism or combination of microorganisms may provide a possible or predicted benefit to a plant. For example, a microorganism may be predicted to improve nitrogen fixation, release phosphate from soil organic matter, release phosphate from inorganic forms of phosphate (e.g., phosphate rock), "fix carbon" in root microspheres, reside in the plant's rhizosphere, thereby helping the plant absorb nutrients from the surrounding soil and then more easily provide them to the plant, increase the number of nodules on the plant's 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, trigger a plant defense response such as ISR (induced systemic resistance) or SAR (systemic acquired resistance) that helps plants resist the invasion and spread of pathogenic microorganisms, compete with microorganisms harmful to plant growth or health through antagonism or competitive use of resources such as nutrients or space, change the color of one or more parts of the plant, or change the plant's chemical profile, its odor, taste, or one or more other qualities.

[0151] The microorganisms of the present disclosure may be isolated in the form of substantially pure cultures or mixed cultures. They may be concentrated, diluted, or provided at the natural concentrations at which they are found in the source material. For example, microorganisms derived from saline sediments may be isolated for use in the present disclosure by suspending the sediment in fresh water and allowing the sediment to settle to the bottom. After a suitable period of settling, the water containing most of the microorganisms may be decanted and applied directly to plant growth medium, or it may be concentrated by filtering or centrifugation, diluted to an appropriate concentration to remove most of the salts, and then applied to plant growth medium. As a further example, microorganisms derived from mineralized or toxic sources may be similarly treated to minimize potential damage to plants and recovered for application to plant growth material.

[0152] In some embodiments, a mixed population of microorganisms is used in the methods of the present disclosure.

[0153] Microbial genome modification In some embodiments, the microorganism may have its genome modified in some way to provide an improved trait of interest in a legume crop, for example, improved nitrogen fixation.

[0154] Various methods are known in the art for modifying polynucleotides within cells (including, but not limited to, any polynucleotide sequence contained within a cell, including genomic, chromosomal, and plasmid DNA). Briefly, a single-strand or double-strand break is introduced into a target polynucleotide (the subject of modification), which can result in an insertion of at least one nucleotide, a deletion of at least one nucleotide, a substitution of at least one nucleotide, or any combination of the foregoing, according to the desires of the practitioner.

[0155] Single- or double-strand breaks (SSBs or DSBs) can be achieved in any of several ways, including the use of chemicals or radiation, as a result of a homologous recombination process, the introduction of specific or non-specific nucleases, or any combination of the foregoing.

[0156] Enzymes that effect polynucleotide cleavage are known in the art and may include (but are not limited to) restriction endonucleases, meganucleases, TALENs, zinc fingers, or Cas endonucleases.

[0157] In some aspects, the present disclosure relates to isolated genetically modified microorganisms that have improved nitrogen fixation activity compared to ungenetically modified variants of the same microbial species or strain.

[0158] Glutamine (Gln) is a universal nitrogen signal in all free-living diazotrophs (see, e.g., Wang et al., PLOS Genetics, 2018). Gram-negative bacteria, such as Klebsiella and Pseudomonas, have well-characterized nitrogen pathways, allowing for easier and more predictable gene delivery and expression in genome-modified strains. In the Gram-negative bacterium Klebsiella, NifL is a negative regulator of the nif operon. When intracellular glutamine is high (nitrogen excess), NifL forms a repressor complex to inactivate nif operon expression. In the Gram-negative bacterium Azospirillum, NifA activates transcription of the nif operon. Expression of nifA is regulated by glutamine via ntrB phosphorylation of ntrC. Nitronase is post-transcriptionally inactivated.

[0159] In contrast, Gram-positive bacteria such as the Paenibacillus described herein are more difficult to transform and have less well-studied nitrogen fixation pathways.

[0160] Therefore, successful cell modification leading to higher nitrogen fixation capacity for Gram-positive bacteria such as Paenibacillus is not only surprising but also highly needed in agricultural biotechnology. The sporulation ability of Paenibacillus increases the commercial potential of products containing gene-edited Paenibacillus strains that improve nitrogen fixation in crop plants.

[0161] In Gram-positive bacteria, the nif operon controls the nitrogen fixation pathway via GlnR. Binding of GlnR to site I activates Nif expression, whereas binding of GlnR to site II represses Nif expression. Therefore, the gene target for improving nitrogen fixation in Paenibacillus is the Nif activator / repressor GlnR and its binding site.

[0162] Within the genus Paenibacillus, there are two distinct subgroups, subgroup I and subgroup II, each containing a different operon composition. Subgroup I Paenibacillus, such as Paenibacillus polymyxa, contains nifB, nifH, nifD, nifK, nifE, nifN, nifZ, hesA, and nifV, in that order. Subgroup II Paenibacillus, such as Paenibacillus graminis, contains nifB, nifH, nifD, nifK, nifE, nifV, nifZ, orf1, hesA, and nifV, in that order.

[0163] In some embodiments, the Paenibacillus microorganism has a genetic modification to the nif gene, which encodes an enzyme involved in nitrogen fixation activity by the microorganism. In other embodiments, the isolated microorganism has a genetic modification to glnR, a gene encoding the protein GlnR, which is involved in sensing local ammonia concentrations and regulating expression of the nif gene. In particular embodiments, the isolated microorganism has a genetic modification to both the nif gene and the glnR gene.

[0164] In some embodiments, the disclosure relates to an isolated genetically modified microorganism comprising one or more genetic modifications selected from a genetic modification to an endogenous glnR gene encoding GlnR and a genetic modification to an upstream (5') regulatory region or a GlnR binding region of an endogenous nif gene, wherein the genetic modification to the endogenous glnR gene encoding GlnR provides a mutant glnR gene that produces a GlnR protein variant; the genetic modification to the 5' regulatory region sequence provides improved binding affinity for GlnR compared to a 5' regulatory region sequence that is not genetically modified; and the one or more genetic modifications provide the microorganism with improved nitrogen fixation activity compared to a microbial strain that is not genetically modified.

[0165] In some embodiments, the genetic modification to the endogenous glnR gene is characterized by providing a mutant glnR gene that produces a GlnR protein variant. In some embodiments, the genetic modification to the glnR gene produces a mutant gene that can be transcribed and translated to produce a modified version of the GlnR protein. In other words, in some embodiments, the isolated microorganisms described herein are not modified to knock out or delete the glnR gene or otherwise prevent production of the GlnR protein. In certain embodiments, the GlnR variant protein retains one or more functions of the unmodified endogenous wild-type GlnR protein. For example, in some embodiments, the variant protein maintains the ability to recognize and bind to a recognition element or 5' regulatory region sequence within the nif gene. In certain embodiments, the GlnR variant protein maintains the ability to regulate expression of the nif gene, particularly with respect to activating or upregulating expression of the nif gene.

[0166] While the present disclosure excludes isolated microorganisms that have been genetically modified to prevent production of the GlnR protein, for example, by knocking out or deleting the glnR gene, the present disclosure encompasses genetically modified microorganisms in which the glnR gene has been deleted and replaced with a recombinant glnR gene. In some embodiments, the recombinant glnR gene may be derived from a different microbial species or strain than the microorganism into which the recombinant gene is incorporated. In some embodiments, the recombinant glnR gene may be a non-native or synthetic gene. In some embodiments, the recombinant glnR gene may encode a variant of the GlnR protein that more efficiently regulates nif expression, for example, by binding with higher affinity to a GlnR recognition element in the 5' regulatory region of nif or by complexing with higher affinity to additional proteins required for nif transcription.

[0167] In some embodiments, the genetic modification of the endogenous glnR gene comprises a truncation, meaning that the gene encodes a variant of the GlnR protein lacking one or more amino acid residues compared to the protein encoded by the native or endogenous gene. Those skilled in the art will understand that genetic modification to produce a truncated mutant gene encoding GlnR can be achieved by several molecular biology methods commonly known in the art. For example, in some embodiments, a truncated mutant gene encoding a GlnR protein can be prepared by inserting a stop codon into the gene upstream of the endogenous stop codon of the native glnR gene. Alternatively, in some embodiments, a truncated mutant glnR gene can be prepared by gene editing the endogenous glnR gene to delete the DNA sequence encoding the amino acids to be excluded in the truncated variant of the GlnR protein.

[0168] In some embodiments, the truncation modification of the glnR gene comprises a truncation that includes a deletion of a portion of the endogenous glnR gene comprising the C-terminal domain of the GlnR protein. In certain embodiments, the deletion of a portion of the C-terminal domain of the GlnR protein is the result of a deletion of a portion of the endogenous gene encoding GlnR that specifically encodes the C-terminal domain. In certain embodiments, the deletion of a portion of the C-terminal domain of the GlnR protein is the result of an insertion of a stop codon immediately upstream of the portion of the endogenous gene encoding GlnR that specifically encodes the C-terminal domain. In certain embodiments, the portion of the endogenous glnR gene encoding GlnR that is modified in the isolated microorganism comprises a portion encoding the last approximately 25 C-terminal amino acids of the GlnR protein.

[0169] Without being bound by theory, the C-terminal domain of the GlnR protein is associated with sensing local ammonia concentrations, for example, by forming a complex with the protein glutamine synthetase. Under conditions of elevated local ammonia concentrations, GlnR binds to and forms a complex with glutamine synthetase, which ultimately represses the transcription of the nif gene, thereby reducing the nitrogen fixation activity of the microorganism. Therefore, by truncating the GlnR protein to delete the C-terminal domain, the variant protein is no longer able to form a complex with glutamine synthetase, which means that this variant has a reduced ability to negatively regulate the expression of the nif gene. As a result, microorganisms with a C-terminal truncated variant of the GlnR protein can maintain the expression of the nif gene even under conditions of elevated local ammonia concentrations in the environment surrounding the microorganism.

[0170] Genetic modifications to endogenous glnR genes that produce mutant glnR genes encoding GlnR variant proteins characterized by improved binding affinity for recognition elements or 5' regulatory region sequences within the endogenous nif gene compared to the wild-type GlnR protein are also contemplated by the present disclosure. In some embodiments, a mutant glnR gene comprising a truncation of a portion of the glnR gene encoding the C-terminal domain of the GlnR protein produces a GlnR variant protein with improved binding affinity for recognition elements or 5' regulatory region sequences within the endogenous nif gene compared to the wild-type GlnR protein. In other embodiments, the mutant glnR gene can comprise a mutation in a portion of the glnR gene encoding the DNA-binding domain of GlnR. In certain of these embodiments, mutations to the portion of the glnR gene encoding the DNA-binding domain of GlnR produce mutant glnR genes encoding GlnR protein variants with improved binding affinity for recognition elements or 5' regulatory region sequences in the endogenous nif gene, e.g., by modifying specific amino acid residues required for interaction of the DNA-binding domain with nucleotides in the recognition element or 5' regulatory region sequence in the nif gene.

[0171] The endogenous gene encoding nif contains at least two 5' regulatory region sequences that the GlnR protein can recognize and bind to. The first 5' regulatory region sequence is located upstream of the transcription start site within the 5' regulatory region of the gene. Without being bound by theory, binding of GlnR to the first 5' regulatory region sequence is associated with activated or upregulated expression of the nif gene. Thus, genetic modifications to the first 5' regulatory region sequence, characterized by providing improved binding affinity for GlnR, provide enhanced or upregulated expression of the nif gene compared to unmodified microorganisms of the same species or strain, thereby improving the nitrogen fixation activity of the isolated microorganism. The second 5' regulatory region sequence is located downstream of the transcription start site. Without being bound by theory, binding of GlnR to the second 5' regulatory region sequence is associated with repression or downregulation of expression of the nif gene.

[0172] In some embodiments, the isolated microorganism comprises a genetic modification to a 5' regulatory region sequence within the endogenous nif gene, wherein the 5' regulatory region sequence is located upstream of the transcription start site of the nif gene.

[0173] In some embodiments, the 5' regulatory region sequence is located upstream of the transcription start site of the nif gene.

[0174] In some embodiments, the isolated genetically modified microorganism described herein also comprises a genetic modification to a second 5' regulatory region sequence within the endogenous nif gene. In some embodiments, the isolated genetically modified microorganism described herein also comprises a genetic modification to a second 5' regulatory region sequence within the endogenous nif gene, wherein the second 5' regulatory region sequence is located downstream of the transcription start site within the endogenous nif gene.

[0175] In some embodiments, the native second 5' regulatory region sequence within the endogenous nif gene is capable of being recognized and bound by the GlnR protein. In some embodiments, the genetic modification to the second 5' regulatory region sequence within the endogenous nif gene is characterized by reduced negative regulation or repression of expression of the endogenous nif gene by GlnR. In some embodiments, the genetic modification to the second 5' regulatory region sequence within the endogenous nif gene comprises a genetic modification that generates a 5' regulatory region sequence that has reduced affinity for the GlnR protein compared to the native second 5' regulatory region sequence. In some embodiments, the genetic modification to the second 5' regulatory region sequence within the endogenous nif gene comprises a genetic modification that generates a 5' regulatory region sequence that cannot be recognized or bound by the GlnR protein. For example, in certain embodiments, the genetic modification to the second 5' regulatory region sequence within the endogenous nif gene comprises deleting or knocking out the second 5' regulatory region sequence from the endogenous nif gene. In certain other embodiments, the genetic modification to the second 5' regulatory region sequence in the endogenous nif gene comprises replacing the second 5' regulatory region sequence in the endogenous nif gene with a DNA sequence that is unrecognizable by GlnR. The DNA sequence that is unrecognizable by GlnR is not limited to, and includes, but is not limited to, any sequence that has no affinity for GlnR protein or has reduced affinity for GlnR protein compared to the native second 5' regulatory region sequence.

[0176] In some embodiments, an isolated genetically modified microorganism disclosed herein comprises both a genetic modification to the endogenous glnR gene encoding GlnR and a genetic modification to a 5' regulatory region sequence within the endogenous nif gene.

[0177] In some embodiments, the genetic modification to the 5' regulatory region sequence in the endogenous nif gene comprises replacing the 5' regulatory region sequence with a DNA sequence that provides improved binding affinity for GlnR compared to the 5' regulatory region sequence. Those skilled in the art will appreciate that replacing the 5' regulatory region sequence in the endogenous nif gene can be achieved by a variety of molecular biology methods commonly known in the art. For example, in some embodiments, the 5' regulatory region sequence in the endogenous nif gene is replaced via site-directed mutagenesis or related processes to mutate specific nucleotides in the 5' regulatory region sequence and generate a DNA sequence that provides improved binding affinity for GlnR. In other embodiments, the 5' regulatory region sequence in the endogenous nif gene is replaced via gene editing methods to excise the native 5' regulatory region sequence and insert a recombinant DNA sequence that provides improved binding affinity for GlnR.

[0178] In some embodiments, the DNA sequence characterized by providing improved binding affinity for GlnR is derived from a second 5' regulatory region sequence within the endogenous nif gene. In other words, the 5' regulatory region sequence within the endogenous nif gene can be replaced with a second 5' regulatory region sequence from the endogenous nif gene located at an alternative site within the gene. For example, in some embodiments, the DNA sequence characterized by providing improved binding affinity for GlnR is derived from a second 5' regulatory region sequence within the endogenous nif gene, where the second 5' regulatory region sequence is located downstream of the transcription start site within the endogenous nif gene. In certain embodiments, the isolated genetically modified microorganism of the present disclosure comprises a genetic modification to a 5' regulatory region sequence within the endogenous nif gene, where the 5' regulatory region sequence is located upstream of the transcription start site of the endogenous nif gene, and the genetic modification to the 5' regulatory region sequence comprises replacing the 5' regulatory region sequence with a DNA sequence derived from the second 5' regulatory region sequence within the endogenous nif gene. In certain embodiments, the isolated genetically modified microorganism of the present disclosure comprises a genetic modification to a 5' regulatory region sequence within an endogenous nif gene, wherein the 5' regulatory region sequence is located upstream of the transcription start site of the endogenous nif gene, and wherein the genetic modification to the 5' regulatory region sequence comprises replacement of the 5' regulatory region sequence with a DNA sequence derived from a second 5' regulatory region sequence within the endogenous nif gene, wherein the second 5' regulatory region sequence within the endogenous nif gene is located downstream of the transcription start site within the endogenous nif gene.

[0179] In another embodiment, the DNA sequence characterized by providing improved binding affinity for GlnR is a non-native DNA sequence, meaning that the DNA sequence is not known to naturally occur within the endogenous nif gene as a binding site for GlnR. Non-native DNA sequences can be designed de novo using techniques and methods commonly known in the art, such as binding assays designed to test the interaction of GlnR with specific DNA sequences. For example, binding assays based on techniques including, but not limited to, fluorescence polarization and surface plasmon resonance can be used to determine the affinity of GlnR for a particular DNA sequence. Thus, a large number of variable DNA sequences can be screened for GlnR binding affinity, and the sequence exhibiting the highest affinity can be incorporated into the 5' regulatory region of the endogenous nif gene to provide regulated regulation of the nif gene by GlnR.

[0180] In some embodiments, DNA sequences characterized by providing improved binding affinity for GlnR are recognized and bound by GlnR with a dissociation constant or Kd that is about 2-fold to about 25-fold lower than the dissociation constant of the complex between GlnR and the native 5' regulatory region sequence. In other embodiments, DNA sequences characterized by providing improved binding affinity for GlnR are recognized and bound by GlnR with a dissociation constant or Kd that is about 1.1-fold to about 25-fold lower than the dissociation constant of the complex between GlnR and the native 5' regulatory region sequence. In some embodiments, a DNA sequence characterized by providing improved binding affinity for GlnR is recognized and bound by GlnR with a dissociation constant that is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, or about 25-fold lower than the dissociation constant of the complex between GlnR and the native 5' regulatory region sequence. In one particular embodiment, a DNA sequence characterized by providing improved binding affinity for GlnR is recognized and bound by GlnR with a dissociation constant that is about 17-fold lower than the dissociation constant of the complex between GlnR and the native 5' regulatory region sequence.

[0181] In some embodiments, an isolated genetically modified microorganism described herein comprises a genetic modification to an endogenous glnR gene encoding GlnR and a genetic modification to a 5' regulatory region sequence within the endogenous nif gene, wherein the genetic modification to the endogenous glnR gene comprises truncating a portion of the gene encoding the C-terminal domain of GlnR, and the genetic modification to the 5' regulatory region sequence within the nif gene comprises replacing the 5' regulatory region sequence upstream of the transcription start site with a DNA sequence that is recognized and bound by GlnR with higher binding affinity compared to the native 5' regulatory region sequence.

[0182] In some embodiments, the isolated genetically modified microorganism described herein comprises a genetic modification to an endogenous glnR gene encoding GlnR and a genetic modification to a 5' regulatory region sequence within the endogenous nif gene, wherein the genetic modification to the endogenous glnR gene comprises truncating a portion of the gene encoding the C-terminal domain of GlnR, and the genetic modification to the 5' regulatory region sequence within the nif gene comprises knocking out or deleting a 5' regulatory region sequence downstream of the transcription start site.

[0183] In some embodiments, the isolated genetically modified microorganism described herein comprises a genetic modification to an endogenous glnR gene encoding GlnR and a genetic modification to a 5' regulatory region sequence within the endogenous nif gene, wherein the genetic modification to the endogenous glnR gene comprises truncating a portion of the gene encoding the C-terminal domain of GlnR; the genetic modification to the 5' regulatory region sequence within the nif gene comprises replacing the 5' regulatory region sequence upstream of the transcription start site with a DNA sequence that is recognized and bound by GlnR with higher binding affinity compared to the native 5' regulatory region sequence; and the genetic modification to the 5' regulatory region sequence within the nif gene also comprises knocking out or deleting the 5' regulatory region sequence downstream of the transcription start site.

[0184] In some embodiments, the isolated genetically modified microorganism described herein comprises a genetic modification to a 5' regulatory region sequence within an endogenous nif gene, wherein the genetic modification to the 5' regulatory region sequence within the nif gene comprises replacing the 5' regulatory region sequence upstream of the transcription start site with a DNA sequence that is recognized and bound by GlnR with a higher binding affinity compared to the native 5' regulatory region sequence, and wherein the genetic modification to the 5' regulatory region sequence within the nif gene also comprises knocking out or deleting the 5' regulatory region sequence downstream of the transcription start site.

[0185] In some embodiments, the isolated genetically modified microorganism of the disclosure is characterized by having constitutive expression of nif genes. In some embodiments, the isolated genetically modified microorganism of the disclosure is characterized by having constitutive expression of nif genes regardless of local nitrogen or ammonia concentrations. For example, in certain embodiments, the isolated genetically modified microorganism is characterized by having constitutive expression of nif genes under nitrogen-limited conditions. In certain embodiments, the isolated genetically modified microorganism is characterized by having constitutive expression of nif genes under nitrogen-replete conditions. In certain embodiments, the isolated genetically modified microorganism is characterized by having constitutive expression of nif genes under nitrogen-limited or nitrogen-replete conditions.

[0186] The present disclosure also encompasses isolated, genetically modified microorganisms in which the microorganism lacks endogenous nif and / or glnR genes and has been genetically edited to incorporate exogenous nif and / or glnR genes. The exogenous nif and / or glnR genes incorporated into the isolated microorganism can include one or more of the genetic modifications to the nif and / or glnR genes described herein. For example, the exogenous glnR gene incorporated into the microorganism can encode a GlnR variant protein comprising a truncation of the C-terminal domain of the protein and / or be characterized as being capable of binding with enhanced affinity to a recognition element or 5' regulatory region sequence within the nif gene. The exogenous nif gene incorporated into the isolated microorganism can include one or more recognition elements or 5' regulatory region sequences that can be recognized and bound by GlnR with enhanced or diminished affinity. In addition to the exogenous nif and / or glnR genes, accessory genes generally known in the art to improve nitrogen fixation capacity can also be incorporated into a microorganism lacking the endogenous nif and / or glnR genes. The additional incorporation of these accessory genes can significantly improve the nitrogen fixation capacity of the microorganism over the incorporation of the exogenous nif and glnR genes alone.

[0187] Further descriptions of various nitrogen fixation cluster and control region editing are provided in WO2022204062A1, published September 29, 2022, and incorporated herein by reference.

[0188] microbial consortia In some aspects, the present disclosure provides a microbial consortium comprising a combination of at least any two microorganisms, one of which is a Paenibacillus strain listed in Table 1a, Table 1b, or Table 1c. In some embodiments, the Paenibacillus strain comprises a polynucleotide sequence sharing at least 90% identity with any one or more of SEQ ID NOs: 1-12. In some embodiments, the Paenibacillus strain is a species selected from the group consisting of Polymyxa, Tritici, Albidus, Anaericanus, Azotifigens, Borealis, Donghaensis, Ehimensis, Graminis, Jilunlii, Odorifer, Panacisoli, Phoenicis, Pocheonensis, Rhizoplanae, Silage, Taohuashanense, Thermophilus, Typhae, and Wynnii. In some embodiments, the Paenibacillus strain is of Subgroup I. In some embodiments, the Paenibacillus strain is of Subgroup II.

[0189] In certain embodiments, a consortia of the present disclosure includes two, three, four, five, six, seven, eight, nine, ten, or more microorganisms, wherein the microorganisms of the consortia are different microbial species or different strains of a microbial species.

[0190] Microbial production composition In some cases, a microorganism of the present disclosure may produce one or more compounds and / or may have one or more activities, for example, one or more of: production of a metabolite, production of a plant hormone such as an auxin, production of acetoin, production of an antimicrobial compound, production of a siderophore, production of a polyketide, production of a phenazine, production of a cellulase, production of a pectinase, production of a chitinase, production of a glucanase, production of a xylanase or a protease or an organic acid or a lipopeptide or a polynucleotide or a polypeptide, nitrogen fixation, phosphate mineral solubilization, or any combination and / or plurality of the foregoing.

[0191] For example, the microorganisms of the present disclosure may produce a plant hormone selected from the group consisting of auxin, cytokinin, gibberellin, ethylene, brassinosteroids, and abscisic acid.

[0192] Thus, "metabolites produced" by the microorganisms of the present disclosure are intended to refer to any molecule (such as a small molecule, vitamin, mineral, protein, nucleic acid, lipid, fat, carbohydrate, etc.) produced by the microorganism. The exact mechanism by which the microorganisms of the present disclosure confer beneficial traits to a given plant species is often unknown. In some instances, it is hypothesized that the microorganism produces a metabolic product that is beneficial to the plant. Thus, in some embodiments, a cell-free or inactivated preparation of a microorganism is beneficial to a plant because the microorganism need not be viable to confer a beneficial trait to the given plant species, as long as the preparation is produced by the microorganism and contains a metabolic product that is beneficial to the plant.

[0193] In one embodiment, the microorganisms of the present disclosure can produce auxin (e.g., indole-3-acetic acid (IAA)). Auxin production can be assayed. Many of the microorganisms described herein can be capable of producing the plant hormone auxin, indole-3-acetic acid (IAA), when grown in culture. Auxin plays an important role in altering plant physiology, including the extent of root growth.

[0194] Thus, in one embodiment, the microorganisms of the present disclosure are present as a population located on the surface or within the tissues of a given plant species. The microorganisms may produce a composition, such as a metabolite, in an amount effective to cause a detectable increase in the amount of the composition found on or within the plant compared to a reference plant that has not been treated with the microorganisms of the present disclosure or a cell-free or inert preparation. The composition produced by the microbial population may be beneficial to the plant species.

[0195] Such microbially produced compositions may be present in the cell culture broth or medium in which the microorganisms grow, or may include exudates produced by the microorganisms. As used herein, "exudate" refers to one or more compositions excreted by or extracted from one or more microbial cells. As used herein, "broth" refers to the aggregate composition of the cell culture medium after microbial cells have been placed in the medium. The composition of the broth may change over time during different stages of microbial growth and / or development. Broths and / or exudates may improve the traits of the plants with which they are associated.

[0196] Microbial-induced traits in plants The present disclosure utilizes microorganisms to impart beneficial properties (or beneficial traits) to desirable plant species, such as agronomic species of interest. In this disclosure, the terms "beneficial property," "beneficial trait," or "trait of interest" are used interchangeably to refer to a phenotype or genetic characteristic of a desired plant of interest that is modulated by application of a microorganism or microbial consortium described herein. As noted above, in some embodiments, it is highly likely that a metabolic product produced by a given microorganism is ultimately responsible for modulating or imparting a beneficial trait to a given plant.

[0197] There are numerous beneficial traits that can be modulated by 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 enhanced growth, increased yield, increased nitrogen use efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, improved water use efficiency, increased pathogen resistance, modification of plant architecture that does not necessarily affect plant yield but rather is related to plant functionality, increasing the production of a desired metabolite in the plant, etc.

[0198] In aspects, the microorganisms taught herein provide a wide range of agricultural applications, including improved grain, fruit, and flower yield, improved growth of plant parts, improved ability to utilize nutrients (e.g., nitrogen, phosphate, and the like), improved disease resistance, bio-pesticidal effects including improved resistance to fungi, insects, and / or nematodes, improved survivability in extreme climates, and improved phenotypic characteristics of other desirable plants.

[0199] In some embodiments, the genetically modified plant relative to a reference plant may exhibit 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, improved health, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved nutrient utilization (e.g., phosphate, potassium, etc.), improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, grain mass, grain moisture content, metal tolerance, number of ears, number of grains per ear, number of pods, nutritional enhancement, pathogen resistance (e.g., to impair pathogen survival), The isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to regulate or modify plant characteristics such as reduced pathogen levels (via excretion of metabolites), pest and disease resistance, improved photosynthetic capacity, salt tolerance, greening, improved plant vigor, increased mature seed dry weight, increased mature seed fresh weight, 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.

[0200] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to negatively regulate certain plant characteristics. For example, in some embodiments, the microorganisms of the present disclosure can reduce a phenotypic trait of interest, as this functionality may be desirable in some applications. For example, the microorganisms of the present disclosure may have the ability to reduce root growth or reduce root length. Or, the microorganisms may have the ability to reduce shoot growth or slow the rate at which a plant grows, as these modulations of plant traits may be desirable in certain applications.

[0201] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to confer resistance to nematode stress to the plants.

[0202] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to provide a biostimulant effect to the plants, hi some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to provide disease resistance to the plants.

[0203] Agricultural composition In some embodiments, the microorganisms of the present disclosure are combined with agricultural compositions. Agricultural compositions generally refer to organic and inorganic compounds, which may include compositions that promote the cultivation of microorganisms and / or plant elements; compositions involved in formulating microorganisms for application to plant elements (such as, but not limited to, wetting agents, compatibilizers (also referred to as "compatibilizers"), antifoaming agents, detergents, sequestering agents, drift reducing agents, neutralizing agents and buffers, corrosion inhibitors, dyes, odorants, spreading agents (also referred to as "spreading agents"), penetration aids (also referred to as "penetrating agents"), adhesives (also referred to as "stickers" or "binders"), dispersants, thickeners (also referred to as "thickeners"), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, etc.); compositions involved in providing protection to plant elements or plants (such as, but not limited to, insecticides, nematicides, fungicides, bactericides, herbicides, etc.), and other compositions that may be of interest for specific applications.

[0204] In some embodiments, the agricultural compositions of the present disclosure are solid. When a solid composition is used, it may be desirable to include one or more carrier materials with the active isolated microorganisms or consortia. In some embodiments, the present disclosure teaches the use of carriers including, but not limited to, silica, silica gel, silicates, talc, kaolin, atta clay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, mineral earth such as calcium sulfate, magnesium sulfate, magnesium oxide, synthetic soil-like materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea, and urea, plant-based products such as cereal meal, tree bark meal, wood meal, and nut shell meal, cellulose powder, attapulgite, montmorillonite, mica, vermiculite, synthetic silica, and synthetic calcium silicate, or compositions thereof.

[0205] growth composition In some embodiments, a growth and development promoting composition is provided to the microorganisms and / or plant elements. Exemplary compositions include liquids (broths, media, etc.) and / or solids (soil, nutrients, etc.). Various organic or inorganic compounds, alone or in combination with plant elements, such as, but not limited to, amino acids, vitamins, minerals, carbohydrates, simple sugars, lipids, can be added to the growth composition to promote the health of the microorganisms.

[0206] Pharmaceutical Composition One or more compositions may be combined in addition to the microorganism or microbially produced composition for various use, stability, activity, and / or storage reasons. The additional compositions may be referred to as "formulation components."

[0207] In some embodiments, the agricultural compositions disclosed herein may be formulated as a liquid, solid, gas, or gel.

[0208] Thus, 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 hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, sodium ammonium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate.

[0209] In some embodiments, the present disclosure teaches that 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 compositions thereof; lubricants such as magnesium stearate, sodium stearate, talc, or polyethylene glycol, or compositions thereof; antifoaming agents such as silicone emulsions, long chain alcohols, phosphate esters, acetylenic diols, fatty acids, or organofluorine compounds; and complexing agents such as salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid, or salts of polyphosphoric acids, or compositions thereof.

[0210] In some embodiments, the agricultural composition includes a surfactant. In some embodiments, the surfactant is added to a liquid agricultural composition. In other embodiments, the surfactant is added to a solid formulation, particularly a solid formulation designed to be diluted with a carrier before application. Thus, in some embodiments, the agricultural composition includes a surfactant. Surfactants may be used alone to improve the biological performance of microorganisms against their targets, or may be used with other additives, such as mineral oil or vegetable oil as an adjuvant to spray tank mixes. The type of surfactant used for biological enhancement largely depends on the nature and mode of action of the microorganism. Surfactants can be anionic, cationic, or nonionic in nature and can be used as emulsifiers, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactants are nonionic surfactants, such as alkyl ethoxylates, linear fatty alcohol ethoxylates, and fatty amine ethoxylates. Surfactants conventionally used in the formulation art and which may be used in the present formulations are described in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, NJ, 1998, and in Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., New York, 1980-81.In some embodiments, the present disclosure provides alkali metal, alkaline earth metal, or ammonium salts of aromatic sulfonic acids, such as lignosulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid, and dibutylnaphthalenesulfonic acid, as well as alkali metal, alkaline earth metal, or ammonium salts of fatty acids, alkyl ethers, lauryl ethers, fatty alcohol sulfates, and fatty alcohol glycol ether sulfates of arylsulfonic acids; condensates of sulfonated naphthalene with formaldehyde and derivatives thereof; condensates of phenol and formaldehyde with naphthalene or naphthalenesulfonic acid; condensates of formaldehyde with phenol or phenolsulfonic acid; condensation product of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctylphenol, ethoxylated octylphenyl ether, or ethoxylated nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohol, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenol, salt of phosphoric acid-containing triarylphenol ethoxylate, lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin sulfite waste liquor, or methylcellulose, or a composition thereof.

[0211] In some embodiments, the present disclosure provides salts of alkyl sulfates such as diethanolammonium lauryl sulfate, alkylaryl sulfonates such as calcium dodecylbenzene sulfonate, alkylphenol-alkylene oxide adducts such as nonylphenol-C18 ethoxylate, alcohol-alkylene oxide adducts such as tridecyl alcohol-C16 ethoxylate, soaps such as sodium stearate, alkylnaphthalene-sulfonates such as sodium dibutyl-naphthalene sulfonate, dialkyl esters of sulfosuccinates such as sodium di(2-ethylhexyl) sulfosuccinate. Other suitable surfactants are taught, including esters, sorbitol esters such as sorbitol oleate, quaternary amines such as lauryltrimethylammonium chloride, polyethylene glycol esters of fatty acids such as polyethylene glycol stearate, block copolymers of ethylene oxide and propylene oxide, salts of mono- and di-alkyl phosphate esters, vegetable oils such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, and the like, and esters, particularly the methyl esters, of the above vegetable oils.

[0212] In some embodiments, the agricultural composition includes a wetting agent. A wetting agent is a substance that, when added to a liquid, increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agricultural chemical formulations: to increase the wetting rate of powders in water during processing and manufacturing to create concentrates for soluble liquids or suspension concentrates, and to reduce the wetting time of wettable powders and improve water penetration 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 granules, are sodium lauryl sulfate, dioctyl sodium sulfonate, alkylphenol ethoxylates, and fatty alcohol ethoxylates.

[0213] In some embodiments, the agricultural composition of the present disclosure includes a dispersant. A dispersant is a substance that adsorbs to the surface of particles and helps maintain the dispersion of the particles and prevent them from reagglomerating. In some embodiments, dispersants are added to the agricultural composition of the present disclosure to facilitate dispersion and suspension during manufacturing and to ensure that the particles redisperse 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 particle surfaces and provide an electrostatic or steric barrier against particle reagglomeration. In some embodiments, the most commonly used surfactants are anionic surfactants, nonionic surfactants, or a mixture of the two.

[0214] In some embodiments of wettable powder formulations, the most common dispersants are sodium lignosulfonates. In some embodiments, suspension concentrates use polyelectrolytes such as sodium naphthalene sulfonate formaldehyde condensates to provide excellent adsorption and stabilization. In some embodiments, tristyrylphenol ethoxylate phosphate esters are also used. In some embodiments, alkylarylethylene oxide condensates and EO-PO block copolymers may be combined with anionic surfactants as dispersants in suspension concentrates.

[0215] In some embodiments, the agricultural compositions of the present disclosure include polymeric surfactants. In some embodiments, these polymeric surfactants have a very long hydrophobic "backbone" and numerous ethylene oxide chains that form the "teeth" of the surfactant "comb." In some embodiments, these high molecular weight polymers can impart excellent long-term stability to suspension concentrates because the hydrophobic backbone provides numerous anchoring points to the particle surface. In some embodiments, examples of dispersants that can be used in the agricultural compositions of the present disclosure include sodium lignosulfonate, sodium naphthalenesulfonate formaldehyde condensate, tristyrylphenol ethoxylate phosphate ester, fatty alcohol ethoxylate, alkyl ethoxylate, EO-PO block copolymer, and graft copolymer.

[0216] In some embodiments, the agricultural compositions of the present disclosure include an emulsifier. An emulsifier is a substance that stabilizes the suspension of droplets of one liquid phase in another. Without an emulsifier, the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier mixtures include an alkylphenol or aliphatic alcohol having 12 or more ethylene oxide units and an oil-soluble calcium salt of dodecylbenzenesulfonic acid. A hydrophilic-lipophilic balance ("HLB") value in the range of 8 to 18 will generally provide an emulsion with good stability. In some embodiments, the addition of a small amount of an EO-PO block copolymer surfactant may improve emulsion stability.

[0217] In some embodiments, the agricultural compositions of the present disclosure include a solubilizing agent. A solubilizing agent is a surfactant that forms micelles in water at concentrations above the critical micelle concentration. These micelles can then dissolve or solubilize water-insoluble substances within the hydrophobic interior of the micelles. The types of surfactants commonly used for solubilization are nonionics: sorbitan monooleate, sorbitan monooleate ethoxylate, and methyl oleate.

[0218] In some embodiments, the agricultural compositions of the present disclosure include an organic solvent. Organic solvents are primarily used in the formulation of emulsifiable concentrates, ULV formulations, and, to a lesser extent, granular formulations. Sometimes, solvent mixtures 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 the high molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as cosolvents to prevent crystallization of the formulation when the anti-pest agent is emulsified in water. Alcohols may be used as cosolvents to increase solvency.

[0219] In some embodiments, agricultural compositions include a gelling agent. Thickeners or gelling agents are primarily used in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheological or flow properties of the liquid and prevent separation and settling of dispersed particles or droplets. Thickeners, gelling agents, and anti-settling agents are broadly divided into two categories: water-insoluble particulates and water-soluble polymers. Clay and silica can be used to create suspension concentrate formulations. In some embodiments, agricultural compositions include one or more thickening agents, including, but not limited to, montmorillonite, e.g., bentonite, magnesium aluminum silicate, and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickening agents. The most commonly used types of polysaccharides are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Some embodiments utilize xanthan, and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickening agents, including, but not limited to, guar gum, locust bean gum, carrageenan, alginate, methylcellulose, sodium carboxymethylcellulose (SCMC), and hydroxyethylcellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents, such as modified starch, polyacrylic acid, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0220] In some embodiments, aqueous formulations can foam due to the presence of surfactants that reduce interfacial tension during mixing operations during production or spray tank application. Therefore, in some embodiments, antifoam agents are often added either during the production stage or before filling into bottles / spray tanks to reduce foaming tendencies. There are generally two types of antifoam agents: silicone antifoams and non-silicone antifoams. Silicones are typically aqueous emulsions of dimethylpolysiloxane, while non-silicone antifoams are water-insoluble oils such as octanol and nonanol, or silica. In both cases, the function of the antifoam agent is to remove surfactants from the air-water interface.

[0221] In some embodiments, the agricultural composition comprises a preservative.

[0222] In some embodiments, the agricultural composition may be formulated as a soil drench, foliar spray, dip treatment, in-furrow treatment, soil amendment, granule, broad-spectrum treatment, post-harvest disease control treatment, or seed treatment. In some embodiments, the agricultural composition may be applied alone or in a rotational application program with other agricultural products.

[0223] In some embodiments, the agricultural compositions may be compatible with tank-mixing. In some embodiments, the agricultural compositions may be compatible with tank-mixing with other agricultural products. In some embodiments, the agricultural compositions may be compatible with equipment used in ground, aerial, and irrigation applications.

[0224] In some embodiments, the agricultural composition may be applied to genetically modified seeds or plants.

[0225] protective composition Furthermore, individual microorganisms, microbial consortia, or microbial communities developed according to the disclosed methods can be combined with known active substances available in the agricultural space, such as pesticides, herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, miticides, plant growth regulators, rodenticides, algae control agents, biocontrol agents, or beneficial agents. Furthermore, the microorganisms, microbial consortia, or microbial communities developed according to the disclosed methods can be combined with known fertilizers. Such combinations may exhibit synergistic properties. Furthermore, individual microorganisms, microbial consortia, or microbial communities developed according to the disclosed methods can be combined with inert ingredients. In some embodiments, the microorganisms of the present disclosure are combined with bioactive agents.

[0226] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods can be combined with biopesticides that function as herbicides, bactericides, fungicides, insecticides, virucides, miticides, nematicides, acaricides, rodenticides, and / or algaecides. Such biopesticides can be, but are not limited to, macroorganisms (e.g., beneficial nematodes and the like), microorganisms (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).

[0227] Insecticides and bio-pesticides In some embodiments, the agricultural compositions of the present disclosure include a pesticide for use in combination with the microorganisms taught. In some embodiments, the agricultural compositions of the present disclosure include a biopesticide for use in combination with the microorganisms taught.

[0228] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods can be combined with known pesticides in the agricultural space, such as herbicides, fungicides, fungicides, insecticides, virucides, miticides, nematicides, acaricides, rodenticides, and / or algaecides.

[0229] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods can be combined with known pesticides in the agricultural space, such as bio-pesticides that function as herbicides, fungicides, fungicides, insecticides, virucides, miticides, nematicides, acaricides, rodenticides, and / or algaecides.

[0230] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients: active ingredients including macroorganisms (e.g., beneficial nematodes and the like), microorganisms (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).

[0231] In some embodiments, the individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods are resistant to at least one of the following: an acetamide selected from the group consisting of acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, mefenacet, metalaclor, metazachlor, napropamide, naproanilide, petoxamide, pretilachlor, propachlor, and thenylchlor; an amino acid derivative selected from the group consisting of bilanaphos, glufosinate, and sulfosate; clodinafop, cyhalofop; aryloxyphenoxypropionates selected from the group consisting of quizalofop-butyl, fenoxaprop, fluazifop, haloxifop, metamifop, propaquizafop, quizalofop, and quizalofop-p-tefuryl, diquat and paraquat, asulam, butyrate, carbetamide, desmedipham, dimepyrate, eptam (EPTC), esprocarb, molinate, orbencarb, phenmedipham, prosulfocarb, pyributicarb, thiobencarb, and triallate, butoroncarb, aryloxyphenoxypropionates selected from the group consisting of quizalofop-p-tefuryl, diquat and paraquat, (thio)carbamates selected from the group consisting of benzophenone-4, benzophenone-4, benzophenone-4-ol ... cyclohexanediones selected from the group consisting of xyzim, clethodim, cycloxydim, profoxydim, sethoxydim, tepraloxydim, and tralkoxydim; dinitroanilines selected from the group consisting of benfluralin, ethoflurane, oryzalin, pendimethalin, prodiamine, and trifluralin; diphenyl ethers selected from the group consisting of acifluorfen, aclonifen, bifenox, diclofop, ethoxyfen, fomesafen, lactofen, and oxyfluorfen; bomoxynil, dichlobenil, and hydroxybenzonitriles selected from the group consisting of ioxynil, imidazolinones selected from the group consisting of imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, and imazethapyr, phenoxyacetic acids selected from the group consisting of clomeprop, 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, fluthiacet, norflurazon, and pyridate,Pyridines selected from the group consisting of aminopyralid, clopyralid, diflufenican, dithiopyr, fluridone, fluroxypyr, picloram, picolinafen, and thiazopyr, azimsulfuron, bensulfuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl, nicosulfuron, oxasulfuron , primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometholone, sulfosulfuron, thifensulfuron, triasulfuron, triberon, trifloxysulfuron, triflusulfuron, tritosulfuron, and sulfonylureas selected from the group consisting of 14(2-chloro-6-propyl-imidazole[1,2]-blpyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea, ametriene, atrazine, cyanazine, dimethamethrine, etiodin, hexazinone, and metamitro triazines selected from the group consisting of chlortoluron, dymron, diuron, fluometuron, isoprotronturon, linuron, mesabentiazuron, and tebuthiuron; bispyribac-sodium, cloransulam-methyl, diclosulam, florasulam, flucarbazone, flumetsulam, metosulam, ortho-sulfamuron, penoxulam, propoxycarbazone, pyribambenz-propyl, pyribambenzyl ... tetrabenzoxazuron, tetrabenzoxazuron, tetrabenzoxazuron, tetrabenzoxazuron, tetrabenzoxazuron, tetrabenzoxazuron, tetrabenzo Acetolactate synthase inhibitors selected from the group consisting of benzoxime, pyriftalid, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyroxasulfone, and pyroxsulam, as well as amicarbazone, aminotriazole, anilofos, beflubutamide, benazolin, bencarbazone, benfuresate, benzofenap, bentazon, benzobicyclon, bromacil, bromobutide, butafenacil, butamifos, cafenstrole, carfentrazone, cinidon-ethyl, chlorthal, cinmethylin, clomazone, cumyluron,Cyprosulfamide, dicamba, difenzoquat, diflufenzopyr, Drechslera monoceras, endothall, ethofumesate, etobenzanide, fentrazamide, fluororac-pentyl, flumioxazin, flupoxam, fluorochloridone, flutamone, indanofan, isoxaben, isoxaflutole, lenacil, propanil, propyzamide, quinclorac, quinmerac, mesotrione, methylarsonic acid, naptalam, oxadiardil, oxadiazon, oxaziclomefon, pentoxazone , pinoxaden, pyraclonil, pyraflufen-ethyl, pyrasulfotole, pyrazoxyfen, pyrazolinate, quinoclamine, saflufenacil, sulcotrione, sulfentrazone, terbacil, tefuryltrione, tembotrione, thiencarbazone, topramezone, 4-hydroxy-3-[2-(2-methoxy-ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-bicyclol[3.2.1]oct-3-ene- 2-one, (3-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydro-2H-pyrimidin-1-yl)-phenoxyl]-pyridin-2-yloxy)-acetic acid ethyl ester, 6-amino-5-chloro-2-cyclopropyl-pyrimidine-4-carboxylic acid methyl ester, 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, 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-phenyl)-pyridine-2-carboxylic acid methyl ester, and a compound selected from the group consisting of 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridine-2-carboxylic acid methyl ester.

[0232] In some embodiments, the individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods are selected from the group consisting of acephate, azamethiphos, azinphos-methyl, chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, diazinon, dichlorvos, dicrotophos, dimethoate, disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion, methamidophos, methidathion, methyl-parathion, mevinphos, monocrotophos, oxydemeton-methyl, paraoxon, parathion ... organo(thio)phosphates selected from the group consisting of thion, phenthoate, phosalone, phosmet, phosphamidon, phorate, phoxim, pirimiphos-methyl, profenofos, prothiofos, sulprofos, tetrachlorvinphos, terbufos, triazophos, and trichlorfon, alanycarb, aldicarb, bendiocarb, benfuracarb, carbaryl, carbofuran, carbosulfan, fenoxycarb, furathiocarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb, and trichlorfon; Carbamates selected from the group consisting of azamates, allethrin, bifenthrin, cyfluthrin, cyfalothrin, cyphenothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, imiprothrin, lambda-cyhalothrin, permethrin, prallethrin, pyrethrins I and II, resmethrin, silafluofen, taufluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin a) a chitin synthesis inhibitor which is a benzoyl urea selected from the group consisting of chlorfluazuron, cilamadine, diflubenzuron, flucycloxalone, hexaflumuron, lufenuron, novaluron, teflubenzuron, triflumuron, buprofezin, diofenolan, hexythiazox, etoxazole, and clofentadine; b) a chitin synthesis inhibitor which is a benzoyl urea selected from the group consisting of halofenozide, methoxyfenozide, tebufenozide,and azadirachtin; c) an ecdysone antagonist selected from the group consisting of pyriproxyfen, methoprene, and fenoxycarb; or d) a lipid biosynthesis inhibitor selected from the group consisting of spirodiclofen, spiromesifen, and spirotetramat; an insect growth regulator selected from the group consisting of clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, acetamiprid, thiacloprid, and 1-(2-chloro-thiazol-5-ylmethyl)-2-nitrimino-3,5-dimethyl-[1,3,5]triazinane; and endosulfan. GABA antagonist compounds selected from the group consisting of ethiprole, fipronil, vaniliprole, pyrafluprole, pyriprole, and 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfinamoyl-1H-pyrazole-3-c-albothioic acid amide; macrocyclic lactone insecticides selected from the group consisting of abamectin, emamectin, milbemectin, lepimectin, spinosad, and spintrum; mitochondrial electron transport inhibitor (METI) I acaricides selected from the group consisting of fenazaquin, pyridaben, tebufenpyrad, tolfenpyrad, and flufenerim; METI I acaricides selected from the group consisting of acequinocyl, fluaciprim, and hydramethylnon. Compounds II and III may be combined with an insecticide selected from the group consisting of oxidative phosphorylation inhibitors selected from the group consisting of chlorfenapyr, cyhexatin, diafenthiuron, fenbutatin oxide, and propargite, sodium channel blockers selected from the group consisting of cryomazine, piperonyl butoxide, indoxacarb, and metaflumizone, and compounds selected from the group consisting of benclothiaz, bifenazate, cartap, flonicamid, pyridalyl, pymetrozine, sulfur, thiocyclam, flubendiamide, chlorantraniliprole, thiadipyr (HGW86), cyenopyrafen, flupyrazophos, cyflumetofen, amidoflumet, imicyaphos, bistrifluron, and pyrifluquinazone.

[0233] In some embodiments, the present invention teaches the synergistic use of a microorganism, or microbial consortium, of the present disclosure with known pesticides in agricultural spaces, such as herbicides, fungicides, fungicides, insecticides, virucides, acaricides, nematicides, miticides, rodenticides, and / or algaecides.

[0234] In some embodiments, the present invention teaches the synergistic use of the microorganisms, or microbial consortia, of the present disclosure with known pesticides in agricultural spaces, such as biopesticides that function as herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, miticides, rodenticides, and / or algaecides.

[0235] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a pesticide, an additive effect on the 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 the plant phenotypic trait of interest is observed.

[0236] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, an additive effect on the desired plant phenotypic trait 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 the desired plant phenotypic trait is observed.

[0237] The synergistic effect obtained by the methods taught can be quantified according to Colby's 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, incorporated herein by reference in its entirety. Thus, by "synergistic" is intended an ingredient whose presence increases the desired effect beyond the amount added.

[0238] The isolated microorganisms and consortia of the present disclosure can synergistically increase the effectiveness of agriculturally active pesticide compounds, as well as agricultural adjunct pesticide compounds.

[0239] The isolated microorganisms and consortia of the present disclosure can synergistically increase the efficacy of agriculturally active biopesticidal compounds, as well as agriculturally adjunct biopesticidal compounds.

[0240] Plant Growth Regulators and Biostimulants In some embodiments, the agricultural compositions of the present disclosure include plant growth regulators and / or biostimulants used in combination with the microorganisms of the present teachings.

[0241] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the methods of the present disclosure can be combined with known plant growth regulators in the agricultural space, such as auxins, gibberellins, cytokinins, ethylene generators, growth inhibitors, and growth retardants.

[0242] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients: ancymidol, butralin, alcohol, chloromequat chloride, cytokinin, daminozide, etepohon, flurpurmidol, gibberellic acid, gibberellin mixtures, indole-3-butyric acid (IBA), maleic hydrazide, mefluidide, mepiquat chloride, mepiquat pentaborate, naphthaleneacetic acid (NAA), 1-napthaleneacetemide (NAD), n-decanol, placlobutrazol, prohexadione calcium, trinexapac-ethyl, uniconazole, salicylic acid, abscisic acid, ethylene, brassinosteroids, jasmonates, polyamines, nitric oxide, strigolactone, or karrikin, among others.

[0243] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods can be combined with known seed inoculants in the agricultural space, such as QUICKROOTS®, VAULT®, RHIZO-STICK®, NODULATOR®, DORMAL®, and SABREX®, among others. In some embodiments, a Bradyrhizobium inoculant is utilized in combination with any single microorganism or microbial consortium disclosed herein. In certain aspects, synergistic effects are observed when one of the aforementioned inoculants, such as QUICKROOTS® or Bradyrhizobium spp., is combined with a microorganism or microbial consortium taught herein.

[0244] In some embodiments, the agricultural compositions of the present disclosure comprise a plant growth regulator, which comprises kinetin, gibberellic acid, and indole butyric acid along with copper, manganese, and zinc.

[0245] In some embodiments, the present disclosure provides a method for treating or preventing ulcers and ulcers in the rectum, including but not limited to, Abide®, A-Rest®, Butralin®, Fair®, Royaltac M®, Sucker-Plucker®, Off-Shoot®, Contact-85®, Citadel®, Cycocel®, E-Pro®, Conklin®, Culbac®, Cytoplex®, Early Harvest®, Foli-Zyme®, Goldengro®, Happygro®, Incite®, Megagro®, Ascend®, Radiate®, Stimulate®, Suppress®, Validate®, X-Cyte®, B-nine®, Compress®, Dazide®, Boll®, Buster®, BollD®, Cerone®, Cotton Quik®, Ethrel®, Finish®, Flash®, Florel®, Mature®, MFX®, Prep®, Proxy®, Quali-Pro®, SA-50®, Setup®, Super Boll®, Whiteout®, Cutless®, Legacy®, Mastiff®, Topflor®, Ascend®, Cytoplex®, Ascend®, Early Harvest®, Falgro®, Florgib®, Foli-Zyme®, GA3®, GibGro®, Green Sol®, Incite®, N-Large®, PGRIV®, Pro-Gibb®, Release®, Rouse®, Ryzup®, Stimulate®, BVB®, Chrysal®, Fascination®, Procone®, Fair®, Rite-Hite®, Royal®, Sucker Stuff®, Embark®, Sta-Lo®, Pix®, Pentia®, DipN Grow®, Goldengro®, Hi-Yield®, Rootone®, Antac®, FST-7®, Royaltac®, Bonzi®, Cambistat®, Cutdown®, Downsize®, Florazol®, Paclo®, Paczol®, Piccolo®, Profile®, Shortstop®, Trimmit®, Turf Enhancer®, Apogee®, Armor Agricultural compositions are taught that include one or more commercially available plant growth regulators, including, but not limited to, Tech®, Goldwing®, Governor®, Groom®, Legacy®, Primeraone®, Primo®, Provair®, Solace®, T-Nex®, T-Pac®, Concise®, and Sumagic®.

[0246] In some embodiments, the present invention teaches the synergistic use of a microorganism or microbial consortium of the present disclosure with a plant growth regulator and / or plant growth stimulant, such as a plant hormone or chemical that affects the production or disruption of a plant growth regulator.

[0247] In some embodiments, the present invention teaches that plant hormones can include auxins (e.g., indole acetic acid IAA), gibberellins, cytokinins (e.g., kinetin), abscisic acid, ethylene (and its production regulated by ACC synthase and disrupted by ACC deaminase).

[0248] In some embodiments, individual microorganisms, or microbial consortia or communities developed according to the disclosed methods can be combined with biostimulants, which can be, but are not limited to, microorganisms, plant extracts, seaweed, acids, biochar, etc.

[0249] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods can be combined with fertilizer, which can be organic (e.g., compost, blood, fish, etc.), nitrogenous (e.g., nitrate, ammonium, urea, etc.), phosphate, and potassium. Such fertilizers can also contain micronutrients, including, but not limited to, sulfur, iron, zinc, etc.

[0250] In some embodiments, the present invention teaches additional plant growth-promoting chemicals such as humic acids, fulvic acids, amino acids, polyphenols, and protein hydrolysates that can act synergistically with the microorganisms and microbial consortia disclosed herein.

[0251] Thus, in some embodiments, the present disclosure provides for applying a microorganism of the present teachings in combination with Ascend® to any crop. Further, the present disclosure provides for applying a microorganism of the present teachings in combination with Ascend® to any crop using any method or application rate.

[0252] In some embodiments, the present disclosure teaches agricultural compositions comprising biostimulants.

[0253] As used herein, the term "biostimulant" refers to any substance that acts to stimulate the growth of microorganisms that may be present in soil or other plant growth media.

[0254] The level of microorganisms in the soil or growing medium directly correlates with plant health. Because microorganisms feed on biodegradable carbon sources, plant health also correlates with the amount of organic matter in the soil. While fertilizers provide nutrients for plant growth and development, in some embodiments, biostimulants provide biodegradable carbon, e.g., molasses, carbohydrates, e.g., sugars, for microorganism growth and proliferation. Unless expressly specified otherwise, biostimulants may include a single component or a combination of several different components capable of promoting microbial activity or plant growth and development, where the promotion is due to the effect of one or more of the components acting independently or in combination.

[0255] In some embodiments, biostimulants are compounds that generate non-nutritional plant growth responses. In some embodiments, many of the important benefits of biostimulants are based on their ability to affect hormonal activity. Plant hormones (phytohormones) are chemical messengers that regulate normal plant development and responses to the environment. Root and shoot growth, as well as other growth responses, are regulated by plant hormones. In some embodiments, compounds in biostimulants can alter the hormonal status of plants and have a significant impact on the growth and health of the plants. Thus, in some embodiments, the present disclosure teaches sea kelp, humic acid, fulvic acid, and B vitamins as common components of biostimulants. In some embodiments, the biostimulants of the present disclosure enhance antioxidant activity, thereby improving the plant's defense system. In some embodiments, antioxidants included in biostimulants include vitamin C, vitamin E, and amino acids such as glycine.

[0256] In other embodiments, biostimulants can act to stimulate the growth of microorganisms present in soil or other plant growth media. Previous studies have shown that certain biostimulants, including certain organic seed extracts (e.g., soybeans), can stimulate the growth of microorganisms contained in a microbial inoculant when used in combination with the inoculant. Thus, in some embodiments, the present disclosure teaches one or more biostimulants that, when used in conjunction with a microbial inoculant, can enhance the populations of both indigenous and inoculant microorganisms. For a review of some common uses of biostimulants, see Calvo et al., 2014, Plant Soil 383:3-41.

[0257] Combinations of plant elements, microorganisms, and agricultural compositions In some embodiments, the present disclosure teaches that individual microorganisms, or microbial consortia, or microbial communities, including, for example, any one or more microorganisms, including the genome-edited Paenibacillus strains described herein, or any combination of the foregoing, optionally in combination with any agricultural composition, can be applied to plant elements for improved plant phenotype.

[0258] Isolated microorganisms or communities or consortia (generally, interchangeably, "microorganism(s)") may be applied to heterologous plant elements to create artificial mixtures. A microorganism is considered heterologous to a plant element if it is not normally associated with the plant element in nature, or if found at all, is applied in an amount different from that found in nature. In some embodiments, a microorganism may be naturally found in one part of a plant but not another, and the introduction of the microorganism into another part of the plant is considered a heterologous association.

[0259] It is contemplated that the microorganisms, either isolated or associated with plants or plant components, may be further associated with one or more agricultural compositions as described above.

[0260] Microorganisms and plant elements, microorganisms and agricultural compositions, and man-made mixtures of microorganisms and plant elements and agricultural compositions are contemplated (generally "synthetic compositions," which are compositions that include components not typically found in nature).

[0261] Plant Element Processing In some embodiments, the present disclosure also relates to the discovery that treating plant elements with one or more combinations of the microorganisms or agricultural compositions of the present disclosure before sowing or planting the plant elements can enhance desirable traits in the plant, such as plant growth, plant health, and / or plant resistance to pests and diseases.

[0262] Thus, in some embodiments, the present disclosure teaches the use of one or more microorganisms or microbial consortia as a plant element treatment. The plant element treatment can be a plant element coating applied directly to untreated and "bare" plant elements. However, the plant element treatment can also be a plant element protective film applied to plant elements that have already been coated with one or more previous plant element coatings or plant element treatments. The previous plant element treatments can include one or more active compounds, chemical or biological, and one or more inactive ingredients.

[0263] The term "plant element treatment" generally refers to the application of a substance to plant elements before or while the plant elements are planted in soil. Treating plant elements with the microorganisms and other agricultural compositions of the present disclosure has the advantage that the treatment occurs at the site where the plant elements will be planted shortly before the plant elements germinate and emerge.

[0264] In other embodiments, the present disclosure also teaches that the use of plant element treatments minimizes the amount of microorganisms or agricultural compositions required to successfully treat plants, and further limits worker contact with the microorganisms and compositions compared to application techniques such as application to the soil or application to emerged plant elements.

[0265] Additionally, in some embodiments, the present disclosure teaches that the microorganisms disclosed herein are important for enhancing the early stages of plant life (e.g., the first 30 days after emergence of plant elements). Thus, in some embodiments, delivery of the microorganisms and / or compositions of the present disclosure as plant element treatments positions the microorganisms at the site of action at a time critical for their activity.

[0266] In some embodiments, the microbial compositions of the present disclosure are formulated as plant element treatments. In some embodiments, it is believed that plant elements can be substantially uniformly coated with one or more of the microbial and / or agricultural compositions disclosed herein using conventional mixing, spraying, or a combination of these methods through the use of treatment application equipment specifically designed and manufactured to accurately, safely, and efficiently coat plant elements with the plant element treatment product. Such equipment uses various types of coating technologies, such as rotary coaters, drum coaters, fluidized bed technology, spouted beds, rotary misters, or combinations thereof. Liquid plant element treatments, such as those disclosed herein, can be applied through either a spinning "atomizer" disk or a spray nozzle, which distributes the plant element treatment evenly over the plant elements as it moves through the spray pattern. In some embodiments, the plant elements are then mixed or rolled for an additional period to further distribute the treatment and dry.

[0267] The plant elements may or may not be germinated before being coated with the microbial composition to increase uniformity of germination and seedling emergence. In an alternative embodiment, the dry powder formulation can be metered and sprinkled onto the moving plant elements and mixed until the dry powder formulation is completely distributed.

[0268] In some embodiments, a plant element has at least a portion of its surface area coated with a microbiological composition according to the present disclosure. In some embodiments, a plant element coating comprising a microbial composition is applied directly to a bare plant element. In some embodiments, a plant element protective film comprising a microbial composition is applied to a plant element already having a plant element coating applied thereon. In some aspects, a plant element can have a plant element coating comprising, for example, clothianidin and / or Bacillus firmus I-1582, onto which the present composition will be applied as a plant element protective film. In some aspects, a microbial composition of the present teachings is applied as a plant element protective film to a plant element that has already been treated with PONCHO™ VOTiVO™. In some aspects, a plant element can have a plant element coating comprising, for example, metalaxyl, and / or clothianidin, and / or Bacillus firmus I-1582, onto which the present composition will be applied as a plant element protective film. In some embodiments, the microbial composition of the present teachings is applied as a protective coating to plant elements that have already been treated with ACCELERON™.

[0269] In some embodiments, the plant elements treated with the microorganisms have a microbial spore concentration or microbial cell concentration of 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 per plant element.

[0270] In some embodiments, the plant elements treated with the microorganisms have a microbial spore concentration or microbial cell concentration of 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 per plant element.

[0271] In some embodiments, the plant elements treated with the microorganisms have a microbial spore or microbial cell concentration of 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 per plant element.

[0272] In some embodiments, the plant elements treated with the microorganisms have a microbial spore 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 element.

[0273] In some embodiments, the microbially treated plant elements have a microbial spore concentration or microbial cell concentration of about 10^5 to 10^9 per plant element.

[0274] In some embodiments, the plant elements treated with the microorganisms have a microbial spore or microbial cell concentration of at least about 1x10^3, or 1x10^4, or 1x10^5, or 1x10^6, or 1x10^7, or 1x10^8, or 1x10^9 per plant element.

[0275] In some embodiments, the amount of one or more of the microorganisms and / or agricultural compositions applied to the plant elements depends on the final formulation and the size or type of plant or plant element being utilized. In some embodiments, one or more of the microorganisms are present in an amount of from 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 is from about 5% w / w to about 65% w / w or from 10% w / w to about 60% w / w by weight of the total formulation.

[0276] In some embodiments, the plant elements may also have more spores or microbial cells per plant element, such as, for example, 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 element.

[0277] In some embodiments, the thickness of the plant element coating of the present disclosure is at most 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 μ 0μm, 390μm, 400μm, 410μm, 420μm, 430μm, 440μm, 450μm, 460μm, 470μm, 480μ m, 490μm, 500μm, 510μm, 520μm, 530μm, 540μm, 550μm, 560μm, 570μm, 580μm, 590μm, 600μm, 610μm, 620μm, 630μm, 640μm, 650μm, 660μm, 670μm, 680μm, 69 0μm, 700μm, 710μm, 720μm, 730μm, 740μm, 750μm, 760μm, 770μm, 780μm, 790μm , 800μm, 810μm, 820μm, 830μm, 840μm, 850μm, 860μm, 870μm, 880μm, 890μm, 9 00μm, 910μm, 920μm, 930μm, 940μm, 950μm, 960μm, 970μm, 980μm, 990μm, 100 0μm, 1010μm, 1020μm, 1030μm, 1040μm, 1050μm, 1060μm, 1070μm, 1080μm, 10 90μm, 1100μm, 1110μm, 1120μm, 1130μm, 1140μm, 1150μm, 1160μm, 1170μm, 11 80μm, 1190μm, 1200μm, 1210μm, 1220μm, 1230μm, 1240μm, 1250μm, 1260μm, 1 270μm, 1280μm, 1290μm, 1300μm, 1310μm, 1320μm, 1330μm, 1340μm, 1350μm, 1 360μm, 1370μm, 1380μm, 1390μm, 1400μm, 1410μm, 1420μm, 1430μm, 1440μm, 1450μm, 1460μm, 1470μm, 1480μm, 1490μm, 1500μm, 1510μm, 1520μm, 1530μm,1540μm, 1550μm, 1560μm, 1570μm, 1580μm, 1590μm, 1600μm, 1610μm, 1620μm, 1630μm, 1640μm, 1650μm, 1660μm, 1670μm, 1680μm, 1690μm, 1700μm, 1710μm ,1720μm,1730μm,1740μm,1750μm,1760μm,1770μm,1780μm,1790μm,1800μm,1810μm,1820μm,1830μm,1840μm,1850μm,1860μm,1870μm,1880μm,1890μm m, 1900μm, 1910μm, 1920μm, 1930μm, 1940μm, 1950μm, 1960μm, 1970μm, 1980μm, 1990μm, 2000μm, 2010μm, 2020μm, 2030μm, 2040μm, 2050μm, 2060μm, 2070 μm, 2080μm, 2090μm, 2100μm, 2110μm, 2120μm, 2130μm, 2140μm, 2150μm, 2160μm, 2170μm, 2180μm, 2190μm, 2200μm, 2210μm, 2220μm, 2230μm, 2240μm, 225 0μm, 2260μm, 2270μm, 2280μm, 2290μm, 2300μm, 2310μm, 2320μm, 2330μm, 2340μm, 2350μm, 2360μm, 2370μm, 2380μm, 2390μm, 2400μm, 2410μm, 2420μm, 24 30μm, 2440μm, 2450μm, 2460μm, 2470μm, 2480μm, 2490μm, 2500μm, 2510μm, 2520μm, 2530μm, 2540μm, 2550μm, 2560μm, 2570μm, 2580μm, 2590μm, 2600μm, 2 610μm, 2620μm, 2630μm, 2640μm, 2650μm, 2660μm, 2670μm, 2680μm, 2690μm, 2700μm, 2710μm, 2720μm, 2730μm, 2740μm, 2750μm, 2760μm, 2770μm, 2780μm, 2790μm, 2800μm, 2810μm, 2820μm, 2830μm, 2840μm, 2850μm, 2860μm, 2870μm, 2880μm, 2890μm, 2900μm, 2910μm, 2920μm, 2930μm, 2940μm, 2950μm, 2960μm,It can be 2970 μm, 2980 μm, 2990 μm, or 3000 μm.

[0278] In some embodiments, the plant element coating 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 thick.

[0279] In some embodiments, the plant element coating of the present disclosure comprises 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%, 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%, 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%, 50%, 51%, 51.5%, 52%, 52.5%, 53%, 53.5%, 54 4%, 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 The ratio may be 0.5%, 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%.

[0280] In some embodiments, the microbial spores and / or cells can be coated onto the plant elements freely, or can be formulated in a liquid or solid composition before being coated onto the plant elements. For example, a solid composition containing the microorganisms can be prepared by mixing a solid carrier with a spore or cell suspension until the solid carrier is impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

[0281] In some other embodiments, the solid or liquid microbial compositions of the present disclosure are contemplated to further contain functional agents, such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the produce, or combinations thereof.

[0282] Plant element coating methods and compositions known in the art can be particularly useful when they are modified by the addition of one of the embodiments of the present disclosure. Such coating methods and apparatus for their application 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, each of which is incorporated herein by reference.

[0283] Plant element 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, each of which is incorporated herein by reference.

[0284] In some embodiments, various additives can be added to plant element treatment formulations containing the compositions of the present invention. Binders can be added, including those composed of adhesive polymers, which can be natural or synthetic, that have no phytotoxic effects on the plant elements being coated. Binders can be selected from polyvinyl acetate, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, cellulose, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose; polyvinylpyrrolidone; starch, modified starch, dextrin, maltodextrin, alginates, and polysaccharides, including 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; polyhydroxyethyl acrylate; methylacrylamide monomer; and polychloroprene.

[0285] Any of a variety of colorants may be employed, including organic chromophores classified as azo, acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene, including nitroso, nitro, monoazo, bisazo, and polyazo. Other additives that may be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc.

[0286] A polymer or other dust suppressant may be applied to retain the treatment on the surface of the plant elements.

[0287] In some specific embodiments, in addition to the microbial cells or spores, the coating can further include a layer of adhesive. 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, celluloses such as methylcellulose, hydroxymethylcellulose, and hydroxymethylpropylcellulose, dextrin, alginate, sugar, molasses, polyvinylpyrrolidone, polysaccharides, proteins, fats, oils, gum arabic, gelatin, syrup, and starch. Further examples can be found, for example, in U.S. Pat. No. 7,213,367, incorporated herein by reference.

[0288] Various additives, such as adhesives, dispersants, surfactants, and nutrients and buffering ingredients, can also be included in the plant element treatment. Other conventional plant element treatments include, but are not limited to, coating agents, wetting agents, buffering agents, and polysaccharides. At least one agriculturally acceptable carrier can be added to the plant element treatment, such as water, a solid, or a dry powder. Dry powders can be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon, and various phosphorus compounds.

[0289] In some embodiments, the plant element coating composition can include at least one filler, which can be an organic or inorganic, natural or synthetic component, with which the active ingredient is combined to facilitate its application to the plant element. In aspects, the filler is an inert solid such as clay, a natural or synthetic silicate, silica, resin, wax, solid fertilizer (e.g., ammonium salt), a natural earth mineral such as kaolin, clay, talc, lime, quartz, attapulgite, montmorillonite, bentonite, or diatomaceous earth, or a synthetic mineral such as silica, alumina, or a silicate, particularly aluminum silicate or magnesium silicate.

[0290] In some embodiments, the plant element treatment agent may contain the following ingredients: other insecticides, including compounds that act only below ground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of these materials; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxy, urea, and benzoic acid; benzoxazines, benzhydryl derivatives, N,N -herbicide safeners such as diallyldichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives, chemical fertilizers, biological fertilizers, 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 can be added as a separate layer on the plant element or, alternatively, can be added as part of the plant element coating composition of the present disclosure.

[0291] In some embodiments, the formulations used to treat plant elements of the present disclosure can be in the form of suspensions, emulsions, slurries of particles in an aqueous medium (e.g., water), wettable powders, wettable granules (dry flowable), and dry granules. When formulated as a suspension or slurry, the concentration of the active ingredient in the formulation can be from about 0.5% to about 99% by weight (w / w), or 5-40%, or as otherwise formulated by one of skill in the art.

[0292] As noted above, other conventional non-active or inactive ingredients can be incorporated into the formulation. Such inactive ingredients include, but are not limited to, conventional adhesives, e.g., dispersants such as methylcellulose, which act as hybrid dispersants / adhesives for use in treating plant elements, polyvinyl alcohol, lecithin, polymeric dispersants (e.g., polyvinylpyrrolidone / vinyl acetate), thickeners (e.g., clay thickeners to improve viscosity and reduce settling of particle suspensions), emulsion stabilizers, surfactants, antifreeze compounds (e.g., urea), dyes, colorants, etc. Additional inactive ingredients useful in the present disclosure can be found in McCutcheon's, vol. 1, "Emulsifiers and Detergents," MC Publishing Company, Glen Rock, NJ, USA, 1996, incorporated herein by reference.

[0293] The plant element coating formulations of the present disclosure can be applied to the plant elements by a variety of methods, including, but not limited to, mixing in a container (e.g., a bottle or bag), mechanical dusting, tumbling, spraying, and dipping. A variety of active or inactive materials can be used to contact the plant elements with the microbial compositions according to the present disclosure.

[0294] In some embodiments, the amount of microorganism or agricultural composition used to treat the plant elements will vary depending on the type of plant element and the type of active ingredient, but the treatment involves contacting the plant element with an agriculturally effective amount of a composition of the present invention.

[0295] As discussed above, an effective amount refers to that amount of a composition of the invention sufficient to affect beneficial or desired results. An effective amount can be administered in one or more administrations.

[0296] In some embodiments, in addition to the coating layer, the plant elements may be treated with one or more of the following ingredients: fungicides and other pesticides, including herbicides, herbicide antidotes, fertilizers, and / or biocontrol agents. These ingredients may be added as a separate layer or may be added to the coating layer.

[0297] In some embodiments, the plant element coating agent of the present disclosure can be applied to the plant elements using various techniques and machines, such as fluidized bed technology, roller milling, rotary electrostatic plant element processors, and drum coaters. Other methods, such as spouted beds, can also be useful. The seed plant elements can be pre-sized before coating. After coating, the plant elements are typically dried and then sent to a sizing machine for sizing. Such techniques are known in the art.

[0298] In some embodiments, the microbially treated plant elements may be enveloped in an overcoating film to protect the coating. Such overcoatings are known in the art and may be applied using fluidized bed and drum film coating techniques.

[0299] In another embodiment of the present disclosure, a composition according to the present disclosure can be introduced into plant elements using solid matrix priming. For example, a certain amount of the composition of the present disclosure can be mixed with a solid matrix material, and then the plant elements can be contacted with the solid matrix material for a period of time to allow the composition to be introduced into the plant elements. The plant elements can then be separated from the solid matrix material and stored or used as desired, or the mixture of the solid matrix material and plant elements can be stored or planted as is. Solid matrix materials useful in the present disclosure include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the composition of the present invention over a period of time and releasing the composition into or onto the plant elements. It is useful to ensure that the composition of the present disclosure and the solid matrix material are compatible with each other. For example, the solid matrix material should be selected so that it can release the composition at a reasonable rate, for example, over minutes, hours, or days.

[0300] In some embodiments, the present disclosure teaches that individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods can be combined with any plant biostimulant.

[0301] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available biostimulants, including, but not limited to, Vitazyme®, Diehard™ Biorush®, Diehard™ Biorush® Fe, Diehard™ Soluble Kelp, Diehard™ Humate SP, Phocon®, Foliar Plus™, Plant Plus™, Accomplish LM®, Titan®, Soil Builder™, Nutri Life, Soil Solution™, Seed Coat™, PercPlus™, Plant Power®, CropKarb®, Thrust™, Fast2Grow®, Baccarat®, and Potente®, among others.

[0302] In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure exhibits an additive effect on a desired phenotypic trait in a plant when combined with an active chemical, while in other embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure exhibits a synergistic effect on a desired phenotypic trait in a plant when combined with an active chemical.

[0303] In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure is combined with a fertilizer to exhibit an additive effect on a desired phenotypic trait in a plant, while in other embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure is combined with a fertilizer to exhibit a synergistic effect on a desired phenotypic trait in a plant.

[0304] In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure is combined with a plant growth regulator to exhibit an additive effect on a phenotypic trait of interest in the plant. In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure is combined with a plant growth regulator to exhibit a synergistic effect. In some aspects, a synergistic effect is observed for one or more phenotypic traits of interest when Ascend® and a microorganism of the present disclosure are combined.

[0305] In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure exhibits an additive effect on a phenotypic trait of interest in a plant when combined with a biostimulant, hi some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure exhibits a synergistic effect when combined with a biostimulant.

[0306] The synergistic effect obtained by the methods taught can be quantified according to Colby's 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, incorporated herein by reference in its entirety. Thus, by "synergistic" is intended an ingredient whose presence increases the desired effect beyond the amount added.

[0307] The isolated microorganisms and consortia of the present disclosure can synergistically enhance the efficacy of agriculturally active compounds and can also synergistically enhance the efficacy of agriculturally adjunct compounds.

[0308] In other embodiments, synergistic effects are observed when a microorganism or microbial consortium identified according to the methods of the present disclosure is combined with a fertilizer.

[0309] Furthermore, in certain embodiments, the present disclosure utilizes synergistic interactions to define microbial consortia, i.e., in certain aspects, the present disclosure mixes certain isolated microbial species that act synergistically together into consortia that confer beneficial traits to plants or that correlate with increased beneficial plant traits.

[0310] To improve the activity of known active agricultural compounds, agricultural compositions developed according to the present disclosure can be formulated with certain adjuvants. This has the advantage that the amount of active ingredient in the formulation can be reduced while maintaining the efficacy of the active compound, thereby keeping costs as low as possible and complying with any official regulations. In individual cases, it may also be possible to broaden the spectrum of action of the active compound, since if treatment with a specific active ingredient without the addition of a specific adjuvant is not successful, adding the specific adjuvant together with the microbial isolates and consortia disclosed herein can actually successfully treat plants. Also, in individual cases, a suitable formulation can enhance the performance of the active compound when environmental conditions are not favorable.

[0311] Such adjuvants that can be used in agricultural compositions can be adjuvants. Adjuvants often take the form of surface-active compounds or salt-like compounds. Depending on their mechanism of action, adjuvants can be broadly classified as modifiers, activators, fertilizers, pH buffers, etc. Modifiers affect the wetting, adhesion, and spreading properties of the formulation. Activators break down the waxy cuticle of plants, improving both short-term (over minutes) and long-term (over hours) penetration of the active ingredient into the cuticle. Fertilizers such as ammonium sulfate, ammonium nitrate, or urea can improve the absorption and solubility of the active ingredient and reduce its antagonistic behavior. pH buffers are conventionally used to achieve an optimal pH in the formulation.

[0312] In some embodiments, the plant element is a plant reproductive element (e.g., a seed, tuber, bulb, and / or shoot). In some embodiments, the plant element is other than a plant reproductive element (e.g., a leaf, stem, and / or root). In some embodiments, a plurality of plant elements are associated with a microorganism described herein.

[0313] In some embodiments, the plant or plant element becomes associated with one or more microorganisms described herein through indirect means, such as, but not limited to, treatment of the growth medium in which the plant or plant element is placed.

[0314] For further embodiments of the agricultural compositions of the present disclosure, see "Chemistry and Technology of Agrochemical Formulations," edited by D.A. Knowles, 1998, copyright Kluwer Academic Publishers, Inc., which is incorporated herein by reference.

[0315] Plant and Agronomic Benefits A wide variety of plants, including those cultivated in agriculture, may be able to benefit from the application of microorganisms such as those described herein, including single microorganisms, consortia, and / or compositions produced therefrom or including any of the foregoing. Any number of a wide variety of plants, including mosses, lichens, and algae, may be used in the methods of the present disclosure. In embodiments, the plants have economic, social, or environmental value. For example, the plants may include plants used as food crops, fiber crops, oil crops, plants used in forestry, plants used in the pulp and paper industry, plants used as feedstock for biofuel production, and plants used as ornamentals.

[0316] The genetically modified microorganisms disclosed herein have applications in improving nitrogen fixation in plants. In some embodiments, such plants include those lacking natural nitrogen-fixing symbionts (e.g., non-legume crops), such as, but not limited to, wheat, maize (corn), rice, and vegetables. In some embodiments, such plants include plants that would benefit from additional nitrogen fixation, including plants that may be associated with nitrogen-fixing counterparts (e.g., legume crops).

[0317] How to apply Microorganisms may be applied to plants, seedlings, cuttings, propagules, etc., and / or to the growth medium containing the plants using any suitable technique known in the art.

[0318] However, by way of example, the microorganisms, consortia, or compositions comprising them and / or compositions produced therefrom may be applied to plants, seedlings, cuttings, propagules, or the like by spraying, coating, dusting, or any other method known in the art.

[0319] In another embodiment, the isolated microorganism, consortia or compositions comprising the same may be applied directly to plant seeds before sowing.

[0320] In another embodiment, the isolated microorganism, consortia or compositions comprising the same may be applied directly to plant seeds as a seed coating.

[0321] In one embodiment of the present disclosure, the isolated microorganism, consortia or compositions comprising the same are provided in the form of granules, or plugs, or soil drenches applied to plant growth media.

[0322] In other embodiments, the isolated microorganism, consortia or compositions comprising the same are provided in the form of a foliar application, such as a foliar spray composition or a foliar liquid composition, which may be applied to the growing plant or to a growing medium, such as soil.

[0323] In some embodiments, the isolated microorganism, consortia, or compositions comprising same are provided in a form selected from a soil drench, a foliar spray, a dip treatment, an in-furrow treatment, a soil amendment, a granular formulation, a broad-spectrum 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.

[0324] In some embodiments, the isolated microorganisms, consortia, or compositions comprising same may be compatible with tank-mixing. In some embodiments, the agricultural compositions may be compatible with tank-mixing with other agricultural products. In some embodiments, the agricultural compositions may be compatible with equipment used in ground, aerial, and irrigation applications.

[0325] In another embodiment, the isolated microorganisms, consortia containing the same, or compositions may be formulated as granules and applied together during planting of the seeds, or the granules may be applied after planting, or the granules may be applied before planting.

[0326] In some embodiments, the isolated microorganisms, consortia comprising the same, or compositions are applied to plants or growing media as topical and / or drench applications to improve crop growth, yield, and quality, which topical application may be through the use of a dry mix or dry powder or dusting composition, or may be a liquid-based formulation.

[0327] In embodiments, isolated microorganisms, consortia, or compositions comprising them can be formulated as, among other things, (1) solutions, (2) wettable powders, (3) dusts, (4) soluble powders, (5) emulsifiable concentrates or suspensions, (6) seed dressings or coatings, (7) tablets, (8) water-dispersible granules, (9) water-soluble granules (slow- or fast-release), (10) microencapsulated granules or suspensions, (11) irrigation components, and (12) components of fertilizers, pesticides, and other compatibility improvers. In certain aspects, the compositions can be diluted in an aqueous medium before conventional spray application. The compositions of the present disclosure can be applied to soil, plants, seeds, the rhizosphere, rhizosheath, or other areas where applying a microbial composition would be beneficial. Furthermore, ballistic methods can be used as a means to introduce endophytic microorganisms.

[0328] In some embodiments, the composition is sprayed onto the leaves of plants. The composition can be sprayed onto the leaves of plants in the form of an emulsion concentrate or suspension concentrate, a solution, or a foliar spray. Application of the composition can be carried out in a laboratory, a growth chamber, a greenhouse, or outdoors.

[0329] In another embodiment, the microorganisms can be inoculated onto the plant by cutting the root or stem and exposing the plant surface to the microorganisms by spraying, dipping, or painting with a liquid microbial suspension, or gel, or powder.

[0330] In another embodiment, the microorganisms may be injected directly into leaf or root tissue, or directly inoculated into leaf or root cuttings, or otherwise excised embryos or radicles or coleoptiles. These inoculated plants may then be exposed to growth medium containing additional microorganisms, although this is not required.

[0331] In other embodiments, microorganisms may be transferred to plants by any one or combination of grafting, explant insertion, aspiration, electroporation, wounding, root pruning, inducing stomatal opening, or any physical, chemical, or biological treatment that provides an opportunity for the microorganism to enter plant cells or intercellular spaces, particularly if the microorganism is not culturable. Those skilled in the art will readily recognize the many alternative techniques that can be used.

[0332] In one embodiment, the microorganisms penetrate plant parts such as roots, stems, leaves, and / or plant reproductive parts (becoming endophytic) and / or grow on the surface of roots, stems, leaves, and / or plant reproductive parts (becoming epiphytic) and / or grow within the plant rhizosphere. In one embodiment, the microorganisms form a symbiotic relationship with the plant.

[0333] Certain non-limiting aspects are provided below.

[0334] Aspect 1: An isolated genetically modified microorganism comprising one or more genetic modifications selected from a genetic modification to an endogenous glnR gene encoding GlnR and a genetic modification to a 5' regulatory region sequence within an endogenous nif gene, wherein the genetic modification to the endogenous glnR gene encoding GlnR provides a mutant glnR gene that produces a GlnR protein variant; the genetic modification to the 5' regulatory region sequence provides an improved binding affinity for GlnR compared to a 5' regulatory region sequence that is not genetically modified; and the one or more genetic modifications provide the microorganism with improved nitrogen fixation activity compared to a microbial strain that is not genetically modified.

[0335] While the present invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Various modifications, alterations, substitutions, and improvements of the present disclosure that will readily occur to those skilled in the art, including certain alterations, alterations, substitutions, and improvements, are also part of this disclosure. For example, the following specific examples may illustrate the methods and embodiments described herein using specific plants, but the principles of these examples may be applied to any plant. Therefore, it will be understood that the scope of the present invention is encompassed not only by the specific examples exemplified below, but also by the embodiments listed herein.

[0336] All cited patents and publications referenced in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each was individually and specifically incorporated by reference. [Example]

[0337] The methods and compositions presented herein improve one or more characteristics of plants, e.g., nitrogen fixation in agricultural crops, based on utilizing the disclosed isolated microorganisms, consortia, communities, and / or compositions comprising and / or produced by microorganisms or consortia or communities.

[0338] The abbreviation "uL" means "microliter" and "ug" means "microgram."

[0339] Example 1: Microbial Cultivation, Sequencing, and Target Selection Paenibacillus strains 8619, 17899, 55083, 68890, and 77155, as well as any of the genetically modified strains described above, were grown in culture medium to obtain sufficient cell growth.

[0340] Subsamples of each of these strains were then aseptically transferred to nitrogen-free growth medium and incubated under microaerobic conditions for 72 hours.

[0341] Isolates of interest were grown to mid-logarithmic phase in R2A medium. DNA was extracted with the Qiagen Powersoil DNA Extraction Kit, and sequencing libraries were constructed with the iGenomix RipTide kit according to the manufacturer's instructions. Sequencing was performed on an Illumina HiSeq using PE150. Raw Illumina reads were trimmed to Q15 with Trimmomatic v38 (Bolger AM, Lohse M, and Usadel B. (2014). Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, btu170) and assembled with SPAdes (Prjibelski A, Antipov D, Meleshko D, Lapidus A, and Korobeynikov A. (2020). Using SPAdes de novo assembler. Curr. Protoc. Bioinform. 70, e102) using default parameters. Assembled contigs were analyzed for purity with a <5% contamination cutoff using BinSantity 0.5.4 (Graham ED, Heidelberg JF, and Tully BJ. (2017) BinSanity: unsupervised clustering of environmental microbial assemblies using coverage and affinity propagation. PeerJ 5:e3035). The largest bin was extracted and annotated with Prokka 1.8 (Seemann T. (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30(14):2068-9). 16S rDNA sequences were identified using Prokka 1.8, and sequences were extracted directly from the .ffn files.Taxonomy was assigned by GTDB-tk using default parameters in the April 2021 database (Pierre-Alain Chaumeil, Aaron J Mussig, Philip Hugenholtz, Donovan H Parks, GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database, Bioinformatics, Volume 36, Issue 6, 15 March 2020, Pages 1925-1927).

[0342] A selection of Paenibacillus strains was characterized as subgroup I or subgroup II according to the method of Xie et al. (2014) (Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization, Evolution and Expression of the Nitrogen Fixation Genes. 10(3)). The nif gene cluster, consisting of nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, and nifV, is highly conserved among 15 N2-fixing Paenibacillus strains, but there is some variation in the DNA sequence of the nif cluster, which can be divided into two subgroups: subgroup I and subgroup II. The nine genes of the nif gene cluster, nifBHDKENXhesAnifV, are contiguous within subgroup I, while a 261- to 561-bp ORF with an unknown predicted product is located between nifX and hesA within subgroup II. The Paenibacillus species P. polymyxa and P. tritici are examples of subgroup I. The Paenibacillus species P. albidus, P. anaericanus, P. azotifigens, P. borealis, P. donghaensis, P. ehimensis, P. graminis, P. jilunlii, P. odorifer, P. panacisoli, P. phoenicis, P. pocheonensis, P. rhizoplanae, P. silage, P. taohuashanense, P. thermophilus, P. typhae, and P. wynnii are examples of subgroup II.

[0343] Various polynucleotide editing targets in the Paenibacillus genome were selected to increase nitrogen fixation in the absence of exogenously applied nitrogen, in the presence of minimal added nitrogen (e.g., ammonium), and in the presence of added nitrogen. Several approaches were developed, including turning off negative regulation of the nif operon in the presence of environmental nitrogen and promoting transcription during all conditions.

[0344] GlnR In Paenibacillus bacteria, the nif operon controls the nitrogen fixation pathway via GlnR, and nif operon gene transcription is regulated by ammonium and oxygen. We constructed a GlnR knockout (removing the entire coding region from ATG to M156 by homologous recombination, leaving a stop codon in the resulting sequence) and a C25 truncation (removing the DNA encoding the last 25 amino acids of glnR) to assess their effects on nitrogen fixation.

[0345] CueR Like GlnR, CueR is an HTH-type transcriptional regulator. It is also located immediately upstream of the nrgA (ammonium transporter) gene and shares the same bidirectional transporter. It may be a novel transcriptional regulator.

[0346] In many bacterial species, CueR is generally recognized as a regulator of the Cue copper efflux system, activating gene expression in response to high levels of intracellular copper. In our Paenibacillus polymyxa, the CueR ORF shares a bidirectional promoter with the ammonium transporter NrgA. CueR, like GlnR, is an HTH-type transcriptional regulator, and its proximity to NrgA in Paenibacillus polymyxa suggests that it may also be involved in the transcription of the ammonium transporter. In this case, removal of CueR misregulates NrgA expression in response to nitrogen levels, reducing ammonium transport into the cell. The reduced ammonium level forces cells to utilize atmospheric nitrogen through the expression of nitrogenase.

[0347] The locus CM8619_hybrid_04839 was extracted from the CM8619_hybrid_CE assembly and analyzed for identity of MerR-family HTH-regulated genes. All orthologous Paenibacillus sp. Y412MC10 MerR-family HTH-regulated genes were retrieved from the KEGG orthology (KO) database. A BLAST database was constructed using MerR orthologous genes, and a bidirectional BLAST search was performed against the putative MerR gene glnaRnt using blastp. The top hit was a 62% identity hit with CueR. The Paenibacillus sp. Y412MC10 assembly was retrieved from NCBI, and the gene landscape of the CueR region was matched to that of CM8619, which has nrgA immediately upstream and a zinc metalloprotease downstream.

[0348] orf1 In the native nif cluster of Paenibacillus odorifer CM17899, the orf1 gene overlaps the downstream sequence of nifX with the first 17 nucleotides of orf1. In the orf1 knockout, these nucleotides were preserved, and the gene sequence from nucleotide A18 to the final nucleotide G567 was deleted without interruption. The deletion results in a significantly truncated orf1 expression product with a sequence of seven amino acids.

[0349] GlnA Glutamine synthetase (GS), encoded by glnA in the glnRA operon, directly interacts with the C-terminal domain of GlnR and regulates GlnR activity.

[0350] GlnR binding sites I and II Binding of GlnR to site I activates Nif expression, while binding of GlnR to site II represses Nif expression. Furthermore, GlnR has a high affinity for binding to site II. Several approaches to increase expression of the nif operon and increase nitrogen fixation were explored: inactivation of site I (substitution of the last six nucleic acids of the native GlnR-binding site I sequence), deletion of site II, inactivation of site II (substitution of the last six nucleic acids of the native GlnR-binding site II sequence), duplication of site II sequence and insertion into site I (e.g., substitution of site I), and combinations of edits involving any one or more of the foregoing.

[0351] Example 2: Compilation of Paenibacillus strains Editing a Paenibacillus microorganism to produce an engineered strain that confers improved characteristics to a plant with which it is associated can be achieved by nucleotide insertion, nucleotide deletion, nucleotide substitution, and / or any combination or plurality of the foregoing. The net effect can be one of upregulation, downregulation, knockout (of the function of the target polynucleotide and / or its encoded RNA or protein), and / or any combination or plurality of the foregoing.

[0352] The difficulty of working with Gram-positive bacteria such as Paenibacillus is well known, so the successful editing and association with plants, which confers improved benefits to plants, compared to docile Gram-negative bacteria such as Klebsiella, is surprising and unexpected.

[0353] The Gram-positive gene editing vector pMiniMad2 was obtained from the Bacillus Genetic Stock Center and modified by insertion of the TraJ transfer origin originally from vector pKVM4 between the SalI and BamHI restriction sites, resulting in the mobilizable Gram-positive gene editing vector pMMmob.

[0354] Editing vector assembly Polymerase chain reaction (PCR) was performed using appropriate primers with Q5 high-fidelity polymerase to amplify upstream and downstream homology arms with the appropriate Gibson assembly overhangs from purified genomic DNA from the target strain. PCR products were run on an agarose gel to confirm the appropriate size. Bands were excised and purified from the gel.

[0355] The backbone vector pMMmob was digested with the restriction enzymes EcoRI and BamHI in Cutsmart buffer for at least 30 minutes at 37°C. The digest was run on an agarose gel to confirm the appropriate size. The digested backbone was purified from the gel.

[0356] Gibson assembly was performed by combining approximately 100 ng of digested pMMmob with the insert in a 10 μl volume at a backbone:insert:insert molar ratio of 1:3:3, followed by the addition of 10 μl of 2× Gibson reagent. The reaction was incubated at 50°C for 60 minutes and then used for transformation into E. coli DH5α.

[0357] One to five microliters of Gibson assembly mixture was added to 50 μl of freshly thawed, chemically competent E. coli DH5α cells and finger-vortexed. The cell-plasmid mixture was incubated on ice for 30 minutes, heat-shocked at 42°C for 30 seconds, and then returned to ice for an additional 5 minutes. 1 mL of SOC (super-optimal catabolic) medium was added, and the cells were incubated at 37°C with shaking at 200 RPM for 60-90 minutes and then harvested. Dilutions of the harvest culture were plated onto LB agar plates supplemented with 100 ng / μL ampicillin and incubated overnight at 37°C.

[0358] The region of the plasmid containing the assembled insert was amplified from several recovered colonies by colony PCR using GoTaq polymerase, and the PCR products were run on an agarose gel to confirm the expected size of the product. PCR products of the appropriate size were sent for Sanger sequencing to confirm proper assembly and the lack of off-target mutations in the editing cassette.

[0359] Colonies confirmed to harbor the correct plasmid were inoculated into LB broth supplemented with 100 ng / uL ampicillin and grown overnight at 37°C with 200 RPM shaking. Plasmids were purified from the overnight cultures and transformed into the mating donor strain E. coli BW29472 by electroporation.

[0360] 1 μL of purified plasmid was combined with 50 μL of freshly thawed E. coli BW29472 electrocompetent cells and incubated on ice for 5 minutes. The cell-plasmid mixture was transferred to a 1 mm electroporation cuvette pre-chilled on ice. An electroporator was used, 1800 V, 25 μF, and 200 Ω charge applied to the cuvette. The sample was immediately resuspended in 1 mL of SOC medium supplemented with 0.3 mM 2,6-diaminopimelic acid (DAP). The resuspended cells were incubated at 37°C for 60-90 minutes with shaking at 200 RPM and then allowed to recover. Dilutions of the recovered culture were plated onto LB agar plates supplemented with 100 ng / μL ampicillin and 0.3 mM 2,6-diaminopimelic acid and incubated overnight at 37°C. The recovered transformants were used as donor strains for conjugation.

[0361] Joining The recipient strain was inoculated into 5 mL of tryptic soy broth (TSB) medium in a 50 mL conical tube and grown overnight at 30° C. with 200 RPM shaking. The donor E. coli BW29427 harboring the mobilized plasmid was inoculated into 5 mL of LB medium supplemented with 100 μg / μL ampicillin and 0.3 mM 2,6-diaminopimelic acid (DAP) and grown overnight at 37° C. with 200 RPM shaking.

[0362] One ml aliquots of the overnight donor and recipient cultures were spun down, washed with sterile water, combined, and spotted onto the surface of LB agar plates supplemented with 0.3 mM DAP for conjugative mating. Mating plates were incubated overnight at 25°C (a temperature permissive for replication of pMMmob in the Gram-positive recipient strain).

[0363] The mating mixture was resuspended by adding 1 ml of sterile water over the top of the spot and agitated with a sterile L-spreader. The resuspension was collected in a microcentrifuge tube, washed, and resuspended in 100 μl of sterile water. The enriched cells were spread onto TSA plates supplemented with MLS (25 μg / ml lincomycin, 1 μg / ml erythromycin) without DAP and incubated at 25°C for 48-72 hours until transconjugant colonies appeared.

[0364] Plasmid integration The recovered transconjugants were inoculated into 5 mL of TSB medium supplemented with MLS and grown for 48 h at 25°C with 200 RMP shaking or until turbid. Dilutions of the culture were plated onto TSA+MLS plates and incubated overnight at 37°C (the restrictive temperature for plasmid replication) until integrated colonies appeared.

[0365] Plasmid excision The integrated colonies were inoculated into 5 mL of TSB medium supplemented with MLS and incubated overnight at 37°C with shaking at 200 RPM. 5 μl of the overnight culture was diluted into 5 mL of fresh TSB medium without antibiotics and grown overnight at 25°C with shaking at 200 RPM. The 5 μl overnight culture was subcultured twice into 5 mL of fresh TSB at 25°C with shaking at 200 RPM, for a total of three rounds of subculturing. A dilution of the third round of overnight culture was plated onto R2A plates lacking antibiotics and incubated overnight at 30°C.

[0366] Plasmid excision was confirmed by picking individual colonies from the R2A plates and replating them in a grid format onto agar plates with and without MLS. Both plates were incubated at 30°C for 24-48 hours until colonies appeared. Colonies that grew when plated on the plates without MLS but not on the plates with MLS confirmed that the plasmid had been excised and lost.

[0367] Check your edits The edited region was amplified from the putatively edited strains by colony PCR, and the presence of proper editing was confirmed by band size when run on an agarose gel (if possible) and / or by Sanger sequencing. PCR assay for the MLS resistance cassette confirmed the absence of the plasmid backbone. Colonies that yielded the MLS cassette band were confirmed to be non-properly edited.

[0368] The sequence results were analyzed to distinguish colonies that had excised to wild type from properly edited colonies, and the sequences were examined for off-target mutations.

[0369] Colonies sequenced as having appropriate edits without off-target mutations were given a modified strain designation, added to the modified strain library, and made available to the project team for bioassay analysis.

[0370] The following edits were delivered to one or more parental lines and made available for in vitro and plant evaluation.

[0371] GlnR binding site II inactivation Substitution of nucleotides bound by GlnR during repression of Nif expression with nucleotides that do not bind GlnR results in increased nitrogenase activity. This edit prevents repression of the nif pathway in response to excess nitrogen levels, analogous to removing an off switch.

[0372] The sequence "ATCGAT" was inserted between approximately 1000 base pairs of native genomic sequence upstream, including the seventh to final nucleotide of GlnR binding site II, and approximately 1000 base pairs of native genomic sequence downstream, including but not including the final base pair of GlnR binding site II.

[0373] GlnR binding site II duplication This replacement of the native site I sequence with the native site II sequence is intended to increase nif expression and increase the binding affinity of GlnR for the activation sequence. Under all conditions, GlnR has a higher binding affinity for the site II sequence. Because the activating versus repressing activity of GlnR appears to depend on the position of binding, increasing the binding affinity for the position involved in activation led to increased nif expression.

[0374] An editing cassette was constructed by inserting the native GlnR binding site II sequence between approximately 1000 base pairs of native genomic sequence upstream, but not including, the first nucleotide of GlnR binding site I, and approximately 1000 base pairs downstream, but not including the last base pair of GlnR binding site I.

[0375] GlnR binding site II inactivation and duplication These edits were predicted to work synergistically by preventing GlnR dimers from binding to the site II "off switch" and enhancing the "on switch" by increasing its binding efficiency. Together, these edits ensure that GlnR can only act as a strong positive influence on nif operon gene transcription.

[0376] An editing cassette was constructed by inserting the native GlnR binding site II sequence between the genomic sequence of a strain previously edited with GlnR binding site II inactivation approximately 1000 base pairs upstream, but not including, the first nucleotide of GlnR binding site I, and the genomic sequence of a strain previously edited with GlnR binding site II inactivation approximately 1000 base pairs downstream, but not including, the last base pair of GlnR binding site I.

[0377] GlnR C25 cleavage The alpha-helical C25 region allows GlnR to exist primarily as a monomer in cells when folded over the dimerization site. The C-terminal region does not fold and promote dimerization until feedback-inhibited glutamine synthase (FBI-GS) interacts with GlnR monomers under conditions of nitrogen excess. GlnR dimers can then tightly bind to binding site II for strict repression of nif expression. Removal of the C-terminal 25 amino acids disrupts this regulatory interaction, preventing GlnR dimerization and binding from being governed by nitrogen levels. This would allow increased nif expression under conditions of nitrogen excess.

[0378] The editing cassette was constructed by inserting approximately 500–1300 (strain-dependent) base pairs of native genomic sequence upstream of, but not including, the 25th codon from the C-terminus of the glnR open reading frame, and assembled with 500–900 (strain-dependent) base pairs of native genomic sequence downstream of, but including, the stop codon of the glnR open reading frame.

[0379] GlnR C25 frameshift truncation The A76 nucleotide sequence between nucleotides A332 and T409 in the glnR gene was deleted, destroying the native stop codon and resulting in a frameshift mutation, which resulted in the addition of 13 amino acids to the GlnR protein before reaching the stop mutation.

[0380] glnA SNP Glutamine synthetase, encoded by glnA, is a key component of the regulatory apparatus of the Paenibacillus nif cluster. Single nucleotide polymorphisms (SNPs) introduced into the sequence can affect the regulatory function of the protein, resulting in a net increase in nitrogen fixation activity. The GlnA SNP described herein is a substitution of an adenosine residue for the cytosine at position 164 of the open reading frame.

[0381] The editing cassette was constructed by assembling the native genome sequence approximately 500–100 base pairs upstream of the SNP site with the native genome sequence approximately 500–1000 base pairs downstream of the SNP site, with the adenosine residue substitution in between.

[0382] GlnR C25 frameshift truncation and glnA SNP The A76 nucleotide sequence between nucleotides A332 and T409 in the glnR gene was deleted, destroying the native stop codon and resulting in a frameshift mutation. This resulted in the addition of 13 amino acids to the GlnR protein before reaching the stop mutation. Concomitantly, a single base was changed in the adjacent glnA gene: nucleotide C164 was changed to A, resulting in a serine-to-tyrosine mutation at amino acid position 55 of the GlnA protein.

[0383] To design an editing cassette deleting the last 25 amino acids, primers were designed to amplify a nucleotide arm approximately 500 nucleotides upstream and including G333 of glnR from Paenibacillus odorifer CM17899, and approximately 500 nucleotides downstream and including T409, resulting in a clean 75-nucleotide deletion from the glnR gene and a 25-amino acid deletion from the GlnR protein while retaining the native stop codon. An error in primer design resulted in the upstream homology region extending from and including A332. This error resulted in an editing cassette that delivered a 76-nucleotide deletion, destroyed the native stop codon, and caused a frameshift mutation.

[0384] During construction of the editing cassette, a single nucleotide polymorphism (SNP) was introduced during polymerase chain reaction (PCR) to amplify the downstream homology arm in the glnA gene. This SNP could have been introduced by random error due to the Q5 high-fidelity polymerase used in the PCR reaction or by a SNP present in the copy of P. polymyxa CM17899 genomic DNA used in the reaction. This nucleotide change was not based on a known mutation in the GlnA gene of Paenibacillus spp. and was non-directed. During integration and removal of the editing plasmid into P. polymyxa CM17899, this off-target modification was left in the resulting homologous region. These edits produced surprising positive results in plants associated with the edited microorganism.

[0385] CueR Knockout The editing cassette was constructed by assembling approximately 1000 base pairs of native genomic sequence upstream of, but not including, the start codon of the cueR open reading frame with 1000 base pairs of native genomic sequence downstream of, but including the stop codon of the cueR open reading frame.

[0386] CueR C25 disconnection The editing cassette was constructed by assembling approximately 1000 base pairs of native genomic sequence upstream, but not including, the codon 25 from the C-terminus of the cueR open reading frame, with 1000 base pairs of native genomic sequence downstream, but including, the stop codon of the cueR open reading frame.

[0387] Orf1 knockout This was expected to indicate a decrease in nif activity due to reduced oxygen tolerance. We speculate that this edit demonstrates the potential value of inserting this gene into strains lacking it. However, if this edit shows increased nif expression, it may be for the following reasons.

[0388] orf1 is an open reading frame found in the nif cluster of some Paenibacillus strains (referred to as subcategory II) but not in others (referred to as subcategory I). It is predicted to function in the presence of high oxygen levels. Its absence in many high-performing strains may indicate that it is redundant; in most cases, nitrogen assimilation is more efficient in its absence. This may be through removing the metabolic burden of expression of this ORF or through redundant activity of the expression product itself.

[0389] The editing cassette was constructed by assembling approximately 1000 base pairs of native genomic sequence upstream, including, the stop codon of the nifX open reading frame with 1000 base pairs of native genomic sequence downstream, but not including, the stop codon of the Orf1 open reading frame.

[0390] In the native nif cluster of Paenibacillus odorifer CM17899, the orf1 gene overlaps the downstream sequence of nifX with the first 17 nucleotides of orf1. In the orf1 knockout, these nucleotides were preserved, and the gene sequence from nucleotide A18 to the final nucleotide G567 was deleted without interruption. The deletion results in a significantly truncated orf1 expression product with a sequence of seven amino acids.

[0391] Using standard molecular cloning techniques and Gibson assembly, an editing cassette was assembled in which homologous arms containing native P. odorifer CM17899 sequences, including approximately 1,000 nucleic acids upstream of and including the final nucleotide of the nifX gene, and approximately 1,000 nucleic acids downstream of and not including the final nucleotide of orf1, were seamlessly assembled into an intact homologous recombination vector. The resulting editing vector was delivered to CM17899, and the resulting transformants were cultured in a manner sufficient to induce integration of the entire plasmid, after which undesired genetic material was removed, leaving only the desired edit. Sanger sequencing of the edited region confirmed the presence of the desired modification.

[0392] GlnR binding site II inactivation, CueR knockout These edits take a different approach to preventing downregulation of nif expression by preventing transcriptional repression through binding of glnR to site II and potentially downregulating NrgA expression, thus preventing ammonium accumulation and triggering downregulation.

[0393] NrgA knockout In Paenibacillus species, the nrgA gene is the primary ammonium transporter. Nitrogen fixation is tightly regulated, being repressed by excessive intracellular ammonium levels. To reduce the amount of ammonium available to repress nitronase expression, nrgA was knocked out by seamlessly removing the entire open reading frame from the first to the last nucleotide, leaving the immediate surrounding sequences intact, but none of the subsequent gene sequences.

[0394] Using standard molecular cloning techniques and Gibson assembly, an editing cassette was assembled in which homologous arms containing native P. polymyxa CM8619 sequences, including approximately 1,000 nucleic acids upstream of the nrgA gene but not including the first nucleotide, and approximately 1,000 nucleic acids downstream of nrgA but not including the final nucleotide, were seamlessly assembled into an intact homologous recombination vector. The resulting editing vector was delivered to CM8619, and the resulting transformants were cultured in a manner sufficient to induce integration of the entire plasmid, after which undesired genetic material was removed, leaving only the desired edit. Sanger sequencing of the edited region confirmed the presence of the desired modification.

[0395] Promoter Swaps and Insertions PLH-77 and PLH-77-d are constitutive promoters isolated from Paenibacillus polymyxa SC2-M1, derived from an isolated form of pepper plant rhizosphere bacteria in Guizhou, China. Promoter pLH77 was identified and reported by Li, et al. (2019), and a cloned version of pLH77-d was described.

[0396] Example 3: Cloning The cloning vector was assembled by introducing the editing cassette (described above) into the pMMmob backbone. pMMmob [oriBsTs traJ ecol1 mls amp] is a derivative of the plasmid pMiniMAD2 obtained from the Bacillus genetic stock center. pMMmob was digested with the restriction enzymes BamH1 and EcoR1, run on a 10% agarose gel, and purified.

[0397] The upstream and downstream homologous regions were amplified from genomic DNA extracts by PCR using a proofreading polymerase, and primers were designed to add flanking sequences for subsequent Gibson assembly. For constructs with added sequences between the flanking homologous arms, the sequence introduction was achieved by inclusion in the primer flanking sequences. PCR products were run on a 10% agarose gel and purified.

[0398] The backbone and insert were combined at a 1:3 backbone:insert molar ratio, combined with Gibson assembly reagent, and incubated at 50°C for 60 minutes for plasmid assembly. The Gibson assembly mixture was then transformed into chemically competent DH5α E. coli. Transformants were recovered on LB+100µg / µL ampicillin plates.

[0399] Proper assembly of the plasmid was confirmed by restriction digest analysis and PCR of the insert region. The editing cassette was sequenced using Sanger sequencing to confirm the absence of off-target mutations.

[0400] Confirmed plasmids were extracted from overnight DH5α cultures, transformed into electrocompetent BW29472 E. coli by electroporation, recovered on LB + 100ug / uL ampicillin + 300uM diaminopimelic acid plates, and conjugated into the host strain.

[0401] Example 4: Gene editing of Paenibacillus spp. Scarless homologous recombination This is a general protocol for gene editing in Paenibacillus using a temperature-sensitive, scarless homologous recombination plasmid, and was used for the editing described herein. This protocol was developed for editing CM8619 and may be broadly applicable to other Paenibacillus isolates with modifications. This protocol requires the pre-assembly of one or more editing vectors designed for the desired edit using a pMMmob backbone hosted in an E. coli donor strain and one or more Paenibacillus recipient strains confirmed for sensitivity to the relevant antibiotic resistance markers.

[0402] Joining The desired recipient strain was grown overnight in an appropriate growth medium. The donor strain was grown overnight in an appropriate growth medium supplemented with the relevant antibiotic marker to maintain the mobilizable plasmid. Aliquots of the overnight cultures were washed, combined, and plated onto appropriate agar media to grow both strains. These plates were incubated overnight at a temperature permissive for plasmid replication in the recipient strain.

[0403] The mating mixture was collected, washed, and replated onto agar plates supplemented with the appropriate antibiotic marker to select for transconjugant recipient strains. Plates were incubated overnight at a temperature permissive for plasmid replication until transconjugant colonies appeared.

[0404] Built-in Transconjugant colonies were grown overnight in liquid culture at a temperature permissive for plasmid replication in the presence of a selectable marker. Dilutions of the liquid culture were plated onto agar plates supplemented with a selective antibiotic and incubated overnight at the restrictive temperature for plasmid replication. Colonies recovered under these conditions were presumed to have integrated the edited plasmid by homologous recombination.

[0405] excision Integrating colonies were inoculated into liquid cultures and grown to turbidity at a temperature permissive for plasmid replication. They were then subcultured into fresh medium lacking antibiotics and grown again overnight at the permissive temperature. This serial subculture was repeated two to three times, and dilutions of the final subculture were plated onto agar plates lacking antibiotics.

[0406] Recovered colonies were assayed for loss of the plasmid by replating them on antibiotic-containing and antibiotic-lacking media. Colonies that grew in the absence of antibiotic but not in its presence confirmed that the plasmid had been excised and lost and were identified as putative edited strains.

[0407] confirmation Putatively edited strains were screened to determine whether the edit was successfully delivered or whether the edited strain reverted to wild-type by amplifying the edited region by polymerase chain reaction (PCR). PCR products were analyzed by gel electrophoresis and Sanger sequencing to confirm the appropriate product size and sequence for editing. Colonies that confirmed successful delivery of the edit were examined by Sanger sequencing to confirm that no off-target mutations had been added to the edited region, and the absence of the plasmid backbone was confirmed by lack of growth in antibiotic-containing medium.

[0408] Strains that passed all validation steps were assigned a qualified strain designation, added to the qualified isolate library, and provided to the bioassay team for testing.

[0409] Other methods Alternatively, any other method known in the art can be used to effect any one or more of the polynucleotide editing techniques described herein, including, but not limited to, targeted and / or homing nucleases, restriction endonucleases, zinc finger nucleases, meganucleases, Cas endonucleases, TAL effector nucleases, guided nucleases, random site mutations, blind editing, chemical mutagenesis, or radiation mutagenesis. Generally, a double-strand break is created at or near the target site to be edited, which 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, or chemical modification of at least one nucleotide. For the purposes of editing Paenibacillus strains described herein, any technique desirable to the practitioner can be used to achieve the final result.

[0410] Example 5: Identification and storage of microorganisms Sequencing preparation and long-term storage for microbial identification was performed by the following method.

[0411] Day 1: Using a 10 uL sterile tip, transfer the colony from the plate to a flask containing the appropriate liquid growth medium. Place the isolate on a shaker at room temperature and incubate for 2 days.

[0412] Day 3: The tubes may become cloudy after 2 days on a shaker. All samples are analyzed by PCR. Vortex each tube and collect 50 μL of sample from each vortexed tube and dispense into a 96-well plate. Using a multichannel pipette, dispense 15 μL of the 50 μL sample into a new 96-well plate. The 96-well plate containing 35 μL of each sample will be used for phenotypic analysis, and the 96-well plate containing 15 μL of each sample will be used for PCR analysis. The 27F / 1492R primers generally are used for 16S PCR analysis because they yield better results than PB36 / 38. Appropriate negative controls should be included on the plate and analyzed by PCR. The plate is analyzed by PCR using an Eppendorf thermocycler. Once PCR is complete, run the gel using standard gel electrophoresis techniques. This is important because most isolates ideally grow well in their long-term storage location by day 3. PCR and gel electrophoresis analysis are used to confirm that the isolate contains bacteria and not other microorganisms. For isolates that do not pass PCR or have a clear broth, vortex the tube and streak it onto a Petri dish using a loop. After a few days, check to see if anything has grown or if the tube is contaminated. For isolates that pass PCR, dispense 600 μL of 50% glycerol into a 2 ml screw-cap tube and add 1200 μL of bacterial culture, so that the broth is stocked in 20% glycerol. Store the glycerol stock at -80°C and record a gel image of the PCR sample.

[0413] Day 4: Check the Petri dish for streaky isolates that failed PCR for growth. (During this time, the 2 ml broth tube will remain on the shaker.) If there is growth on the dish and the colonies appear to be successfully isolated, dispense 600 μL of the broth-glycerol mixture into a small tube and place both tubes in their respective -80 boxes. Isolates may fail the PCR check for any of the following reasons: the primers may not work on all bacteria; the isolate is actually a fungus; the isolate is very adherent and therefore does not homogenize in the broth; the isolate produces excessive EPS and therefore requires dilution before PCR setup; or the isolate grows slowly. Continue checking the dish over the next few days to ensure that only a single bacterial species has been isolated. If contamination is observed, prepare a new isolate. The viability of the prepared glycerol stock should be verified.

[0414] Example 6: Microbial formulation Microorganisms identified according to the previous examples can be formulated with additional components for application via methods such as, but not limited to, seed treatment, root drench, root wash, seedling dip, foliar spray, soil inoculation, in-furrow application, lateral fertilization, soil pre-treatment, wound inoculation, drip tape irrigation, pollinator-mediated vector transfer, injection, osmotic priming, hydroponics, aquaponics, and aeroponics. Formulations containing the microorganisms are prepared for agricultural use as liquid, solid, or gas formulations. Application to plants can be accomplished, for example, as a powder for surface deposition on plant leaves, as a spray on the whole plant or selected plant elements, as part of a drip onto the soil or roots, or as a coating on plant elements prior to planting. Such examples are intended to be illustrative and not limiting of the scope of the invention.

[0415] Media ingredients for exemplary microbial preparations are shown in Tables 2a-2d below. Add all contents along with 50% of the final volume of water required and stir the solution at elevated temperature until dissolved. After all contents are dissolved, use sterile RO water to bring the solution to the final desired volume. Field test preparations are typically performed using quadruplicate formulations. [Table 4]

[0416] Table 2b: Exemplary media components of microbial formulations [Table 5]

[0417] Table 2c: Exemplary media components of microbial formulations [Table 6]

[0418] Table 2d: Exemplary media components of microbial formulations [Table 7]

[0419] The procedure for mixing the TIX formulation is as follows: Weigh all dry ingredients into a 50 mL tube. Vortex the ingredients well to ensure the xanthan gum is "separated" from the other carbon sources. Add approximately half of the total sterile RO water to the mixture and vortex. Use the long end of an L-shaped spreader to break up clumps as much as possible. Heat some sterile RO water in the microwave to hot water bath temperature (45-50°C). Add the remaining sterile RO water to the mixture and vortex. Repeat step 4 and vortex as necessary until a clear solution without lumps is obtained. Sediment any air bubbles created during the mixing process by using the centrifuge on "high speed spin" for 5-10 seconds. Remember to counterbalance the formulation (TIX) tube. Allow the formulation to cool to room temperature. 1. Mix within the microbial consortium. Vortex to ensure homogeneity. Ideally, add microorganisms at a concentration of 10^9 CFU / ml to the formulation.

[0420] For testing in field trials, the formulation is applied to plants or plant elements.

[0421] Example 7: Application of microorganisms to plant elements and their cultivation Microbial compositions (comprising one or more isolated microorganisms, whether single strains, consortia, communities, combinations, or any combination of the foregoing) were prepared according to the previous examples, including one or more microorganisms, optionally in combination with one or more additional microorganisms disclosed herein.

[0422] Microbial compositions for application In some methods, the microbial composition was dried and applied directly to the plant elements.

[0423] In some methods, the microbial composition was suspended in a liquid formulation for application to the plant elements.

[0424] In some methods, the microbial composition is combined with another composition, such as, but not limited to, a carrier, a wetting agent, a stabilizer, or a salt. In some methods, the other composition comprises a molecule that induces additional agriculturally beneficial effects in the plant to which the microbial composition is applied. Examples of other compositions include, but are not limited to, herbicides, fungicides, bactericides, pesticides, insecticides, nematicides, biostimulants, and the like.

[0425] Applicable Type The microbial compositions are applied to plant elements at a time during development appropriate for the desired result, for example, in a pre-plant soil drench / furrow formulation, as a seed or other reproductive element treatment, as a post-plant reproductive element application, as a post-plant furrow, drip, or drench application, as a direct application to plant elements (e.g., roots, leaves, stems), or as an application to harvested plant elements (e.g., fruit or grain). Combinations of application types are also tested.

[0426] How to apply The microbial composition is applied (inoculated) to the plant or plant element or plant product (before planting, after planting, before harvest, or after harvest). This is accomplished, for example, by applying the agricultural composition to a hopper, spreader, or tank that contains the microbial composition and is configured to spread it widely.

[0427] A seed coating of the microbial composition was applied to one or more seeds of a crop plant. Once the isolated microorganism was applied as a seed coating, the seeds were planted and cultivated according to established practices for that crop.

[0428] Alternatively, the microbial compositions have been applied to the soil for the benefit of plants present in the soil. Methods of soil application include in-furrow treatment, drench, and drip application.

[0429] Alternatively, the microbial composition was applied to the surface of the plant or plant part after emergence.

[0430] Alternatively, the microbial composition was applied to material obtained from the plant after harvest.

[0431] Control plots of plants to which no isolated microorganisms were applied were also planted. Plants associated with the microbial composition show improvement in the traits of interest.

[0432] The application method may be carried out according to any protocol known in the art.

[0433] The plant elements, plants, or growth medium (eg, soil) can be further inoculated with a disease or pest depending on the purpose of the test.

[0434] An exemplary, non-limiting protocol for irrigating tomato plants is given below. 1. Ten days after planting, carefully separate the plants into six rows for each treatment. The plants are delicate and the leaves can tear easily. Ensure that the plants are as uniform as possible in size and overall appearance (the purpose of thinning is to maintain a homogenous plant population). If there are not enough plants per row, transplant them. See Step 3 for transplanting guidelines. 2. Begin thinning the pots to one plant per pot. Remove any smaller plants, unhealthy ones, or ones that are deformed in any way. If there are two or more healthy plants per pot, the excess can be transplanted into another pot. Use the remaining soil prepared from the initial planting or from pots where the seeds did not germinate. 3. To transplant: If some pots did not germinate, they can be filled with plants from another container. To do this, simply scoop out the excess plants with a scoop (trying to scoop out as much of the root mass as possible without disturbing the other plants) and place them in the hole made in the empty pot. Firm the soil around the plants with slight finger pressure. 4. Arrange the pots into 6 pot lines (one pot line per treatment) and take 4 RL98 trays. Once finished, look at all the treatments and allow for several pot rotations to ensure some treatments are free of all large plants and other treatments have all large plants. 5. Change gloves if necessary. Label each pot with the Avery labels you prepared in advance. Treatments should be labeled in columns of six replicates, i.e., 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, etc. This will make it easier to find all the replicates for each treatment. 6. Two weeks after planting (approximately four days after thinning and labeling), receive treatment from the microbiology team. Arrange the prepared plant trays on the table. Collect Combi-Tip, Repeater, and RO water. (Note: Plants should be lightly watered on the day of treatment) 7. Mix the microbial solution by inverting the tube / container (microbial treatment) 2-3 times or gently shaking. Set the Combitip to dispense 2 ml. Collect the treatment fluid in the Combitip and dispense the first step back into the tube. Ensure that the treatment corresponds to the row of plants being treated. Once confirmed, gently dispense 2 ml of treatment onto the topsoil of each pot, close to the stem, but avoiding direct contact with the stem and leaves. 8. Discard the Combitip and repeat step 6 for all treatments. For the inoculated controls (IC or InoCon) and untreated controls (UTC), apply RO water instead of treatment.

[0435] Once all treatments have been applied, the plants are placed back into the growth chamber for (optional inoculation), growth and evaluation.

[0436] Visualization of microorganisms associated with plant elements Individual microorganisms can be tagged with fluorescent proteins according to methods known in the art. Microscopic image analysis demonstrated that the microorganisms disclosed herein are found associated with various plant tissues.

[0437] Example 8: In vitro testing The wild-type and genome-edited strains were evaluated for root colonization, acetylene reduction activity, biofilm formation, turbidity (OD at 600 nm), oxygen tolerance, and gene expression. Strain IDs are listed.<ParentStrain#-Edit#> The results are given as: Unless otherwise specified, protocols were performed using methods known in the art. The results are shown in Tables 3a-3k.

[0438] ARA by GC-FID for Gram-positive strains Ensure all equipment and materials are sterilized. Sealed device containers are wrapped in foil before autoclaving so that they can be unwrapped in the anaerobic chamber pass box and enter the anaerobic chamber sterile. Seal vial openings with foil before sterilization. A loose "seal" is required to allow gas exchange within the pass box. 1. Streak the isolate from -80°C and incubate at 30°C or 25°C until colonies are observed. 2. Spread one plate per isolate and incubate at 25°C or 30°C until a bacterial lawn is observed. 3. Harvest plates and balance to approximately 0.3 OD600 for each isolate to normalize the inoculum. 4. Prepare the anaerobic chamber by cleaning the surface and passing a sealing device, ensuring the container allows gas exchange. Add 5.30 mL of NF11 / vial (3 doses per isolate). 6. Add 150uL of inoculum / vial balanced to an OD600 of 0.3 using sterile water. 7. Pass the vials through the anaerobic chamber and seal under anaerobic conditions; include an empty vial (with foil "cap") for adding an anaerobic indicator for QC purposes. 8. Place the vial at 30°C and 200 rpm for 5 hours. After 9.5 hours, working in a fume hood, remove 10% (4 mL) from the headspace of each vial and replace it with an equal volume of acetylene gas. NOTE: Acetylene gas is highly reactive and explosive, so the bag should be kept in the fume hood while working. 10. Incubate at 30°C and 200 rpm for 48 hours. 11. At 48 hours (or other known time point), a 1 mL headspace sample is taken and placed in a GC collection tube. 12. Run the sample on the GC using instrument method "Split 4" for ethylene analysis, which measures the acetylene peak and ethylene peak. 13. Quantify the amount of gas by peak area. 14. Obtain an OD600 measurement of 200 ul of culture and TVC of the culture. 15. Analyze ethylene gas as the percentage conversion of acetylene to ethylene. This provides an estimate of total conversion.

[0439] The volume of gas (ethylene) produced can be quantified either by using calibration points in the Chromeleon or by calculating from the peak area %. In this case, the acetylene + ethylene peak area % should be 100%. From the known amount of acetylene added, the ethylene produced can be determined in mL. 1M gas is 24 dm3 or 24,000 ml. Therefore, 1 mM gas is 24 ml.

[0440] To calculate how many mM ethylene were produced, divide the amount by 24: mM ET = ml / 24.

[0441] To calculate RATE:mM / hour / CFU, you need to calculate mM as above and know the volume of headspace sampled (if using the calibration calculation, for example, 1 mL of headspace sampled has x mM gas, but the total headspace is 6 mL, so the total ethylene produced is 6 x mM). If calculating using only peak area %, the above step is not necessary and you only need to know the amount of acetylene added. You need to know the time of incubation with acetylene. To calculate CFU / mL, you need to perform a TVC and then multiply the CFU value by the number of mL incubated, e.g., 4 mL (gneg) or 30 mL (gpos).

[0442] Rate = Total ethylene mM / (Time (hr) x Total CFU)

[0443] ARA with oxygen tolerance test protocol Ensure all equipment and materials are sterilized. Sealed device containers are wrapped in foil before autoclaving so that they can be unwrapped in the anaerobic chamber pass box and enter the anaerobic chamber sterile. Seal vial openings with foil before sterilization. A loose "seal" is required to allow gas exchange within the pass box. 1. Streak the isolate from -80°C and incubate at 30°C or 25°C until colonies are observed. 2. Spread one plate per isolate and incubate at 25°C or 30°C until a bacterial lawn is observed. 3. Harvest plates and balance to approximately 0.3 OD600 for each isolate to normalize the inoculum. 4. Prepare the anaerobic chamber by cleaning the surface and passing a sealing device, ensuring the container allows gas exchange. 5. After clarification, load NF11 medium into the anaerobic chamber. Add 20 g / L agar to the NF11 and place on a hot plate. Boil briefly to dissolve the agar. After the agar is dissolved, pour 30 mL of warmed agar into the 70 mL container, pouring it over its side and tilting it to maximize the surface area. 6. Add 150uL of inoculum / vial balanced to an OD600 of 0.3 using sterile water. Try to maximize the surface area exposed to the inoculum. 7. Pass the vials through the anaerobic chamber and seal under anaerobic conditions; include an empty vial (with foil "cap") for adding an anaerobic indicator for QC purposes. 8. To adjust the oxygen level, after sealing the vial, remove a thin needle syringe and remove some of the anaerobic air from the vial, replacing it with 100% pure medical-grade oxygen. 9. This assay has been run under various oxygen conditions, ranging from 0% oxygen to 22% oxygen, and can be increased to much higher oxygen conditions by manually adding oxygen. For example, to achieve 5% oxygen, 2.2 mL of anaerobic gas is manually removed and 2 mL of 100% pure oxygen is added back into the assay. 10. Place the vial in a 30°C incubator for 5 hours. After 11.5 hours, working in a fume hood, remove 10% (4 mL) from the headspace of each vial and replace it with an equal volume of acetylene gas. NOTE: Acetylene gas is highly reactive and explosive, so the bag should be kept in the fume hood while working. 12. Incubate at 30°C and 200 rpm for 48 hours. At 13.48 hours (or other known time point), a 1 mL headspace sample is taken and placed in a GC collection tube. 14. Run the sample on the GC using instrument method "Split 4" for ethylene analysis, which measures the acetylene peak and ethylene peak. 15. Quantify the amount of gas by peak area. 16. Analyze ethylene gas as the percentage conversion of acetylene to ethylene. This provides an estimate of total conversion.

[0444] The volume of gas (ethylene) produced can be quantified either by using calibration points in the Chromeleon or by calculating from the peak area %. In this case, the acetylene + ethylene peak area % should be 100%. From the known amount of acetylene added, the ethylene produced can be determined in mL. 1 M gas is 24 dm3 or 24,000 ml. Therefore, 1 mM gas is 24 ml. To calculate how many mM ethylene was produced, divide the volume by 24: mM ET = ml / 24.

[0445] To calculate RATE:mM / hour / CFU, you need to calculate mM as above. You need to know the amount of headspace sampled (if using the calibration calculation, for example, 1 mL of headspace sampled has x mM gas, but the total headspace is 6 mL, so the total ethylene produced is 6 x mM). If calculating using only peak area %, the above steps are not necessary, but you need to know how much acetylene was added. You need to know the incubation time with acetylene. To calculate CFU / mL, you need to perform a TVC, then multiply the CFU value by the number of mL cultured, e.g., 4 mL (gneg) or 30 mL (gpos).

[0446] Rate = Total ethylene mM / (Time (hr) x Total CFU)

[0447] Root colonization Bacterial strains were prepared with a GFP gene integrated into their genome using techniques known in the art. Seeds were treated with the strain, and the inoculated seeds were dropped into phytagel tubes using sterile techniques. The tubes were placed in a suitable growth chamber, covered, and allowed to germinate for 5 days. Root tissue was separated from the seeds and shoots using EtOH and flame-sterilized tweezers and scalpels. All root tissues were cut at the same focal plane and pressed at the same level into 0.8% water agar in a square plate for imaging. The same was done for shoot tissue. Plant tissues were imaged for bacterial colonization using a fluorescent microscope.

[0448] Biofilm assay protocol This protocol is based on the publication "Effects of an EPS Biosynthesis Gene Cluster of Paenibacillus polymyxa WLY78 on Biofilm Formation and Nitrogen Fixation under Aerobic Conditions" (Chen 2021). Materials: Sterile 3 mL glass tubes, "Biofilm Broth (BFB)" medium, 0.1% crystal violet in water, 40% acetic acid. Seven days produced the best overall biofilm results. Some isolates performed better over five days and began to degrade at this point. Prepare using sterile technique.

[0449] The BFB recipe includes 5g / L KH2PO4, 5g / L K2HPO4, 0.86g / L monosodium glutamate, 0.1g / L yeast extract, and 1g / L NH4Cl (pH 7). Filter-sterilized after autoclaving: 36g / L glucose, 0.03g / L MgSO4.7H2O, 0.02g / L CaCl2.2H2O, and 1ml / L trace element solution.

[0450] The method steps are as follows: 1. Streak the isolate from -80°C. 2. Prepare spread plates for each isolate. Autoclave 3.3 mL glass tubes (3 per isolate) in a tube rack and use foil as a cover. 4. Collect the diffusion plate and balance to an OD600 of approximately 0.3. 5. Fill each tube with 1 mL of BFB. 6. Inoculate 10 uL / tube of spread plate harvest. 7. Place the foil cover back on the tube and incubate undisturbed at 30°C for 7 days. After 8.7 days, first remove the culture from the tube using a long (1250 uL) pipette tip and collect the culture into a 2 mL snap-cap tube. 9. Add water to the culture to reach a final volume of 1 mL. Take an OD600 reading. 10. Rinse the glass tube using RO water, filling it approximately halfway, holding the tube, sealing the top, and shaking to remove excess cellular material. Rinse several times. 11. Use a long pipette tip to remove excess water. Add 12.1 mL / tube of 0.1% crystal violet solution and incubate at room temperature for 10 minutes. 13. Remove the crystal violet solution by pipetting into a waste container (e.g., a 50 mL Falcon tube) and discard in an incineration waste bin. 14. Rinse the glass tube until the water runs clear. 15. Allow the glass tube to dry (usually overnight). Add 16.1 mL of 40% acetic acid solution to dissolve the stained biofilm ring. 17. Take an OD570 reading. 18. Normalize OD570 to OD600 (if applicable).

[0451] result Note: Blank spaces in the table indicate untested items. Table 3a: Wild-type (unedited) Paenibacillus strains [Table 8]

[0452] Table 3b: GlnR C25 cleavage [Table 9]

[0453] Table 3c: GlnR binding site II duplication and inactivation [Table 10]

[0454] Table 3d: GlnR binding site II inactivation [Table 11]

[0455] These data indicate that, although difficult to achieve, improved nitrogen fixation capabilities can be conferred on Paenibacillus strains by modifying various sites within the microbial genome.

[0456] Example 9: In plant testing The edited microorganisms described above were tested on soybeans.

[0457] Soybean plants were associated with the wild-type and / or edited microorganisms described above and tested in field trials with or without the addition of Bradyrhizobium. Association can be achieved by any one or more of the following: seed treatment, foliar treatment, in-furrow application, irrigation, and lateral application.

[0458] Replicates of soybean plants were treated with the microorganisms described herein and grown according to standard field testing methods known in the art. Data are presented in Tables 4a and 4b with and without Bradyrhizobium addition ("with Brady" and "without Brady," respectively). Table 4a: 2022 Soybean Field Test Data [Table 12]

[0459] Table 4b: 2023 Soybean Field Test Data [Table 13-1] [Table 13-2]

[0460] These data indicate that, although difficult to achieve, for plant improvement, Paenibacillus strains can be endowed with improved nitrogen fixation capabilities that will benefit soybean plants by modifying various sites within the microbial genome.

Claims

1. 1. A synthetic composition comprising: a plant element; and a Paenibacillus bacterium heterologously disposed on said plant element, wherein said Paenibacillus bacterium comprises an edit at one or more loci in its genome, wherein said 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: glnR, GlnR binding site I, GlnR binding site II, nrgA, orf1, cueR, and / or any combination or plurality of edits at any one or more of said genomic loci; The synthetic composition, wherein the Paenibacillus bacterium exhibits an improved phenotype compared to a Paenibacillus bacterium that does not comprise the edit, and the improved phenotype is selected from the group consisting of increased acetylene reduction ability, improved biofilm formation, increased turbidity in culture, higher nitrogen fixation tolerance to oxygen levels, and any multiple and / or combination of the foregoing.

2. 2. The synthetic composition of claim 1, wherein the edits are selected from those set forth in Table 1b, and any multiple and / or combination of the foregoing.

3. 2. The synthetic composition of claim 1, wherein the Paenibacillus bacterium comprises a sequence selected from the group consisting of SEQ ID NOs: 1-12.

4. 2. The synthetic composition of claim 1, wherein the Paenibacillus bacterium is of a species selected from the group consisting of polymyxa, tritici, albidus, anaericanus, azotifigens, borealis, donghaensis, ehimensis, graminis, jilunlii, odorifer, panacisoli, phoenicis, pocheonensis, rhizoplanae, sillage, taohuashanense, thermophilus, typhae, and wynnii.

5. 2. The synthetic composition of claim 1, wherein the Paenibacillus bacterium is of subgroup I.

6. 2. The synthetic composition of claim 1, wherein the Paenibacillus bacterium is of subgroup II.

7. 10. The composite composition of claim 1, further comprising a formulation component and / or an agricultural composition.

8. 10. The synthetic composition of claim 1, wherein the Paenibacillus bacteria are present at a concentration of at least about 10^2 CFU / mL in a liquid formulation or at a concentration of at least about 10^2 CFU / gram in a non-liquid formulation.

9. 10. The synthetic composition of claim 1, further comprising at least one additional microorganism.

10. 2. The synthetic composition of claim 1, wherein the plant element is a seed.

11. 2. The synthetic composition of claim 1, wherein the plant element is a seed containing a transgene.

12. 10. The synthetic composition of claim 1, wherein the plant element is a leaf.

13. 2. The synthetic composition of claim 1, wherein the plant element is a root.

14. 10. The synthetic composition of claim 1, wherein the plant element is a whole plant.

15. The synthetic composition of claim 1 , wherein the plant elements are plant reproductive elements.

16. 10. The synthetic composition of claim 1, wherein the formulation component is selected from the group consisting of a compound that improves the stability of the microorganism, a preservative, a carrier, a surfactant, an anticomplex agent, and any combination thereof.

17. 10. The synthetic composition of claim 1, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, an insecticide, a herbicide, a micronutrient, a macronutrient, nitrogen, phosphorous, potassium, or any multiple and / or combination of the foregoing.

18. 10. The plurality of synthetic compositions of claim 1, wherein the synthetic compositions are substantially confined within an object selected from the group consisting of a tube, a bottle, a jar, an ampoule, a package, a vessel, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

19. 20. The plurality of composite compositions of claim 18, wherein the composite composition is at a temperature below 0 degrees Celsius.

20. 10. The synthetic composition of claim 1, wherein said plant components are obtained from a dicotyledonous plant.

21. 21. The synthetic composition of claim 20, wherein the plant is a legume.

22. 21. The synthetic composition of claim 20, wherein the plant is a seed.

23. 10. The synthetic composition of claim 1, wherein the agricultural composition comprises a growing medium.

24. 24. The synthetic composition of claim 23, wherein the growing medium comprises soil.

25. 10. The plurality of synthetic compositions of claim 1, wherein the plurality of synthetic compositions are disposed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

26. 1. A method for improving the health, yield, and / or vigor of a plant, said method comprising: (a) associating the plant element with a Paenibacillus bacterium that comprises an edit at one or more loci in 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: glnR, GlnR binding site I, GlnR binding site II, nrgA, orf1, cueR, and / or any combination or multiple edits at any one or more of said genomic loci; (b) placing the elements of the crop plant in a medium that supports plant growth; (c) growing a plant derived from said element of said 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 not obtained from elements associated with the Paenibacillus bacterium of (a); The method wherein the plant is a soybean plant.

27. 27. The method of claim 26, wherein the one or more characteristics of (d) include improved nitrogen fixation, increased biomass, increased leaf area, increased plant height, increased root area, increased shoot nitrogen composition, increased greenness, increased NDVI, increased NPCI, increased PSRI, increased CCI, increased yield, and any combination of the foregoing.

28. 27. The method of claim 26, further comprising at least one additional microorganism.

29. 27. The method of claim 26, wherein said associating said crop plant elements with Paenibacillus bacteria comprising an edit at one or more loci in their genome is accomplished by a method selected from the group consisting of in-furrow application, soil-drenched application, lateral dressing application, and any combination of the foregoing.

30. 27. The method of claim 26, wherein said associating said crop plant element with Paenibacillus bacteria that contain an edit at one or more loci in its genome is accomplished by coating said plant element with a liquid formulation of said bacteria.

31. 27. The method of claim 26, wherein said associating said crop plant element with Paenibacillus bacteria that contain an edit at one or more loci in its genome is accomplished by coating said plant element with a substantially non-liquid formulation of said bacteria.

32. 27. The method of claim 26, wherein the plant element is a seed.

33. 27. The method of claim 26, wherein the plant element is a leaf.

34. 27. The method of claim 26, wherein the plant element is a root.

35. 27. The method of claim 26, wherein the plant element is a whole plant.

36. 37. The modified Paenibacillus bacterium of claim 36, wherein the Paenibacillus bacterium exhibits an improved phenotype compared to a Paenibacillus bacterium that does not comprise the edit, the improved phenotype being selected from the group consisting of increased acetylene reduction ability, improved biofilm formation, increased turbidity in culture, higher nitrogen fixation tolerance to oxygen levels, and any combination of the foregoing.

37. 1. A modified Paenibacillus bacterium, wherein the Paenibacillus bacterium comprises an edit at one or more loci in 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: glnR, GlnR binding site I, GlnR binding site II, nrgA, orf1, cueR, and / or any combination or multiple edits at any one or more of said genomic loci.

38. 37. The modified Paenibacillus bacterium of claim 36, wherein the edits are selected from the edits listed in Table 1b, and any multiple and / or combination of the foregoing.

39. 37. The modified Paenibacillus bacterium of claim 36, wherein the Paenibacillus bacterium comprises a sequence selected from the group consisting of SEQ ID NOs: 1-12.

40. 37. The modified Paenibacillus bacterium of claim 36, wherein the Paenibacillus bacterium is of a species selected from the group consisting of polymyxa, tritici, albidus, anaericanus, azotifigens, borealis, donghaensis, ehimensis, graminis, jilunlii, odorifer, panacisoli, phoenicis, pocheonensis, rhizoplanae, sillage, taohuashanense, thermophilus, typhae, and wynnii.

41. 37. The modified Paenibacillus bacterium of claim 36, wherein the Paenibacillus bacterium is of subgroup I.

42. 37. The modified Paenibacillus bacterium of claim 36, wherein the Paenibacillus bacterium is of subgroup II.

43. 37. A substantially pure composition comprising the modified Paenibacillus bacterium of claim 36.

44. 37. A bacterial culture comprising the modified Paenibacillus bacterium of claim 36.

45. 37. A fermentation culture comprising the modified Paenibacillus bacterium of claim 36.

46. 37. An agricultural composition comprising the modified Paenibacillus bacterium of claim 36 and an agriculturally acceptable carrier.

47. 40. The agricultural composition of claim 39, further comprising a plant or plant component, wherein the modified Paenibacillus bacterium is present in the agricultural composition in an amount effective to confer an improved phenotype on the plant.

48. 50. The agricultural composition of claim 49, wherein the improved phenotype is an increase in the health, weight, height, yield, and / or vigor of the plant.

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