Agriculturally beneficial microorganisms, microbial compositions, and consortia

Abstract

The present disclosure includes novel strains of microorganisms, microbial consortia, and agricultural compositions containing them. Furthermore, the present disclosure teaches methods of utilizing the described microorganisms. In methods of imparting beneficial properties to target plant species, including microbial consortia and agricultural compositions containing them, the present disclosure provides methods of increasing desirable plant traits in agriculturally important crop species. In certain aspects, the present disclosure provides methods of increasing desirable plant traits in agriculturally important crop species.

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 / 301,480, filed January 20, 2022, and U.S. Provisional Patent Application No. 63 / 311,394, filed February 17, 2022, all of which are incorporated by reference in their entireties.

[0002] Reference to Electronically Submitted Sequence Listing An official copy of the Sequence Listing will be submitted electronically as an XML formatted Sequence Listing with filename 22041-WO-PCT.xml, 52,257 bytes in size, created on December 16, 2022, and submitted concurrently with the specification. The Sequence Listing contained in this XML formatted document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates generally to the field of biology, and more particularly to microorganisms and microbial compositions for plant improvement. [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 developing countries of the world, where one in six children is underweight. The lack of available food can be attributed to many socio-economic 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 2050. The United Nations estimates that agricultural yields must increase by 70-100% to feed the projected world population in 2050.

[0005] These staggering world population and malnutrition figures highlight the importance of agricultural efficiency and productivity to sustain a growing world population. The technological advances achieved by modern continuous crop agriculture have been remarkable, resulting in unprecedented crop yields. However, despite advances made through technological innovations such as genetically modified crops and novel insecticidal and herbicidal compounds, improved crop yields are required 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 best observed yields and results elsewhere) could be closed. That is, if all farmers, wherever they are in the world, could achieve the highest yields expected in their area, a large part of the world's food production shortfall could be addressed. But the problem of how to achieve high yields in different lands around the world is difficult.

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

[0008] It is therefore 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 of increasing crop yields and imparting beneficial traits to desired plant species. Summary of the Invention

[0010] The present invention also provides an agriculturally acceptable microbial consortium comprising at least two of the disclosed microorganisms, and a method for using the consortium in agricultural applications. In some embodiments, the genome modification of microorganisms (individuals, consortia, and / or communities) is contemplated for the improvement of microbial traits and the improvement of microbial-associated plants. ...

[0011] The present disclosure addresses this important problem of how to improve crop yields, thereby narrowing the global yield gap, as well as providing methods for imparting other beneficial traits to plant species.

[0012] The solutions provided by the present disclosure to increased crop yields and yield increases are not detrimental to earth resources because they do not rely on increased water consumption or increased synthetic chemical inputs to the system. Rather, the present disclosure utilizes microorganisms to impart beneficial traits, including increased yield, to desirable plants.

[0013] Thus, the present disclosure provides an environmentally sustainable solution that allows farmers to increase yields of important crops and is not dependent on the increased use of synthetic herbicides and synthetic pesticides.

[0014] In embodiments, the present disclosure provides efficient and broadly applicable agricultural platforms that utilize microorganisms and microbial consortia (multiple microorganisms, in some aspects multiple microorganisms that improve plant health or a desirable phenotype, such as an agronomic trait with which it is associated) that promote one or more desirable plant characteristics.

[0015] The microorganisms disclosed herein improve the performance of plants, such as crop plants, by both direct and indirect mechanisms. In some embodiments, the microorganisms are symbiotic with the plants. In some embodiments, the microorganisms produce compounds (e.g., metabolites, toxins, proteins, lipopeptides, or other compositions) that benefit 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, to 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 are contemplated beyond the exemplary non-limiting embodiments listed above.

[0016] 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.

[0017] In some embodiments, the single microorganism is mixed with one or more other microorganisms of different species or strains, whether they are taxonomically identifiable species or taxonomically identifiable strains. In certain embodiments, the combination of two or more microorganisms forms a consortia or consortium. The terms consortia and consortium are used interchangeably.

[0018] 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 have functional properties that are 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 have functional properties that are not possessed by any one member of the consortium when viewed individually.

[0019] In some embodiments, the functional property possessed by the microbial consortia 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 utilization efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, enhanced water utilization 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 are also contemplated, including adverse effects on nematodes, insects, or other pests.

[0020] In some embodiments, these individual microorganisms do not have the ability to confer these beneficial traits to plants when they exist in nature. Rather, in some embodiments, functional compositions are developed that have properties and functional attributes that do not exist in nature by artificially mixing these microorganisms into a consortium. In some embodiments, the consortium may include microorganisms that have been genetically edited, modified, or modified through the modification of genetic material, including DNA, RNA, proteins, and / or combinations thereof, by techniques known to those skilled in the art.

[0021] 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.

[0022] In some embodiments, the microorganism is a strain of Bacillus thuringiensis that has nematicidal activity. In some aspects, the strain further comprises a toxin that is active against other pests, such as Coleoptera and / or Lepidoptera. In some aspects, the strain has activity against multiple pests.

[0023] In some embodiments, the Bacillus thuringiensis strain is referred to herein as strain 39400, which is deposited in accordance with the Budapest Treaty as NRRL B-68090, deposited on January 26, 2022.

[0024] In some embodiments, the Bacillus thuringiensis strain is referred to herein as strain 42901.

[0025] Any of the strains disclosed herein may be further mixed with one or more additional microorganisms that may form a microbial consortium. A microbial consortium is a mixture of one or more individual microorganisms, and in certain embodiments, the microbial consortium comprises two microorganisms, or three microorganisms, or four microorganisms, or five microorganisms, or six microorganisms, or seven microorganisms, or eight microorganisms, or nine microorganisms, or ten microorganisms, or more than ten microorganisms.

[0026] Another object of the present disclosure relates to the use of isolated microorganisms and microbial consortia as plant growth promoters. In another aspect, the isolated microorganisms and microbial consortia function as growth regulators, for example, they can disrupt normal senescence and cause biomass increase.

[0027] Yet another object of the present disclosure relates to the use of the isolated microorganisms and microbial consortia as soil health enhancers and plant health enhancers. In other aspects, the isolated microorganisms and microbial consortia function as biostimulants.

[0028] A further object of the present disclosure relates to the use of the isolated microorganisms and microbial consortia as pesticides. In other aspects, the isolated microorganisms and microbial consortia function as biofungicides. In other aspects, the isolated microorganisms and microbial consortia function as bionematicides.

[0029] Another objective of the present disclosure is to design microbial consortia that can perform multidimensional activities in the same way. In certain embodiments, the microorganisms that compose the consortia act synergistically. In embodiments, the effect that the microbial consortia has on a particular plant trait is greater than the effect observed when any one particular microbial member of the consortia is used alone. That is, in some embodiments, the consortia exhibits an effect on a desired plant trait that is greater than the sum of the effects compared to the effect seen when any one particular member of the consortia is used alone.

[0030] In some aspects, the consortia establish other plant-microbe interactions, for example, by serving as primary colonizers or founder populations that chart the trajectory of future microbiome development.

[0031] In embodiments, the present disclosure is directed to synergistic combinations (or mixtures) of microbial isolates.

[0032] In some embodiments, the consortia taught herein provide a wide range of agricultural applications, including improved grain, fruit, and flower yields, improved plant part growth, improved ability to utilize nutrients (e.g., nitrogen, phosphate, and the like), improved disease resistance, biopesticidal effects including improved resistance to fungi, insects, and nematodes, improved ability to survive in extreme climates, and other desirable plant phenotypic characteristics. Importantly, such benefits to the plant and / or adverse effects against targeted pests and / or pathogens are obtained without any harmful side effects to the environment.

[0033] In some aspects, individual microorganisms or consortia comprising microorganisms of the present disclosure can be combined into agriculturally acceptable compositions.

[0034] 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, adhesion promoters, binders, dispersants, thickeners, stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, fertilizers, insecticides, herbicides, inert carriers, polymers, and the like.

[0035] In one embodiment of the disclosure, the microorganism (including an isolated single species, or a composition thereof, such as a strain, consortia, or metabolites) is provided to a seed in the form of a seed coating or other application. In an embodiment, 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 disclosure teaches a method of treating a seed comprising applying an isolated bacterial strain or microbial consortia to the seed. In certain embodiments, the isolated bacterial strain or microbial consortia 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 adapted for tank mixing. In some embodiments, the agricultural composition may be adapted for tank mixing with other agricultural products. In some embodiments, the agricultural compositions may be compatible with equipment used in ground, aerial, and irrigation applications.

[0036] 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.

[0037] In one embodiment of the disclosure, the microorganisms are provided in the form of granules, plugs, or soil drench applied to the plant medium. In another embodiment, 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 medium, such as soil.

[0038] 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 may 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.

[0039] 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.

[0040] In embodiments, the agricultural compositions of the present disclosure may be formulated as, among others, (1) solutions, (2) wettable powders, (3) dusts, (4) soluble powders, (5) concentrated emulsifiable concentrates or suspensions, (6) seed dressings, (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 may be diluted in an aqueous medium before conventional spray treatment. The compositions of the present disclosure may be applied to soil, plants, seeds, root zones, rhizosheath, or other areas where it is beneficial to apply a microbial composition.

[0041] Yet another object of the present disclosure relates to agricultural compositions that are formulated to provide a high colony forming unit (CFU) bacterial population or consortia. In some aspects, these agricultural compositions have adjuvants that provide adequate shelf life. In embodiments, the CFU concentration of the agricultural compositions of the present teachings is higher than the concentration when the microorganism exists in nature, i.e., outside the method 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 the seed as a seed coating at a concentration of 10^5 to 10^9 CFU. In another aspect, the microbial cells are applied on top of 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.

[0042] In an embodiment, the present disclosure is directed to an agricultural microbial formulation that promotes plant growth. In an embodiment, the present disclosure provides the isolated microorganisms of the present teachings and consortia containing the same, which are 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 containing isolated microorganisms can be found in U.S. Patent No. 7,097,830, which is incorporated herein by reference.

[0043] The disclosed microbial formulations can reduce the need for nitrogen-containing fertilizers, solubilize minerals, provide bio-pesticidal 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.

[0044] 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.

[0045] In some embodiments, agriculturally acceptable compositions containing 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.

[0046] 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.

[0047] 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 a desired plant species.

[0048] In some embodiments, the isolated biologically pure microorganisms of the present disclosure and / or the 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 WO2012125050A1, and (2) International Patent Application No. PCT / NZ2013 / 000171, published on March 27, 2014 as WO2014046553A1, each of which is incorporated herein by reference in its entirety for all purposes.

[0049] 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 the embodiments of the present disclosure are selected from among the members of the microorganisms present in the database. In certain aspects, the microorganisms utilized in the embodiments of the present disclosure are selected from the microorganisms present in the database based on certain characteristics of the microorganism.

[0050] 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.

[0051] Some embodiments described herein are methods for preparing agricultural seed compositions or seed coatings, comprising contacting a surface of a seed with a formulation comprising a purified microbial population, comprising at least one isolated microorganism that is heterologous to the seed or that is rarely present on the seed. Further embodiments involve preparing agricultural plant compositions, comprising contacting a surface of a plant with a formulation comprising a purified microbial population, comprising at least one isolated microorganism that is heterologous to the plant. In other aspects, the formulation or microorganism is introduced into the interior of the seed, for example, into the cotyledons or other seed tissues, such as the embryo.

[0052] In some embodiments, application of the isolated microorganisms, microbial consortia, exudates, metabolites, and / or agricultural compositions of the present disclosure to seeds or plants modulates agronomically important traits. Agronomically significant 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 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, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root structure, increased seed weight, enhanced 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 significant traits are modulated. In some embodiments, the modulation is a positive effect on one of the aforementioned agronomic traits.

[0053] In some embodiments, relative to a reference plant, the altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical resistance, 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, 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 ears, number of kernels per ear, number of pods, nutritional enhancement, pathogen resistance. 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, pest resistance, improved photosynthetic capacity, salt tolerance, greening, improved 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 regulation of metabolite levels, detectable regulation of transcript levels, and detectable regulation of the proteome.

[0054] 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 agent, a herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

[0055] The methods described herein may 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.

[0056] The methods described herein can include contacting a seed or plant with a composition comprising a metabolite produced by a single microorganism or microbial consortia 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 microbial consortia 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 microbial consortia 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 microbial consortia 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 microbial consortia 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 microbial consortia disclosed herein. In some embodiments, the method comprises contacting a seed or plant with a composition comprising at least 100 g of a metabolic product produced by a single microorganism or a microbial consortia disclosed herein. In some embodiments, the method comprises contacting a seed or plant with a composition comprising at least 1 kg of a metabolic product produced by a single microorganism or a microbial consortia disclosed herein. In some embodiments, the method comprises contacting a seed or plant with a composition comprising more than 1 kg of a metabolic product produced by a single microorganism or a microbial consortia disclosed herein.

[0057] In some embodiments of the method described herein, the isolated microorganism of the present disclosure is present in the formulation in an effective amount to be detectable in and / or on the target tissue of agricultural plants.For example, the microorganism is detected in and / or on the target tissue of plants in an amount of 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. Alternatively, or in addition, 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 in addition, 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 a 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 a formulation of the present disclosure has not been applied.

[0058] In some embodiments of the methods described herein, one or more metabolites isolated from a microorganism or consortia of the present disclosure are present in the formulation in an amount effective to be detectable in and / or on a target tissue of an agricultural plant, for example, 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 in addition, 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 in addition, 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 of a plant to which a 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 a formulation of the present disclosure has not been applied.

[0059] 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.

[0060] 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 vessel, a bag, a box, a bottle, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

[0061] In some embodiments, 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 crop's products. Thus, in one embodiment, the present disclosure provides an artificial mixture consisting of a seed of a first plant and a preparation of a microorganism coated on the surface of the seed of the first plant, where the microorganism is present on the surface of the seed at a higher level than is present on the surface of an uncoated, reference seed. In another embodiment, the present disclosure provides an artificial mixture consisting of a portion of a first plant and a preparation of a microorganism coated on the surface of the portion of the first plant, where the microorganism is present on the surface of the portion of the first plant at a higher level than is present on the surface of an uncoated, reference plant. The aforementioned methods can be used alone or in conjunction with plant breeding and plant gene transfer techniques.

[0062] In some embodiments, the bacterial strain can be Bacillus thuringiensis, deposited under NRRL accession number B-68090 on Jan. 26, 2022. In some embodiments, the bacterial strain can be Bacillus thuringiensis comprising a sequence that shares at least 95%, 96%, 97%, 98%, 99%, or greater than 99% identity with one or more of SEQ ID NOs: 1-19. In some embodiments, the bacterial strain can be isolated from its native environment and optionally substantially purified.

[0063] 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 mutant 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.

[0064] 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. In some embodiments, the isolated bacterial strains of the present disclosure comprise a polynucleotide sequence that shares at least 95% sequence identity with any of SEQ ID NOs: 1-19.

[0065] In some embodiments, acellular or inactivated preparations of the isolated bacterial strains of the present disclosure, or mutants of the aforementioned isolated bacterial strains, are contemplated. In some embodiments, acellular or inactivated preparations of the isolated bacterial strains of the present disclosure, or mutants of the aforementioned isolated bacterial strains, or genetically edited, modified, or modified variants are contemplated. In some embodiments, a metabolic product produced by the isolated bacterial strains of the present disclosure, or mutants of the aforementioned isolated bacterial strains, or genetically modified variants are contemplated. In some embodiments, a metabolic product produced by the isolated bacterial strains of the present disclosure, or mutants or genetically modified variants of the aforementioned isolated bacterial strains, are contemplated.

[0066] In some embodiments, the agricultural composition comprises 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.

[0067] In some embodiments, the 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, the 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.

[0068] In some embodiments, the present disclosure teaches a method of cultivating a plant having at least one beneficial trait. In some embodiments, the method comprises applying an isolated bacterial strain or microbial consortia to a seed of the plant, sowing or planting the seed, and cultivating the plant. In certain embodiments, the isolated bacterial strain or microbial consortia is applied as an agricultural composition further comprising an agriculturally acceptable carrier.

[0069] In some embodiments, the microbial consortia has substantially similar morphological and physiological characteristics to the microbial consortia of the present disclosure. In some embodiments, the microbial consortia has substantially similar genetic characteristics to the microbial consortia of the present disclosure. In some embodiments, the microbial consortia is in a substantially pure culture. In some embodiments, the progeny of any microorganism of the microbial consortia is contemplated. In some embodiments, mutants of any microorganism of the microbial consortia are contemplated. In some embodiments, gene-edited, modified, or modified variants of any microorganism of the microbial consortia are contemplated. In some embodiments, cell-free or inactivated preparations of the microbial consortia, or mutants or gene-edited, modified, or modified variants of any microorganism in the microbial consortia are contemplated. In some embodiments, metabolites produced by the microbial consortia, or mutants or gene-edited, modified, or modified variants of any microorganism in the microbial consortia are contemplated.

[0070] 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.

[0071] 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 Table 1A.

[0072] The present disclosure can be more fully understood from the following detailed description and the accompanying drawings and sequence listing, which form a part of this specification. [Brief description of the drawings]

[0073] [Figure 1] Figure 1 shows a stained microscopy image of Bacillus thuringiensis strain 39400. Dark dots indicate crystallized protein and light dots indicate spores.

[0074] [Figure 2A] FIG. 2A is a diagram of the life cycle of C. elegans, a model organism for nematode research in agricultural crops.

[0075] [Figure 2B] FIG. 2B shows the reduction of C. elegans nematodes over time in bioassay treatments with strain 39400 compared to the control (no microbial treatment).

[0076] [Diagram 3] FIG. 3 shows that strain 39400 reduced the number of hatched root-knot nematode eggs compared to the control and to a similar extent as the other strains tested.

[0077] [Figure 4] 4A and 4B show the effect of dilution of Bt strain 39400 on both size (FIG. 4A) and survival (FIG. 4B) of Spodoptera litura.

[0078] [Diagram 5] FIG. 5 shows that line 39400 provided over a 15% improvement in wheat yield, measured in bushels per acre.

[0079] [Figure 6] FIG. 6 shows that strain 39400 increased corn growth under nematode pressure by more than 3% when applied as a seed treatment.

[0080] [Figure 7] FIG. 7 shows that strain 39400 increased corn growth under nematode pressure by more than 2% when applied as an in-furrow treatment.

[0081] [Figure 8] FIG. 8 shows that strain 39400 has activity against Coleoptera, as shown by enhanced activity against S. litura in dilution assays (left bars) compared to the commercial biological control (right bars).

[0082] [Figure 9] FIG. 9 shows that strain 39400 has activity against Coleoptera, as shown by improved activity against T. molitor (middle bar), activity greater than the water control (left bar), and activity comparable to the commercial biological control (right bar). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] The sequence descriptions and sequence listing accompanying this specification 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. The microorganisms described in this application have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL), an international depository authority located at 1815 North University Street, Peoria, Illinois, 61604, USA.

[0084] The deposit was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0085] The deposit is made in accordance with and to satisfy the standards set forth in 37 CFR §§1.801-1.809 and the US Manual of Patent Examining Procedures §§2402-2411.05.

[0086] [Table 1]

[0087] Although the following terms are believed to be well understood by those of skill in the art, the following are set forth to facilitate explanation of the subject matter of the present disclosure.

[0088] The term "a" or "an" refers to one or more of that entity, i.e., it 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 is present, unless the context clearly requires that there is only one of that element.

[0089] 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 domains, Bacteria and Archaea, and Eukaryotes and Protista. In some embodiments, the present disclosure refers to the "microorganisms" of Tables 1-2, or various other tables or paragraphs present in this disclosure. This characterization can refer not only to the identified taxonomic bacterial genera of the tables, but also to the identified taxonomic bacterial species, and to the various novel and newly identified bacterial strains of the tables.

[0090] As used herein, the term "microbe" or "microorganism" refers to any microbial species or taxon, including, but not limited to, archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasma, 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.

[0091] As used herein, the term "bacterium" or "bacteria" 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 have been redesignated for various reasons (such as, but not limited to, the evolving field of whole genome sequencing), and such nomenclature redesignations are understood to be 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).).

[0092] The term "16S" refers to the DNA sequence of the bacterial 16S ribosomal RNA (rRNA) sequence. 16S rRNA gene sequencing is an established method for studying bacterial phylogeny and taxonomy. As used herein, the term "fungus or fungi" generally refers to any organism from the kingdom Fungi. Historical taxonomic classification of fungi is by morphological presentation. Beginning in the mid-1800s, it was recognized that some fungi have a polymorphic 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, JW 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 evident that taxonomic classification based on molecular phylogenetics was not compatible with morphology-based nomenclature (Shenoy, BD, Jeewon, R. and Hyde, KD (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 result of designating "one fungus" = "one name" (Hawksworth, DL Managing and coping with names of pleomorphic fungi in a period of transition. IMA Fungus 3,15-24 (2012)).

[0093] 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 ITS sequences and some by LSU sequences. It is understood that both are equally descriptive and accurate for determining taxonomy.

[0094] The term "microbial consortia" or "microbial consortium" refers to a portion 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.

[0095] 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 have to perform a common function or be related to, cause, or correlate with a recognizable parameter or plant phenotypic trait.

[0096] The term "rapid microbial sorting" or "AMS" is used interchangeably with the term "directed 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 the aforementioned species.

[0097] As used herein, "isolate," "isolated," "isolated microorganism," and like terms are intended to mean that one or more microorganisms are separated from at least one of the substances to which they are attached in a particular environment (e.g., soil, water, plant tissue).

[0098] Thus, an "isolated microorganism" does not exist in its natural environment. Rather, through various techniques described herein, the microorganism is 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 a spore (or other form of the strain) attached to an agricultural carrier.

[0099] In certain embodiments of the present disclosure, the isolated microorganism exists as an isolated and biologically pure culture. It is understood by those skilled in the art that an isolated and biologically pure culture of a particular microorganism represents that other organisms are substantially absent from the culture (for scientific reasons) and that only the particular microorganism in question is present in the culture. The culture may contain various concentrations of the microorganism. The present disclosure points out that isolated and biologically pure microorganisms are often "necessarily different from less pure or pure material." See, for example, In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins). See also In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microorganisms). See also Parke-Davis & Co. v. HK Mulford & Co., 189 F. 95 (SDNY 1911), which was affirmed in part at 196 F. 496 (2d Cir. 1912) and reversed in part (Learned Hand discussing purified adrenaline). Each of these cases is incorporated herein by reference. Additionally, in some aspects, the present disclosure prescribes certain quantitative measurements of concentration or purity limits that must be found in isolated and 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 microorganisms that exist 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 vitamin B12 produced by microorganisms).

[0100] As used herein, an "individual isolate" should be understood to mean a composition or culture that predominantly contains a single genus, species, or strain of microorganisms after separation from one or more other microorganisms. The phrase should not be understood to represent the extent to which the microorganism has been isolated or purified. However, an "individual isolate" can contain substantially only one genus, species, or strain of microorganisms.

[0101] The term "growth medium" as used herein is 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 in solid plant support systems, and tissue culture gels. It should be understood that such media may be used alone or in combination with one or more other media. The medium may be used with or without the addition of exogenous nutrients and physical support systems for the roots and leaves.

[0102] In one embodiment, the growth medium is a natural 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, water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0103] The media may be amended or enriched with additional compounds or components, such as components that may aid in the interaction of certain groups of microorganisms with plants and with each other and / or the selection of said groups of microorganisms. For example, antibiotics (such as penicillin) or sterilizing agents (such as quaternary ammonium salts and oxidizing agents) may be present, and / or the physical conditions (salts, plant nutrients (such as organic and inorganic minerals, such as phosphorus, nitrogenous salts, ammonia, potassium, and trace nutrients such as cobalt and magnesium), pH, and / or temperature) may be modified.

[0104] 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. "Plant elements" are intended to refer to either whole plants or plant components, which may include differentiated and / or undifferentiated tissues, such as, but are not limited to, plant tissues, parts, and cell types. In one embodiment, the plant element is one of the following: whole plants, seedlings, meristems, ground tissues, vascular tissues, skin tissues, seeds, leaves, roots, shoots, stems, flowers, fruits, stolon, bulbs, tubers, corms, shoots, shoots, tumor tissues, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, callus tissues). The term "plant organ" refers to a plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant. As used herein, "plant part" is synonymous with "part" of a plant and refers to any part of a plant, which can include distinct tissues and / or organs, and may be used synonymously with the term "tissue" throughout.

[0105] "Progeny" includes any descendant of an organism produced through sexual or asexual reproduction.

[0106] 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 the plants themselves. Grain is intended to mean mature seeds produced by commercial growers for purposes other than seed growth or propagation. Progeny, variants, and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts contain the introduced polynucleotide.

[0107] Similarly, "plant propagation element" is generally intended to refer to any part of a plant that can initiate other plants through either sexual or asexual propagation of the plant, such as, but not limited to, a seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, stem, or sprout. A plant element can be in a plant or in a plant organ, tissue culture, or cell culture.

[0108] The term "monocotyledonous" or "monocotyledonous plant" refers 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 progeny thereof.

[0109] The terms "dicotyledonous" and "dicot" refer to the subclass of angiosperms also known as "dicots," whose seeds typically contain two embryonic leaves or cotyledons. The terms include reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0110] 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.

[0111] As used herein, "improved" should be broadly understood to include an improvement in a certain characteristic of a plant 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 skilled 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 that demonstrates that one value (e.g., average treatment value) is different from another value (e.g., average control value) can be raised to the level of "improvement."

[0112] As used herein, "inhibiting and suppressing" and such terms should not be construed as requiring complete inhibition or suppression, although this may be desired in some embodiments.

[0113] As used herein, the term "genotype" refers to the genetic makeup of an individual cell, a cell culture, a tissue, an organism (e.g., a plant), or a group of organisms.

[0114] 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 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, as compared to an isogenic plant that does not contain the modification resulting from the methods or compositions herein.

[0115] "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.

[0116] As used herein, the term "molecular marker", "marker" or "genetic marker" refers to an indicator used in a method for visualizing differences in the characteristics of nucleic acid sequences. 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, that define specific genetic and chromosomal locations. Mapping molecular markers in the vicinity of an allele is a technique that can be performed by those skilled in the art of molecular biology.

[0117] 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 include other characteristics of a plant, including, but not limited to, water use efficiency, nutrient use efficiency, yield, mechanical harvestability, fruit maturity, storage time, pest / disease resistance, early plant maturity, stress tolerance, and the like. A trait may be dominantly or recessively inherited, or may be partially or incompletely dominantly inherited. A trait 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.

[0118] 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.

[0119] As used herein, a "synthetic nucleotide sequence" or "synthetic polynucleotide sequence" is a nucleotide sequence that is not known to occur in nature or 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.

[0120] As used herein, the term "nucleic acid" refers to any length of polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, or their analogs. The term refers to the primary structure of the molecule, and thus includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. 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.

[0121] As used herein, the term "gene" refers to any fragment of DNA associated with biological function. Thus, genes include, but are not limited to, coding sequences and / or regulatory sequences required for the expression of coding sequences. Genes can also include non-expressed DNA fragments, such as those that form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a desired source or synthesis from known or predicted sequence information, and can include sequences designed to have desired parameters.

[0122] As used herein, the terms "homologous" or "homologue", "homolog" or "ortholog" are known in the art and refer to related sequences that have 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 is unaffected by changes 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 as compared to the original unmodified fragment. It is therefore understood that the present disclosure encompasses sequences other than the specific exemplary sequences, as will be appreciated by those 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 the 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 may be indicated in any one of a number of ways, including, but not limited to, (a) degree of sequence identity and / or (b) same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (eds. FMA Usubel et al., 1987) Supplement 30, section 7.718, Table 7.71.Some alignment programs are MacVector (Oxford Molecular, Oxford, UK), ALIGN Plus (Scientific and Educational Software, PA) and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, MI), using default parameters.

[0123] 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.

[0124] 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.

[0125] As used herein, the term "at least a portion" or "fragment" of a nucleic acid or polypeptide refers to a portion having a minimal size characteristic of such sequence, or any fragment of the full-length molecule that is somewhat larger than the portion, 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 genetic regulatory element. A biologically active portion of a genetic regulatory element can be prepared by isolating a portion of one of the polynucleotides of the present disclosure that contains the genetic regulatory element and evaluating the activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, etc., up to the full-length polypeptide. The length of the portion 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, it is 20 nucleotides. A portion of a polypeptide useful as an epitope can be as long as 4 amino acids. A portion of a polypeptide that performs the function of a full-length polypeptide will generally be longer than 4 amino acids.

[0126] The term "primer" as used herein refers to an oligonucleotide that, when placed under conditions inducing the synthesis of a primer extension product, i.e., in the presence of nucleotides and a polymerization agent such as DNA polymerase and appropriate temperature and pH, is capable of annealing to an amplification target to allow DNA polymerase to attach and thereby serve as a starting point for DNA synthesis. (Amplification) primers are preferably single-stranded to maximize the efficiency of amplification. The primers are preferably oligodeoxyribonucleotides. The primers must be long enough 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.

[0127] The term "stringency" or "stringent hybridization conditions" refers to hybridization conditions that affect hybrid stability, such as temperature, salt concentration, pH, formamide concentration, etc. These conditions are empirically optimized to maximize specific binding of a primer or probe to its target nucleic acid sequence and minimize non-specific binding. The terms used include reference to conditions under which a probe or primer hybridizes to its target sequence at a higher degree of detection (e.g., at least 2-fold over background) than to other sequences. Stringent conditions are sequence-dependent and will vary under different circumstances. Larger sequences hybridize specifically at higher temperatures. Stringent conditions are generally selected to be about 5°C lower than the melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm is the temperature (under a defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Stringent conditions are typically conditions with 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., more than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency conditions or "reduced stringency conditions" include hybridization in a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. and washing in 2×SSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C. and washing in 0.1×SSC at 60° C.Hybridization methods are well known in the art and are described, for example, by Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, stringent conditions are hybridization in 0.25 M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 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 5xSSC containing 0.1% (weight / volume) sodium dodecyl sulfate at 55°C to 65°C.

[0128] In some embodiments, a cell or organism has at least one heterologous trait. As used herein, the term "heterologous trait" refers to a phenotype conferred to a cell or organism by an exogenous molecule or another organism (e.g., a microorganism), DNA segment, heterologous polynucleotide, or heterologous nucleic acid.

[0129] 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 increasing expression of endogenous products in plants using the methods and compositions of the present disclosure.

[0130] An "artificial mixture" may include a mixture of a plant and a microorganism of the present disclosure. The mixture may be achieved, for example, by coating the surface of a seed of a plant, such as an agricultural plant, or a host plant tissue (root, stem, leaf, etc.) with a microorganism of the present disclosure. Additionally, an "artificial mixture" may include a mixture of different strains or species of microorganisms. An artificial mixture has at least one variable that distinguishes it from any mixture occurring in nature. The variable may be, in particular, a concentration of microorganisms on a 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 has structural and / or functional properties that are not present when the individual components of the mixture are considered in isolation.

[0131] 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 plant specimen from which it is obtained. That is, the microorganism is found in nature in association with said plant. In some embodiments of applying an endogenous microorganism to a plant, the endogenous microorganism is applied at a level different 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.

[0132] 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 derived from the plant specimen from which it is obtained. That is, the microorganism is not found in association with said plant in nature. 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 have said microorganism. In another example, a microorganism normally associated with a corn plant would be considered foreign to a wheat plant that naturally lacks said microorganism.

[0133] A composition is "heterogeneously 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 in the plant element, seedling, plant, plant growth medium, or treatment formulation, in a combination not found in nature in that plant variety, in that plant development stage, in that plant tissue, in that abundance, or in that growing 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 in or on the plant, the presence of the microorganism at different periods, e.g., developmental stages, times, seasons of the plant or plant element, and combinations thereof. In some embodiments, "heterogeneously 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 disposed" means that the 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 the microorganism is normally found at the flowering stage of a plant and not at other stages, the microorganism applied at the seedling stage may be considered heterologously disposed. In some embodiments, the microorganism is heterologously disposed and the microorganism is normally found in the root tissue of the plant element but not in the leaf tissue, and the microorganism is applied to the leaf. In another non-limiting example, if the microorganism is naturally found in the mesophyll layer of the leaf tissue, but is applied to the epidermis layer, the microorganism would be considered heterologously disposed. In some embodiments, "heterologously disposed" means that the 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 disposed" means that the microorganism being applied is in a concentration, number, or amount of 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 times higher, 1.5-2 times higher, 2 times higher, 2-3 times higher, 3 times higher, 3-5 times higher, 5 times higher, 5-7 times higher, 7 times higher, 7-10 times higher, 10 times higher, or even more than 10 times 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 cypress tree 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 quantity.

[0134] A microorganism can also be "heterologously distributed" on a given plant tissue. This means that the microorganism is distributed on plant tissue where it is not found in nature. For example, if a given microorganism occurs only on the roots of a given plant in nature, the microorganism can be exogenously applied to the above-ground tissue of the plant, thereby "heterologously distributing" the microorganism on said plant tissue. Thus, a microorganism is considered to be heterologously distributed when it does not have a naturally occurring microorganism or does not naturally have a microorganism present in the number being applied.

[0135] The compositions and methods herein may provide "modulated" "agronomic traits" or "agronomically important traits" to the host plant, 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 resistance, 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 structure, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased seed protein content, and increased seed protein content, compared to isogenic plants grown from seeds without the use of the aforementioned seed treatment formulations. The effects of the present invention may include: increased root length, increased yield, increased yield under limited water conditions, grain mass, grain moisture content, metal resistance, ear number, number of grains per ear, pod number, nutritional enhancement, pathogen resistance, pest resistance, improved photosynthetic capacity, salt tolerance, greening, improved vigor, increased mature seed dry weight, increased mature seed fresh weight, increased mature seed number per plant, chlorophyll content, increased pod number 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 changes in metabolite levels, detectable changes in transcript levels, and detectable changes in proteome. By the term "modulated" it is intended to refer to changes in agronomic traits that are altered by the presence of microorganisms, exudates, broths, metabolites, and the like. In an embodiment, the modulation provides for the impartation of a beneficial trait.

[0136] 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.

[0137] By way of example, the microorganisms include those of the phylum Proteobacteria (such as the genera Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Duganella, Delftia, Bradyrhizobium, Sinorhizobium, and Halomonas), Firmicutes (such as the genera Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetobacterium), Actinobacteria (such as the genera Brevibacterium, Janibacter, Streptomyces, Rhodococcus, Microbacterium, Curtobacterium, Cellulomonas, Nocardioides, and the like), and the fungal Ascomycota (such as Trichoderma, Ampelomyces, Coniothyrium, Paecoelomyces, Penicillium, Cladosporium, Hypocrea, Beauveria, Metarhizium, Verticillium, Cordyceps, Pichia, and Candida), Basidiomycota (such as Coprinus, Corticium, and Agaricus) and Oomycota (such as Pythium), and Mucormycota (such as Mucor, Mortierella), as well as the genera Orbiliacea / Arturobotrys, Lysinibacillus, Microbacterium, Talaromyces, Arthrobacter, Kosaconia, Massilia, Novosphingobium, and Tumebacillus.

[0138] In certain embodiments, the microorganism is an endophyte or epiphyte, or a microorganism that resides in the plant rhizosphere or rhizocyst, 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.

[0139] In one embodiment, the microorganism is an endophyte. Endophytes may benefit the host plant by preventing pathogenic organisms from infecting the host plant. Endophytes infect plant tissues over a wide area, creating a "barrier effect" in which the endophyte in that area outcompetes pathogenic organisms and prevents them from establishing themselves. Endophytes may also produce chemicals that inhibit the growth of competitors, including pathogenic organisms.

[0140] In certain embodiments, the microorganism is non-culturable, which should be understood to mean that the microorganism is not known to be culturable or is difficult to culture using methods known to those of skill in the art.

[0141] The microorganisms of the present disclosure may be collected or obtained from any source, or may be contained within and / or attached to material collected from any source.

[0142] In one embodiment, the microorganisms are obtained from any common terrestrial environment, including its soil, plants, fungi, animals (including invertebrates), and other biota, including the sediments, water, and biota of lakes and rivers, from marine environments, their biota and sediments (e.g., seawater, marine mud, marine plants, marine invertebrates (e.g., sponges), marine vertebrates (e.g., fish)), from the terrestrial and marine geosphere (topsoil and rocks, e.g., fractured subsurface rock, sand, and clay), the cryosphere and its meltwaters, the atmosphere (e.g., filtered airborne dust, clouds, and raindrops), urban, industrial, and other man-made environments (e.g., concrete, roadside gutters, roof surfaces, accumulated organic matter and minerals on road surfaces).

[0143] In another embodiment, the microorganisms are collected from a source that may be favorable for the selection of suitable microorganisms. By way of example, the source may be a particular environment in which other plants are desirable to grow, or a particular environment that is considered to be associated with a terroir. In another example, the source may be a plant with one or more desirable traits, such as a plant that grows naturally in a particular environment, or a plant that grows naturally under particular conditions of interest. By way of example, a particular plant may naturally grow in sandy soil or saline sand, or at extreme temperatures, or with little water, or a particular plant may be resistant to a particular pest or disease present in that environment. It may be desirable for a commercial crop to be cultivated under such conditions, especially if such conditions are the only conditions available, for example, in a particular geographic location. As other examples, microorganisms may be collected from commercial crops grown in such environments, or more specifically from individual crops that best exhibit the traits of interest among the crops grown in any particular environment, such as the plants that grow fastest among crops grown in soils with limited salt content, or the plants that are least damaged among crops exposed to severe insect or disease epidemics, or plants that have desired amounts of certain particular metabolic substances and other compounds, including fiber content, oil content, etc., or plants that exhibit a desired color, taste, or odor. Microorganisms may be collected from the plants of interest, or from any material occurring in the environment of interest, including fungi and other animal and plant biota, soil, water, sediment, and other components of the environment mentioned previously. In certain embodiments, the microorganisms are individual isolates separated from different environments.

[0144] In one embodiment, the microorganisms or mixtures of microorganisms used in the methods of the present disclosure may be selected from existing collections of individual microbial species or strains based on some knowledge of their possible or anticipated benefits to a given plant. For example, microorganisms may be expected 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, live in the rhizosphere of plants, thereby helping plants absorb nutrients from the surrounding soil and then making these more readily available 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 plant defense responses such as ISR (induced systemic resistance) or SAR (systemic acquired resistance) that help plants resist the invasion and spread of pathogenic microorganisms, compete with microorganisms harmful to plant growth or health by 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.

[0145] In one embodiment, the microorganism or mixture of microorganisms is selected from an existing collection of individual microbial species or strains that does not provide knowledge of possible or expected benefits to a plant, such as a collection of unidentified microorganisms isolated from plant tissue without any knowledge of their ability to improve plant growth or health, or a collection of microorganisms collected for their potential to produce compounds that may lead to pharmaceutical development.

[0146] In one embodiment, the microorganisms are obtained from the source sample (e.g., soil, rock, water, air, dust, plants or other organisms) in which they naturally occur. The microorganisms may be provided in any suitable form considering the intended use in the method of the present disclosure. However, by way of example only, the microorganisms may be provided as an aqueous suspension, gel, homogenate, granules, powder, mud, living organisms, or desired material.

[0147] The microorganisms of the present disclosure may be isolated in substantially pure or mixed cultures. They may be concentrated, diluted, or provided at the natural concentrations at which they are found in the original sample. For example, microorganisms from a saline sediment 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 either directly added to the plant growth medium or concentrated by filtering or centrifugation, diluted to a suitable concentration, and most of the salts removed to be added to the plant growth medium. As a further example, microorganisms from mineralized or toxic sources may be similarly treated to recover the microorganisms for addition to the plant culture material in order to minimize potential damage to the plant.

[0148] In another embodiment, the microorganisms are used in a crude form that is not isolated from the original sample in which they occur in nature. For example, the microorganisms are provided in combination with the original sample in which they occur, for example, as soil, or the roots, seeds or leaves of a plant. In this embodiment, the original sample may contain one or more species of the microorganism.

[0149] In some embodiments, a mixed population of microorganisms is used in the methods of the present disclosure.

[0150] In embodiments of the present disclosure in which microorganisms are isolated from a source sample (e.g., a material in which the microorganism occurs in nature), any one or combination of a number of standard techniques readily understood by those skilled in the art may be used. However, by way of example, these techniques generally employ processes that allow a solid or liquid culture of a single microorganism to be obtained in substantially pure form, usually by physical separation on the surface of a solid microbial growth medium or by volumetric dilution isolation into a liquid microbial growth medium. These processes may include isolation from the material, where a dry material, liquid suspension, sludge or homogenate is spread in a thin layer on a suitable solid gel growth medium, or serial dilutions of the material are made in a sterile medium and inoculated into a liquid or solid growth medium.

[0151] Although not required, in one embodiment, a material containing microorganisms can be pretreated prior to isolation by enriching the material for microorganisms in order to grow all microorganisms in the material or to select for a portion of the microbial population (e.g., by pasteurizing the sample to select for microorganisms that are resistant to heat exposure (e.g., Bacillus subtilis)), or by exposing the sample to low concentrations of organic solvents or sterilants (e.g., household bleach) to enhance the survival of spore-forming or solvent-resistant microorganisms. Microorganisms can then be isolated from the enriched material or material treated for selective survival as above.

[0152] In one embodiment of the present disclosure, endophytic or epiphytic microorganisms are isolated from plant material. Any number of standard techniques known in the art may be used, and microorganisms may be isolated from any suitable tissue of the plant, including, for example, roots, stems and leaves, and plant reproductive tissue. By way of example, conventional methods for isolation from plants typically include aseptic cutting of the desired plant material (e.g., roots or stems, leaves), surface sterilization with a suitable solution (e.g., 2% sodium hypochlorite), and then placing the plant material in a nutrient medium for microbial culture (see, for example, Strobel G and Daisy B (2003) Microbiology and Molecular Biology Reviews 67(4):491-502; Zinniel DK et al. (2002) Applied and Environmental Microbiology 68(5):2198-2208).

[0153] In one embodiment of the present disclosure, the microorganisms are isolated from root tissue. Other methods for isolating microorganisms from plant material are described in more detail below.

[0154] In one embodiment, the microbial population is exposed to selection pressure (either prior to the method or at any stage of the method), for example by exposing the microorganisms to pasteurization prior to addition to the (preferably sterile) plant growth medium, it may be possible to increase the probability that plants selected for the desired trait will be populated with spore-forming microorganisms that can more readily survive adverse conditions, in commercial storage areas, or when applied to seeds as a coating.

[0155] In certain embodiments described herein above, the microorganisms may be used in a crude form, without the need to isolate the microorganisms from the plant or medium. For example, plant material or medium containing the microorganisms identified as beneficial to the selected plant may be obtained and used as a crude source of microorganisms for subsequent processes or as a crude source of microorganisms at the end of the process. For example, whole plant material may be obtained and optionally treated, such as mulched or crushed. Alternatively, individual tissues or parts of the selected plant (such as leaves, stems, roots, and seeds) may be separated from the plant and optionally treated, such as mulched or crushed. In certain embodiments, one or more parts of the plant having the second set of one or more microorganisms attached thereto may be removed from the one or more selected plants, and the parts may be grafted onto one or more plants used at any stage of the plant breeding method if the method is performed in successive iterations.

[0156] Exemplary Microorganisms In aspects, the present disclosure provides isolated microorganisms, including new strains of the identified microbial species presented in Table 1A.

[0157] In other aspects, the disclosure provides isolated whole microbial cultures of the species and strains identified in Table 1 A. These cultures may contain microorganisms at various concentrations.

[0158] In an aspect, the present disclosure provides agricultural uses of a microorganism selected from Table 1A.

[0159] In some embodiments, microorganisms of the genus Bacillus are used in agriculture to impart one or more beneficial properties to plant species.

[0160] Additionally, the present disclosure relates to microorganisms having characteristics substantially similar to the characteristics of the microorganisms identified in Table 1A.

[0161] The isolated microbial species, and novel strains of such species, identified in this disclosure are capable of conferring beneficial properties or traits, such as agronomically important traits, to target plant species.

[0162] For example, the isolated microorganisms or microbial consortia described in Table 1A can improve plant health and vigor, which can be quantitatively measured, for example, by measuring the effect that the microbial application has on the phenotypic or genotypic traits of the plant.

[0163] Sourcing microorganisms The microorganisms of the present disclosure were obtained from various locations within New Zealand and the United States, among other locations.

[0164] Microbial isolation and culture The microorganisms in Table 1A have been identified using standard microscopic techniques to characterize the phenotype of the organism and then use it to identify the organism to a taxonomically recognized species.

[0165] Isolation, identification, and culture of the microorganisms of the present disclosure can be accomplished using standard microbiological techniques. Examples of such techniques can be found in Gerhardt, P. (ed.) Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, DC (1994), and Lennette, EH (ed.) Manual of Clinical Microbiology, Third Edition. American Society for Microbiology, Washington, DC (1980), each of which is incorporated by reference.

[0166] Isolation can be achieved by streaking specimens onto solid media (e.g., nutrient agar plates) to obtain single colonies characterized by the phenotypic traits described herein above (e.g., gram positive / negative, ability to sporulate aerobically / anaerobicly, cell morphology, carbon source metabolism, acid / base production, enzyme secretion, metabolic secretions, etc.) and to reduce the chance of working with contaminated cultures.

[0167] For example, the isolated bacteria of the present disclosure can be obtained by repeatedly subculturing the biological sample and streaking on solid medium after each subculturing to obtain individual colonies to obtain biologically pure isolates. Methods for preparing, thawing and growing freeze-dried bacteria are generally known, see, for example, Gherna, RL and CAReddy. 2007. Culture Preservation, p 1019-1033. In CAReddy, TJ Beveridge, JA Breznak, GA Marzluf, TM Schmidt, and LR Snyder, eds. American Society for Microbiology, Washington, DC, 1033 pages, which are incorporated herein by reference. Thus, freeze-dried liquid preparations and cultures that have been stored long-term at -70°C in a solution containing glycerol are contemplated for use in providing the liquid preparations of the present invention.

[0168] The bacteria of the present disclosure can be grown in liquid medium under aerobic conditions. The medium for growing the bacterial strains of the present disclosure includes a carbon source, a nitrogen source, and inorganic salts, as well as specially required substances such as vitamins, amino acids, nucleic acids, etc. Examples of suitable carbon sources that can be used to grow the bacterial strains include, but are not limited to, starch, peptone, yeast extract, amino acids, sugars such as glucose, arabinose, mannose, glucosamine, maltose, salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid, alcohols such as ethanol and glycerol, oils or fats such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil, etc. The amount of carbon source added varies depending on the type of carbon source and is typically between 1 gram and 100 grams per liter of medium. The medium preferably contains glucose, starch, and / or peptone as the main carbon source at a concentration of 0.1-5% (weight / volume). Examples of suitable nitrogen sources that can be used for the growth of the bacterial strains of the present invention include, but are not limited to, amino acids, yeast extract, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, or combinations thereof. The amount of nitrogen source varies depending on the type of nitrogen source and is typically between 0.1 and 30 grams per liter of medium. The inorganic salts of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferrous chloride, ferrous chloride, manganese sulfate, manganese chloride, zinc sulfate, zinc chloride, copper sulfate, calcium chloride, sodium chloride, calcium carbonate, and sodium carbonate can be used alone or in combination. The amount of inorganic acid varies depending on the type of inorganic salt and is typically between 0.001 and 10 grams per liter of medium. Examples of specially required substances include, but are not limited to, vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, dried yeast, and combinations thereof.Cultivation can be carried out at a temperature that allows the growth of those bacterial strains, typically between 20°C and 46°C. In some aspects, the temperature range is 30°C to 37°C. For optimal growth, in some embodiments, the medium can be adjusted to a pH of 7.0 to 7.4. It will be appreciated that commercially available media, such as nutrient broth or nutrient agar available from Difco, Detroit, MI, can also be used to cultivate the bacterial strains. It will be appreciated that the incubation time can vary depending on the type of medium used and the concentration of sugar as the main carbon source.

[0169] In an embodiment, the culture continues for between 24 and 96 hours. The bacterial cells thus obtained are isolated using methods well known in the art. Examples include, but are not limited to, membrane filtration and centrifugation. The pH may be adjusted using sodium hydroxide or the like, and the culture may be dried using a freeze dryer until the moisture content is equal to or less than 4%. A microbial co-culture can be obtained by growing each of the strains described herein above. It is understood that the microbial strains may be cultured together when compatible culture conditions can be used.

[0170] Microbial Identification Microorganisms can be classified into genera based on polyphasic taxonomy (Vandamme et al. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996, 60:407-438), which incorporates all available phenotypic and genotypic data into a consensus classification. One accepted genotypic method for species definition is based on global genomic relatedness, whereby strains with approximately 70% or more relatedness using DNA-DNA hybridization under standard conditions with a ΔTm (difference in melting temperature between homologous and heterologous hybrids) of 5°C or less are considered to be members of the same species. Thus, populations with a higher proportion than the aforementioned 70% threshold can be considered to be variants of the same species.

[0171] For bacterial microorganisms, 16S rRNA sequences are often used to determine taxonomy and distinguish between species, whereby if a 16S rRNA sequence has less than a certain percent sequence identity with a reference sequence, the two organisms from which the sequences were obtained are said to be of different species.

[0172] Thus, multiple microorganisms can be considered to be of the same species if they have at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across their 16S or 16S rRNA or rDNA sequences. In some embodiments, a given microorganism can be considered to be of the same species if it has at least 95% identity.

[0173] Furthermore, microbial strains of a species can be defined as microbial strains that have at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across their 16S rRNA sequences.

[0174] The 23S rRNA sequence may be compared to the reference sequence. In some aspects, a microorganism may be considered to be the same strain if it has at least 95% identity. In some embodiments, "substantially similar genetic characteristics" refers to a microorganism having at least 95% identity.

[0175] For fungal microorganisms, ITS (internal transcribed sequences) are often used for taxonomic identification. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the widest range of fungi, has the most clearly defined barcode gap between inter- and intra-species variation, and has been proposed as a formal fungal identification sequence (Schoch et al., PNAS April 17, 2012 109(16)6241-6246).

[0176] In one embodiment, the microbial strains of the present disclosure include those comprising a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs:1-19.

[0177] In one embodiment, the microorganisms of the present disclosure include those that contain a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs:1-19.

[0178] In one embodiment, a microbial consortium of the present disclosure includes two or more microorganisms comprising a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs:1-19.

[0179] In one embodiment, a microbial consortium of the present disclosure comprises two or more microbial strains, at least one of which comprises a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs:1-19.

[0180] In one embodiment, the microbial consortium of the present disclosure comprises two or more microbial strains, at least one of which comprises a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs:1-19, and at least one of the microorganisms is optionally selected from Table 1A.

[0181] It is often not possible to assign unculturable microorganisms to a definitive species without a determined phenotype, but they can be designated as candidates (candidatus) within a genus if their 16S rRNA sequences conform to the identity principle with known species.

[0182] One approach is to look at the distribution of a large number of strains of closely related species in sequence space and identify clusters of strains that are well resolved from other clusters. This approach has been developed by assessing clustering patterns using concatenated sequences of multiple core (housekeeping) genes and is called multilocus sequence analysis (MLSA) or multilocus sequence phylogenetic analysis. MLSA has been used successfully to investigate clustering patterns among a large number of strains that are assigned to very closely related species by current taxonomic methods, to examine relationships among a small number of strains within a genus or within a broader taxonomic group, and to address specific taxonomic questions. More generally, the method can be used to ask whether bacterial species exist, i.e., to observe whether large populations of similar strains always fall into well-separated clusters, or to observe whether genetic continuity exists where in some cases no clear separation into clusters is observed.

[0183] To more accurately determine genus, phenotypic traits such as morphological, biochemical, and physiological traits are determined and compared to reference genus prototypes. Colony morphology may include color, shape, pigmentation, slime production, etc. Cellular traits are described for shape, size, Gram reaction, extracellular material, presence of endospores, presence and location of flagella, motility, and inclusions. Biochemical and physiological traits describe the growth of the organism in a range of temperature, pH, salinity, and atmospheric conditions, growth in the presence of different single carbon sources, and growth in the presence of different single nitrogen sources. One of skill in the art will reasonably identify phenotypic traits that define the genus of the present disclosure. For example, colony color, morphology, and texture on specific agar (e.g., YMA) have been used to identify species of Rhizobium.

[0184] In one embodiment, the bacterial microorganisms taught herein were identified utilizing 16S rRNA gene sequences. It is known in the art that 16S rRNA contains hypervariable regions that can provide species / strain specific signature sequences useful for identifying bacteria. In the present disclosure, many of these microorganisms were identified via partial (500-1200 bp) 16S rRNA sequence signatures. In aspects, each strain represents a pure colony isolate selected from an agar plate. Selection was based on any defining morphological characteristics of the colonies on the agar medium to represent the diversity of organisms present. In embodiments, the media used was R2A, PDA, nitrogen-free semi-solid medium, or MRS agar. Colonies of each of the "selected" isolates were described after 24 hours of incubation and entered into our database. Sequence data was then obtained for each of the isolates.

[0185] Phylogenetic analysis using the 16S rRNA gene was used to define "substantially similar" species within a common genus, and also to define "substantially similar" strains of a given taxonomic species. Additionally, physiological and / or biochemical traits of the isolates were recorded that could be used to highlight both minor and important differences between strains that may act to the plant's advantage.

[0186] Microbial Consortia In aspects, the disclosure provides a microbial consortium comprising a combination of at least any two microorganisms, one of which is Bacillus thuringiensis strain 39400, strain 42901, or any strain comprising at least one polynucleotide sequence sharing at least 95%, 96%, 97%, 98%, 99%, or greater than 99% identity to one or more of SEQ ID NOs:1-19.

[0187] In certain embodiments, a consortia of the present disclosure includes two microorganisms, or three microorganisms, or four microorganisms, or five microorganisms, or six microorganisms, or seven microorganisms, or eight microorganisms, or nine microorganisms, or ten or more microorganisms, where the microorganisms of the consortia are different microbial species or different strains of a microbial species.

[0188] Microbial production composition In some cases, a microorganism of the present disclosure may produce one or more compounds and / or have one or more activities, such as, for example, 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 one or more of any combination and / or plurality of the foregoing.

[0189] For example, the microorganisms of the present disclosure may produce a plant hormone selected from the group consisting of auxins, cytokinins, gibberellins, ethylene, brassinosteroids, and abscisic acid.

[0190] Thus, the term "metabolites produced" by the microorganisms of the present disclosure refers to any molecule (small molecule, vitamins, minerals, proteins, nucleic acids, lipids, fats, carbohydrates, etc.) produced by said microorganisms. 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 microorganisms produce metabolic products that are 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 does not need to be alive to confer beneficial traits to a given plant species, as long as the preparation is produced by the microorganism and contains metabolic products that are beneficial to the plant.

[0191] In one embodiment, the microorganisms of the present disclosure may produce auxins (e.g., indole-3-acetic acid (IAA)). Auxin production may be assayed. Many of the microorganisms described herein may be capable of producing the plant hormone auxin, indole-3-acetic acid (IAA), when grown in culture. Auxins play an important role in altering plant physiology, including the extent of root growth.

[0192] Thus, in one embodiment, the microorganisms of the present disclosure are present as a population located on the surface or in the tissue of a given plant species. The microorganisms can 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 in the plant, when compared to a reference plant that is not treated with the microorganisms of the present disclosure or the acellular or inactive preparation. The composition produced by the microbial population can be beneficial to the plant species.

[0193] Such microbial production compositions may be present in the cell culture broth or medium in which the microorganism is grown, or may include exudates produced by the microorganism. 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 are placed in the medium. The composition of the broth may change over time during different stages of growth and / or development of the microorganism. The broth and / or exudates may improve the traits of the plant with which they become associated.

[0194] Microbial-induced traits in plants The present disclosure utilizes microorganisms to impart beneficial traits (or beneficial traits) to desirable plant species, e.g., agronomic species of interest. In the present disclosure, the terms "beneficial trait" or "beneficial trait" are used interchangeably to refer to the modulation of a desirable plant phenotype or genetic trait of interest by application of a microorganism or microbial consortium as described herein. As mentioned above, in some embodiments, it is highly likely that the metabolic products produced by a given microorganism are ultimately responsible for modulating a beneficial trait or imparting a beneficial trait to a given plant.

[0195] 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 attributes 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 metabolite of interest in the plant, etc.

[0196] In embodiments, the microorganisms taught herein provide a wide range of agricultural applications, including improved crop, fruit, and flower yields, improved plant part growth, 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 other desirable plant phenotypic characteristics. In some embodiments, the nematodes include root-knot nematodes. In some embodiments, the insects may be of the genus Diabrotica.

[0197] In some embodiments, the genetic traits that may be improved relative to a reference plant include altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical resistance, 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 structure, 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 kernels per ear, number of pods, nutritional enhancement, pathogen resistance, (e.g., metabolism that impairs pathogen survival), The isolated microorganisms, co-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 (through excretion of products), pest resistance, improved photosynthetic capacity, salt tolerance, greening, improved 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 regulation of metabolite levels, detectable regulation of transcript levels, and detectable regulation of the proteome.

[0198] 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 phenotypic traits 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, they may have the ability to reduce shoot growth, or slow down the speed at which plants grow, as these modulations of plant traits may be desirable in certain applications.

[0199] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to confer resistance to nematode stress to the plants.

[0200] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to provide biostimulation (biostimulatory effect) to the plants. In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to provide disease resistance to the plants.

[0201] 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"), defoamers, detergents, sequestering agents, drift reducing agents, neutralizing and buffering agents, corrosion inhibitors, dyes, odorants, spreading agents (also referred to as "spreading agents"), penetration aids (also referred to as "penetrating agents"), adhesion agents (also referred to as "sticking agents" or "binding agents"), dispersants, thickening agents (also referred to as "thickening agents"), stabilizing agents, 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.), as well as other compositions that may be of interest for specific applications.

[0202] In some embodiments, the agricultural composition of the present disclosure is a solid. When a solid composition is used, it may be desirable to include one or more carrier materials with the active isolated microorganism 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, products of plant origin 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.

[0203] growth composition In some embodiments, a composition that promotes growth and development 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 can be added to the growth composition to promote the health of the microorganisms, either alone or in combination with the plant elements, such as, but not limited to, amino acids, vitamins, minerals, carbohydrates, simple sugars, lipids.

[0204] Pharmaceutical Composition One or more compositions may be combined in addition to the microorganism or a composition made from a microorganism for various uses, stability, activity, and / or storage reasons. The additional compositions may be referred to as "formulation components."

[0205] In some embodiments, the agricultural composition of the present disclosure is liquid.Thus, in some embodiments, the present disclosure teaches that the agricultural composition 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 dihydrogen diphosphate, ammonium dihydrogen monophosphate, sodium ammonium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate.

[0206] In some embodiments, the present disclosure teaches that the agricultural composition may include binders such as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, 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.

[0207] In some embodiments, the agricultural composition comprises a surfactant. In some embodiments, the surfactant is added to a liquid agricultural composition. In other embodiments, the surfactant is added to a solid formulation, particularly a solid formulation designed to be diluted with a carrier before application. Thus, in some embodiments, the agricultural composition comprises a surfactant. The surfactant may be used alone to improve the biological performance of the microorganism on the target, or may be used with other additives, for example, mineral or vegetable oils as adjuvants to spray tank mixes. The type of surfactant used for biological enhancement largely depends on the nature and mode of action of the microorganism. The surfactant may be anionic, cationic, or nonionic in nature and may be used as an emulsifier, a wetting agent, a suspending agent, or for other purposes. In some embodiments, the surfactant is a nonionic surfactant, such as alkyl ethoxylates, linear fatty alcohol ethoxylates, and fatty amine ethoxylates. Surfactants conventionally used in the art of formulations 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 an improved method for the preparation of a condensate of sulfonated naphthalene with formaldehyde and its derivatives, a condensate of phenol and formaldehyde with naphthalene or naphthalene sulfonic acid, a condensate of formaldehyde with phenol or phenol ... The use of surfactants including condensates of acids, condensates 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, salts of phosphoric acid-containing triarylphenol ethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin sulfite waste liquor, or methylcellulose, or compositions thereof, is taught.

[0208] In some embodiments, the present disclosure provides salts of alkyl sulfates such as diethanolammonium lauryl sulfate, alkylarylsulfonates such as calcium dodecylbenzenesulfonate, 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-naphthalenesulfonate, dialkyl esters of sulfosuccinates such as sodium di(2-ethylhexyl)sulfosuccinate, and the like. 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, as well as the esters, particularly the methyl esters, of the above vegetable oils.

[0209] In some embodiments, the agricultural composition comprises a wetting agent. A wetting agent is a substance that, when added to a liquid, increases the spreading or penetrating power of the liquid by lowering 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 make concentrates for soluble liquids or suspension concentrates, and to reduce the wetting time of wettable powders and improve the penetration of water into water-dispersible granules during mixing of the product with water in a spray tank or other container. In some embodiments, examples of wetting agents used in the agricultural compositions of the present disclosure, including wettable powders, suspension concentrates, and water-dispersible granules, are sodium lauryl sulfate, dioctyl sodium sulfonate, alkylphenol ethoxylates, and fatty alcohol ethoxylates.

[0210] 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 to protect the dispersion state of the particles and prevents them from re-agglomerating. 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 the spray tank. In some embodiments, dispersants are used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants used as dispersants have the ability to strongly adsorb onto the particle surface and provide an electrostatic or steric barrier against particle re-agglomeration. In some embodiments, the most commonly used surfactants are anionic surfactants, nonionic surfactants, or a mixture of the two.

[0211] 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 very good adsorption and stabilization. In some embodiments, tristyrylphenol ethoxylate phosphate esters are also used. In some embodiments, alkylaryl ethylene oxide condensates and EO-PO block copolymers may be combined with anionic surfactants as dispersants for suspension concentrates.

[0212] 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 many ethylene oxide chains that form the "teeth" of the surfactant "comb". In some embodiments, these high molecular weight polymers can impart very good long-term stability to suspension concentrates because the hydrophobic backbone has many anchoring points on the particle surface. In some embodiments, examples of dispersants used in the agricultural compositions of the present disclosure include sodium lignosulfonate, sodium naphthalene sulfonate formaldehyde condensate, tristyrylphenol ethoxylate phosphate ester, fatty alcohol ethoxylate, alkyl ethoxylate, EO-PO block copolymers, and graft copolymers.

[0213] In some embodiments, the agricultural composition of the present disclosure includes an emulsifier. An emulsifier is a substance that stabilizes the suspension of droplets of one liquid phase in another liquid phase. Without the 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 with 12 or more ethylene oxide units, and an oil-soluble calcium salt of dodecylbenzene sulfonic acid. A hydrophilic lipophilic balance ("HLB") value in the range of 8 to 18 will usually 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.

[0214] In some embodiments, the agricultural composition of the present disclosure comprises a solubilizer. A solubilizer 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 inside the hydrophobic portion of the micelle. The types of surfactants commonly used for solubilization are non-ionic substances: sorbitan monooleate, sorbitan monooleate ethoxylate, and oleic acid methyl ester.

[0215] In some embodiments, the agricultural composition of the present disclosure includes 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 co-solvents to prevent crystallization of the formulation of the anti-pest agent when it is emulsified in water. Alcohols may be used as co-solvents to increase the solvency.

[0216] In some embodiments, the agricultural composition includes a gelling agent. Thickening agents or gelling agents are primarily used in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and prevent separation and settling of dispersed particles or droplets. Thickening agents, gelling agents, and anti-settling agents are broadly classified into two categories: water-insoluble particulates and water-soluble polymers. Clays and silicas can be used to create suspension concentrate formulations. In some embodiments, the agricultural composition includes 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), 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.

[0217] In some embodiments, aqueous formulations may foam due to the presence of surfactants that reduce interfacial tension during mixing operations during production or during spray tank application. Thus, in some embodiments, antifoaming agents are often added either during the production stage or before filling into bottles / spray tanks to reduce foaming tendency. There are generally two types of antifoaming agents: silicone antifoaming agents and non-silicone antifoaming agents. Silicones are usually aqueous emulsions of dimethylpolysiloxanes, while non-silicone antifoaming agents are water-insoluble oils such as octanol and nonanol, or silica. In both cases, the function of the antifoaming agent is to displace surfactants from the air-water interface.

[0218] In some embodiments, the agricultural composition comprises a preservative.

[0219] 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 rotation program with other agricultural products.

[0220] 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 for ground, aerial, and irrigation applications.

[0221] In some embodiments, the agricultural composition may be applied to genetically modified seeds or plants.

[0222] protective composition Furthermore, the individual microorganisms, or 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, acaricides, plant growth regulators, rodenticides, algaecides, biocontrol agents, or beneficial agents. Furthermore, those 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, the 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 also combined with bioactive agents.

[0223] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods may be combined with bio-pesticides that function as herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, acaricides, rodenticides, and / or algaecides. Such bio-pesticides may be, but are not limited to, macro-organisms (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).

[0224] Insecticides and bio-insecticides In some embodiments, the agricultural compositions of the present disclosure include a pesticide for use in combination with the microorganisms taught in the present disclosure. In some embodiments, the agricultural compositions of the present disclosure include a bio-pesticide for use in combination with the microorganisms taught in the present disclosure.

[0225] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods may be combined with known pesticides in the agricultural space, such as herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, miticides, rodenticides, and / or algaecides.

[0226] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods may be combined with known pesticides in the agricultural space, such as bio-pesticides that function as herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, miticides, rodenticides, and / or algaecides.

[0227] For example, in some embodiments, the present disclosure teaches agricultural compositions that include 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).

[0228] 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 acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, mefenacet, metalaclor, metazachlor, napropamide, naproanilide, petoxamide, pretilachlor, propachlor, and thenylchlor, acetamides selected from the group consisting of vilanaphos, glufosinate, and sulfosate, clodinaf, aryloxyphenoxypropionates selected from the group consisting of cycloprop, cyhalofop-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 ( thio)carbamates, cyclohexanediones selected from the group consisting of butroxydim, clethodim, cycloxydim, profoxydim, sethoxydim, tepraloxydim, and tralkoxydim, dinitroanilines selected from the group consisting of benfluralin, ethofluralin, oryzalin, pendimethalin, prodiamine, and trifluralin, diphenyl ethers selected from the group consisting of acifluorfen, aclonifen, bifenox, diclofop, ethoxyfene, fomesafen, lactofen, and oxyfluorfen. hydroxybenzonitriles selected from the group consisting of ter, vomoxinil, dichlobenil, and 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; chloridazon, flufenpyr-ethyl, fluthiacet, norflurazon;and pyrazines selected from the group consisting of pyridate, aminopyralid, clopyralid, diflufenican, dithiopyr, fluridone, fluroxypyr, picloram, picolinafen, and thiazopyr, pyridines selected from the group consisting of azimsulfuron, bensulfuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metul sulfonylureas selected from the group consisting of 14(2-chloro-6-propyl-imidazol[1,2]-pyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea, ametriene, atrazine, cyanazine, dimethysulfuron, sulphonylurea ... triazines selected from the group consisting of tametryn, etiodin, hexazinone, metamitron, metribuzin, prometryn, simazine, terbuthylazine, terbutryn, and triaziflam; urea compounds selected from the group consisting of chlorotoluron, dymuron, diuron, fluometuron, isoprotronturon, linuron, mesabentiazuron, and tebuthiuron; bispyribac-sodium, cloransulam-methyl, diclosulam, florasulam, flucarbazone, flumetuslam, metosulam, ortho-sulfamuron, penoxsulam, promethamine, squalene ... acetolactate synthase inhibitors selected from the group consisting of propylcarbazone, pyribambenz-propyl, pyribenzoxim, pyriftalid, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyroxasulfone, and piroxulam, 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, endothal, ethofumesate, etobenzanide, fentrazamide, fluororac-pentyl, flumioxazin, flupoxam, fluorochloridone, furutamone, indanofan, isoxaben, isoxaflutole, lenacil, propanil, propyzamide, quinclorac, quinmerac, mesotrione, methylarsonic acid, naptalam, oxadiardil, oxadiazon, oxaziclomefone, pentoxazone , pinoxaden, pyraclonil, pyraflufen-ethyl, pyrasulfotole, pyrazoxyfene, pyrazolinate, quinoclamine, saflufenacil, sulcotrione, sulfentrazone, terbacil, tefuryltrione, tembotrione, thiencarbazone, topramezone, 4-hydroxy-3-[2-(2-methoxy-ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-bicyclo[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 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridine-2-carboxylic acid methyl ester.

[0229] 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, parathiol, ... an organo(thio)phosphate selected from the group consisting of phenthoate, phosalone, phosmet, phosphamidon, phorate, phoxim, pirimiphos-methyl, profenofos, prothiofos, sulprofos, tetrachlorvinphos, terbuphos, triazophos, and trichlorfon; alanycarb, aldicarb, bendiocarb, benfuracarb, carbaryl, carbofuran, carbosulfan, fenoxycarb, furathiocarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb, and triaza; carbamates selected from the group consisting of mates, 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, tau-fluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin, pyrethroids selected from the group consisting of profluthrin, and dimefluthrin; a) chitin synthesis inhibitors, which are benzoyl ureas selected from the group consisting of chlorfluazuron, cilamadine, diflubenzuron, flucycloxalone, hexaflumuron, lufenuron, novaluron, teflubenzuron, triflumuron, buprofezin, diofenolan, hexythiazox, etoxazole, and clofentadine; b) halofenozide, methoxyfenozide, tebufenozide,and azadirachtin; c) an insect growth regulator selected from the group consisting of a juvenile hormone mimetic 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; a nicotinic receptor agonist / antagonist compound 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; 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 inhibitors (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.

[0230] In some embodiments, the present invention teaches the synergistic use of the microorganisms, or microbial consortia, of the present disclosure with known pesticides in the agricultural space, such as herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, acaricides, rodenticides, and / or algaecides.

[0231] In some embodiments, the present invention teaches the synergistic use of the microorganisms, or microbial consortia, of the present disclosure with known pesticides in the agricultural space, such as bio-pesticides that function as herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, miticides, rodenticides, and / or algaecides.

[0232] 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.

[0233] 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 plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, a synergistic effect on the plant phenotypic trait of interest is observed.

[0234] 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, RS, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations," 1967 Weeds, vol. 15, pp. 20-22, which is incorporated herein by reference in its entirety. Thus, by "synergistic" we mean an ingredient whose presence increases a desired effect beyond the amount added.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] For example, in some embodiments, the present disclosure provides a method for treating a psoriasis comprising administering to a patient, among other things, the following active ingredients: ancymidol, butralin, alcohol, chloromequat chloride, In one embodiment, the present invention teaches agricultural compositions that include one or more of the following active ingredients: methyl methacrylate (MMA), ...

[0240] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods may be combined with known seed inoculants in the agricultural space, such as QUICKROOTS®, VAULT®, RHIZO-STICK®, NODULATOR®, DORMAL®, 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, is combined with a microorganism or microbial consortium taught herein.

[0241] In some embodiments, the agricultural compositions of the present disclosure include plant growth regulators, which include kinetin, gibberellic acid, and indole butyric acid along with copper, manganese, and zinc.

[0242] In some embodiments, the present disclosure provides a method and system for the treatment of various conditions, 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 Tech®, Goldwing®, Governor®, Groom®, Legacy®, Primeraone®, Primo®, Provair®, Solace®, T-Nex®, T-Pac®, Concise®, and Sumagic®.

[0243] 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 a plant growth stimulant, such as a plant hormone or a chemical that affects the production or interruption of a plant growth regulator.

[0244] 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).

[0245] In some embodiments, the individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods can be combined with biostimulants, which can be, but are not limited to, microorganisms, plant extracts, plant extracts, seaweed, acids, biochar, etc.

[0246] In some embodiments, individual microorganisms, or microbial consortia, or microbial communities developed according to the disclosed methods may be combined with fertilizers, which may be organic (e.g., compost, blood, fish, etc.), nitrogenous (e.g., nitrates, ammonium, urea, etc.), phosphates, and potassium. Such fertilizers may also contain micronutrients, including, but not limited to, sulfur, iron, zinc, etc.

[0247] 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.

[0248] Thus, in some embodiments, the present disclosure provides for applying a microorganism of the present teachings in combination with Ascend® to any crop. Additionally, 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.

[0249] In some embodiments, the present disclosure teaches agricultural compositions comprising biostimulants.

[0250] 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 media.

[0251] The level of microorganisms in the soil or medium directly correlates with the health of the plant. Because microorganisms feed on biodegradable carbon sources, plant health also correlates with the amount of organic matter in the soil. While fertilizers provide nutrients to grow and develop plants, in some embodiments, biostimulants provide biodegradable carbon, e.g., molasses, carbohydrates, e.g., sugars, to grow and develop microorganisms. Unless otherwise expressly specified, biostimulants may include a single component or a combination of several different components capable of promoting microbial activity or plant growth and development, the promotion being due to the effect of one or more of those components acting independently or in combination.

[0252] In some embodiments, biostimulants are compounds that generate non-nutritional plant growth responses. In some embodiments, many 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 modify the hormonal state of a plant and have a significant effect on the growth and health of the plant. 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 defense system of the plant. In some embodiments, vitamin C, vitamin E, and amino acids such as glycine are antioxidants included in the biostimulant.

[0253] In other embodiments, biostimulants may act to stimulate the growth of microorganisms present in soil or other plant media. Previous studies have shown that certain biostimulants, including certain organic seed extracts (e.g., soybean), can stimulate the growth of microorganisms contained in a microbial inoculant when used in combination with the microbial inoculant. Thus, in some embodiments, the present disclosure teaches one or more biostimulants that can enhance the population of both native and inoculant microorganisms when used with a microbial inoculant. For a review of some popular uses of biostimulants, see Calvo et al., 2014, Plant Soil 383:3-41.

[0254] Combination 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, Bacillus thuringiensis strain 39400 or strain 42901, or any one or more microorganisms comprising a polynucleotide sharing at least 95% identity to any one of SEQ ID NOs:1-19, or any combination of the foregoing, may be applied to plant elements, optionally in combination with any agricultural composition, for improvement of the plant phenotype.

[0255] Isolated microorganisms or communities or consortia (generally, and synonymously, "microbes" or "microbes") may be applied to heterologous plant elements to create man-made 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, is applied in an amount different from that found in nature. In some embodiments, a microorganism may be found naturally in one part of a plant but not another, and the introduction of the microorganism to another part of the plant is considered a heterologous association.

[0256] It is contemplated that the microorganisms, either isolated or associated with plants or plant components, may further be associated with one or more agricultural compositions as described above.

[0257] Microorganisms and plant elements, microorganisms and agricultural compositions, and man-made mixtures of microorganisms and plant elements and agricultural compositions are contemplated (broadly, "synthetic compositions," which are compositions that include components not typically found in nature).

[0258] Plant Element Processing In some embodiments, the present disclosure also relates to the discovery that desirable plant traits, such as plant growth, plant health, and / or plant resistance to pests and diseases, can be enhanced by treating plant elements with one or more combinations of the microorganisms or agricultural compositions of the present disclosure prior to sowing or planting the plant elements.

[0259] Thus, in some embodiments, the present disclosure teaches the use of one or more of the microorganisms or microbial consortia as a plant element treatment. The plant element treatment may be a plant element coating that is applied directly to untreated and "bare" plant elements. However, the plant element treatment may also be one or more previous plant element coatings or plant element protective films that are applied to plant elements that have already been coated with a plant element treatment. The previous plant element treatment may include one or more active compounds, chemical or biological, and one or more inactive ingredients.

[0260] 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 in the area where the plant elements are to be planted shortly before the plant elements germinate and emerge.

[0261] 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 a plant, 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.

[0262] Also, in some embodiments, the present disclosure teaches that the microorganisms disclosed herein are important for enhancing early stages in the life cycle of a plant (e.g., the first 30 days after the emergence of plant elements). Thus, in some embodiments, delivery of the microorganisms and / or compositions of the present disclosure as plant element treatments places the microorganisms at the site of action at a time critical for their activity.

[0263] In some embodiments, the microbial compositions of the present disclosure are formulated as plant element treatments. In some embodiments, it is believed that the 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 combinations of these methods through the use of treatment application equipment that is specifically designed and manufactured to coat the plant elements with the plant element treatment product accurately, safely, and efficiently. Such equipment uses various types of coating techniques, such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mist, or combinations thereof. Liquid plant element treatments, such as the plant element treatments of the present disclosure, can be applied through either a spinning "atomizer" disk or a spray nozzle, which distributes the plant element treatment evenly to the plant elements as it moves through the spray pattern. In embodiments, the plant elements are then mixed or rolled for an additional period of time to further distribute the treatment and dry.

[0264] The plant elements may or may not be germinated prior to coating 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.

[0265] In some embodiments, the 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 coating comprising a microbial composition is applied to a plant element that already has a plant element coating applied thereon. In some aspects, the plant element may have a plant element coating comprising, for example, clothianidin and / or Bacillus firmus I-1582, onto which the composition will be applied as a plant element protective coating. In some aspects, a microbial composition of the present teachings is applied as a plant element protective coating to a plant element that has already been treated with PONCHO™ VOTiVO™. In some aspects, the plant element may have a plant element coating comprising, for example, metalaxyl, and / or clothianidin, and / or Bacillus firmus I-1582, onto which the composition will be applied as a plant element protective coating. In some embodiments, the microbial compositions of the present teachings are applied as a protective coating to plant elements that have already been treated with ACCELERON™.

[0266] In some embodiments, plant elements treated with microorganisms have a microbial spore 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] In some embodiments, the microbially treated plant components have a microbial spore concentration, or microbial cell concentration, of about 10^5 to 10^9 per plant component.

[0271] 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.

[0272] 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, as well as the size or type of the plant or plant elements being utilized. In some embodiments, the one or more of the microorganisms are present in a proportion of about 2% w / w to about 80% w / w of the total formulation. In some embodiments, the one or more of the microorganisms employed in the composition are 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.

[0273] 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.

[0274] 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, 480μm 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 may be 2970 μm, 2980 μm, 2990 μm, or 3000 μm.

[0275] In some embodiments, the plant element coating of the present disclosure may 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.

[0276] 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%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 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 .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%.

[0277] In some embodiments, the microbial spores and / or cells can be coated freely on the plant elements, or can be formulated in a liquid or solid composition before being coated on the plant elements. For example, a solid composition containing the microorganism can be prepared by mixing a solid carrier with a suspension of spores until the solid carrier is impregnated with the spore or cell suspension. The mixture can then be dried to obtain the desired particles.

[0278] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents, such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the produce, or combinations thereof.

[0279] Plant element coating methods and compositions known in the art may 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.

[0280] 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.

[0281] In some embodiments, various additives can be added to the plant element treatment formulations containing the compositions of the present invention. Binders can be added, including those comprised of adhesive polymers that can be natural or synthetic and have no phytotoxic effects on the plant elements being coated. Binders can be selected from cellulose, including polyvinyl acetate, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose, polysaccharides, including polyvinylpyrrolidone, starch, modified starch, dextrin, maltodextrin, alginates, and chitosan, fats, oils, proteins, including gelatin and zein, gum arabic, shellac, vinylidene chloride and vinylidene chloride copolymers, calcium lignosulfonate, acrylic copolymers, polyvinyl acrylate, polyethylene oxide, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomer, and polychloroprene.

[0282] 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, bisazos, and polyazos. Other additives that may be added include micronutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc.

[0283] A polymer or other dedusting agent may be applied to retain the treatment on the surface of the plant elements.

[0284] In some particular 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, cellulose such as polyvinyl acetate, polyvinyl acetate copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, 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.

[0285] 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, solids, or dry powders. Dry powders can be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon, and various phosphorus compounds.

[0286] In some embodiments, the plant element coating composition can include at least one filler, which is an organic or inorganic, natural or synthetic component, with which the active ingredient is combined to facilitate its application to the plant element. In an aspect, the filler is an inert solid such as clay, natural or synthetic silicate, silica, resin, wax, solid fertilizer (e.g., ammonium salts); natural earth minerals such as kaolin, clay, talc, lime, quartz, attapulgite, montmorillonite, bentonite, or diatomaceous earth, or synthetic minerals such as silica, alumina, or silicates, especially aluminum silicate or magnesium silicate.

[0287] 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-diamines, benzoates, benzoyl esters, benzoic acids ... The plant element coating composition may further include one or more of herbicide safeners such as allyl dichloroacetamide, 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 components may be added as a separate layer on the plant element or, alternatively, may be added as part of the plant element coating composition of the present disclosure.

[0288] In some embodiments, the formulations used to treat plant elements of the present disclosure may 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 active ingredient in the formulation may 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.

[0289] As mentioned 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, such as dispersants such as methylcellulose, 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. Other 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, which is incorporated herein by reference.

[0290] The plant element coating formulations of the present disclosure may 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 immersion. A variety of active or inactive materials can be used to contact the plant elements with the microbial composition according to the present disclosure.

[0291] 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.

[0292] As discussed above, an effective amount means 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.

[0293] 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 safeners, fertilizers, and / or biocontrol agents. These ingredients may be added as a separate layer or may be added to the coating layer.

[0294] In some embodiments, the plant element coating agent of the present disclosure can be applied to plant elements using various techniques and machines, such as fluidized bed technology, roller mill method, rotary electrostatic plant element treatment machine, and drum coater. Other methods, such as spouted bed, 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.

[0295] In some embodiments, the microbially treated plant elements may be wrapped 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.

[0296] In another embodiment of the present disclosure, the composition according to the present disclosure can be introduced into the plant elements by using solid matrix priming. For example, a quantity of the composition of the present invention 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 the 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 invention and the solid matrix material are compatible with each other. For example, the solid matrix material should be selected so that the solid matrix material can release the composition at a reasonable rate, for example over minutes, hours, or days.

[0297] 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.

[0298] 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.

[0299] 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 an active chemical agent, while in other embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure exhibits a synergistic effect on a phenotypic trait of interest in a plant when combined with an active chemical agent.

[0300] In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure has an additive effect on a phenotypic trait of interest in a plant when combined with a fertilizer, while in other embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure has a synergistic effect on a phenotypic trait of interest in a plant when combined with a fertilizer.

[0301] In some embodiments, an additive effect on a phenotypic trait of interest in a plant is observed when a microorganism or microbial consortium identified according to the methods of the present disclosure is combined with a plant growth regulator. In some embodiments, a synergistic effect is observed when a microorganism or microbial consortium identified according to the methods of the present disclosure is combined with a plant growth regulator. 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.

[0302] In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure exhibits additive effects on the desired phenotypic traits of the plant when combined with a biostimulant. In some embodiments, a microorganism or microbial consortium identified according to the methods of the present disclosure exhibits synergistic effects when combined with a biostimulant.

[0303] 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, RS, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations, 1967 Weeds, vol. 15, pp. 20-22, which is incorporated herein by reference in its entirety. Thus, by "synergistic" we mean an ingredient whose presence increases a desired effect beyond the amount added.

[0304] 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.

[0305] In other embodiments, synergistic effects are observed when the microorganisms or microbial consortia identified according to the methods of the present disclosure are combined with fertilizer.

[0306] Moreover, in certain embodiments, the present disclosure utilizes synergistic interactions to define microbial consortia, i.e., in certain aspects, the present disclosure mixes together certain isolated microbial species that act synergistically to confer beneficial traits to plants or into consortia that correlate with increased beneficial plant traits.

[0307] In order to improve the activity of known active agricultural compounds, the 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, thus keeping costs as low as possible and complying with any official regulations. It may also be possible to broaden the scope of action of the active compound, since in individual cases, when treatment with a specific active ingredient without the addition of a certain adjuvants is not successful, the addition of the certain adjuvants together with the microbial isolates and consortia of the present disclosure can actually succeed in treating the plant. Also, the performance of the active compound can be enhanced by appropriate formulations when environmental conditions are not favorable in individual cases.

[0308] Such auxiliary agents that may be used in agricultural compositions may be adjuvants. Adjuvants often take the form of surface-active or salt-like compounds. Adjuvants may be broadly classified as modifiers, activators, fertilizers, pH buffers, etc., depending on their mechanism of action. Modifiers affect the wetting, adhesion, and spreading properties of the formulation. Activators break down the waxy cuticle of the plant and improve the penetration of the active ingredient into the cuticle in both the short term (over minutes) and long term (over hours). Fertilizers such as ammonium sulfate, ammonium nitrate, or urea improve the absorption and solubility of the active ingredient and may reduce the antagonistic behavior of the active ingredient. pH buffers are conventionally used to bring the formulation to an optimal pH.

[0309] For further embodiments of the agricultural compositions of the present disclosure, see "Chemistry and Technology of Agrochemical Formulations," edited by DA Knowles, 1998, published by Kluwer Academic Publishers, Inc., which is incorporated herein by reference.

[0310] 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 above. Any number of a wide variety of plants, including mosses and lichens and algae, may be used in the methods of the present disclosure. In some 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.

[0311] In other embodiments, the plants may be economically, socially, or environmentally undesirable plants, such as weeds. The following is a non-limiting list of examples of plant types to which the methods of the present disclosure may be applied:

[0312] edible crops Cereals, such as maize, rice, wheat, barley, sorghum, millet, oats, rye, triticale, and buckwheat;

[0313] Leafy vegetables, e.g. cruciferous plants such as cabbage, broccoli, bok choy, and arugula; salad vegetables such as spinach, watercress, and lettuce;

[0314] Fruiting and flowering vegetables, e.g. avocado, sweet corn, artichoke, cucumbers, e.g. pumpkin, cucumber, melon, zucchini, pumpkin, nightshade vegetables / fruits, e.g. tomato, eggplant, and pepper;

[0315] Legumes, such as peanuts, groundnuts, peas, soybeans, beans, lentils, chickpeas, okra,

[0316] Bulbous and stem vegetables, such as asparagus, celery, Allium crops, such as garlic, onions, and leeks;

[0317] Root and tuber vegetables, such as carrots, beets, bamboo shoots, cassava, yams, ginger, Jerusalem artichoke, parsnips, radishes, potatoes, sweet potatoes, taro, turnips, and wasabi;

[0318] Sugar crops, including sugar beet (Beta vulgaris) and sugar cane (Saccharum officinarum);

[0319] Crops grown for the production of non-alcoholic beverages and stimulants, such as coffee, black, herbal, and green tea, cocoa, marijuana, and tobacco;

[0320] Fruit vegetables such as berries (e.g. kiwifruit, grapes, currants, gooseberries, guavas, feijoas, pomegranates), citrus fruits (e.g. oranges, lemons, limes, grapefruit), pseudofruits (e.g. bananas, cranberries, blueberries), aggregate fruits (blackberries, raspberries, boysenberries), compound fruits (e.g. pineapple, figs), stone fruits (e.g. apricots, peaches, cherries, plums), pome fruits (e.g. apples, pears) and other fruit vegetables such as strawberries, sunflower seeds,

[0321] Culinary and medicinal herbs, such as rosemary, basil, laurel, coriander, mint, dill, hypericum, foxglove, aloe vera, rose hips, and hemp;

[0322] Spices, such as black pepper, cumin, cinnamon, nutmeg, ginger, cloves, saffron, cardamom, mace, paprika, masala, and star anise-producing crops;

[0323] Nuts, e.g. almonds and walnuts, Brazil nuts, cashews, coconuts, chestnuts, macadamia nuts, pistachios; crops grown for the production of peanuts, pecan nuts;

[0324] Crops grown for the production of beer, wine and other alcoholic beverages, such as grapes and hops,

[0325] Oil crops, such as soybean, peanut, cotton, olive, sunflower, sesame, lupin species and cruciferous crops (e.g. canola / rapeseed), and edible fungi, such as mushrooms, shiitake mushrooms and oyster mushrooms;

[0326] Plants used in pastoral agriculture Clover spp., Medicago spp., and Lotus spp.; white clover (T. repens), red clover (T. pratense), Caucasian clover (T. ambidum), subterranean clover (T. subterraneum), alfalfa / lucerne (Medicago sativum), annual alfalfa, Medicago tallowii, alfalfa, sainfoin (Onobrychis viciifolia), Lotus corniculatus, Lotus pedunculatus,

[0327] Seed legumes / beans, including peas (Pisum sativum), beans (Phaseolus vulgaris), broad beans (Vicia faba), mung beans (Vigna radiata), cowpeas (Vigna unguiculata), chickpeas (Cicer arietum), lupins (Lupinus spp.), cereals, including maize / cane (Zea mays), sorghum (Sorghum spp.), millet (Panicum miliaceum, P. sumatrense), rice (Oryza sativa indica, Oryza sativa japonica), wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale), triticale (Triticum x Secale), oats (Avena sativa),

[0328] Forage and amenity grasses: cool season grasses such as Lolium spp., Festuca spp., Agrostis spp., Lolium perenne, Lolium hybridum, Lolium multiflorum, Festuca arundinacea, Festuca pratensis, Festuca rubra, Festuca ovina, Lolium x Festuca crosses, Dactylis glomerata, Kentucky bluegrass (Poa pratensis, Poa palustris, Poa nemoralis, Poa trivialis, Poa compresa, Bromus spp., Phleum spp., Arrhenatherum elatius, Agropyron spp., Avena strigosa, Setaria italic,

[0329] Tropical grasses such as Phalaris spp., Brachiaria spp., Eragrostis spp., Panicum spp., Paspalum notatum, Brachypodium spp., and grasses used in biofuel production such as switchgrass (Panicum virgatum) and Miscanthus spp.;

[0330] Fiber crops processed wood fibre products such as cotton, hemp, jute, coconut, sisal, flax (Linum species), New Zealand flax (Phormium species), plantation and natural forest species harvested for paper, and softwood and hardwood forest species;

[0331] Trees and shrubs used for forestation and biofuel crops Pines (Pinus spp.), firs (Pseudotsuga spp.), spruces (Picea spp.), cypresses (Cupressus spp.), acacias (Acacia spp.), alder (Alnus spp.), oaks (Quercus spp.), redwoods (Sequoiadendron spp.), willows (Salix spp.), birch (Betula spp.), cedars (Cedurus spp.), ash (Fraxinus spp.), larch (Larix spp.), Eucalyptus spp., bamboos (Bambuseae spp.) and poplars (Populus spp.).

[0332] Plants grown for conversion to energy, biofuels, or industrial products by extractive, biological, physical, or biochemical processes: For the production of biofuels, i.e. chemical, physical (e.g. thermal or catalytic), or biochemical (e.g. enzymatic pretreatment during the production of biofuels, industrial solvents or chemical products, e.g. ethanol or butanol, propanediol, or other fuels or industrial materials, including oil-producing plants such as oil palm, jatropha, corn, soybean, cotton, linseed, latex-producing plants such as Hevea brasiliensis, Panama Laver tree, Castilla elastica, sugar crops (e.g. beet, sugarcane), starch-producing crops (e.g. C3 and C4 cereal and tuber crops), forest trees (e.g. pine, eucalyptus), and cellulose crops such as grasses (Graminaceous and Poaceous) plants such as bamboo, switchgrass, miscanthus, etc. or plants used as direct or indirect feedstocks after biological (e.g. microbial fermentation) transformation, with or without the production of biochar, crops used for energy, biofuel, or industrial chemical production via gasification and / or microbial or catalytic conversion of gases to biofuels or other industrial feedstocks such as solvents or plastics (e.g. conifers, eucalyptus, tropical or broadleaf forest trees, graminaceous and poaceous crops such as bamboo, switchgrass, miscanthus, sugarcane, or hemp, or conifers such as poplar, willow, and biomass crops used for the production of biochar,

[0333] Crops that produce natural products useful for the pharmaceutical, agro-food, and functional cosmetics industries Crops producing pharmaceutical precursors or pharmaceutical compounds or compounds and materials for functional foods and functional cosmetics, e.g. star anise (shikimic acid), Japanese knotweed (resveratrol), kiwi fruit (soluble fiber, proteolytic enzymes),

[0334] Horticultural, ornamental and institutional plants grown for their aesthetic or environmental qualities; Flowers such as roses, tulips, and chrysanthemums,

[0335] Ornamental shrubs such as boxwood, hebe, rose, rhododendron, and ivy;

[0336] Amenity plants such as Platanus, Choisia, Escallonia, Euphorbia, and Carex;

[0337] Mosses such as Sphagnum,

[0338] Plants cultivated for bioremediation, In certain embodiments, the microorganisms of the present disclosure are sprayed onto hybrid plants to increase the beneficial traits of the hybrid plants. In other embodiments, the microorganisms of the present disclosure are applied onto genetically modified plants to increase the beneficial traits of the GM plants. The microorganisms taught herein can be sprayed onto hybrid and GM plants to maximize the elite genetics and trait technology of these plants.

[0339] It should be understood that the plant may be provided in the form of a seed, seedling, cutting, vegetative propagule, or any other plant material or tissue capable of growing. In one embodiment, the seed may be surface sterilized with a substance such as sodium hypochlorite or mercuric chloride to remove surface contaminating microbial contaminants. In one embodiment, the vegetative propagule is propagated in axenic culture, for example as axenic plantlets in tissue culture, before being placed in a plant growth medium.

[0340] How to apply The microorganisms may be applied to plants, seedlings, cuttings, vegetative propagules, etc., and / or to the growth medium containing the plants, using any suitable technique known in the art.

[0341] However, by way of example, isolated microorganisms, consortia, or compositions comprising and / or produced therefrom may be applied to plants, seedlings, cuttings, propagules, and the like by spraying, coating, dusting, or any other method known in the art.

[0342] In another embodiment, the isolated microorganism, a consortium comprising the same or a composition may be applied directly to the plant seeds before sowing.

[0343] In another embodiment, the isolated microorganism, a consortium comprising the same or a composition may be applied directly to the plant seed as a seed coating.

[0344] 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 drench that are applied to the plant medium.

[0345] In other embodiments, the isolated microorganism, consortia comprising the same or compositions are provided in the form of a foliar application, 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 a medium, such as soil.

[0346] 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 may be applied alone or in a rotational spray program.

[0347] 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 for ground, aerial, and irrigation applications.

[0348] In another embodiment, the isolated microorganism, consortia or compositions containing the same may be formulated as granules and applied together during planting of the seeds. Alternatively, the granules may be applied after planting. Alternatively, the granules may be applied before planting.

[0349] In some embodiments, the isolated microorganisms, consortia comprising the same or compositions are applied to plants or media as topical and / or irrigation applications to improve crop growth, yield, and quality, which topical application may be via the use of a dry mix or dry powder or dusting composition, or may be in a liquid-based formulation.

[0350] In embodiments, the isolated microorganisms, consortia, or compositions comprising them may be formulated as, among others, (1) solutions, (2) wettable powders, (3) dusts, (4) soluble powders, (5) concentrated 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 may be diluted in aqueous media prior to conventional spray application. The compositions of the present disclosure may be applied to soil, plants, seeds, rhizosphere, rhizosheath, or other areas where it is beneficial to apply a microbial composition. Furthermore, ballistic methods may be utilized as a means to introduce endophytic microorganisms.

[0351] In an embodiment, the composition is sprayed on the leaves of plants.The composition can be sprayed on the leaves of plants in the form of a concentrated emulsion or suspension, a solution, or a foliar spray.The application of the composition can be carried out in a laboratory, a growth chamber, a greenhouse, or outdoors.

[0352] 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.

[0353] 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 roots or coleoptiles. These inoculated plants may then be exposed to a growth medium containing additional microorganisms, however, this is not necessary.

[0354] 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 numerous alternative techniques that may be used.

[0355] In one embodiment, the microorganism penetrates parts of the plant, such as the roots, stems, leaves and / or reproductive parts of the plant (becoming endophytic) and / or grows on the surface of the roots, stems, leaves and / or reproductive parts of the plant (becoming epiphytic) and / or grows within the plant rhizosphere. In one embodiment, the microorganism forms a symbiotic relationship with the plant.

[0356] Although the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details may be made thereto without departing from the spirit and scope of the present invention. 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. Thus, it will be understood that the scope of the present invention is encompassed by the embodiments listed herein, and not only by the specific examples exemplified below.

[0357] The present disclosure enables a person skilled in the art to make and use the invention provided herein according to multiple various embodiments. Various modifications, alterations, substitutions, and improvements of the present disclosure, including certain modifications, alterations, substitutions, and improvements, that are easily conceived by a person skilled in the art, are also part of the present disclosure. Thus, the foregoing description illustrates the findings provided herein by way of example. Furthermore, the foregoing description and examples are illustrative of the present invention, and not limiting thereof. Thus, the scope of the present invention is set forth in the appended claims.

[0358] 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. EXAMPLES

[0359] The methods and compositions presented herein improve one or more characteristics of a plant, e.g., a plant such as an agricultural crop, based on utilizing the disclosed isolated microorganisms, communities, consortia, and / or compositions comprising and / or produced by the microorganisms or consortia or communities.

[0360] Example 1: Microbial isolation and identification Bacillus thuringiensis strain 39400, described herein, was isolated from soil around clover (Trifolium repens) roots collected in Napier, NZ in 2006. As detailed herein, strain 39400 is a spore-forming, highly effective root-colonizing microorganism that has been demonstrated to positively impact yield performance in a variety of crops and to have a positive impact on nematode control.

[0361] In strain 39400, multiple parasitic forms were identified with different crystal shapes, including cuboids and dipyramids.

[0362] The isolation procedure was as follows: the dried soil fractions were soaked in 50% EtOH overnight in sterile Eppendorf tubes, diluted up to four times, and spread on TSA plates to isolate spore-forming bacteria.

[0363] Bacillus thuringiensis strain 42901 described herein was isolated as an endophyte from rye. Rye seeds were treated with multiple isolated bacterial and fungal strains, including Bacillus thuringiensis strain 39400, and grown under a variety of conditions. Strain 42901 was isolated from adult rye plants.

[0364] Isolation of microorganisms can be carried out according to any method known in the art. One illustrative, non-limiting example is given below.

[0365] Approximately 4 cm sections are prepared from various plant element segments and placed in zip-lock plastic bags. For example, if corn is the plant and the root is the desired tissue, sections are taken from (1) the primary root, (2) the seminal root, (3) the crown root, and (4) the prop root to ensure various tissues for isolation.

[0366] The tissue is then sterilized as follows: Ensure that the tissue is free of soil in a 50 mL Falcon tube with 25 mL of sterile reverse osmosis (RO) water by washing and vortexing. This may require multiple washes to remove the soil from the roots and may involve the use of tweezers. Using tweezers, immerse the root tissue in 70% ethanol in a 50 mL Falcon tube for 10 seconds. Immediately remove the root system from the ethanol bath, shake off excess liquid, and immerse it in 1.5% NaOCl in a 50 mL Falcon tube for 3 minutes. Transfer the root system to a sterile 50 ml Falcon tube and wash six times with sterile RO water by immersing the plant material in clean, sterile water six times.

[0367] Extraction of plant tissue associated microorganisms may be performed by culturing, partial culturing, and selection of the supernatant liquid from the previous step. Alternatively, transfer the sterilized and washed tissue to a petri dish and cut strips using sterile scissors. Transfer approximately 500 μL of this tissue to a sterile 2 ml tube and add 100 μL of medium to soften by immersion using a sterile instrument such as tweezers or the back end of an L-shaped spreader. These pieces of root tissue may be added to freshly poured (warm but not hot) medium for embedding in agar. Look for colonies growing from the "cut" ends of the plant material. Add 1 ml of 10 mM sterile potassium phosphate buffer at pH 7 per 500 μL of tissue. Mix the solution well. Use 50 ul of this solution in 450 μL of phosphate buffer to create 10x, 100x, and 1000x dilutions by serial dilution. Using a sterile L-shaped spreader, spread 100-150 µL of this solution onto an appropriate isolation plate and incubate.

[0368] Isolation of spore-forming bacteria may be performed as follows: Take an aliquot of the undiluted solution from above and pipette into a sterile Eppendorf tube. Seal the tube and heat in a 60°C water bath for 20 minutes to enrich for spore-forming bacteria. Create 10x, 100x, and 1000x dilutions by serial dilution using 50ul of this solution in 450µL of phosphate buffer. Spread 100-150µL of this solution on a suitable isolation plate using a sterile L-shaped spreader and incubate.

[0369] Sequencing preparation for microbial identification and long-term storage can be performed by the following methods.

[0370] Day 1: Using a 10 µL 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.

[0371] 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 is used for phenotypic analysis and the 96-well plate containing 15 μL of each sample is used for PCR analysis. The 27F / 1492R primers are used for 16S PCR analysis as they generally give better results than PB36 / 38. Appropriate negative controls should be included in the plate and analyzed by PCR. The plate is analyzed by PCR using an Eppendorf thermocycler. Once PCR is complete, the gel is run using standard gel electrophoresis techniques. This is important as most isolates are grown well where they should ideally be stored long term on day 3. PCR and gel electrophoresis analysis are used to confirm that the isolates contain bacteria and not other microorganisms. For isolates that do not pass PCR or have a clear broth, vortex the tube and streak onto a Petri dish using a loop. After a few days, check to see if anything has grown or if the tube has been contaminated. For isolates that pass PCR, aliquot 600ul of 50% glycerol into a 2ml screw-cap tube and add 1200µL of bacterial culture so that the broth is preserved in 20% glycerol. Store the glycerol stock at -80°C and record an image of the gel of the PCR sample.

[0372] Day 4: Check the Petri dish for streaky isolates that failed the PCR for growth. (During this time, the 2ml broth tube will remain on the shaker.) Once there is growth on the dish and colonies appear to have been successfully isolated, dispense 600ul of the broth-glycerol mixture into a small tube and place both tubes into their respective -80 boxes. Isolates may fail the PCR check for any of the following reasons: 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 is slow growing. Over the next few days, continue to check the dish 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.

[0373] Bioinformatics analysis Illumina reads were trimmed with Trimmomatic to a quality score of Q20. Pacbio raw reads and trimmed Illumina reads were co-assembled using Unicycler with default parameters to balance the requirements of chromosome assembly and short circular plasmid assembly. To check the quality of the assembly, trimmed Illumina reads were mapped to the assembly and manually inspected for breaks / misassemblies and SNPs. Genomes were annotated with Prokka and genes of interest were identified by annotation. 16S was confirmed by BLAST against the NCBI database. Cry protein identity was confirmed by BtToxin_Digger as there are no Cry genes in NCBI.

[0374] Strains 42901 and 39400 share identical 16S sequences (14 in total in each genome), as well as identical sequences for five different Cry proteins, as shown in the sequences and in Table 1A, however, there are some differences, as shown in Table 1B below. [Table 2]

[0375] Example 2: Microbial formulation The 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 wash, seedling dipping, foliar application, soil inoculation, in-furrow application, lateral fertilization application, soil pre-treatment, wound inoculation, drip tape irrigation, vector-mediated via pollinators, injection, osmotic priming, hydroponics, aquaponics, aeroponics, etc. Formulations containing the microorganisms are prepared for agricultural use as liquid, solid, or gas formulations. Application to plants is 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 droplets on the soil or roots, or as a coating on plant elements prior to planting. Such examples are intended to be illustrative and not limiting to the scope of the present invention.

[0376] The media ingredients for an exemplary microbial preparation are shown in Table 2 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, bring the solution to the final desired volume using sterile RO water. Field test preparations are typically performed using quadruplicate formulations. [Table 3]

[0377] The procedure for mixing the TIX formulation is as follows: Weigh all dry ingredients into a 50mL tube. Vortex ingredients well to ensure that the xanthan gum is "separated" through 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 in the mixing process by using the centrifuge on "high speed spin" for 5-10 seconds. Remember to counter balance the formulation (TIX) tube. Allow the formulation to cool to room temperature. 1. Mix in microbial consortium. Vortex to ensure homogeneity. Ideally, microorganisms at a concentration of 10^9 CFU / ml are added to the formulation.

[0378] For testing in field trials, the formulation is applied to plants or plant elements.

[0379] Example 3: Application of microorganisms to plant elements and their cultivation Microbial compositions (comprising one or more isolated microorganisms, whether a single strain, a consortium, a community, a combination, or any combination of the foregoing) are prepared according to the previous examples. Microbial compositions include one or more microorganisms, optionally in combination with one or more additional microorganisms disclosed herein.

[0380] Microbial compositions for application In some methods, the microbial composition is dried and applied directly to the plant elements.

[0381] In some methods, the microbial composition is suspended in a liquid formulation for application to the plant elements.

[0382] In some methods, the microbial composition is combined with another composition, such as, but not limited to, a carrier, a wetting agent, a stabilizer, a salt, etc. In some methods, the other composition comprises a molecule that introduces additional agriculturally beneficial results to the plant to which the microbial composition is applied. The other composition includes, but is not limited to, for example, a herbicide, a fungicide, a bactericide, a pesticide, an insecticide, a nematicide, a biostimulant.

[0383] Applicable Type The microbial compositions are applied to the plant elements at the time during development appropriate for the desired result, for example, in a pre-plant soil drench / in-furrow formulation, as a seed or other propagation element treatment, as a post-plant propagation element application, as a post-plant in-furrow, drop, or drench application, as a direct application to the plant elements (e.g., roots, leaves, stems), as an application to harvested plant elements (e.g., fruit or grains), etc. Combinations of application types are also tested.

[0384] How to apply The microbial composition is applied (inoculated) to the plant or plant element or plant product (pre-planting, post-planting, pre-harvest, or post-harvest). This can be accomplished, for example, by applying the agricultural composition to a hopper or spreader or tank that contains the microbial composition and is configured to spread it widely.

[0385] A seed coating of the microbial composition is applied to one or more seeds of a crop plant. Once the isolated microorganism has been applied as a seed coating, the seeds are planted and grown according to established practices for that crop.

[0386] Alternatively, the microbial composition is applied to soil for the benefit of plants present in the soil. Methods of soil application include in-furrow treatments, flooding, and drop application.

[0387] Alternatively, the microbial composition is applied to the surface of the plant or plant part after emergence.

[0388] Alternatively, the microbial composition is applied to material obtained from the plant after harvest.

[0389] Control plots of plants to which the isolated microorganisms were not applied are also planted. Plants associated with the microbial composition show improvement in the desired trait.

[0390] The application method may be carried out according to any protocol known in the art.

[0391] 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.

[0392] 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 in size and overall appearance as possible (the purpose of thinning is to continue a homogenous plant population). If there are not enough plants per row, transplant. See step 3 for transplanting guidelines. 2. Begin thinning the pots down to one plant per pot. Remove any plants that are smaller, unhealthy, or 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 first planting or from pots where the seeds did not germinate. 3. To transplant: If some pots did not sprout, they can be filled with plants from another container. To do this, simply scoop out the extra 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. With slight finger pressure, firm the soil around the plants. 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 a few pot rotations to ensure some treatments are free of all the large plants and other treatments have all the large plants. 5. Change gloves if necessary. Label each pot with the pre-prepared Avery labels. Treatments should be labeled in columns of 6 replicates i.e. 1-1, 1-2, 1-3 to 1-6 etc. This will make it easier to find all the replicates for each treatment. 6. Two weeks after planting (approximately 4 days after thinning and labeling), obtain treatments from the microbiology team. Arrange the trays of prepared plants 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 shake. Set the Combitip to dispense 2ml. Collect the treatment fluid into the Combitip and dispense the first step back into the tube. Ensure that the treatment you have corresponds to the row of plants being treated. Once confirmed, gently dispense 2ml 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. Once all treatments have been applied, the plants are placed back into the growth chamber for (optional inoculation), growth and evaluation.

[0393] 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 a variety of plant tissues.

[0394] Example 4: Improved crop yield and resistance to nematode, lepidopteran, and coleopteran pests Increased plant / crop yield is achieved, in some embodiments, by association of the plant with one or more microorganisms disclosed herein, without the need for added chemical fertilizers. In some embodiments, the microorganisms act as "biostimulants," i.e., agents that promote plant health, growth, vigor, and / or production ("yield").

[0395] In some embodiments, "yield" can be determined by, for example, but not limited to, biomass, seed size, seed weight, leaf composition, fiber production of one or more crop products.

[0396] In some embodiments, the increase in yield is statistically significant. In other embodiments, the increase in biomass is not statistically significant, but is still quantifiable and relevant to crop improvement. Other parameters related to yield, such as but not limited to plant vigor, NDVI score, photosynthetic capacity, nutrient utilization, and stress tolerance, may be improved by the addition of the microorganisms disclosed herein. In some cases, the microorganisms may stimulate plant health and further improve crop yield. Comparable yields under microbial and control conditions are one desired outcome when other parameters are improved.

[0397] The improvement in plant health or yield may be under relatively stress-free conditions. In other cases, the improvement may be in plants grown under any number of abiotic or biotic stresses, including drought stress, salt stress, heat stress, cold stress, low nutrient stress, nematode stress, insect herbivory stress, fungal pathogen stress, multiple pathogen stress, or viral pathogen stress.

[0398] It is expected that plants grown from seeds treated with the agricultural composition will exhibit quantitatively higher yields and / or improved health and / or improved stress tolerance than control plants.

[0399] The yield from the treated plants is about 1-10% greater, 10-20% greater, 20-30% greater, 30-40% greater, 40-50% greater, 50-60% greater, 60-70% greater, 70-80% greater, 80-90% greater, or more. The biomass from the treated plants is equal to about 1 bushel per acre increase, or about 2 bushels per acre increase, or about 3 bushels per acre increase, or about 4 bushels per acre increase, or about 5 bushels per acre increase, or more, over the control. In some cases, the yield improves under normal or typical conditions in the absence of biotic and / or abiotic stress factors. In some cases, the yield is normal under conditions of biotic and / or abiotic stress. In some cases, the yield is increased under conditions of biotic and / or abiotic stress.

[0400] The microorganisms described herein improve the health and / or yield of various crop plants, including those described below. The yield and / or plant health of a particular harvested material is improved under normal conditions, as well as under conditions of abiotic stress (e.g., drought, application of herbicides, application of insecticides, reduction or elimination of applied nutrients such as nitrogen, phosphorus, potassium, etc.) or biotic stress (e.g., presence of insects, larvae, nematodes, fungal diseases, bacterial diseases, viral diseases).

[0401] Bacillus thuringiensis (Bt) strain 39400 Bt strain 39400, when associated with crop plants, can confer nematode, coleoptera, and lepidoptera resistance to the plants.

[0402] C. elegans MOA assay To investigate the mechanism of action of the nematicidal microbes developed by the applicants, the model organism, the soil-dwelling free-living nematode C. elegans, was used in several different assays. The life cycle of the nematode is shown in Figure 2A. First, developmental assays were performed to determine activity. Worm larvae were fed the microbes. During this time, the development and fecundity of the worms were tracked. How quickly the nematodes reached reproductive age and the resulting population of offspring determined how these microbes affected the nematodes.

[0403] The mortality of the adult worms was then assessed: the adults were exposed to the microorganisms and the mortality was followed, which indicated the toxicity of the microorganisms to the nematodes.

[0404] Nematodes were also tested in chemotaxis assays. Two different stimuli (e.g., microbial, chemical, metabolic, etc.) were introduced to the worms. Based on the movement of the nematodes in response to these stimuli, a chemotaxis index, repulsion or attraction, was calculated.

[0405] The results of these assays determined the mechanism of action of Bt strain 39400. As shown in Figure 2B, strain 39400 reduced the total nematode population by more than 99% in in vitro assays.

[0406] Root-knot nematode (RKN) assay Seeds of susceptible crops were planted in pots containing a 1:1 mixture of soil and sand two weeks prior to treatment application. Excess seeds were planted per pot and thinned to one seedling per pot.

[0407] Two weeks after planting, microbial treatments were sprayed into the soil at the base of the plants at 1x10^7 CFU / mL per plant, with the amount being sufficient to cover the root zone in the pot.

[0408] The plants were not watered either immediately before or after treatment (no large amounts of water were given on those days either before or after treatment). Normal watering was resumed the following day.

[0409] Nematodes were applied at the desired concentration 48 hours after treatment application, eg, in some cases, J2 stage was used instead of eggs to reduce variability.

[0410] On the morning of nematode application, plants were sprayed with water prior to nematode application. Four weeks after nematode application, shoots were harvested and their dry weights were measured, and roots were washed and assessed for nematodes.

[0411] Roots were stained for egg masses and egg masses, galls, eggs, etc. were counted (galls, eggs, egg masses, cysts, females, etc.).

[0412] Dry root weight was obtained and the number of nematodes per gram of dry root weight was calculated.

[0413] Positive and negative controls included: untreated (no nematode inoculation, no microbial treatment, only water immersion), inoculated control (no nematode inoculation, no microbial treatment, only water immersion), positive control (known nematicide product against the nematode tested - ideally having both chemical and biological nematicides as positive controls in the same experiment), negative microbial control - using E. coli DH5 alpha or other available microorganisms that did not have nematicidal activity.

[0414] As shown in FIG. 3, strain 39400 reduced egg hatching by approximately 35% and inhibited molting and larval viability.

[0415] Strain 39400 was further tested on cucumber and showed excellent reduction in the number of galls per gram of root as well as the number of eggs per root, as shown in Table 3. [Table 4]

[0416] Additional growth chamber studies (2 mL irrigation treatment of approximately 1e6-1e7 CFU / mL) showed that strain 39400 reduced RKN counts by 58% in cucumber and 60% in tomato. Greenhouse studies showed that strain 39400 reduced RKN counts in tomato by 46%.

[0417] Strain 39400 showed root colonization in both cucumber and wheat.

[0418] Coleoptera assay As shown in Figures 8 and 9, strain 39400 demonstrated activity against Coleoptera.

[0419] Lepidoptera assay (Spodoptera sp.) The effect of strain 39400 on size and mortality was investigated on Spodoptera litura (tobacco bark beetle) larvae. The results are shown in Table 4. [Table 5]

[0420] To investigate the effect of microbial concentration on the size and viability of Spodoptera litura, a dilution of strain 39400 was performed, and the results are shown in Figures 4A and 4B, respectively.

[0421] Grain Field Testing Wheat (microorganisms applied as seed treatment, 2 trials, 6 replicates) and maize (microorganisms applied as seed treatment, 8 trials, 6 replicates or in-furrow treatment, 2 trials, 6 replicates) were grown in field trials following standard protocols.

[0422] Due to unusually hot and drought conditions, nematode infestations were not observed in the wheat trials until approximately 35 days after planting. However, as shown in Figure 5, plants treated with strain 39400 showed improved yield compared to the biological and chemical controls, as well as all other microorganisms tested.

[0423] Corn seeds treated with line 39400, as well as corn plants treated in-furrow with 39400, both showed improved average yield (bu / ac) compared to both biological and chemical controls, as well as the other lines tested (Figures 6 and 7, respectively).

[0424] Additional field trials were conducted in 2022 for corn and soybean.

[0425] Corn trials were conducted at seven locations with six replicates each. Corn seeds were treated with strain 39400 at planting. Nematode counts were conducted 35-45 days after planting (DPP). The average yield for strain 39400 was 189.07 bushels / acre (chemical control was 184.74), with an IOC of 4.01 (chemical control was 0.32), %IOC of 1.81, and win rate of 71% (chemical control had -0.21, win rate of 43%), with an average nematode reduction of -34.66% and win rate of 100% (chemical control was -26.42%, win rate of 83.3%).

[0426] Soybean trials were conducted at multiple locations with six replicates each. Soybean seeds were treated with strain 39400 at planting. Nematode counts were conducted 45 days after planting (DPP) 350. Across the four locations, strain 39400 had an average yield of 67.27 bu / ac (chemical control was 69.66), an IOC of 2.5 (chemical control was 4.89), and a 5 IOC of 3.78% (chemical control was 7.43%). Across the eleven locations, strain 39400 showed an average reduction in nematode populations of -16% (80% success rate).

[0427] Vegetable Field Test For root-knot nematodes (RKN), two field trials with six replicates of tomato were conducted using drench / drip treatments at a rate of 8 qt / A, with treatments at 0, 7, and 14 days after planting. Strain 39400 reduced the number of RKN per 100 cc of soil by 62% compared to the untreated control.

[0428] Example 5: Activity comparison of different strains Different dilutions of strain 39400 and strain 42901 were prepared and tested in the Spodoptera assay described above. Despite identical sequences of the 16S and Cry protein genes, the two strains exhibited different activities between dilutions and compared to the commercial biological Bacillus thuringiensis control, as shown in Table 5. [Table 6]

[0429] Both strain 39400 and strain 42901 performed better than the commercial Bt control.

Claims

1. 1. A synthetic composition comprising: a. Microbial cells, exudates therefrom, or culture broths therefrom, wherein the microbial cells are: i. a microbial cell comprising a 16S sequence that shares at least 97% identity with any one or more of SEQ ID NOs: 1-14; ii. A microbial cell comprising a nucleotide sequence that shares at least 97% identity with at least one sequence selected from the group consisting of SEQ ID NOs: 15-19; iii. Microbial cells obtained or derived from Bacillus thuringiensis strain 39400, deposited as NRRL B-68090; iv. microbial cells obtained or derived from Bacillus thuringiensis strain 42901, and v. a Bacillus thuringiensis strain comprising at least one genomic mutation described in Table 1B, and b. at least one heterologous composition selected from the group consisting of plant components, formulation ingredients, agricultural compositions, and any combination thereof; The composite composition, wherein the microorganism is present at a concentration of at least about 10^2 CFU / mL in a liquid formulation or at least about 10^2 CFU / gram in a non-liquid formulation, and may further comprise at least one additional microorganism.

2. 10. The synthetic composition of claim 1, wherein the plant element is a seed, a seed containing a transgene, a leaf, a root, a whole plant, or a combination or combinations thereof.

3. the formulation ingredients are selected from the group consisting of compounds that improve the stability of the microorganism, preservatives, carriers, surfactants, anticomplex agents, and any combination thereof; 10. The synthetic composition of claim 1, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, an insecticide, a herbicide, or any combination thereof.

4. 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 jar, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

5. 10. The synthetic composition of claim 1, wherein the plant elements are obtained from a plant selected from the group consisting of corn, soybean, wheat, cotton, cucumber, tomato, bell pepper, potato, strawberry, orange, lemon, lime, apple, snap pea, zucchini, pea, lettuce, broccoli, celery, cauliflower, sorghum, and canola.

6. 10. The synthetic composition of claim 1, wherein the agricultural composition comprises a growing medium, and the growing medium may comprise soil.

7. 7. The plurality of synthetic compositions of claim 6, 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.

8. 1. A synthetic composition comprising: a. an exudate or culture broth of a plurality of cells, said cells comprising: i. a microbial cell comprising a 16S sequence that shares at least 97% identity with any one or more of SEQ ID NOs: 1-14; ii. A microbial cell comprising a nucleotide sequence that shares at least 97% identity with at least one sequence selected from the group consisting of SEQ ID NOs: 15-19; iii. Microbial cells obtained or derived from Bacillus thuringiensis strain 39400, deposited as NRRL B-68090; iv. microbial cells obtained or derived from Bacillus thuringiensis strain 42901, and v. a Bacillus thuringiensis strain comprising at least one genomic mutation described in Table 1B; and at least one heterologous composition selected from the group consisting of plant elements, formulation ingredients, agricultural compositions, and any combination thereof; b. may further comprise at least one additional microorganism; Synthetic composition.

9. 1. A method for modulating an agronomically important trait in a plant obtained or derived from a plant element, the method comprising treating said plant element with a formulation comprising microbial cells, an exudate therefrom, or a culture broth therefrom, wherein said microbial cells a. a microbial cell comprising a 16S sequence that shares at least 97% identity with any one or more of SEQ ID NOs: 1-14; b. A microbial cell comprising a nucleotide sequence that shares at least 97% identity with at least one sequence selected from the group consisting of SEQ ID NOs: 15-19; c. Microbial cells obtained or derived from Bacillus thuringiensis strain 39400, deposited as NRRL B-68090; d. Microbial cells obtained or derived from Bacillus thuringiensis strain 42901, and e. A Bacillus thuringiensis strain comprising at least one genomic alteration described in Table 1B; the bacterium has a detrimental effect on nematodes, coleoptera, and / or lepidoptera, may further comprise at least one additional microorganism, method.

10. 10. The method of claim 9, wherein the microbial cells, exudate therefrom, or culture broth therefrom are present in an amount capable of providing a benefit to a plant derived from the plant element compared to a plant derived from a plant element that has not been treated with the microbial cells or exudate therefrom.

11. 1. A method for reducing the impact of pest insects or nematodes on a plant, comprising introducing microbial cells, exudates therefrom, or culture broths therefrom to plant elements of said plant, wherein said microbial cells: a. a microbial cell comprising a 16S sequence that shares at least 97% identity with any one or more of SEQ ID NOs: 1-14; b. A microbial cell comprising a nucleotide sequence that shares at least 97% identity with at least one sequence selected from the group consisting of SEQ ID NOs: 15-19; c. Microbial cells obtained or derived from Bacillus thuringiensis strain 39400, deposited as NRRL B-68090; d. Microbial cells obtained or derived from Bacillus thuringiensis strain 42901, and e. A Bacillus thuringiensis strain comprising at least one genomic alteration described in Table 1B; the microbial cell is heterologous to the plant element; introducing into said plant elements is achieved by a method selected from the group consisting of in-furrow treatment, soil drench treatment, lateral application treatment, coating said plant elements with a formulation of said microorganism or exudates therefrom, and a combination thereof, and / or a combination thereof; The method may further comprise at least one additional microorganism.

12. 12. The method of claim 11, wherein the plant element is a seed, a leaf, a root, a whole plant, or a plurality and / or combination thereof.

13. 1. A method for modulating an agronomically important trait in a harvested product, the method comprising introducing microbial cells, an exudate therefrom, or a culture broth therefrom to said harvested product and / or to an organism from which said harvested product is obtained, wherein said microbial cells a. a microbial cell comprising a 16S sequence that shares at least 97% identity with any one or more of SEQ ID NOs: 1-14; b. A microbial cell comprising a nucleotide sequence that shares at least 97% identity with at least one sequence selected from the group consisting of SEQ ID NOs: 15-19; c. Microbial cells obtained or derived from Bacillus thuringiensis strain 39400, deposited as NRRL B-68090; d. Microbial cells obtained or derived from Bacillus thuringiensis strain 42901, and e. A Bacillus thuringiensis strain comprising at least one genomic mutation described in Table 1B.

14. 14. The method of claim 13, further comprising at least one additional microorganism.

15. 14. The method of claim 13, wherein the harvested produce is a fruit, a vegetable, a seed, a fiber, a plurality of these, or a combination thereof.

16. An isolated bacterial strain comprising a polynucleotide sequence that shares at least 97% sequence identity with any one of SEQ ID NOs: 1-19.

17. 1. An agricultural composition comprising: a) the isolated bacterial strain of claim 16, and b) comprising an agriculturally acceptable carrier; the bacterial strain is present in the agricultural composition in an amount effective to produce an improved phenotype in a plant with which it is associated; The agricultural composition is formulated as a seed coating, foliar spray, soil drench, dip treatment, in-furrow treatment, soil amendment, granule, broad-spectrum treatment, or post-harvest disease control treatment.

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