Intergeneric diazotrophic microorganisms for use in agriculture

Abstract

The disclosure relates to genetically modified microorganisms, particularly those comprising intergeneric modification(s), for the improvement of phenotypes of plants, for example nitrogen availability for plants. Included are novel strains of the microorganisms, microbial consortia, and agricultural compositions comprising the same. Furthermore, the disclosure teaches methods of utilizing the described microorganisms, microbial consortia, and agricultural compositions comprising the same, in methods for imparting beneficial properties to target plant species. In particular aspects, the disclosure provides methods of increasing desirable plant traits in agronomically important species, for example nitrogen fixation, utilization, regulation, uptake, acquisition, tolerance, and / or processing in plants.

Description

Docket # 24032-WO- PCTINTERGENERIC DIAZOTROPHIC MICROORGANISMS FOR USE IN AGRICULTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Serial No. 63 / 730,394 filed 10 December 2024, herein incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to isolated and biologically pure microorganisms that have application, inter alia, in agriculture. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into agriculturally acceptable compositions. Also disclosed are methods of using the isolated microorganisms or agriculturally acceptable compositions in agricultural applications.BACKGROUND

[0003] According to the United Nations World Food Program, there are close to 900 million malnourished people in the world. The malnourishment epidemic is particularly striking in the developing nations of the world, where one in six children is underweight. The paucity of available food can be attributed to many socioeconomic factors; however, regardless of ultimate cause, the fact remains that there is a shortage of food available to feed a growing world population, which is expected to reach 9 billion people by 2050. The United Nations estimates that agricultural yields must increase by 70-100% to feed the projected global population in 2050.

[0004] These startling world population and malnutrition figures highlight the importance of agricultural efficiency and productivity, in sustaining the world’s growing population. The technological advancements achieved by modern row crop agriculture, which has led to never- before-seen crop yields, are impressive. However, despite the advancements made by technological innovations such as genetically engineered crops and new novel pesticidal and herbicidal compounds, there is a need for improved crop performance, in order to meet the demands of an exponentially increasing global population.Docket # 24032-WO- PCT

[0005] Scientists have estimated that if the global agricultural “yield gap” (which is the difference between the best observed yield and results elsewhere) could be closed, then worldwide crop production would rise by 45-70%. That is, if all farmers, regardless of worldwide location, could achieve the highest attainable yield expected for their respective regions, then a great majority of the deficiencies in worldwide food production could be addressed. However, solving the problem of how to achieve higher yields across a heterogenous worldwide landscape is difficult.

[0006] Often, yield gaps can be explained by inadequate water, substandard farming practices, inadequate fertilizers, and the non-availability of herbicides and pesticides. However, to vastly increase the worldwide use of water, fertilizers, herbicides, and pesticides, would not only be economically infeasible for most of the world, but would have negative environmental consequences.

[0007] Thus, meeting global agricultural yield expectations, by simply scaling up current high- input agricultural systems — utilized in most of the developed world — is simply not feasible.

[0008] There is therefore an urgent need in the art for improved methods of increasing crop performance and imparting beneficial traits to desired plant species.

[0009] The technology described herein include environmental nitrogen fixing bacteria that have been gene edited, to increase the amount of atmospheric nitrogen that is fixed.

[0010] These gene edited bacterial strains impart improved phenotypes to plants, for example crop plants, to enhance the amount of nitrogen made available to the plant and increase the health and productivity of the plant.SUMMARY

[0011] Included are isolated and biologically pure microorganisms that have application, inter alia, in agriculture. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into agriculturally acceptable compositions. Further provided are agriculturally beneficial microbial consortia, comprising at least two members of the disclosed microorganisms, as well as methods of utilizing said consortia in agricultural applications. In some aspects, genomic modification of the microbes (individual, consortia, and / or communities) are contemplated, for the improvement of microbial traits and the improvement of microbe-associated plants.Docket # 24032-WO- PCT

[0012] Herein are presented genetically engineered microorganisms for the improvement of nitrogen fixation capability.

[0013] The present disclosure addresses this important issue of how to improve plant performance, thereby closing the worldwide yield gap, along with providing ways of imparting other beneficial traits to plant species. The novel edited strains described herein improve plant performance by enabling the plant for increased and / or improved nitrogen availability, fixation, uptake, acquisition, tolerance, distribution, regulation, processing, and / or any plurality and / or combination of any of the preceding.

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

[0015] In some embodiments, the plant is a leguminous crop plant.

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

[0017] In some embodiments, the plant is a dicot. In some embodiments, the plant is selected from the group consisting of: soybean, cotton, canola, rapeseed, a legume (e.g., pea, bean, lentil, peanut), mint, lettuce, tomato, pepper, eggplant, brinjal, broccoli, carrot, cauliflower, potato, kale, spinach, beet, radish, cauliflower, squash, mustard, berry, pepper, greens, pole beans, muskmelon, cucumber, basil, grape, and okra. In some embodiments, the plant is a tree, such as apple, peach, pear, cherry, almond, walnut, lemon, lime, orange. In some embodiments, the plant is a fruit, such as blackberry, strawberry, blueberry, raspberry.

[0018] The solution to increasing crop performance and increasing yield proffered by the present disclosure is not detrimental to the earth’s resources, as it does not rely upon increased water consumption or increased input of synthetic chemicals into a system. Rather, the present disclosure utilizes microbes to impart beneficial properties, including increased yields, to desirable plants.

[0019] The disclosure therefore offers an environmentally sustainable solution that allows farmers to increase yields of important crops, which is not reliant upon increased utilization of synthetic herbicides and pesticides.Docket # 24032-WO- PCT

[0020] In embodiments, the disclosure provides for an efficient and broadly applicable agricultural platform utilizing microbes and microbial consortia (a plurality of microbes, in some aspects a plurality that improves the health or desired phenotype of the plant, such as an agronomic trait, with which it is associated) that promote one or more desirable plant properties.

[0021] The microbes disclosed herein improve the performance of plants, such as crop plants, by both direct and indirect mechanisms. In some aspects, the microbe becomes symbiotic with the plant. In some aspects, the microbe produces a compound (e.g., a metabolite, a toxin, a protein, a lipopeptide, or other composition) that confers a benefit to the plant or that the plant can use for improved characteristics. In some aspects, the microbe improves the solubility of one or more compositions, such as a nutrient, thereby benefitting the plant. In some aspects, the microbe imparts a tolerance to the plant to an exogenous substance such as an herbicide or a pesticide. In some aspects, the microbe produces a composition that is detrimental to a plant pest, such as an insect. In some aspects, the microbe fixes Nitrogen, thereby improving the nutritional status of the plant. Other aspects beyond the exemplary non-limiting aspects listed above are contemplated.

[0022] In some embodiments, a single microbe is utilized. In some aspects, the single microbe is isolated and purified. In some aspects, the single microbe is a taxonomic species of bacteria. In some aspects, the single microbe is an identifiable strain of a taxonomic species of bacteria. In some aspects, the single microbe is a novel, newly discovered strain of a taxonomic species of bacteria.

[0023] In some aspects, a single microbe — whether a taxonomically identifiable species or strain — is combined with one or more other microbes of a different species or strain. In certain aspects, the combination of two or more microbes forms a consortia or consortium. The terms consortia and consortium are utilized interchangeably.

[0024] In certain aspects, the disclosure provides for the development of highly functional microbial consortia that help promote the development and expression of a desired phenotypic or genotypic plant trait. In some embodiments, the consortia of the present disclosure possess functional attributes that are not found in nature, when the individual microbes are living alone. That is, in various embodiments, the combination of particular microbial species into consortia, leads to the microbial combination possessing functional attributes that are not possessed by any one individual member of the consortia when considered alone.Docket # 24032-WO- PCT

[0025] In some embodiments, this functional attribute possessed by the microbial consortia is the ability to impart one or more beneficial properties to a plant species, for example: increased growth, increased yield, increased nutrient utilization (e.g, nitrogen, phosphate, and the like), increased nitrogen utilization efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, enhanced water use efficiency, increased pathogen resistance, modifications to plant architecture that don’t necessarily impact plant yield, but rather address plant functionality, etc. Further contemplated are beneficial properties of pest resistance and / or tolerance, comprising an adverse effect against a nematode, insect, or other pest.

[0026] The ability to impart these beneficial properties upon a plant is not possessed, in some embodiments, by the individual microbes as they would occur in nature. Rather, in some embodiments, it is by the hand of man combining these microbes into consortia that a functional composition is developed, said functional composition possessing attributes and functional properties that do not exist in nature. In some embodiments, the consortia may include microbes that have been genetically edited, altered, or modified through the modification of cellular compositions, including DNA, RNA, proteins and / or combinations of the same, via techniques known to those of ordinary skill in the art.

[0027] However, in other embodiments, the disclosure provides for individual isolated and biologically pure microbes that are capable of imparting beneficial properties upon a desired plant species, without the need to combine said microbes into consortia.

[0028] In some embodiments, the microbe is a strain of the genus Paerribacillus that has been genetically modified to improve nitrogen fixation capabilities.

[0029] The disclosure therefore offers an environmentally sustainable solution that allows farmers to increase yields of important crops that is not reliant upon increased utilization of synthetic fertilizer, herbicides, and / or pesticides. For example, in one aspect, the present disclosure describes isolated microbes that are genetically modified to improve the nitrogen fixation ability of the microbe.

[0030] The present disclosure further relates to agricultural compositions that include one or more strains of the isolated, genetically modified microbes disclosed herein and an agriculturally acceptable carrier. In some embodiments, the agricultural compositions include one or more additional agriculturally beneficial agents (e.g. fertilizers, biofertilizers, bionematicides, biostimulants, synthetic pesticides, and / or synthetic herbicides).Docket # 24032-WO- PCT

[0031] Also disclosed herein are methods of imparting one or more beneficial traits to a plant, where the methods include applying an agriculturally effective amount of one or more of the isolated, genetically modified microbes or agricultural compositions disclosed herein.

[0032] Any strain disclosed herein may further be combined with one or more additional microbes, which may form a microbial consortia. The microbial consortia can be any combination of one or more individual microbes. In certain embodiments, the microbial consortia comprise two microbes, or three microbes, or four microbes, or five microbes, or six microbes, or seven microbes, or eight microbes, or nine microbes, or 10 microbes, or more than 10 microbes.

[0033] Another object of the disclosure is to design a microbial consortium, which is able to perform multidimensional activities in common. In certain aspects, the microbes comprising the consortium act synergistically. In aspects, the effect that the microbial consortium has on a certain plant characteristic is greater than the effect that would be observed had any one individual microbial member of the consortium been utilized singularly. That is, in some aspects, the consortium exhibits a greater than additive effect upon a desired plant characteristic, as compared to the effect that would be found if any individual member of the consortium had been utilized by itself.

[0034] In some aspects, the consortia lead to the establishment of other plant-microbe interactions, e.g., by acting as primary colonizers or founding populations that set the trajectory for the future microbiome development.

[0035] In embodiments, the disclosure is directed to synergistic combinations (or mixtures) of microbial isolates.

[0036] In some aspects, the consortia taught herein provide a wide range of agricultural applications, including: improvements in yield of grain, fruit, and flowers; improvements in growth of plant parts; improved ability to utilize nutrients e.g., nitrogen, phosphate, and the like), improved resistance to disease; biopesticidal effects including improved resistance to fungi, insects, and nematodes; improved survivability in extreme climate; and improvements in other desired plant phenotypic characteristics. Significantly, these benefits to plants and / or adverse effect on targeted pests and / or pathogens can be obtained without any hazardous side effects to the environment.Docket # 24032-WO- PCT

[0037] In some aspects, the individual microbes of the disclosure, or consortia comprising same, can be combined into an agriculturally acceptable composition.

[0038] In some embodiments, the agricultural compositions of the present disclosure include, but are not limited to: wetters, compatibilizing agents, antifoam agents, cleaning agents, sequestering agents, drift reduction agents, neutralizing agents, buffers, corrosion inhibitors, dyes, odorants, spreading agents, penetration aids, sticking agents, binders , dispersing agents, thickening agents, stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, fertilizers, pesticides, nematicides, insecticides, herbicides, inert carriers, polymers, and the like.

[0039] In one embodiment of the present disclosure, the microbes (including isolated single species, or strains, consortia, or compositions thereof, such as compound(s)), are supplied in the form of seed coatings or other applications to the seed. In embodiments, the seed coating may be applied to a naked and untreated seed. In other embodiments, the seed coating may be applied to a previously treated seed. Thus, in some embodiments, the present disclosure teaches a method of treating a seed comprising applying an isolated bacterial strain or a microbial consortium to a seed. In certain embodiments, the isolated bacterial strain or microbial consortium is applied as an agricultural composition including an agriculturally acceptable carrier. In some embodiments, the agricultural compositions may be formulated as: a soil drench, a foliar spray, a dip treatment, an in-furrow treatment, a soil amendment, granules, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural compositions may be applied alone in or in rotation spray programs with other agricultural products. 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.

[0040] In some embodiments, the applied microbes may become endophytic and consequently may be present in the growing plant that was treated and its subsequent offspring. In other embodiments the microbes might be applied at the same time as a co-treatment with seed treatments.

[0041] In one embodiment of the present disclosure, the microbes are supplied in the form of granules, or plug, or soil drench that is applied to the plant growth media. In other embodiments, the microbes are supplied in the form of a foliar application, such as a foliar spray or liquidDocket # 24032-WO- PCT composition. The foliar spray or liquid application may be applied to a growing plant or to a growth media, e.g., soil.

[0042] In other embodiments, the microbes (including isolated single species, or strains, or consortia, or compositions thereof, such as compound(s)) are supplied as fertilizers, pesticides, or other amendments that may be applied to soil. In some embodiments, the microbes are supplied as fertilizers, pesticides, or other amendments that are applied to soil prior to planting. In some embodiments, the microbes are supplied as fertilizers, pesticides, or other amendments that are applied to soil concurrent with planting. In some embodiments, the microbes are supplied as fertilizers, pesticides, or other amendments that are applied to soil after planting.

[0043] In other embodiments of the present disclosure, the microbes (including isolated single species or strains, or consortia) and / or compositions thereof e.g., compound(s)) are supplied in the form of a post-harvest disease control application.

[0044] In embodiments, the agricultural compositions of the disclosure can be formulated as: (1) solutions; (2) wettable powders; (3) dusting powders; (4) soluble powders; (5) emulsions or suspension concentrates; (6) seed dressings, (7) tablets; (8) water-dispersible granules; (9) water soluble granules (slow or fast release); (10) microencapsulated granules or suspensions; (11) as irrigation components, and (12) a component of fertilizers, pesticides, and other compatible amendments, among others. In certain aspects, the compositions may be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure can be applied to the soil, plant, seed, rhizosphere, rhizosheath, or other area to which it would be beneficial to apply the microbial compositions.

[0045] Still another object of the disclosure relates to the agricultural compositions being formulated to provide a high colony forming units (CFU) bacterial population or consortia. In some aspects, the agricultural compositions have adjuvants that provide for a pertinent shelf life. In embodiments, the CFU concentration of the taught agricultural compositions is higher than the concentration at which the microbes would exist naturally, outside of the disclosed methods. In another embodiment, the agricultural composition contains the microbial cells in a concentration of 10A2- 10Al 2 CFU per gram of the carrier or 10A5- 10A9 CFU per gram of the carrier. In an aspect, the microbial cells are applied as a seed coat directly to a seed at a concentration of 10A5- 10A9 CFU. In other aspects, the microbial cells are applied as a seed overcoat on top of anotherDocket # 24032-WO- PCT seed coat at a concentration of 10A5-l 0A9 CFU. Tn other aspects, the microbial cells are applied as a co-treatment together with another seed treatment at a rate of 10A5-10A9 CFU.

[0046] In aspects, the disclosure is directed to agricultural microbial formulations that promote plant growth. In aspects, the disclosure provides for the taught isolated microbes, and consortia comprising same, to be formulated as an agricultural bioinoculant. The taught bioinoculants can be applied to plants, seeds, or soil, or combined with fertilizers, pesticides, and other compatible amendments. Suitable examples of formulating bioinoculants comprising isolated microbes can be found in U.S. Pat. NO 7,097,830.

[0047] The disclosed microbial formulations can: lower the need for nitrogen containing fertilizers, solubilize minerals, provide biopesticidal protection of the plants, protect plants against pathogens (e.g., fungi, insects, and nematodes), and make available to the plant valuable nutrients, such as nitrogen and / or phosphate, thus reducing and eliminating the need for using chemical pesticides and chemical fertilizers.

[0048] In some embodiments, the isolated and biologically pure microbes of the present disclosure can be utilized, in a method of imparting one or more beneficial properties or traits to a desired plant species.

[0049] In some embodiments, the agriculturally acceptable composition containing isolated and biologically pure microbes of the present disclosure can be utilized, in a method of imparting one or more beneficial properties or traits to a desired plant species.

[0050] In some embodiments, the consortia of the present disclosure can be utilized, in a method of imparting one or more beneficial properties or traits to a desired plant species.

[0051] In some embodiments, the agriculturally acceptable composition containing consortia of the present disclosure can be utilized, in a method of imparting one or more beneficial properties or traits to a desired plant species.

[0052] In some aspects, the isolated and biologically pure microbes 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 aspects of the present disclosure is described, for example, in: (1) International Patent Application NO PCT / NZ2012 / 000041, published on September 20, 2012, as International Publication NO WO 2012125050 Al, and (2) International Patent Application NO PCT / NZ2013 / 000171, published on March 27, 2014, as International Publication No. WO 2014046553 Al.Docket # 24032-WO- PCT

[0053] However, in other embodiments, the microbes of the present disclosure are not derived from an accelerated microbial selection process. In some aspects, the microbes utilized in embodiments of the disclosure are chosen from amongst members of microbes present in a database. In particular aspects, the microbes utilized in embodiments of the disclosure are chosen from microbes present in a database based upon particular characteristics of said microbes.

[0054] The present disclosure provides that a plant element or plant part can be effectively augmented, by coating said plant element or plant part with an isolated microbe or microbial consortia, in an amount that is not normally found on the plant element or plant part.

[0055] Some embodiments described herein are methods for preparing an agricultural seed composition, or seed coating, comprising: contacting the surface of a seed with a formulation comprising a purified microbial population that comprises at least one isolated microbe that is heterologous to, or rarely present on the seed. Further embodiments entail preparing an agricultural plant composition, comprising: contacting the surface of a plant with a formulation comprising a purified microbial population that comprises at least one isolated microbe that is heterologous to the plant. In other aspects, the formulation or microbe(s) is(are) introduced into the interior of the seed, for example into the cotyledon or the embryo other seed tissue.

[0056] In some aspects, applying an isolated microbe, microbial consortia, exudate, metabolite, and / or agricultural composition of the disclosure to a seed or plant modulates a trait of agronomic importance. The trait of agronomic importance can be, e.g., disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen utilization, improved resistance to nitrogen stress, improved nitrogen fixation, improved nutrient utilization (e.g., phosphate, potassium, and the like), pest resistance, herbivore resistance, pathogen resistance, reduced pathogen levels (e.g., via the excretion of compound(s) that impair pathogen survival), increased yield, increased yield under water limited conditions, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, increased seed weight, faster seed germination, altered seed carbohydrate composition, altered seed oil composition, number of pods, delayed senescence, stay -green, and altered seed protein composition. In some aspects, atDocket # 24032-WO- PCT least 2, 3, 4, or more traits of agronomic importance are modulated. Tn some aspects, the modulation is a positive effect on one of the aforementioned agronomic traits.

[0057] In some aspects, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to modulate or alter a plant characteristic such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, growth improvement, health enhancement, 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, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutrition enhancement, pathogen resistance, pest resistance, photosynthetic capability improvement, salinity tolerance, stay-green, vigor improvement, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of nonwilted leaves per plant, a detectable modulation in the level of a metabolite, a detectable modulation in the level of a transcript, and a detectable modulation in the proteome relative to a reference plant.

[0058] 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 antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

[0059] The methods described herein can include contacting a seed or plant with at least 100 CFU or spores, at least 300 CFU or spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000 CFU or spores, at least 1,000,000 CFU or spores or more, of the microbes taught herein.Docket # 24032-WO- PCT

[0060] The methods described herein can include contacting a seed or plant with a composition that includes one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes at least 1 mg of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes at least 10 mg of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes at least 100 mg of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes at least 1 g of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes at least 10 g of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes at least 100 g of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes at least 1 kg of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein. In some aspects, the methods include contacting a seed or plant with a composition that includes greater than 1 kg of one or more compounds produced by, or fixed by, a single microbe or microbial consortium disclosed herein.

[0061] In some embodiments of the methods described herein, an isolated microbe of the disclosure is present in a formulation in an amount effective to be detectable within and / or on a target tissue of an agricultural plant. For example, the microbe is detected 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, in and / or on a target tissue of a plant. Alternatively or in addition, the microbes of the disclosure may be present in a formulation in an amount effective to increase the biomass and / or yield of a plant that has had such a formulation applied thereto, by at least 1%, at least 2%, at least 3%, atDocket # 24032-WO- PCT least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more, when compared with a reference agricultural plant that has not had the formulations of the disclosure applied. Alternatively or in addition, the microbes of the disclosure may be present in a formulation in an amount effective to detectably modulate an agronomic trait of interest of a plant that has had such a formulation applied thereto, by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more, when compared with a reference agricultural plant that has not had the formulations of the disclosure applied.

[0062] In some embodiments of the methods described herein, one or more compounds isolated, fixed by, or produced from the microbes or consortia of the disclosure are present in a formulation in an amount effective to be detectable within and / or on a target tissue of an agricultural plant. For example, the compound(s) are detected 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, in and / or on a target tissue of a plant. Alternatively or in addition, the compound(s) present in a formulation in an amount effective to increase the biomass and / or yield of a plant that has had such a formulation applied thereto, by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more, when compared with a reference agricultural plant that has not had the formulations of the disclosure applied. Alternatively or in addition, the compound(s) may be present in a formulation in an amount effective to detectably modulate an agronomic trait of interest of a plant that has had such a formulation applied thereto, by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more, when compared with a reference agricultural plant that has not had the formulations of the disclosure applied.

[0063] In some embodiments, the agricultural compositions taught herein are shelf-stable. In some aspects, the microbes taught herein are freeze-dried. In some aspects, the microbes taught herein are spray -dried. In some aspects, the microbes taught herein are placed in a liquid formulation. In some aspects, the microbes taught herein are present on granules,Docket # 24032-WO- PCT

[0064] Also described herein are a plurality of isolated microbes confined within an object selected from the group consisting of: bottle, jar, ampule, package, vessel, bag, box, bin, envelope, carton, container, silo, shipping container, truck bed, and case.

[0065] In some aspects, combining a selected plant species with a disclosed microbe — operational taxonomic unit (OTU), strain, or composition comprising any of the aforementioned — leads to improved yield from crops and generation of products thereof. Therefore, in one aspect, the present disclosure provides a synthetic combination of a seed of a first plant and a preparation of a microbe(s) that is coated onto the surface of the seed of the first plant, such that the microbe is present at a higher level on the surface of the seed, than is present on the surface of an uncoated reference seed. In another aspect, the present disclosure provides a synthetic combination of a part of a first plant and a preparation of a microbe(s) that is coated onto the surface of the part of the first plant, such that the microbe is present at a higher level on the surface of the part of the first plant, than is present on the surface of an uncoated reference plant part. The aforementioned methods can be used alone, or in parallel with plant breeding and transgenic technologies.

[0066] In some embodiments, the isolated bacterial strain has substantially similar morphological and physiological characteristics as an isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain has substantially similar genetic characteristics as an isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a mutant, naturally occurring or man-made, of an isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a genetically edited, altered, or modified bacterial strain. In some embodiments, an isolated bacterial strain of the present disclosure is in substantially pure culture. In some embodiments, an isolated bacterial strain of the present disclosure is in pure culture. In some embodiments, an isolated bacterial strain of the present disclosure is in a cell fraction, extract or supernatant.

[0067] In some embodiments, progeny and / or mutants of an isolated bacterial strain of the present disclosure are contemplated. In some embodiments, progeny, mutants, and / or genetically modified versions of an isolated bacterial strain of the present disclosure are contemplated.

[0068] In some embodiments, a cell-free or inactivated preparation of an isolated bacterial strain of the present disclosure is contemplated, or a mutant of said isolated bacterial strain. In some embodiments, a cell-free or inactivated preparation of an isolated bacterial strain of the presentDocket # 24032-WO- PCT disclosure is contemplated, or a mutant or genetically edited, altered, or modified variant of said isolated bacterial strain. In some embodiments, a metabolite produced by an isolated bacterial strain of the present disclosure is contemplated, or a mutant of said isolated bacterial strain. In some embodiments, a metabolite produced by an isolated bacterial strain of the present disclosure is contemplated, or a mutant or genetically modified variant of said isolated bacterial strain.

[0069] In some embodiments, an 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 10A2 to 1 x 10A12 CFU per gram. The agricultural composition may be formulated as a seed coating.

[0070] In some embodiments, a method of imparting at least one beneficial trait upon a plant species comprises applying an isolated bacterial strain to the plant or to a growth medium in which said plant is located. In some embodiments, a method of imparting at least one beneficial trait upon 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.

[0071] In some embodiments, the present disclosure teaches a method of growing a plant having at least one beneficial trait. In some embodiments, the method comprises applying an isolated bacterial strain or microbial consortium to the seed of a plant; sowing or planting the seed; and growing the plant. In certain embodiments, the isolated bacterial strain or microbial consortium is applied as an agricultural composition that further includes an agriculturally acceptable carrier.

[0072] In some embodiments, the microbial consortium has substantially similar morphological and physiological characteristics as a microbial consortium of the present disclosure. In some embodiments, the microbial consortium has substantially similar genetic characteristics as a microbial consortium of the present disclosure. In some embodiments, the microbial consortium is in substantially pure culture. In some embodiments, a subsequent generation of any microbe of the microbial consortium is contemplated. In some embodiments, a mutant of any microbe of the microbial consortium is contemplated. In some embodiments, a genetically edited, altered, or modified variant of any microbe of the microbial consortium is contemplated. In some embodiments, a cell-free or inactivated preparation of the microbial consortium, or a mutant or genetically edited, altered, or modified variant of any microbe in the microbial consortium, is contemplated. In some embodiments, a metabolite produced by the microbial consortium, or aDocket # 24032-WO- PCT mutant or genetically edited, altered, or modified variant of any microbe in the microbial consortium, is contemplated.

[0073] In some embodiments, an 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>< 1OA3 to l*10A12 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 upon a plant species comprises applying a microbial consortium to said plant, or to a growth medium in which said plant is located. In some embodiments, a method of imparting at least one beneficial trait upon a plant species, comprising applying the agricultural composition to the plant, or to a growth medium in which said plant is located.

[0074] In some embodiments, the microbe comprises one or more polynucleotides from a different genus of organism. In some embodiments, said one or more polynucleotides comprise one or more genes capable of imparting nitrogen fixation to the recipient microbe.BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The disclosure can be more fully understood from the following detailed description and the accompanying drawings, which form a part of this application.

[0076] FIG. 1 shows the acetylene reduction assay GC chromatogram of Strain 39400-G280 at 5mM NH4 (bottom bold line) compared to the negative control Strain 39400-G279 (top narrow line). Curves here, are displayed without offset and are thus graphed upon the same X and Y axis, with the Y axis representing the picoAmps (pA) of the detected signal and X axis representing time (minutes).DETAILED DESCRIPTION

[0077] While the following terms are believed to be well understood by one of ordinary skill in the art, the following are set forth to facilitate explanation of the presently disclosed subject matter.

[0078] The term “a” or “an” refers to one or more of that entity, i.e., can refer to a plural referent. As such, 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 more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.Docket # 24032-WO- PCT

[0079] As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, Bacteria and Archaea, as well as eukaryotic Fungi and Protists.

[0080] As used herein, the term "microbe" or "microorganism" refers to any species or taxon of microorganism, including, but not limited to, archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, a microbe or microorganism encompasses individual cells (e.g., unicellular microorganisms) or more than one cell (e.g., multi-cellular microorganism). A "population of microorganisms" may thus refer to a multiple cells of a single microorganism, in which the cells share common genetic derivation.

[0081] As used herein, the term "bacterium" or "bacteria" refers in general to any prokaryotic organism, and may reference an organism from either Kingdom Eubacteria (Bacteria), Kingdom Archaebacteria (Archaea), or both. In some cases, bacterial genera or other taxonomic classifications may be in taxonomic flux, have been reassigned due to various reasons (such as but not limited to the evolving field of whole genome sequencing), and / or may be variable based on methodology, and it is understood that such nomenclature variabilities are within the scope of any claimed taxonomy. For example, certain species of the genus Erwinia have been described in the literature as belonging to genus Pantoea (Zhang, Y. and Qiu, S., Examining phylogenetic relationships of Erwinia and Pantoea species using whole genome sequence data. Antonie van Leeuwenhoek 108, 1037-1046 (2015).).

[0082] The term “ 16S” refers to the DNA sequence of the 16S ribosomal RNA (rRNA) sequence of a bacterium. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of bacteria.

[0166] As used herein, the term "fungus" or "fungi" refers in general to any organism from Kingdom Fungi. Historical taxonomic classification of fungi has been according to morphological presentation. Beginning in the mid-1800' s, it was recognized that some fungi have a pleomorphic life cycle, and that different nomenclature designations were being used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association laid out rules for the naming of fungi according to their status as anamorph, teleomorph, or holomorph (Taylor, J.W. One Fungus = One Name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2, 113-120 (2011).). With the development of genomic sequencing, it became evident that taxonomic classification based onDocket # 24032-WO- PCT molecular phylogenetics did not align with morphological -based nomenclature (Shenoy, B.D., Jeewon, R. and Hyde, K.D. (2007). Impact of DNA sequence-data on the taxonomy of anamorphic fungi. Fungal Diversity 26: 1-54.). As a result, in 2011 the International Botanical Congress adopted a resolution approving the International Code of Nomenclature for Algae, Fungi, and Plants (Melbourne Code) (2012), with the stated outcome of designating "One Fungus = One Name" (Hawksworth, D.L. Managing and coping with names of pleomorphic fungi in a period of transition. IMA Fungus 3, 15-24 (2012)).

[0083] The term "Internal Transcribed Spacer" (“ITS”) refers to the spacer DNA (non-coding DNA) situated between the small-subunit ribosomal RNA (rRNA) and large-subunit (LSU) rRNA genes in the chromosome or the corresponding transcribed region in the polycistronic rRNA precursor transcript. ITS gene sequencing is a well-established method for studying phylogeny and taxonomy of fungi. In some cases, the "Large SubUnit" (“LSU”) sequence is used to identify fungi. LSU gene sequencing is a well-established method for studying phylogeny and taxonomy of fungi. Some fungal microbes of the present invention may be described by an ITS sequence and some may be described by an LSU sequence. Both are understood to be equally descriptive and accurate for determining taxonomy.

[0084] The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter or plant phenotypic trait. The community may comprise one or more species, or strains of a species, of microbes. In some instances, the microbes coexist within the community symbiotically.

[0085] The term “microbial community” means a group of microbes comprising two or more species or strains. Unlike microbial consortia, a microbial community does not have to be carrying out a common function, or does not have to be participating in, or leading to, or correlating with, a recognizable parameter or plant phenotypic trait.

[0086] The term “accelerated microbial selection” or “AMS” is used interchangeably with the term “directed microbial selection” or “DMS” and refers to the iterative selection methodology that was utilized, in some embodiments of the disclosure, to derive the claimed microbial species or consortia of said species.Docket # 24032-WO- PCT

[0087] As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, plant tissue).

[0088] Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with an agricultural carrier.

[0089] In certain aspects of the disclosure, the isolated microbes exist as isolated and biologically pure cultures. It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular microbe, denotes that said culture is substantially free (within scientific reason) of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated and biologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g., 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 microbes), see also, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), affirmed in part, reversed in part, 196 F. 496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B 12 produced by microbes), incorporated herein by reference.

[0090] As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can comprise substantially only one genus, species, or strain, of microorganism.Docket # 24032-WO- PCT

[0091] With respect to microbes, the term “modified” or “edited” (and their corresponding terms “modify”, “edit”, etc.) means that the microbe has been changed in some way via a non-naturally occurring means, as compared to the natural state in which it was found. In this context, “modified” or “edited” is synonymous with “engineered”, and indicates that the hand of man was involved with creating the modification. In some cases, the modification includes the change of a polynucleotide within the microbe, for example in its genome. Modifications may include deletion, insertion, replacement, and / or chemical alteration of at least one nucleotide, including any plurality and / or combination of the preceding, and may result in a change in the phenotype of the edited organism (e.g., upregulation of a particular pathway, downregulation of a particular pathway, knockout of a gene or protein function) and / or a change in the phenotype of another, heterologous organism with which the microbe is or becomes associated.

[0092] The term “growth medium” as used herein, is any medium which is suitable to support growth of a plant. By way of example, the media may be natural or artificial including, but not limited to: soil, potting mixes, bark, vermiculite, hydroponic solutions alone and applied to solid plant support systems, and tissue culture gels. It should be appreciated that the media may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients and physical support systems for roots and foliage.

[0093] In one embodiment, the growth medium is a naturally occurring medium such as soil, sand, mud, clay, humus, regolith, rock, or water. In another embodiment, the growth medium is artificial. Such an artificial growth medium may be constructed to mimic the conditions of a naturally occurring medium; 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.

[0094] The medium may be amended or enriched with additional compounds or components, for example, a component which may assist in the interaction and / or selection of specific groups of microorganisms with the plant and each other. For example, antibiotics (such as penicillin) or sterilants (for example, quaternary ammonium salts and oxidizing agents) could be present and / or the physical conditions (such as salinity, plant nutrients (for example organic and inorganic minerals (such as phosphorus, nitrogenous salts, ammonia, potassium and micronutrients such as cobalt and magnesium), pH, and / or temperature) could be amended.Docket # 24032-WO- PCT

[0095] The term “plant” generically includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. As used herein, the term “plant element” refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like, as well as the parts themselves. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.

[0096] A “plant element” is intended to reference either a whole plant or a plant component, which may comprise differentiated and / or undifferentiated tissues, for example but not limited to plant tissues, parts, and cell types. In one embodiment, a plant element is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keiki, shoot, bud, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, callus tissue). The term “plant organ” refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. As used herein, a “plant part” is synonymous to a “portion” of a plant, and refers to any part of the plant, and can include distinct tissues and / or organs, and may be used interchangeably with the term “tissue” throughout.

[0097] Similarly, a “plant reproductive element” is intended to generically reference any part of a plant that is able to initiate other plants via either sexual or asexual reproduction of that plant, for example but not limited to: seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, keiki, or bud. The plant element may be in plant or in a plant organ, tissue culture, or cell culture. Plant “vegetative element” is intended to refer to any non-reproductive portion of a plant related to growth and / or development, for example but not limited to: root, stem, leaf.

[0098] “Progeny” comprises any subsequent generation of an organism, produced via sexual or asexual reproduction.

[0099] “Grain” is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species.Docket # 24032-WO- PCT

[0100] The term “monocotyledonous” or “monocot” refers to the subclass of angiosperm plants also known as “monocotyledoneae”, whose seeds typically comprise only one embryonic leaf, or cotyledon. The term includes references to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same.

[0101] The term “dicotyledonous” or “dicot” refers to the subclass of angiosperm plants also knows as “dicotyledoneae”, whose seeds typically comprise two embryonic leaves, or cotyledons. The term includes references to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same.

[0102] As used herein, the term “cultivar” refers to a variety, strain, or race, of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations.

[0103] As used herein, “improved” should be taken broadly to encompass improvement of a characteristic of a plant, as compared to a control plant, or as compared to a known average quantity associated with the characteristic in question. For example, “improved” plant biomass associated with application of a beneficial microbe, or consortia, of the disclosure can be demonstrated by comparing the biomass of a plant treated by the microbes taught herein to the biomass of a control plant not treated. Alternatively, one could compare the biomass of a plant treated by the microbes taught herein to the average biomass normally attained by the given plant, as represented in scientific or agricultural publications known to those of skill in the art. In the present disclosure, “improved” does not necessarily demand that the data be statistically sig / / / / icant (e.g., p < 0.05); rather, any quantifiable difference demonstrating that one value (e.g., the average treatment value) is different from another (e.g, the average control value) can rise to the level of “improved.”

[0104] As used herein, “inhibiting and suppressing” and like terms should not be construed to require complete inhibition or suppression, although this may be desired in some embodiments.

[0105] As used herein, the term “genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.

[0106] The compositions and methods herein may provide for an improved “agronomic trait” or “trait of agronomic importance” or “trait of agronomic interest” to a plant, which may include, but not be limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency,Docket # 24032-WO- PCT improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, 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 isoline plant not comprising a modification derived from the methods or compositions herein

[0107] “Agronomic trait potential” is intended to mean a capability of a plant element for exhibiting a phenotype, preferably an improved agronomic trait, at some point during its life cycle, or conveying said phenotype to another plant element with which it is associated in the same plant.

[0108] As used herein, the term “molecular marker”, “marker”, or “genetic marker” refers to an indicator that is used in methods for visualizing differences in 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- characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. Mapping of molecular markers in the vicinity of an allele is a procedure which can be performed by the average person skilled in molecular- biological techniques.

[0109] As used herein, the term “trait” refers to a characteristic or phenotype. For example, in the context of some embodiments of the present disclosure, yield of a crop relates to the amount of marketable biomass produced by a plant (e.g., fruit, fiber, grain). Desirable traits may also include other plant characteristics, including but not limited to: water use efficiency, nutrient use efficiency, production, mechanical harvestability, fruit maturity, shelf life, pest / disease resistance, early plant maturity, tolerance to stresses, etc. A trait may be inherited in a dominant or recessive manner, or in a partial or incomplete-dominant manner. A trait may be monogenic ( / .£?., determined by a single locus) or polygenic (i.e., determined by more than one locus) or may also result from the interaction of one or more genes with the environment.Docket # 24032-WO- PCT

[0110] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism (e.g., a plant), or group of organisms which results from the interaction between that individual’s genetic makeup (i.e., genotype) and the environment.

[0111] As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.

[0112] As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms “nucleic acid” and “nucleotide sequence” are used interchangeably.

[0113] As used herein, the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and / or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0114] As used herein, the term “homologous” or “homologue”, “homolog”, or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homologous,” “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant disclosure such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the disclosure encompasses more than the specific exemplary sequences. TheseDocket # 24032-WO- PCT terms describe the relationship between a gene found in one species, subspecies, variety, cultivar or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar or strain. For purposes of this disclosure homologous sequences are compared. “Homologous sequences” or “homologues” or “orthologs” are thought, believed, or known to be functionally related. A 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) the same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania) and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.

[0115] As used herein, the term “nucleotide change” refers to, e.g., nucleotide substitution, deletion, insertion, chemical alteration, or any of the preceding, as is well understood in the art.

[0116] As used herein, the term “protein modification” refers to, e.g., amino acid substitution, amino acid modification, deletion, and / or insertion, as is well understood in the art.

[0117] As used herein, the term “at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full-length molecule, up to and including the full-length molecule. A fragment of a polynucleotide of the 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 disclosure that comprises the genetic regulatory element and assessing activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, going up to the full-length polypeptide. The length of the portion to be used will depend on the particular application. A portion of a nucleic acid useful as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide useful as an epitope may be as short as 4 amino acids. A portion of a polypeptide that performs the function of the full-length polypeptide would generally be longer than 4 amino acids.Docket # 24032-WO- PCT

[0118] The term “primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and composition (A / T vs. G / C content) of primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

[0119] The terms “stringency” or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimized to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na + ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes or primers e.g., 10 to 50 nucleotides) and at least about 60° C for long probes or primers (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or “conditions of reduced stringency” include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C and a wash in 2><SSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, IM NaCl, 1% SDS at 37° C, andDocket # 24032-WO- PCT a wash in 0.1 *SSC at 60° C. Hybridization procedures are well known in the art and are described by e.g., 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% sodium dodecyl sulfate at 45°C, such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by a wash in 5><SSC, containing 0.1% (w / v) sodium dodecyl sulfate, at 55°C to 65°C.

[0120] In some embodiments, the cell or organism has at least one heterologous trait. As used herein, the term “heterologous trait” refers to a phenotype imparted to a cell or organism by an exogenous molecule or other organism (e.g., a microbe), DNA segment, heterologous polynucleotide or heterologous nucleic acid.

[0121] Various changes in phenotype are of interest to the present disclosure, including but not limited to modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, increasing a plant’s yield of an economically important trait e.g., grain yield, forage yield, etc.) and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants using the methods and compositions of the present disclosure

[0122] A “synthetic combination” can include a combination of a plant and a microbe of the disclosure. The combination may be achieved, for example, by coating the surface of a seed of a plant, such as an agricultural plant, or host plant tissue (root, stem, leaf, etc ), with a microbe of the disclosure. Further, a “synthetic combination” can include a combination of microbes of various strains or species. Synthetic combinations have at lest one variable that distinguishes the combination from any combination that occurs in nature. That variable may be, inter alia, a concentration of microbe on a seed or plant tissue that does not occur naturally, or a combination of microbe and plant that does not naturally occur, or a combination of microbes or strains that do not occur naturally together. In each of these instances, the synthetic combination demonstrates the hand of man and possesses structural and / or functional attributes that are not present when the individual elements of the combination are considered in isolation.

[0123] In some embodiments, a microbe can be “endogenous” to a seed or plant. As used herein, a microbe is considered “endogenous” to a plant or seed, if the microbe is derived from the plant specimen from which it is sourced. That is, if the microbe is naturally found associated with said plant. In embodiments in which an endogenous microbe is applied to a plant, then theDocket # 24032-WO- PCT endogenous microbe is applied in an amount that differs from the levels found on the plant in nature. Thus, a microbe that is endogenous to a given plant can still form a synthetic combination with the plant, if the microbe is present on said plant at a level that does not occur naturally.

[0124] In some embodiments, a composition (such as a microbe) can be “heterologous” (also termed “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 microbe is considered “heterologous” to a plant or seed, if the microbe is not derived from the plant specimen from which it is sourced. That is, if the microbe is not naturally found associated with said plant. For example, a microbe that is normally associated with leaf tissue of a maize plant is considered exogenous to a leaf tissue of another maize plant that naturally lacks said microbe. In another example, a microbe that is normally associated with a maize plant is considered exogenous to a wheat plant that naturally lacks said microbe.

[0125] A composition is “heterologously disposed” when mechanically or manually applied, artificially inoculated, associated with, or disposed onto or into a plant element, seedling, plant or onto or into a plant growth medium or onto or into a treatment formulation so that the treatment exists on or in the plant element, seedling, plant, plant growth medium, or formulation in a manner not found in nature prior to the application of the treatment, e.g., said combination which is not found in nature in that plant variety, at that stage in plant development, in that plant tissue, in that abundance, or in that growth environment (for example, drought). In some embodiments, such a manner is contemplated to be selected from the group consisting of the presence of the microbe; presence of the microbe in a different number of cells, concentration, or amount; the presence of the microbe in a different plant element, tissue, cell type, or other physical location in or on the plant; the presence of the microbe at different time period, e.g., developmental phase of the plant or plant element, time of day, time of season, and combinations thereof. In some embodiments, “heterologously disposed” means that the microbe being applied to a different tissue or cell type of the plant element than that in which the microbe is naturally found. In some embodiments, “heterologously disposed” means that the microbe is applied to a developmental stage of the plant element, seedling, or plant in which said microbe is not naturally associated, but may be associated at other stages. For example, if a microbe is normally found at the flowering stage of a plant and no other stage, a microbe applied at the seedling stage may be considered to be heterologously disposed. In some embodiments, a microbe is heterologouslyDocket # 24032-WO- PCT disposed the microbe is normally found in the root tissue of a plant element but not in the leaf tissue, and the microbe is applied to the leaf. In another non-limiting example, if a microbe is naturally found in the mesophyll layer of leaf tissue but is being applied to the epithelial layer, the microbe would be considered to be heterologously disposed. In some embodiments, “heterologously disposed” means that the native plant element, seedling, or plant does not contain detectable levels of the microbe in that same plant element, seedling, or plant. In some embodiments, “heterologously disposed” means that the microbe being applied is at a greater concentration, number, or amount of the plant element, seedling, or plant, than that which is naturally found in said plant element, seedling, or plant. For example, a microbe is heterologously disposed when present at a concentration that is at least 1.5 times greater, between 1.5 and 2 times greater, 2 times greater, between 2 and 3 times greater, 3 times greater, between 3 and 5 times greater, 5 times greater, between 5 and 7 times greater, 7 times greater, between 7 and 10 times greater, 10 times greater, or even greater than 10 times higher number, amount, or concentration than the concentration that was present prior to the disposition of said microbe. In another non-limiting example, a microbe that is naturally found in a tissue of a cupressaceous tree would be considered heterologous to tissue of a maize, wheat, cotton, soybean plant. In another example, a microbe that is naturally found in leaf tissue of a maize, spring wheat, cotton, soybean plant is considered heterologous to a leaf tissue of another maize, spring wheat, cotton, soybean plant that naturally lacks said microbe, or comprises the microbe in a different quantity.

[0126] Microbes can also be “heterologously disposed” on a given plant tissue. This means that the microbe is placed upon a plant tissue that it is not naturally found upon. For instance, if a given microbe only naturally occurs on the roots of a given plant, then that microbe could be exogenously applied to the above-ground tissue of a plant and would thereby be “heterologously disposed” upon said plant tissue. As such, a microbe is deemed heterologously disposed, when applied on a plant that does not naturally have the microbe present or does not naturally have the microbe present in the number that is being applied.

[0127] The compositions and methods herein may provide for a “modulated” “agronomic trait” or “trait of agronomic importance” to a host plant, which may include, but not be limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical tolerance, coldDocket # 24032-WO- PCT tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, growth improvement, health enhancement, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutrition enhancement, pathogen resistance, pest resistance, photosynthetic capability improvement, salinity tolerance, stay-green, vigor improvement, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods 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, a detectable modulation in the level of a metabolite, a detectable modulation in the level of a transcript, and a detectable modulation in the proteome, compared to an isoline plant grown from a seed without said seed treatment formulation. By the term “modulated”, it is intended to refer to a change in an agronomic trait that is changed by virtue of the presence of the microbe(s), exudate, broth, metabolite, etc. In aspects, the modulation provides for the imparting of a beneficial trait.Microbes and Microorganisms

[0128] As used herein the term “microorganism” should be taken broadly. It includes, but is not limited to, prokaryotic Bacteria and Archaea, as well as eukaryotic Fungi and Protists.

[0129] In a particular embodiment, the microorganism is an endophyte, or an epiphyte, or a microorganism inhabiting the plant rhizosphere or rhizosheath. That is, the microorganism may be found present in the soil material adhered to the roots of a plant or in the area immediately adjacent a plant’s roots.

[0130] In one embodiment, the microorganism is an endophyte. Endophytes may benefit host plants by preventing pathogenic organisms from colonizing them. Extensive colonization of the plant tissue by endophytes creates a “barrier effect,” where the local endophytes outcompete and prevent pathogenic organisms from taking hold. Endophytes may also produce chemicals which inhibit the growth of competitors, including pathogenic organisms.Docket # 24032-WO- PCT

[0131] In certain embodiments, the microorganism is unculturable. This should be taken to mean that the microorganism is not known to be culturable or is difficult to culture using methods known to one skilled in the art.

[0132] Microorganisms of the present disclosure may be collected or obtained from any source or contained within and / or associated with material collected from any source.

[0133] In one embodiment, a microorganism or a combination of microorganisms, may provide likely or predicted benefit to a plant. For example, the microorganism may be predicted to: improve nitrogen fixation; release phosphate from the soil organic matter; release phosphate from the inorganic forms of phosphate (e.g., rock phosphate); “fix carbon” in the root microsphere; live in the rhizosphere of the plant thereby assisting the plant in absorbing nutrients from the surrounding soil and then providing these more readily to the plant; increase the number of nodules on the plant roots and thereby increase the number of symbiotic nitrogen fixing bacteria (e.g., Rhizobium species) per plant and the amount of nitrogen fixed by the plant; elicit plant defensive responses such as ISR (induced systemic resistance) or SAR (systemic acquired resistance) which help the plant resist the invasion and spread of pathogenic microorganisms; compete with microorganisms deleterious to plant growth or health by antagonism, or competitive utilization of resources such as nutrients or space; change the color of one or more part of the plant, or change the chemical profile of the plant, its smell, taste or one or more other quality.

[0134] The microorganisms of the disclosure may be isolated in substantially pure or mixed cultures. They may be concentrated, diluted, or provided in the natural concentrations in which they are found in the source material. For example, microorganisms from saline sediments may be isolated for use in this disclosure by suspending the sediment in fresh water and allowing the sediment to fall to the bottom. The water containing the bulk of the microorganisms may be removed by decantation after a suitable period of settling and either applied directly to the plant growth medium, or concentrated by filtering or centrifugation, diluted to an appropriate concentration and applied to the plant growth medium with the bulk of the salt removed. By way of further example, microorganisms from mineralized or toxic sources may be similarly treated to recover the microbes for application to the plant growth material to minimize the potential for damage to the plant.Docket # 24032-WO- PCT

[0135] In some embodiments, a mixed population of microorganisms is used in the methods of the disclosure.Genome Modification of Microbes

[0136] In some embodiments, the microorganism may have its genome altered in some way to provide an improved trait of interest, for example improvement of nitrogen fixation for non- leguminous crops.

[0137] Various methods are known in the art for modification of polynucleotides in a cell (which includes, without limitation, any polynucleotide sequence comprised within the cell, including genomic, chromosomal, and plasmid DNA). Briefly, a single- or double-strand break is introduced into a target polynucleotide (the subject of the modification), the result of which may be an insertion of at least one nucleotide, the deletion of at least one nucleotide, the replacement of at least one nucleotide, or any combination of the preceding, according to the desire of the practitioner.

[0138] The single- or double-strand break (SSB or DSB) may be accomplished by any of a number of methods, including the utilization of chemicals or radiation, a result of the process of homologous recombination, by the introduction of a specific or non-specific nuclease, or by any combination of the preceding.

[0139] Enzymes that effect polynucleotide cleavage are known in the art, and may include (without limitation): restriction endonucleases, meganucleases, TALENs, Zinc Fingers, or Cas endonucleases.Nitrogen Fixation

[0140] One area of interest is in the improvement of nitrogen fixation. Nitrogen gas (N2) is a major component of the atmosphere of Earth. In addition, elemental nitrogen (N) is an important component of many chemical compounds which make up living organisms. However, many organisms cannot use N2 directly to synthesize the chemicals used in physiological processes, such as growth and reproduction. In order to utilize the N2, the N2 must be combined with hydrogen. The combining of hydrogen with N2 is referred to as nitrogen fixation. Nitrogen fixation, whether accomplished chemically or biologically, requires an investment of large amounts of energy. In biological systems, an enzyme known as nitrogenase catalyzes the reaction which results in nitrogen fixation.Docket # 24032-WO- PCT

[0141] In order to utilize elemental nitrogen (N) for chemical synthesis, life forms combine nitrogen gas (N2) available in the atmosphere with hydrogen in a process known as nitrogen fixation. Because of the energy -intensive nature of biological nitrogen fixation, diazotrophs (bacteria and archaea that fix atmospheric nitrogen gas) have evolved sophisticated and tight regulation of the nif gene cluster in response to environmental oxygen and available nitrogen. Nif genes encode enzymes involved in nitrogen fixation (such as the nitrogenase complex) and proteins that regulate nitrogen fixation.

[0142] Two main drivers of universal nitrogen cycling are ammonia-oxi dizing bacteria (AOB), and archaea (AOA). Metabolic pathways usually are common between archaea and bacteria, and almost all genes involved in this process are founder under these domains.

[0143] All nitrogen fixers carry the nif (nitrogen fixation) genes, which encode the nitrogenase complex. Biological nitrogen fixation is catalyzed by nitrogenase, a complex enzyme that has two components, a heterotetrameric core encoded by nif I) and nifK, and a dinitrogenase reductase subunit encoded by nifH. Dinitrogenase reductase transfers reducing equivalents to the core enzyme complex where those reducing equivalents are used to convert N2 into NH3 equivalents.

[0144] The molybdenum nitrogenase is an oxygen sensitive complex dinitrogenase (NifDK heterotetramer), and dinitrogenase reductase (NifH homodimer). MoFe7S9 metal cluster is the active site for dinitrogen reduction for a, however, is some organisms Mo is replaced by either Fe or V, which is called Anf and Vnf, respectively instead of Nif. It has been reported that FeMo nitrogenase has been recognized to be more efficient in binding dinitrogen and reducing it to ammonia compare with alternative nitrogenase (Nif > Vnf > Anf). A discussion of nitrogen fixation and diazotrophic bacteria can be found in Sun et al. Rom. Biotechnol. Lett. Vol. 26 No. 4, pp. 2834-2845 (2021).

[0145] Although Glutamine (Gin) is a universal signal for nitrogen fixation across all bacteria, there are differences in the regulatory mechanisms and nif cluster components in Gram-positive and Gram-negative bacteria.Nitrogen Fixation in Gram-Positive Bacteria

[0146] In Nitrogen-fixing Gram-Positive bacteria, the nif operon controls the nitrogen fixation pathways by involving GlnR. It has been reported in the literature that binding of GlnR to Site IDocket # 24032-WO- PCT activates nif expression, while binding of GlnR to Site IT represses nif expression. Other mechanisms may be involved.

[0147] Within the Paenibacillus genus, are two distinct Subgroups, Subgroup I and Subgroup II, each comprising a different operon composition. Subgroup I Paenibacillus, such as Paenibacillus polymyxa, comprise in this order: nifB, nifH, nifD, nifK, nifE, niJN, nifX, hesA, nifV. Subgroup II Paenibacillus, such as Paenibacillus graminis, comprise in this order: nifB, nifH, nifD, nifK, nifE, nifV, nifX, orfl, hesA, nifV.

[0148] Some Paenibacillus strains are described in, for example, PCT Patent Application Publication No. WO2024123814 published 13 June 2024.

[0149] Most Paenibacillus bacteria utilize the components of the canonical nif cluster, as described above, to execute the various facets of nitrogen fixation, via a protein complex of NifD / NifK and NifE / NifN and the cofactors Molybdenum and Iron. It is known that some bacteria comprise other nitrogen fixation cluster components such as anf (uses Iron only) or vnf (uses Vanadium), forming a protein complex of 6 components instead of 4. nifH

[0150] The nitrogenase enzyme complex consists of the following two conserved proteins: the MoFe protein, composed of subunits encoded by the nifD and nifK genes; and the Fe protein, encoded by the nifH gene. The nitrogenase iron protein gene, nifH, is one of the oldest existing functional genes in the history of gene evolution. The protein encoded by nifH is a dinitrogenase reductase which provides electrons to NifDK for reduction of nitrogen.

[0151] The nucleotide sequences for coding regions of nifHDK genes among all nitrogen-fixing organisms are highly conserved. However, the copy numbers and arrangement of nifH, nifD, and nifK are different among the different diazotrophic bacteria. glnR

[0152] In Paenibacillus bacteria, the nif operon controls the nitrogen fixation pathways through GlnR; nif operon gene transcription is regulated by ammonium and oxygen. The protein encoded by glnR is a nitrogen-responsive, helix-turn-helix transcription factor which regulates expression of many targets including wz / 'genes.

[0153] In Gram-positive bacteria, binding of GlnR to Site I activates Nif expression, while binding of GlnR to Site II represses Nif expression. Further, GlnR has a higher affinity for binding to Site II.Docket # 24032-WO- PCT glnA

[0154] Glutamine synthetase (GS) encoded by glnA within glnRA operon directly interacts with the C-terminal domain of GlnR and then controls the GlnR activity. cueR

[0155] Structurally similar to GlnR, CueR is an HTH-type transcriptional regulator. The cueR gene is also located immediately upstream of the nrgA (ammonium transporter) gene, sharing the same bidirectional transporter. In many bacterial species, CueR is generally recognized as the regulator of the Cue copper efflux system, activating gene expression in response to high levels of intracellular copper. In Paenibacillus polymyxa, the CueR ORF shares a bidirectional promoter with the ammonium transporter NrgA. CueR is HTH-type transcriptional regulator like GlnR, and its proximity to NrgA in Paenibacillus polymyxa indicates it may play a role in transcription of the ammonium transporter as well.

[0156] Other gene(s) with similarity may be present in addition to, or instead of, CueR. orfl

[0157] In the native nif cluster of Paenibacillus odorifer Strain 17899, the orfl gene overlaps the downstream sequence of nifX for the first 17 nucleotides of orfl. glnB

[0158] The protein encoded by glnB is a P-II protein which transduces nitrogen status of the cell and affects expression of nitrogen assimilation genes indirectly in gram negative bacteria. nifB

[0159] A FeMo cofactor biosynthesis protein, the protein encoded by nifB acts as a molecular scaffold which produces iron-sulfur clusters that are cofactors for nitrogenase activity.

[0160] nifK

[0161] The protein encoded by nifK is one subunit of the dinitrogenase which contains the molybdenum-iron catalytic site for nitroge fixation along with NifD. nifD

[0162] The protein encoded by nifD is one subunit of the dinitrogenase which contains the molybdenum-iron catalytic site for nitrogen fixation along with NifK. nifS

[0163] The protein encoded by the nifS gene is a putative cysteine desulfurase NifS.Docket # 24032-WO- PCT nifE and nifN

[0164] The proteins encoded by nifE and nifN make up the Iron-Molyb denum cofactor (FeMo- Co) critical for nitrogen fixation. The FeMo-Co complex bind the active site (nifB) where dinitrogen is converted to ammonia. NifEN is a 200-kDa a202 heterotetramer that contains [Fe- S] clusters. The function of NifEN is essential to FeMo-co biosynthesis. Because of the amino acid sequence similarity between NifEN and NifDK and the observation that NifDK was not required for FeMo-Co biosynthesis, it has been proposed that NifEN could be a molecular scaffold to assemble FeMo-Co. nijV

[0165] A potential homolog to lys20, nijV may endode a homocitrate synthase. hesA

[0166] The HesA protein is encoded by two different genes, hesAl ( heteroyst) and hesA2 (vegetative), and is a shuttle for transporting sulfur and molybdenum cofactors to NifHEN complexes for FeMoco synthesis and maturation. anf senes

[0167] Anf nitrogenase is a Nitrogenase iron-iron protein with a hexameric subunit structure, comprising two alpha, two beta, and two gamma subunits.

[0168] The protein encoded by anfG is the delta-subunit of dinitrogenase enzyme that utilizes only iron as cofactor.

[0169] The protein encoded by anfD is the alpha-subunit of dinitrogenase enzyme that utilizes only iron as cofactor.

[0170] The protein encoded by anfK is the beta-subunit of dinitrogenase enzyme that utilizes only iron as cofactor. vnf genes

[0171] The protein encoded by vnfA is a transcriptional regulator that activates expression of vnf genes required to produce vanadium-dependent nitrogenase.

[0172] The protein encoded by vnfD is the alpha-subunit of vanadium-dependent dinitrogenase.

[0173] In some embodiments, the microorganism may have its genome altered in some way to provide an improved trait of interest, for example improvement of nitrogen fixation.Docket # 24032-WO- PCTNitrogen Fixation in Gram-Negative Bacteria

[0174] The nif cluster architecture in Gram-Negative bacteria is generally larger, and comprises more genes. For example, in Klebsiella pnemoniae, the / / / / operon comprises (in this order): nifQ*, nifB*, nif A *, nifL*, nifF, nifM*, nifV*, nifS*, nifU*, nifX*, nifN*, nifE*, nifY*, nifK*, nifD*, nifH*, nif J (asterisks represent reverse directionality within the cluster).

[0175] As with Gram-positives, the locus composition and architecture can vary between species.

[0176] In some Gram-negative organisms, such as Klebsiella, NifL is the negative regulator of the nif operon. When intracellular glutamine is high (nitrogen excess), NifL forms a repressor complex to inactivate the nif operon expression.

[0177] In some Gram-negative organisms, such as Azospirillum, NifA activates transcription of the nif operon. Expression of nifA is regulated by glutamine through ntrB phosphorylation of ntrC. Nitrogenase is inactivated pos-transcriptionally.

[0178] Other genes that may be involved in Nitrogen fixation, primarily in Gram-negative bacteria, include yggx, which reverses oxygen inactivation in Fe-S proteins. It is part of the SoxRS regulon (but does not need soxR to work). It is thought to protect cells or oxygen-labile enzymes from oxidation damage. Also included are nafll, which is involved in aerobic nitrogen fixation.Modification of Microbes for Imparting / Improving Nitrogen Fixation

[0179] In some embodiments, microbes that lack intrinsic nitrogen-fixation capability are genetically modified with one or more polynucleotides from a nitrogen-fixing strain, to gain the capacity for nitrogen fixation.

[0180] In some embodiments, microbes possessing intrinsic nitrogen-fixation capability are improved via genetic modification with one or more polynucleotides from a nitrogen-fixing strain, to increase the capacity for nitrogen fixation.

[0181] In some embodiments, the isolated, genetically modified microbes of the present disclosure are characterized as having constitutive expression of the / / / / gene. In some embodiments, the isolated, genetically modified microbes of the present disclosure are characterized as having constitutive expression of the / / / / gene regardless of local nitrogen or ammonia concentrations. For example, in certain embodiments, the isolated, genetically modified microbe is characterized as having constitutive expression of the nif gene underDocket # 24032-WO- PCT nitrogen-limiting conditions. In certain embodiments, the isolated, genetically modified microbe is characterized as having constitutive expression of the nif gene under nitrogen-abundant conditions. In certain embodiments, the isolated, genetically modified microbe is characterized as having constitutive expression of the nif gene under nitrogen-limiting or nitrogen-abundant conditions.

[0182] In one aspect, a first bacterium that lacks native nitrogen fixation capabilities is engineered to become a diazotroph by moving the minimum nitrogen fixing gene(s) and / or cluster(s) from a second bacterium required to generate ammonia from atmospheric nitrogen into said strain. In some cases, the first bacterium possesses some nitrogen fixation capabilities but is engineered with polynucleotides from a second bacterium to improve nitrogen fixation capabilities. The condition of lacking nitrogen fixation capability and / or having some level of capability that is not desired is termed lack of sufficient nitrogen fixation activity.

[0183] In some embodiments, the first bacterium is engineered (genetically modified) to comprise one or more polynucleotide(s) encoding polypeptide(s) involved in nitrogen fixation from a plurality of second bacterium sources, wherein at least one of the second bacterium sources are from a different taxonomic genus than the first bacterium. In some cases, the minimum necessary and sufficient gene(s) for nitrogen fixation are placed into the first bacterium. In some cases, nitrogen fixation gene(s) and / or cluster(s) are obtained from a plurality of different second bacteria, for example from different taxonomic species or genera from each other. In some cases, the gene(s) and / or cluster(s) from the plurality of second bacteria are nonidentical.

[0184] In some embodiments, the one or more polynucleotide(s) comprises an a / / / gene.

[0185] In some embodiments, the one or more polynucleotide(s) comprises a vnf gene.

[0186] In some embodiments, the one or more polynucleotide(s) comprises a nifH gene.

[0187] In some embodiments, the one or more polynucleotide(s) comprises a nifL gene.

[0188] In some embodiments, the one or more polynucleotide(s) comprises a nifA gene.

[0189] In some embodiments, the one or more polynucleotide(s) comprises a GlnR gene.

[0190] In some embodiments, the one or more polynucleotide(s) comprises a GlnR binding site.

[0191] In some embodiments, the one or more polynucleotide(s) comprises a cueR gene.

[0192] In some embodiments, the one or more polynucleotide(s) comprises a mer gene.Docket # 24032-WO- PCT

[0193] In some embodiments, the one or more polynucleotide(s) comprises nifB, nifE. nifl). nifK nifE, nifN, nifX, hesA, and nijV. In some cases, the one or more polynucleotides further comprises orfl.

[0194] In some embodiments, the genetic modification of the first bacterium includes one or more of the following: anf4 replicative plasmid insertion; anf5 replicative plasmid insertion; CueR C25 truncation; CueR C25 truncation & GlnR Binding Site II inactivation; CueR C25 truncation & GlnR Binding Site II inactivation and duplication; CueR C25 truncation, DS truncation and SNP, GlnR site II inactivation; CueR C25 truncation, GlnR site II inactivation; CueR Knockout; CueR knockout, GlnR Site II duplication and inactivation; CueR knockout, GlnR site II duplication and truncation; CueR Knockout, GlnR Site II inactivation; CueR knockout, GlnR site II truncation; CueR knockout, Nif PLH77 promoter insertion; CueR knockout, Nif PLH77 promoter insertion, NrgA knockout; CueR knockout, NrgA knockout; GlnR Binding Site II duplication & inactivation; GlnR Binding Site II duplication (into Site II region); GlnR Binding Site II inactivation; GlnR Binding Site II inactivation and duplication & NifH knockout; GlnR C25 frame shift truncation; GlnR C25 frame shift truncation and GlnA SNP; GlnR C25 frame shift truncation and GlnA SNP ; GlnR C25 frame shift truncation and GlnA SNP, NifH knockout; GlnR C25 frame shift truncation and GlnA SNP, Orfl Knockout; GlnR C25 frame shift truncation ; GlnR C25 frame shift truncation, GlnA SNP, GlnR Site II duplication; GlnR C25 frame shift truncation, GlnA SNP, GlnR Site II duplication ; GlnR C25 frame shift truncation, GlnA SNP, GlnR site II duplication and inactivation; GlnR C25 frame shift truncation, GlnA SNP, GlnR site II inactivation ; GlnR C25 frame shift truncation, GlnA SNP, NifH knockout; GlnR C25 frame shift truncation, GlnA SNP, Orfl Knockout; GlnR C25 frame shift truncation, Orfl knockout; GlnR C25 truncation; GlnR C25 truncation and GlnA SNP; GlnR C25 Truncation, GlnR Site II duplication; GlnR C25 Truncation, GlnR Site II inactivation; GlnR Knockout; GlnR Site II duplication; GlnR site II duplication and inactivation; GlnR site II duplication and inactivation, GlnR C25 truncation; GlnR site II duplication and inactivation, NrgA knockout; GlnR site II duplication and truncation; GlnR site II inactivation; GlnR Site II inactivation and duplication, NifH knockout; GlnR site II inactivation, GlnR C25 truncation; GlnR site II inactivation, NrgA knockout; GlnR site II truncation; GlnR site II truncation, GlnR C25 truncation; hesA2 replicative plasmid insertion; insertion of hesA intergenic region; insertion of nifl) intergenic region; insertion of nifE intergenic region; katADocket # 24032-WO- PCT replicative plasmid insertion; Nif PLH77 promoter insertion; Nif PLH77 promoter insertion, CueR KO; Nif PLH77 promoter insertion, GlnR C25 truncation; Nif PLH77 promoter insertion, NrgA knockout; Nif PLH77d promoter insertion , CueR KO; Nif PLH77d promoter insertion; Nif PLH77d promoter insertion, GlnR C25 truncation; nifl replicative plasmid insertion; nif2 replicative plasmid insertion; nif4 replicative plasmid insertion; nifB replicative plasmid insertion; NifH Knockout; NrgA knockout; NrgA knockout, GlnR C25 truncation; NrgA Knockout, GlnR site II truncation; NrgA knockout, Nif PLH77d promotor insertion; Orfl Knockout; Orfl knockout, GlnR C25 frame shift truncation; Orfl knockout, GlnR C25 truncation; orfl replicative plasmid insertion; Site II inactivation and duplication, NrgA Knockout; Site II inactivation, NrgA Knockout; sodA replicative plasmid insertion.

[0195] In some embodiments, the first bacterium is capable of colonizing the roots of a plant.

[0196] In some embodiments, the first bacterium is capable of colonizing a plant reproductive element.

[0197] In some embodiments, the first bacterium is capable of colonizing a root nodule.

[0198] In some embodiments, the first bacterium is capable of colonizing the shoot and / or leaf of a plant.

[0199] In some embodiments, the first bacterium is capable of producing a biofilm. In some cases, the biofilm provides a barrier to oxygen for the first bacterium.

[0200] In some embodiments, the first bacterium comprises higher than average levels of ATP, In one example, the higher than average levels of ATP result from higher than average respiration rates. In one example, the higher than average levels of ATP result from a genome modification. In one example, the higher than average levels of ATP result from down-regulated ATP spilling pathways.

[0201] In some embodiments, the first bacterium is an endophyte.

[0202] In some embodiments, the first bacterium is Gram-negative.

[0203] In some embodiments, the first bacterium is Gram-positive.

[0204] In some embodiments, the first bacterium is of the genus Bacillus.

[0205] In some embodiments, the first bacterium produces one or more composition that impact an insect, a fungus, and / or a nematode.Docket # 24032-WO- PCT

[0206] In some embodiments, the first bacterium produces one or more composition that impact plant growth, health, yield, potassium solubilization, phosphorous solubilization, drought tolerance, and / or water use efficiency.

[0207] In some embodiments, the second bacterium is Gram-negative.

[0208] In some embodiments, the second bacterium is Gram-positive.

[0209] In some embodiments, the second bacterium is of the genus Paenibacilh s, Gorillibacterium, Pseudomonas, Klebsiella, or Kosakonia.Microbial-produced Compositions

[0210] In some cases, the microbes of the present disclosure may produce one or more compounds and / or have one or more activities, e.g., one or more of the following: production of a metabolite, production of a phytohormone such as 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 protease or organic acid or lipopeptide or polynucleotide or polypeptide, nitrogen fixation, mineral phosphate solubilization, or any combination and / or plurality of the preceding.

[0211] For example, a microbe of the disclosure may produce a phytohormone selected from the group consisting of an auxin, a cytokinin, a gibberellin, ethylene, a brassinosteroid, and abscisic acid.

[0212] Thus, a “metabolite produced by” a microbe of the disclosure, is intended to capture any molecule (small molecule, vitamin, mineral, protein, nucleic acid, lipid, fat, carbohydrate, etc.) produced by the microbe. Often, the exact mechanism of action, whereby a microbe of the disclosure imparts a beneficial trait upon a given plant species is not known. It is hypothesized, that in some instances, the microbe is producing a metabolite that is beneficial to the plant. Thus, in some aspects, a cell-free or inactivated preparation of microbes is beneficial to a plant, as the microbe does not have to be alive to impart a beneficial trait upon the given plant species, so long as the preparation includes a metabolite that was produced by said microbe and which is beneficial to a plant.

[0213] Therefore, in an embodiment, the microbes of the disclosure are present as a population disposed on the surface or within a tissue of a given plant species. The microbes may produce a composition, such as a metabolite, in an amount effective to cause a detectable increase in theDocket # 24032-WO- PCT amount of composition that is found on or within the plant, when compared to a reference plant not treated with the microbes or cell-free or inactive preparations of the disclosure. The composition produced by said microbial population may be beneficial to the plant species.

[0214] Such microbial -produced compositions may be present in the cell culture broth or medium / a in which the microbes are grown, or may encompass an exudate produced by the microbes. As used herein, “exudate” refers to one or more compositions excreted by or extracted from one or more microbial cell(s). As used herein, “broth” refers to the collective composition of a cell culture medium after microbial cells are placed in the medium. The composition of the broth may change over time, during different phases of microbial growth and / or development. Broth and / or exudate may improve the traits of plants with which they become associated.Microbial-induced Traits in Plants

[0215] The present disclosure utilizes microbes to impart beneficial properties (or beneficial traits) to desirable plant species, such as agronomic species of interest. In the current disclosure, the terminology “beneficial property”, “beneficial trait”, or “trait of interest”, is used interchangeably and denotes that a desirable plant phenotypic or genetic property of interest is modulated, by the application of a microbe or microbial consortia as described herein. As aforementioned, in some aspects, it may very well be that a metabolite produced by a given microbe is ultimately responsible for modulating or imparting a beneficial trait to a given plant.

[0216] There are a vast number of beneficial traits that can be modulated by the application of microbes of the disclosure. For instance, the microbes may have the ability to impart one or more beneficial properties to a plant species, for example: increased growth, increased yield, increased nitrogen utilization efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, enhanced water use efficiency, increased pathogen resistance, modifications to plant architecture that don’t necessarily impact plant yield, but rather address plant functionality, causing the plant to increase production of a metabolite of interest, etc.

[0217] In aspects, the microbes taught herein provide a wide range of agricultural applications, including: improvements in yield of grain, fruit, and flowers, improvements in growth of plant parts, improved ability to utilize nutrients (e.g., nitrogen, phosphate, and the like), improved resistance to disease, biopesticidal effects including improved resistance to fungi, insects, and / or nematodes; improved survivability in extreme climate, and improvements in other desired plant phenotypic characteristics.Docket # 24032-WO- PCT

[0218] In some aspects, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to modulate or alter a plant characteristic such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, growth improvement, health enhancement, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved nutrient utilization (e.g., phosphate, potassium, and the like), improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutrition enhancement, pathogen resistance, reduced pathogen levels (e.g., via the excretion of compound(s) that impair pathogen survival), pest resistance, photosynthetic capability improvement, salinity tolerance, stay-green, vigor improvement, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of nonwilted leaves per plant, a detectable modulation in the level of a metabolite, a detectable modulation in the level of a transcript, and a detectable modulation in the proteome relative to a reference plant.

[0219] In some aspects, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to modulate in a negative way, a particular plant characteristic. For example, in some aspects, the microbes of the disclosure are able to decrease a phenotypic trait of interest, as this functionality can be desirable in some applications. For instance, the microbes of the disclosure may possess the ability to decrease root growth or decrease root length. Or the microbes may possess the ability to decrease shoot growth or decrease the speed at which a plant grows, as these modulations of a plant trait could be desirable in certain applications.

[0220] In some embodiments, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to impart nematode stress tolerance to plants.Docket # 24032-WO- PCT

[0221] In some embodiments, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to provide biostimulation (biostimulant effects) to plants. In some embodiments, the isolated microbes, consortia, and / or agricultural compositions of the disclosure can be applied to a plant, in order to provide disease tolerance to plants.Agricultural Compositions

[0222] In some embodiments, the microbes of the disclosure are combined with agricultural compositions. Agricultural compositions generally refer to organic and inorganic compounds that can include compositions that promote the cultivation of the microbe and / or the plant element; compositions involved in formulation of microbes for application to plant elements (for example, but not limited to: wetters, compatibilizing agents (also referred to as “compatibility agents”), antifoam agents, cleaning agents, sequestering agents, drift reduction agents, neutralizing agents and buffers, corrosion inhibitors, dyes, odorants, spreading agents (also referred to as “spreaders”), penetration aids (also referred to as “penetrants”), sticking agents (also referred to as “stickers” or “binders”), dispersing agents, thickening agents (also referred to as “thickeners”), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, and the like); compositions involved in conferring protection to the plant element or plant (for example, but not limited to: pesticides, nematicides, fungicides, bactericides, herbicides, and the like); as well as other compositions that may be of interest for the particular application.

[0223] In some embodiments, the agricultural compositions of the present disclosure are solid. Where solid compositions are used, it may be desired to include one or more carrier materials with the active isolated microbe or consortia. In some embodiments, the present disclosure teaches the use of carriers including, but not limited to: mineral earths such as silicas, silica gels, silicates, talc, kaolin, attaclay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, or compositions of these.Docket # 24032-WO- PCTGrowth Compositions

[0224] In some embodiments, a composition is provided to the microbe and / or the plant element that promotes the growth and development. Exemplary compositions include liquid (such as broth, media) and / or solid (such as soil, nutrients). Various organic or inorganic compounds may be added to the growth composition to facilitate the health of the microbe, alone or in combination with the plant element, for example but not limited to: amino acids, vitamins, minerals, carbohydrates, simple sugars, lipids.Formulation Compositions

[0225] One or more compositions, in addition to the microbe(s) or microbial-produced composition, may be combined for various application, stability, activity, and / or storage reasons. The additional compositions may be referred to as “formulation components”.

[0226] In some embodiments, the agricultural compositions disclosed herein may be formulated as a liquid, a solid, a gas, or a gel.

[0227] Thus in some embodiments, the present disclosure teaches that the agricultural compositions disclosed herein can include compounds or salts such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamate.

[0228] In some embodiments, the present disclosure teaches that agricultural compositions can include binders such as: polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone / vinyl acetate copolymers and polyvinyl acetate, or compositions of these; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or compositions of these; antifoams such as silicone emulsions, long-chain alcohols, phosphoric esters, acetylene 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 of these.

[0229] In some embodiments, the agricultural compositions comprise surface-active agents. In some embodiments, the surface-active agents are added to liquid agricultural compositions. InDocket # 24032-WO-PCT other embodiments, the surface-active agents are added to solid formulations, especially those designed to be diluted with a carrier before application. Thus, in some embodiments, the agricultural compositions comprise surfactants. Surfactants are sometimes used, either alone or with other additives, such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the microbes on the target. The types of surfactants used for bioenhancement depend generally on the nature and mode of action of the microbes. The surface-active agents can be anionic, cationic, or nonionic in character, and can be employed as emulsifying agents, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactants are non-ionics such as: alky ethoxylates, linear aliphatic alcohol ethoxylates, and aliphatic amine ethoxylates. Surfactants conventionally used in the art of formulation and which may also be used in the present formulations are described, in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, N.J., 1998, and in Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., New York, 1980- 81. In some embodiments, the present disclosure teaches the use of surfactants including alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids, for example, ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids of aryl sulfonates, of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether sulfates, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenesulfonic acids with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenols, salts of phosphated triarylphenolethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin-sulfite waste liquors or methylcellulose, or compositions of these.

[0230] In some embodiments, the present disclosure teaches other suitable surface-active agents, including salts of alkyl sulfates, such as diethanolammonium lauryl sulfate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C18 ethoxylate; alcohol -alkylene oxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonate salts, such as sodium dibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2-Docket # 24032-WO- PCT ethylhexyl)sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono and dialkyl phosphate esters; vegetable oils such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like; and esters of the above vegetable oils, particularly methyl esters.

[0231] In some embodiments, the agricultural compositions comprise wetting agents. A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank or other vessel to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. In some embodiments, examples of wetting agents used in the agricultural compositions of the present disclosure, including wettable powders, suspension concentrates, and water-dispersible granule formulations are: sodium lauryl sulphate; sodium dioctyl sulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.

[0232] In some embodiments, the agricultural compositions of the present disclosure comprise dispersing agents. A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. In some embodiments, dispersing agents are added to agricultural compositions of the present disclosure to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. In some embodiments, dispersing agents are used in wettable powders, suspension concentrates, and water-dispersible granules.Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to re-aggregation of particles. In some embodiments, the most commonly used surfactants are anionic, non-ionic, or mixtures of the two types.Docket # 24032-WO- PCT

[0233] In some embodiments, for wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. In some embodiments, suspension concentrates provide very good adsorption and stabilization using polyelectrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. In some embodiments, tri styrylphenol ethoxylate phosphate esters are also used. In some embodiments, such as alkyl aryl ethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates.

[0234] In some embodiments, the agricultural compositions of the present disclosure comprise polymeric surfactants. In some embodiments, the polymeric surfactants have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. In some embodiments, these high molecular weight polymers can give very good long-term stability to suspension concentrates, because the hydrophobic backbones have many anchoring points onto the particle surfaces. In some embodiments, examples of dispersing agents used in agricultural compositions of the present disclosure are: sodium lignosulphonates; sodium naphthalene sulphonate formaldehyde condensates; tri styrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alky ethoxylates; EO-PO block copolymers; and graft copolymers.

[0235] In some embodiments, the agricultural compositions of the present disclosure comprise emulsifying agents. An emulsifying agent is a substance, which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.

[0236] In some embodiments, the agricultural compositions of the present disclosure comprise solubilizing agents. A solubilizing agent is a surfactant, which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The typesDocket # 24032-WO- PCT of surfactants usually used for solubilization are non-ionics: sorbitan monooleates; sorbitan monooleate ethoxylates; and methyl oleate esters.

[0237] In some embodiments, the agricultural compositions of the present disclosure comprise organic solvents. Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffinic oils such as kerosene or refined paraffins. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and CIO aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as co-solvents to prevent crystallization of pesticides when the formulation is emulsified into water. Alcohols are sometimes used as co-solvents to increase solvent power.

[0238] In some embodiments, the agricultural compositions comprise gelling agents. Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. In some embodiments, the agricultural compositions comprise one or more thickeners 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 types of polysaccharides most commonly used 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 are not limited to: guar gum; locust bean gum; carrageenan; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0239] In some embodiments, the presence of surfactants, which lower interfacial tension, can cause water-based formulations to foam during mixing operations in production and inDocket # 24032-WO- PCT application through a spray tank. Thus, in some embodiments, in order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before fdling into bottles / spray tanks. Generally, there are two types of anti -foam agents, namely silicones and nonsilicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane, while the nonsilicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.

[0240] In some embodiments, the agricultural compositions comprise a preservative.

[0241] In some embodiments, the agricultural compositions may be formulated as: a soil drench, a foliar spray, a dip treatment, an in-furrow treatment, a soil amendment, granules, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural compositions may be applied alone in or in rotation spray programs with other agricultural products.

[0242] 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.

[0243] In some embodiments, the agricultural compositions may be applied to genetically modified seeds or plants.Protective Compositions

[0244] Further, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known actives available in the agricultural space, such as: pesticide, herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, plant growth regulator, rodenticide, anti-algae agent, biocontrol or beneficial agent. Further, the microbes, microbial consortia, or microbial communities developed according to the disclosed methods can be combined with known fertilizers. Such combinations may exhibit synergistic properties. Further still, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with inert ingredients. Also, in some aspects, the disclosed microbes are combined with biological active agents.Docket # 24032-WO- PCT

[0245] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with biopesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent. Such biopesticides may be, but are not limited to, macrobial organisms (e.g., beneficial nematodes and the like), microbial organisms (e.g., Serenade, Bt, and the like), plant extracts (e.g., Timorex Gold and the like), biochemical (e.g., insect pheromones and the like), and / or minerals and oils (e.g., canola oil).Pesticides and Biopesticides

[0246] In some embodiments, the agricultural compositions of the present disclosure comprise pesticides, used in combination with the taught microbes. In some embodiments, the agricultural compositions of the present disclosure comprise biopesticides, used in combination with the taught microbes.

[0247] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known pesticides in the agricultural space, such as: pesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0248] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known biopesticides in the agricultural space, such as: biopesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0249] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients including: macrobial organisms (e.g., beneficial nematodes and the like), microbial organisms (e.g., Serenade, Bt, and the like), plant extracts (e.g., Timorex Gold and the like), biochemical (e.g., insect pheromones and the like), and / or minerals and oils (e.g., canola oil).

[0250] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with an herbicide selected from the group consisting of: an acetamide selected from the group consisting of acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, mefenacet, metolachlor,Docket # 24032-WO- PCT metazachlor, napropamide, naproanilide, pethoxamid, pretilachlor, propachlor, and thenyl chi or; an amino acid derivative selected from the group consisting of bilanafos, glufosinate, and sulfosate; an aryloxyphenoxypropionate selected from the group consisting of clodinafop, cyhalofop-butyl, fenoxaprop, fluazifop, haloxyfop, metamifop, propaquizafop, quizalofop, and quizalo-fop-P-tefuryl; diquat and paraquat; a (thio)carbamate selected from the group consisting of asulam, butylate, carbetamide, desmedipham, dimepiperate, eptam (EPTC), esprocarb, molinate, orbencarb, phenmedipham, prosulfocarb, pyributicarb, thiobencarb, and triallate; a cyclohexanedione selected from the group consisting of butroxydim, cl ethodim, cycl oxydim, profoxydim, sethoxydim, tepraloxydim, and tralkoxydim; a dinitroaniline selected from the group consisting of benfluralin, ethalfluralin, oryzalin, pendimethalin, prodiamine, and trifluralin; a diphenyl ether selected from the group consisting of acifluorfen, aclonz / en, bifenox, diclofop, ethoxyfen, fomesafen, lactofen, and oxyfluorfen; a hydroxybenzonitrile selected from the group consisting of bomoxynil, dichlobenil, and ioxynil; an imidazolinone selected from the group consisting of imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, and imazethapyr; a phenoxy acetic acid selected from the group consisting of clomeprop, 2,4- dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dichlorprop, MCPA, MCPA-thioethyl, MCPB, and Mecoprop; a pyrazine selected from the group consisting of chloridazon, flufenpyr-ethyl, fluthiacet, norflurazon, and pyridate; a pyridine selected from the group consisting of aminopyralid, clopyralid, diflufenican, dithiopyr, fluridone, fluroxypyr, picloram, picolinafen, and thiazopyr; a sulfonyl urea selected from the group consisting of amidosulfuron, azimsulfuron, bensulfuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron, triflusulfuron, tritosulfuron, and 14(2- chloro-6-propyl-imidazol[l,2]-blpyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea; a triazine selected from the group consisting of ametryn, atrazine, cyanazine, a dimethametryn, ethiozin, hexazinone, metamitron, metribuzin, prometryn, simazine, terbuthylazine, terbutryn, and triaziflam; a urea compound selected from the group consisting of chlorotoluron, daimuron, diuron, fluometuron, isoproturon, linuron, methabenzthiazuron, and tebuthiuron; an acetolactate synthase inhibitor selected from the group consisting of bispyribac-sodium, cloransulam-methyl,Docket # 24032-WO- PCT diclosulam, florasulam, flucarbazone, flumetsulam, metosulam, ortho-sulfamuron, penoxsulam, propoxycarbazone, pyribambenz-propyl, pyribenzoxim, pyriftalid, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyroxasulfone, and pyroxsulam; and a compound selected from the group consisting of amicarbazone, aminotriazole, anilofos, beflubutamid, benazolin, bencarbazone, benfluresate, benzofenap, bentazone, benzobicyclon, bromacil, bromobutide, butafenacil, butamifos, cafenstrole, carfentrazone, cinidon-ethlyl, chlorthal, cinmethylin, clomazone, cumyluron, cyprosulfamide, dicamba, difenzoquat, diflufenzopyr, Drechslera monoceras, endothal, ethofumesate, etobenzanid, fentrazamide, flumiclorac-pentyl, flumioxazin, flupoxam, flurochloridone, flurtamone, indanofan, isoxaben, isoxaflutole, lenacil, propanil, propyzamide, quinclorac, quinmerac, mesotrione, methyl arsonic acid, naptalam, oxadiargyl, oxadiazon, oxaziclomefone, pentoxazone, pinoxaden, pyraclonil, pyraflufen-ethyl, pyrasulfotole, pyrazoxyfen, pyrazolynate, quinoclamine, saflufenacil, sulcotrione, sulfentrazone, terbacil, tefuryltrione, tembotrione, thiencarb azone, topram ezone, 4-hydroxy-3 -[2-(2-m ethoxy - ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-bicyclol[3.2.1]oct-3-en-2-one, (3-[2- chloro-4-fluoro-5 -(3 -methyl-2, 6 -di oxo-4-trifluorom ethyl -3 , 6-dihydro-2H-pyrimidin- 1 -y 1)- 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.

[0251] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with an insecticide selected from the group consisting of: an organo(thio)phosphate selected from the group consisting of acephate, azamethiphos, azinphos-methyl, chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, diazinon, dichlorvos, dicrotophos, dimethoate, disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion, methamidophos, methidathion, methyl-parathion, mevinphos, monocrotophos, oxydemeton-methyl, paraoxon, parathion, phenthoate, phosalone, phosmet, phosphamidon, phorate, phoxim, pirimiphos-methyl, profenofos, prothiofos, sulprophos, tetrachlorvinphos, terbufos, triazophos, and trichlorfon; a carbamate selected from the group consisting of alanycarb, aldicarb, bendiocarb, benfuracarb, carbaryl, carbofuran, carbosulfan, fenoxycarb, furathiocarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb, andDocket # 24032-WO- PCT triazamate; a pyrethroid selected from the group consisting of allethrin, bifenthrin, cyfluthrin, cyhalothrin, cyphenothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta- cypermethrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, imiprothrin, lambda-cyhalothrin, permethrin, prallethrin, pyrethrin I and II, resmethrin, silafluofen, taufluvalinate, tefluthrin, tetramethrin, tralomethrin, transfluthrin, profluthrin, and dimefluthrin; an insect growth regulator selected from the group consisting of a) a chitin synthesis inhibitor wherein said chitin synthesis inhibitor is a benzoylurea selected from the group consisting of chlorfluazuron, cyramazin, diflub enzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, teflubenzuron, triflumuron; buprofezin, diofenolan, hexythiazox, etoxazole, and clofentazine; b) an ecdysone antagonist selected from the group consisting of halofenozide, methoxyfenozide, tebufenozide, and azadirachtin; c) a juvenoid 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 l-(2-chloro-thiazol-5-ylmethyl)-2-nitrimino-3,5-dimethyl-[l,3,5]triazinane; a GABA antagonist compound selected from the group consisting of endosulfan, ethiprole, fipronil, vaniliprole, pyrafluprole, pyriprole, and 5-amino-l-(2,6-dichloro-4-methyl-phenyl)-4-sulfinamoyl-lH- pyrazole-3-c arbothioic acid amide; a macrocyclic lactone insecticide selected from the group consisting of abamectin, emamectin, milbemectin, lepimectin, spinosad, and spinetoram; a mitochondrial electron transport inhibitor (METI) I acaricide selected from the group consisting of fenazaquin, pyridaben, tebufenpyrad, tolfenpyrad, and flufenerim; a METI II and III compound selected from the group consisting of acequinocyl, fluacyprim, and hydramethylnon; chlorfenapyr; an oxidative phosphorylation inhibitor selected from the group consisting of cyhexatin, diafenthiuron, fenbutatin oxide, and propargite; cryomazine; piperonyl butoxide; a sodium channel blocker selected from the group consisting of indoxacarb and metaflumizone; and a compound selected from the group consisting of benclothiaz, bifenazate, cartap, flonicamid, pyridalyl, pymetrozine, sulfur, thiocyclam, flubendiamide, chlorantraniliprole, cyazypyr (HGW86), cyenopyrafen, flupyrazofos, cyflumetofen, amidoflumet, imicyafos, bistrifluron, and pyrifluquinazon.Docket # 24032-WO- PCT

[0252] In some embodiments, the present invention teaches a synergistic use of the presently disclosed microbes or microbial consortia with known pesticides in the agricultural space, such as: pesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0253] In some embodiments, the present invention teaches a synergistic use of the presently disclosed microbes or microbial consortia with known biopesticides in the agricultural space, such as: biopesticides that function as an herbicide, bactericide, fungicide, insecticide, virucide, miticide, nematicide, acaricide, rodenticide, and / or anti-algae agent.

[0254] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a pesticide one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a pesticide one witness a synergistic effect on a plant phenotypic trait of interest.

[0255] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a biopesticide one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a biopesticide one witnesses a synergistic effect on a plant phenotypic trait of interest.

[0256] The synergistic effect obtained by the taught methods can be quantified according to Colby’s formula (i.e., (E) =X+Y-(X*Y / 100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount.

[0257] The isolated microbes and consortia of the present disclosure can synergistically increase the effectiveness of agriculturally active pesticide compounds and also agricultural auxiliary pesticide compounds.

[0258] The isolated microbes and consortia of the present disclosure can synergistically increase the effectiveness of agriculturally active biopesticide compounds and also agricultural auxiliary biopesticide compounds.Docket # 24032-WO- PCTPlant Growth Regulators and Biostimulants

[0259] In some embodiments, the agricultural compositions of the present disclosure comprise plant growth regulators and / or biostimulants, used in combination with the taught microbes.

[0260] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with known plant growth regulators in the agricultural space, such as: auxins, gibberellins, cytokinins, ethylene generators, growth inhibitors, and growth retardants.

[0261] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients including: ancymidol, butralin, alcohols, chloromequat chloride, cytokinin, daminozide, ethepohon, flurprimidol, giberrelic acid, gibberellin mixtures, indole-3 -butryic acid (IB A), maleic hydrazide, mefludide, mepiquat chloride, mepiquat pentaborate, naphthalene-acetic acid (NAA), 1- napthaleneacetemide, (NAD), n-decanol, placlobutrazol, prohexadione calcium, trinexapac-ethyl, uniconazole, salicylic acid, abscisic acid, ethylene, brassinosteroids, jasmonates, polyamines, nitric oxide, strigolactones, or karrikins among others.

[0262] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with seed inoculants known 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 microbe or microbial consortia disclosed here. In particular aspects, a synergistic effect is observed when one combines one of the aforementioned inoculants, e.g., QUICKROOTS® ’ or Bradyrhizobium, with a microbe or microbial consortia as taught herein.

[0263] In some embodiments, the agricultural compositions of the present disclosure comprise a plant growth regulator, which contains: kinetin, gibberellic acid, and indole butyric acid, along with copper, manganese, and zinc.

[0264] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available plant growth regulators, 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®, Happy gro®, Incite®, Megagro®, Ascend®, Radiate®, Stimulate®,Docket # 24032-WO- PCTSuppress®, 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®, PGR IV®, 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®, Antae®, FST-7®, Royaltac®, Bonzi®, Cambistat®, Cutdown®, Downsize®, Florazol®, Paclo®, Paczol®, Piccolo®, Profile®, Shortstop®, Trimmit®, Turf Enhancer®, Apogee®, Armor Tech®, Goldwing®, Governor®, Groom®, Legacy®, Primeraone®, Primo®, Provair®, Solace®, T-Nex®, T-Pac®, Concise®, and Sumagic®.

[0265] In some embodiments, the present invention teaches a synergistic use of the presently disclosed microbes or microbial consortia with plant growth regulators and / or stimulants such as phytohormones or chemicals that influence the production or disruption of plant growth regulators.

[0266] In some embodiments, the present invention teaches that phytohormones can include: Auxins (e.g., Indole acetic acid IAA), Gibberellins, Cytokinins (e.g., Kinetin), Abscisic acid, Ethylene (and its production as regulated by ACC synthase and disrupted by ACC deaminase).

[0267] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with biostimulants. Such biostimulants may be, but are not limited to, microbial organisms, plant extracts, seaweeds, acids, biochar, and the like.

[0268] In some embodiments, the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with fertilizers, which may be organic (e.g., manure, blood, fish, and the like), nitrogen-based (e.g., nitrate, ammonium, urea, and the like), phosphate, and potassium. Such fertilizers may also contain micronutrients including, but not limited to, sulfur, iron, zinc, and the like.

[0269] In some embodiments, the present invention teaches additional plant-growth promoting chemicals that may act in synergy with the microbes and microbial consortia disclosed herein, such as: humic acids, fulvic acids, amino acids, polyphenols and protein hydrolysates.Docket # 24032-WO- PCT

[0270] Thus, in some embodiments, the disclosure provides for the application of the taught microbes in combination with Ascend® upon any crop. Further, the disclosure provides for the application of the taught microbes in combination with Ascend® upon any crop and utilizing any method or application rate.

[0271] In some embodiments, the present disclosure teaches agricultural compositions with biostimulants.

[0272] As used herein, the term “biostimulanf ’ refers to any substance that acts to stimulate the growth of microorganisms that may be present in soil or other plant growing medium.

[0273] The level of microorganisms in the soil or growing medium is directly correlated to plant health. Microorganisms feed on biodegradable carbon sources, and therefore plant health is also correlated with the quantity of organic matter in the soil. While fertilizers provide nutrients to feed and grow plants, in some embodiments, biostimulants provide biodegradable carbon, e.g., molasses, carbohydrates, e.g., sugars, to feed and grow microorganisms. Unless clearly stated otherwise, a biostimulant may comprise a single ingredient, or a combination of several different ingredients, capable of enhancing microbial activity or plant growth and development, due to the effect of one or more of the ingredients, either acting independently or in combination.

[0274] In some embodiments, biostimulants are compounds that produce non-nutritional plant growth responses. In some embodiments, many important benefits of biostimulants are based on their ability to influence hormonal activity. Hormones in plants (phytohormones) are chemical messengers regulating normal plant development as well as responses to the environment. Root and shoot growth, as well as other growth responses are regulated by phytohormones. In some embodiments, compounds in biostimulants can alter the hormonal status of a plant and exert large influences over its growth and health. Thus, in some embodiments, the present disclosure teaches sea kelp, humic acids, fulvic acids, and B Vitamins as common components of biostimulants. In some embodiments, the biostimulants of the present disclosure enhance antioxidant activity, which increases the plant's defensive system. In some embodiments, vitamin C, vitamin E, and amino acids such as glycine are antioxidants contained in biostimulants.

[0275] In other embodiments, biostimulants may act to stimulate the growth of microorganisms that are present in soil or other plant growing medium. Prior studies have shown that when certain biostimulants comprising specific organic seed extracts e.g., soybean) were used in combination with a microbial inoculant, the biostimulants were capable of stimulating growth ofDocket # 24032-WO- PCT microbes included in the microbial inoculant. Thus, in some embodiments, the present disclosure teaches one or more biostimulants that, when used with a microbial inoculant, is capable of enhancing the population of both native microbes and inoculant microbes. For a review of some popular uses of biostimulants, please see Calvo et al., 2014, Plant Soil 383:3-41.Combinations of Plant Elements, Microbes, and Agricultural Compositions

[0276] In some embodiments, the present disclosure teaches that the individual microbes, or microbial consortia, or microbial communities, or any combination of the preceding, for example comprising any one or a plurality of microorganisms that include a edited Paenibacillus strain described herein, may be applied to a plant element, optionally in combination with any agricultural composition, for the improvement of a plant phenotype.

[0277] Isolated microbes or communities or consortia (generally “microbes” or “microbe”, interchangeably) may be applied to a heterologous plant element, creating a synthetic combination. Microbes are considered heterologous to a plant element if they are not normally associated with the plant element in nature, or if found, are applied in amounts different than that found in nature. In some embodiments, the microbes may be found naturally in one part of a plant but not another, and introduction of the microbes to another part of the plant is considered a heterologous association.

[0278] It is further contemplated that the microbe, either isolated or in combination with a plant or plant element, may be further associated with one or more agricultural compositions, such as those described above.

[0279] Synthetic combinations of microbes and plant elements, microbes and agricultural compositions, and microbes and plant elements and agricultural compositions are contemplated (generally “synthetic compositions”, compositions that comprise components not typically found associated in nature).Plant Element Treatments

[0280] In some embodiments, the present disclosure also concerns the discovery that treating plant elements before they are sown or planted with a combination of one or more of the microbes or agricultural compositions of the present disclosure can enhance a desired plant trait, e.g., plant growth, plant health, and / or plant resistance to pests.

[0281] Thus, in some embodiments, the present disclosure teaches the use of one or more of the microbes or microbial consortia as plant element treatments. The plant element treatment can beDocket # 24032-WO- PCT a plant element coating applied directly to an untreated and “naked” plant element. However, the plant element treatment can be a plant element overcoat that is applied to a plant element that has already been coated with one or more previous plant element coatings or plant element treatments. The previous plant element treatments may include one or more active compounds, either chemical or biological, and one or more inert ingredients.

[0282] The term “plant element treatment” generally refers to application of a material to a plant element prior to or during the time it is planted in soil. Plant element treatment with microbes, and other agricultural compositions of the present disclosure, has the advantages of delivering the treatments to the locus at which the plant elements are planted shortly before germination of the plant element and emergence of a plant element.

[0283] In other embodiments, the present disclosure also teaches that the use of plant element treatments minimizes the amount of microbe or agricultural composition that is required to successfully treat the plants, and further limits the amount of contact of workers with the microbes and compositions compared to application techniques such as spraying over soil or over emerging plant element.

[0284] Moreover, in some embodiments, the present disclosure teaches that the microbes disclosed herein are important for enhancing the early stages of plant life (e.g., within the first thirty days following emergence of the plant element). Thus, in some embodiments, delivery of the microbes and / or compositions of the present disclosure as a plant element treatment places the microbe at the locus of action at a critical time for its activity.

[0285] In some embodiments, the microbial compositions of the present disclosure are formulated as a plant element treatment. In some embodiments, it is contemplated that the plant elements can be substantially um / ormly coated with one or more layers of the microbes and / or agricultural compositions disclosed herein, using conventional methods of mixing, spraying, or a combination thereof through the use of treatment application equipment that is specifically designed and manufactured to accurately, safely, and efficiently apply plant element treatment products to plant elements. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists, or a combination thereof. Liquid plant element treatments such as those of the present disclosure can be applied via either a spinning “atomizer” disk or a spray nozzle, which evenly distributes the plant element treatment onto the plant element as it moves though the spray pattern. In aspects,Docket # 24032-WO- PCT the plant element is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying.

[0286] The plant elements can be primed or unprimed before coating with the microbial compositions to increase the u / y' / ormity of germination and emergence. In an alternative embodiment, a dry powder formulation can be metered onto the moving plant element and allowed to mix until completely distributed.

[0287] In some embodiments, the plant elements have at least part of the surface area coated with a microbiological composition, according to the present disclosure. In some embodiments, a plant element coat comprising the microbial composition is applied directly to a naked plant element. In some embodiments, a plant element overcoat comprising the microbial composition is applied to a plant element that already has a plant element coat applied thereon. In some aspects, the plant element may have a plant element coat comprising, e.g., clothianidin and / or Bacillus firmus-I-1582, upon which the present composition will be applied on top of, as a plant element overcoat. In some aspects, the taught microbial compositions are applied as a plant element overcoat to plant elements that have already been treated with PONCHO™ VOTiVO™. In some aspects, the plant element may have a plant element coat comprising, e.g., Metalaxyl, and / or clothianidin, and / or Bacillus firmus-I-1582, upon which the present composition will be applied on top of, as a plant element overcoat. In some aspects, the taught microbial compositions are applied as a plant element overcoat to plant elements that have already been treated with ACCELERON™.

[0288] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A2 to 10A12, 10A2 to 10Al 1, 10A2 to 10A10, 10A2 to 10A9, lA02 to 10A8, 10A2 to 10A7, 10A2 to 10A6, 10A2 to 10A5, 10A2 to 10A4, or 10A2 to 10A3 per plant element.

[0289] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A3 to 10A12, 10A3 to 10Al 1, 10A3 to 10Al 0, 10A3 to 10A9, 10A3 to 10A8, 10A3 to 10A7, 10A3 to 10A6, 10A3 to 10A5, or 10A3 to 10A4 per plant element.

[0290] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A4 to 10A12, 10A4 to 10Al 1, 10A4Docket # 24032-WO- PCT to 10Al 0, 10A4 to 10A9, 10A4 to 10A8, 10A4 to 10A7, 10A4 to 10A6, or 10A4 to 10A5 per plant element.

[0291] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A5 to 10A12, 10A5 to 10Al 1, 10A5 to 10Al 0, 10A5 to 10A9, 10A5 to 10A8, 10A5 to 10A7, or 10A5 to 10A6 per plant element.

[0292] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, from about: 10A5 to 10A9 per plant element.

[0293] In some embodiments, the microorganism-treated plant elements have a microbial spore concentration, or microbial cell concentration, of at least about: 1 x 10A3, or 1 x 10A4, or 1 x 10A5, or 1 x 10A6, or 1 x 10A7, or 1 x 10A8, or 1 x 10A9 per plant element.

[0294] In some embodiments, the amount of one or more of the microbes and / or agricultural compositions applied to the plant element depend on the final formulation, as well as size or type of the plant or plant element utilized. In some embodiments, one or more of the microbes are present in about 2% w / w / to about 80% w / w of the entire formulation. In some embodiments, the one or more of the microbes employed in the compositions is about 5% w / w to about 65% w / w, or 10% w / w to about 60% w / w by weight of the entire formulation.

[0295] In some embodiments, the plant elements may also have more spores or microbial cells per plant element, such as, for example about 10A2, 10A3, 10A4, 10A5, 10A6, 10A7, 10A8, 10A9, 10A10, 10Al l, 10A12, 10A13, 10A14, 10A15, 10A16, or 10A17 spores or cells per plant element.

[0296] In some embodiments, the plant element coats of the present disclosure can be up to 10pm, 20pm, 30pm, 40pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 110pm, 120pm, 130pm, 140pm, 150pm, 160pm, 170pm, 180pm, 190pm, 200pm, 210pm, 220pm, 230pm, 240pm, 250pm, 260pm, 270pm, 280pm, 290pm, 300pm, 310pm, 320pm, 330pm, 340pm, 350pm, 360pm, 370pm, 380pm, 390pm, 400pm, 410pm, 420pm, 430pm, 440pm, 450pm, 460pm, 470pm, 480pm, 490pm, 500pm, 510pm, 520pm, 530pm, 540pm, 550pm, 560pm, 570pm, 580pm, 590pm, 600pm, 610pm, 620pm, 630pm, 640pm, 650pm, 660pm, 670pm, 680pm, 690pm, 700pm, 710pm, 720pm, 730pm, 740pm, 750pm, 760pm, 770pm, 780pm, 790pm, 800pm, 810pm, 820pm, 830pm, 840pm, 850pm, 860pm, 870pm, 880pm, 890pm, 900pm, 910pm, 920pm, 930pm, 940pm, 950pm, 960pm, 970pm, 980pm, 990pm, 1000pm, 1010pm, 1020pm, 1030pm, 1040pm, 1050pm, 1060pm, 1070pm, 1080pm, 1090pm, 1100pm, 1110pm, 1120pm, 1130pm, 1140pm, 1150pm, 1160pm, 1170pm, 1180pm, 1190pm, 1200pm, 1210pm,Docket # 24032-WO- PCT1220pm, 1230pm, 1240pm, 1250pm, 1260pm, 1270pm, 1280pm, 1290pm, 1300pm, 1310pm, 1320pm, 1330pm, 1340pm, 1350pm, 1360pm, 1370pm, 1380pm, 1390pm, 1400pm, 1410pm, 1420pm, 1430pm, 1440pm, 1450pm, 1460pm, 1470pm, 1480pm, 1490pm, 1500pm, 1510pm, 1520pm, 1530pm, 1540pm, 1550pm, 1560pm, 1570pm, 1580pm, 1590pm, 1600pm, 1610pm, 1620pm, 1630pm, 1640pm, 1650pm, 1660pm, 1670pm, 394000pm, 1690pm, 1700pm, 1710pm, 1720pm, 1730pm, 1740pm, 1750pm, 1760pm, 1770pm, 1780pm, 1790pm, 1800pm, 1810pm, 1820pm, 1830pm, 1840pm, 1850pm, 1860pm, 1870pm, 1880pm, 1890pm, 1900pm, 1910pm, 1920pm, 1930pm, 1940pm, 1950pm, 1960pm, 1970pm, 1980pm, 1990pm, 2000pm, 2010pm, 2020pm, 2030pm, 2040pm, 2050pm, 2060pm, 2070pm, 2080pm, 2090pm, 2100pm, 2110pm, 2120pm, 2130pm, 2140pm, 2150pm, 2160pm, 2170pm, 2180pm, 2190pm, 2200pm, 2210pm, 2220pm, 2230pm, 2240pm, 2250pm, 2260pm, 2270pm, 2280pm, 2290pm, 2300pm, 2310pm, 2320pm, 2330pm, 2340pm, 2350pm, 2360pm, 2370pm, 2380pm, 2390pm, 2400pm, 2410pm, 2420pm, 2430pm, 2440pm, 2450pm, 2460pm, 2470pm, 2480pm, 2490pm, 2500pm, 2510pm, 2520pm, 2530pm, 2540pm, 2550pm, 2560pm, 2570pm, 2580pm, 2590pm, 2600pm, 2610pm, 2620pm, 2630pm, 2640pm, 2650pm, 2660pm, 2670pm, 2680pm, 2690pm, 2700pm, 2710pm, 2720pm, 2730pm, 2740pm, 2750pm, 2760pm, 2770pm, 2780pm, 2790pm, 2800pm, 2810pm, 2820pm, 2830pm, 2840pm, 2850pm, 2860pm, 2870pm, 2880pm, 2890pm, 2900pm, 2910pm, 2920pm, 2930pm, 2940pm, 2950pm, 2960pm, 2970pm, 2980pm, 2990pm, or 3000pm thick.

[0297] In some embodiments, the plant element coats of the present disclosure can be 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, or 5mm thick.

[0298] In some embodiments, the plant element coats of the present disclosure can be at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%,23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%,30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%,37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%,44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, or 50% of the uncoated plant element weight.Docket # 24032-WO- PCT

[0299] In some embodiments, the microbial spores and / or cells can be coated freely onto the plant elements or they can be formulated in a liquid or solid composition before being coated onto the plant elements. For example, a solid composition comprising the microorganisms can be prepared by mixing a solid carrier with a suspension of the spores until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

[0300] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents e.g., activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the products or a combination thereof.

[0301] Plant element coating methods and compositions that are known in the art can be particularly useful when they are modified by the addition of one of the embodiments of the present disclosure. Such coating methods and apparatus for their application are disclosed in, for example: U.S. Pat. Nos. 5,916,029; 5,918,413; 5,554,445; 5,389,399; 4,759,945; 4,465,017, and U.S. Pat. App. NO 13 / 260,310.

[0302] Plant element coating compositions are disclosed in, for example: U.S. Pat. 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.

[0303] In some embodiments, a variety of additives can be added to the plant element treatment formulations comprising the inventive compositions. Binders can be added and include those composed of an adhesive polymer that can be natural or synthetic without phytotoxic effect on the plant element to be coated. The binder may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.Docket # 24032-WO- PCT

[0304] Any of a variety of colorants may be employed, including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

[0305] A polymer or other dust control agent can be applied to retain the treatment on the plant element surface.

[0306] In some specific embodiments, in addition to the microbial cells or spores, the coating can further comprise a layer of adherent. The adherent should be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, such as methyl celluloses, hydroxymethyl celluloses, and hydroxymethyl propyl celluloses; dextrins; alginates; sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins; fats; oils; gum arabics; gelatins; syrups; and starches. More examples can be found in, for example, U.S. Pat. NO 7,213,367, incorporated herein by reference.

[0307] Various additives, such as adherents, dispersants, surfactants, and nutrient and buffer ingredients, can also be included in the plant element treatment formulation. Other conventional plant element treatment additives 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 formulation such as water, solids, or dry powders. The dry powders can be derived from a variety of materials such as calcium carbonate, gypsum, vermiculite, talc, humus, activated charcoal, and various phosphorous compounds.

[0308] In some embodiments, the plant element coating composition can comprise at least one filler, which is an organic or inorganic, natural or synthetic component with which the active components are combined to facilitate its application onto the plant element. In aspects, the filler is an inert solid such as clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earths, or synthetic minerals, such as silica, alumina or silicates, in particular aluminum or magnesium silicates.

[0309] In some embodiments, the plant element treatment formulation may further include one or more of the following ingredients: other pesticides, including compounds that act only belowDocket # 24032-WO- PCT the ground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of those materials, and the like; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such as benzoxazine, benzhydryl derivatives, N,N-diallyl di chloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; chemical fertilizers; biological fertilizers; and biocontrol agents such as other naturally-occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These ingredients 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 disclosure.

[0310] In some embodiments, the formulation that is used to treat the plant element in the present disclosure can be in the form of a suspension; emulsion; slurry of particles in an aqueous medium (e.g., water); wettable powder; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation can be about 0.5% to about 99% by weight (w / w), or 5-40%, or as otherwise formulated by those skilled in the art.

[0311] As mentioned above, other conventional inactive or inert ingredients can be incorporated into the formulation. Such inert ingredients include, but are not limited to: conventional sticking agents; dispersing agents such as methylcellulose, for example, serve as combined di spersant / sti eking agents for use in plant element treatments; 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, and the like. Further inert ingredients useful in the present disclosure can be found in McCutcheon’s, vol. 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.

[0312] The plant element coating formulations of the present disclosure can be applied to plant elements by a variety of methods, including, but not limited to: mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert material can be used for contacting plant elements with microbial compositions according to the present disclosure.Docket # 24032-WO- PCT

[0313] In some embodiments, the amount of the microbes or agricultural composition that is used for the treatment of the plant element will vary depending upon the type of plant element and the type of active ingredients, but the treatment will comprise contacting the plant elements with an agriculturally effective amount of the inventive composition.

[0314] As discussed above, an effective amount means that amount of the inventive composition that is sufficient to affect beneficial or desired results. An effective amount can be administered in one or more administrations.

[0315] In some embodiments, in addition to the coating layer, the plant element may be treated with one or more of the following ingredients: other pesticides including fungicides and herbicides; herbicidal safeners; fertilizers and / or biocontrol agents. These ingredients may be added as a separate layer or alternatively may be added in the coating layer.

[0316] In some embodiments, the plant element coating formulations of the present disclosure may be applied to the plant elements using a variety of techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic plant element treaters, and drum coaters. Other methods, such as spouted beds may also be useful. The plant elements may be presized before coating. After coating, the plant elements are typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.

[0317] In some embodiments, the microorganism-treated plant elements may also be enveloped with a fdm overcoating to protect the coating. Such overcoatings are known in the art and may be applied using fluidized bed and drum fdm coating techniques.

[0318] In other embodiments of the present disclosure, compositions according to the present disclosure can be introduced onto a plant element by use of solid matrix priming. For example, a quantity of an inventive composition can be mixed with a solid matrix material and then the plant element can be placed into contact with the solid matrix material for a period to allow the composition to be introduced to the plant element. The plant element can then optionally be separated from the solid matrix material and stored or used, or the mixture of solid matrix material plus plant element can be stored or planted directly. Solid matrix materials which are 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 inventive composition for a time and releasing that composition into or onto the plant element. It is useful to make sure that the inventive composition and the solid matrix material are compatible withDocket # 24032-WO- PCT each other. For example, the solid matrix material should be chosen so that it can release the composition at a reasonable rate, for example over a period of minutes, hours, or days.

[0319] In some embodiments, the present disclosure teaches that the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods can be combined with any plant biostimulant.

[0320] 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.

[0321] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with an active chemical agent one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with an active chemical agent one witness a synergistic effect on a plant phenotypic trait of interest.

[0322] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a fertilizer one witnesses an additive effect on a plant phenotypic trait of interest. In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a fertilizer one witness a synergistic effect on a plant phenotypic trait of interest.

[0323] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a plant growth regulator, one witnesses an additive effect on a plant phenotypic trait of interest. In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a plant growth regulator, one witnesses a synergistic effect. In some aspects, the microbes of the present disclosure are combined with Ascend® and a synergistic effect is observed for one or more phenotypic traits of interest.

[0324] In some embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a biostimulant, one witnesses an additive effect on a plant phenotypic trait of interest. In some embodiments, when the microbe or microbial consortiaDocket # 24032-WO- PCT identified according to the taught methods is combined with a biostimulant, one witnesses a synergistic effect.

[0325] The synergistic effect obtained by the taught methods can be quantified according to Colby’s formula (i.e., (E) =X+Y-(X*Y / 100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount.

[0326] The isolated microbes and consortia of the present disclosure can synergistically increase the effectiveness of agricultural active compounds and also agricultural auxiliary compounds.

[0327] In other embodiments, when the microbe or microbial consortia identified according to the taught methods is combined with a fertilizer one witnesses a synergistic effect.

[0328] Furthermore, in certain embodiments, the disclosure utilizes synergistic interactions to define microbial consortia. That is, in certain aspects, the disclosure combines together certain isolated microbial species, which act synergistically, into consortia that impart a beneficial trait upon a plant, or which are correlated with increasing a beneficial plant trait.

[0329] The agricultural compositions developed according to the disclosure can be formulated with certain auxiliaries, in order to improve the activity of a known active agricultural compound. This has the advantage that the amounts of active ingredient in the formulation may be reduced while maintaining the efficacy of the active compound, thus allowing costs to be kept as low as possible and any official regulations to be followed. In individual cases, it may also possible to widen the spectrum of action of the active compound since plants, where the treatment with a particular active ingredient without addition was insufficiently successful, can indeed be treated successfully by the addition of certain auxiliaries along with the disclosed microbial isolates and consortia. Moreover, the performance of the active may be increased in individual cases by a suitable formulation when the environmental conditions are not favorable.

[0330] Such auxiliaries that can be used in an agricultural composition can be an adjuvant.Frequently, adjuvants take the form of surface-active or salt-like compounds. Depending on their mode of action, they can roughly be classified as modifiers, activators, fertilizers, pH buffers, and the like. Modifiers affect the wetting, sticking, and spreading properties of a formulation. Activators break up the waxy cuticle of the plant and improve the penetration of the active ingredient into the cuticle, both short-term (over minutes) and long-term (over hours). FertilizersDocket # 24032-WO- PCT such as ammonium sulfate, ammonium nitrate or urea improve the absorption and solubility of the active ingredient and may reduce the antagonistic behavior of active ingredients. pH buffers are conventionally used for bringing the formulation to an optimal pH.

[0331] In some embodiments, the plant element is a plant reproductive element (e.g., seed, tuber, bulb, and / or shoot). In some embodiments, the plant element is other than a plant reproductive element (e.g., leaf, stem, and / or root). In some embodiments, a plurality of plant elements are associated with the microbe(s) described herein.

[0332] In some embodiments, the plant or plant element becomes associated with one or more microbes described herein via an indirect method, such as but not limited to treatment of the growth medium in which the plant or plant element is placed.

[0333] For further embodiments of agricultural compositions of the present disclosure, See “Chemistry and Technology of Agrochemical Formulations,” edited by D. A. Knowles, copyright 1998 by Kluwer Academic Publishers.Plants and Agronomic Benefits

[0334] A wide variety of plants, including those cultivated in agriculture, are capable of receiving benefit from the application of microbes, such as those described herein, including single microbes, consortia, and / or compositions produced therefrom, or comprising any of the preceding. Any number of a variety of different plants, including mosses and lichens and algae, may be used in the methods of the disclosure. In embodiments, the plants have economic, social, or environmental value. For example, the plants may include those used as: food crops, fiber crops, oil crops, in the forestry industry, in the pulp and paper industry, as a feedstock for biofuel production, and as ornamental plants.

[0335] The genetically modified microorganisms disclosed herein have application in the improvement of nitrogen fixation in plants. In some embodiments, such plants include those which lack natural nitrogen-fixing symbionts (e.g., non-leguminous crops), such as but not limited to: wheat, maize (corn), rice, and vegetables. In some embodiments, such plants include those that would benefit from additional nitrogen fixation.Methods of Application

[0336] The microorganisms may be applied to a plant, seedling, cutting, propagule, or the like and / or the growth medium comprising said plant, using any technique known in the art.Docket # 24032-WO- PCT

[0337] However, by way of example, a microbe, consortium, or composition comprising the same, and / or a composition produced therefrom, may be applied to a plant, seedling, cutting, propagule, or the like, by spraying, coating, dusting, or any other method known in the art.

[0338] In another embodiment, the isolated microbe, consortia, or composition comprising the same may be applied directly to a plant seed prior to sowing.

[0339] In another embodiment, the isolated microbe, consortia, or composition comprising the same may applied directly to a plant seed, as a seed coating.

[0340] In one embodiment of the present disclosure, the isolated microbe, consortia, or composition comprising the same is supplied in the form of granules, or plug, or soil drench that is applied to the plant growth media.

[0341] In other embodiments, the isolated microbe, consortia, or composition comprising the same are supplied in the form of a foliar application, such as a foliar spray or liquid composition. The foliar spray or liquid application may be applied to a growing plant or to a growth media, e.g., soil.

[0342] In some embodiments, the isolated microbe, consortia, or composition comprising the same are supplied in a form selected from: a soil drench, a foliar spray, a dip treatment, an in furrow treatment, a soil amendment, granules, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural compositions may be applied alone in or in rotation spray programs.

[0343] In some embodiments, the isolated microbe, consortia, or composition comprising the 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.

[0344] In another embodiment, the isolated microbe, consortia, or composition comprising the same may be formulated into granules and applied alongside seeds during planting. Or the granules may be applied after planting. Or the granules may be applied before planting.

[0345] In some embodiments, the isolated microbe, consortia, or composition comprising the same are administered to a plant or growth media as a topical application and / or drench application to improve crop growth, yield, and quality. The topical application may be via utilization of a dry mix or powder or dusting composition or may be a liquid based formulation.Docket # 24032-WO- PCT

[0346] In embodiments, the isolated microbe, consortia, or composition comprising the same can be formulated as: (1) solutions; (2) wettable powders; (3) dusting powders; (4) soluble powders;(5) emulsions or suspension concentrates; (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) as irrigation components, and (12) a component of fertilizers, pesticides, and other compatible amendments, among others. In certain aspects, the compositions may be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure can be applied to the soil, plant, seed, rhizosphere, rhizosheath, or other area to which it would be beneficial to apply the microbial compositions. Further still, ballistic methods can be utilized as a means for introducing endophytic microbes.

[0347] In aspects, the compositions are applied to the foliage of plants. The compositions may be applied to the foliage of plants in the form of an emulsion or suspension concentrate, liquid solution, or foliar spray. The application of the compositions may occur in a laboratory, growth chamber, greenhouse, or in the field.

[0348] In another embodiment, microorganisms may be inoculated into a plant by cutting the roots or stems and exposing the plant surface to the microorganisms by spraying, dipping, or otherwise applying a liquid microbial suspension, or gel, or powder.

[0349] In another embodiment, the microorganisms may be injected directly into foliar or root tissue, or otherwise inoculated directly into or onto a foliar or root cut, or else into an excised embryo, or radicle, or coleoptile. These inoculated plants may then be further exposed to a growth media containing further microorganisms; however, this is not necessary.

[0350] In other embodiments, particularly where the microorganisms are unculturable, the microorganisms may be transferred to a plant by any one or a combination of grafting, insertion of explants, aspiration, electroporation, wounding, root pruning, induction of stomatai opening, or any physical, chemical or biological treatment that provides the opportunity for microbes to enter plant cells or the intercellular space. Persons of skill in the art may readily appreciate a number of alternative techniques that may be used.

[0351] In one embodiment, the microorganisms infdtrate parts of the plant such as the roots, stems, leaves and / or reproductive plant parts (become endophytic), and / or grow upon the surface of roots, stems, leaves and / or reproductive plant parts (become epiphytic) and / or grow in theDocket # 24032-WO- PCT plant rhizosphere. In one embodiment, the microorganisms form a symbiotic relationship with the plant.

[0352] Some non-limiting aspects of the present disclosure include the following:

[0353] Aspect 1 : A synthetic composition comprising:

[0354] (a) a first microbe, wherein the first microbe lacks sufficient nitrogen fixation capability,

[0355] (b) one or more polynucleotide(s) encoding one or more polypeptide(s) involved in nitrogen fixation obtained from a second bacterium;

[0356] wherein the first microbe and the second microbe are from different taxonomic categories;

[0357] wherein the first microbe comprises the one or more polynucleotides of (b); and

[0358] wherein the first microbe comprising the one or more polynucleotides of (b) displays an improved phenotype as compared to a microbe not comprising the one or more polynucleotides of (b), wherein the improved phenotype is selected from the group consisting of: increased acetylene reduction capability, improved biofilm formation, increased turbidity in culture, greater nitrogen fixation, improved tolerance to oxygen levels, increased release of nitrogenous compounds, and any combination and / or plurality of the preceding.

[0359] Aspect 2: The synthetic composition of Aspect 1 :, wherein the first microbe is selected from the group consisting of: archaebacterium, bacterium, fungus.

[0360] Aspect 3: The synthetic composition of Aspect 1 :, wherein the second microbe is selected from the group consisting of: archaebacterium, bacterium, fungus.

[0361] Aspect 4: The synthetic composition of Aspect 1 :, wherein the different taxonomic categories are selected from the group consisting of: kingdom, phylum, class, order, family, and genus.

[0362] Aspect 5: The synthetic composition of Aspect 1 :, wherein the one or more polynucleotides of (b) comprises a gene(s) selected from the group consisting of: nifQ, nifB, nifA, nifL, nifF, nifM, nifV, nifS, nifU, nifX, nifN, nifE, nifY, nifK, nifD, nifH, nifl, nifB, nifH, nifHl nifD, nifK, nifE, nifV, nifX, orfl, hesA, hesA2, nifV, nifW, glnR, cueR, glnA, anfD, anfG, anfK vnfA, vnfD, vnfG, vnfK yggx, nifU, sox, and sodA.

[0363] Aspect 6: The synthetic composition of Aspect 1 :, wherein the one or more polynucleotides comprise a nif cluster.Docket # 24032-WO- PCT

[0364] Aspect 7: The synthetic composition of Aspect 1 :, further comprising a formulation component and / or an agricultural composition.

[0365] Aspect 8: The synthetic composition of Aspect 1 wherein the bacterium is present at a concentration of at least about 10A2 CFU / mL in a liquid formulation, or at least about 10A2 CFU / gram in a non-liquid formulation.

[0366] Aspect 9: The synthetic composition of Aspect 1 further comprising at least one additional microbe.

[0367] Aspect 10: The synthetic composition of Aspect 1 further comprising a plant or plant element.

[0368] Aspect 11 : The synthetic composition of Aspect 10:, wherein the plant or plant element comprises a transgene.

[0369] Aspect 12: The synthetic composition of Aspect 10:, wherein the plant element is obtained from vegetative tissue.

[0370] Aspect 13: The synthetic composition of Aspect 10:, wherein the plant element is a plant reproductive element.

[0371] Aspect 14: The synthetic composition of Aspect 1 :, wherein the formulation component is selected from the group consisting of: a compound that improves the stability of the microbe, a preservative, a carrier, a surfactant, an anticomplex agent, and any plurality and / or combination thereof.

[0372] Aspect 15: The synthetic composition of Aspect 1 :, wherein the agricultural composition is selected from the group consisting of: a fungicide, a nematicide, a bactericide, an insecticide, an herbicide, a micronutrient, a macronutrient, Nitrogen, Phosphorous, Potassium, and any plurality and / or combination of the preceding.

[0373] Aspect 16: A plurality of synthetic compositions of Aspect 1 :, wherein said synthetic compositions are substantially confined within an object selected from the group consisting of: a tube, a petri dish, a bottle, ajar, an ampule, a package, a vessel, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

[0374] Aspect 17: The synthetic composition of Aspect 10:, wherein the plant element is obtained from a monocot plant.

[0375] Aspect 18: The synthetic composition of Aspect 10:, wherein the plant element is obtained from a di cot plant.Docket # 24032-WO- PCT

[0376] Aspect 19: The synthetic composition of Aspect 15:, wherein the agricultural composition comprises a growth medium.

[0377] Aspect 20: The synthetic composition of Aspect 19:, wherein the growth medium comprises soil.

[0378] Aspect 21 : A plurality of synthetic compositions of Aspect 20:, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

[0379] Aspect 22: A method of improving a characteristic of a plant, the method comprising:

[0380] (a) associating an element of the plant with a synthetic composition, wherein the synthetic composition comprises:

[0381] i. a first microbe, wherein the first microbe lacks sufficient nitrogen fixation capability; and

[0382] ii. one or more polynucleotide(s) encoding one or more polypeptide(s) involved in nitrogen fixation obtained from a second microbe;

[0383] wherein the first microbe and the second microbe are from different taxonomic categories;

[0384] (a) placing the element of the crop plant in a medium that supports plant growth;

[0385] (b) growing a plant from the element of the crop plant;

[0386] (c) assessing one or more characteristics of the plant, wherein at least one of said characteristics is improved, as compared to the same characteristic of a plant not obtained from an element associated with the microbe of (a).

[0387] Aspect 23 : The method of Aspect 22:, wherein the first microbe is selected from the group consisting of: archaebacterium, bacterium, fungus.

[0388] Aspect 24: The method of Aspect 22:, wherein the second microbe is selected from the group consisting of: archaebacterium, bacterium, fungus.

[0389] Aspect 25: The method of Aspect 22:, wherein the different taxonomic categories are selected from the group consisting of: kingdom, phylum, class, order, family, and genus.

[0390] Aspect 26: The method of Aspect 22:, wherein the one or more characteristics of (d) includes an improvement of nitrogen fixation, increase in biomass, increase in leaf area, increase in plant height, increase in root area, increase in shoot nitrogen composition, increase inDocket # 24032-WO- PCT greenness, increase in ND VI, increase in NPCI, increase in PSRI, increase in CCT, increase in yield, and any combination of the preceding.

[0391] Aspect 27: The method of Aspect 22:, further comprising at least one additional microbe.

[0392] Aspect 28: The method of Aspect 22:, wherein the associating an element of the crop plant with a bacterium comprising an edit in one or more loci of its genome is accomplished by a method selected from the group consisting of: in-furrow application, soil drench application, side-dress application, and any combination of the preceding.

[0393] Aspect 29: The method of Aspect 22:, wherein the associating an element of the crop plant with a bacterium comprising an edit in one or more loci of its genome is accomplished by coating said plant element with a liquid formulation of the bacterium.

[0394] Aspect 30: The method of Aspect 22:, wherein the associating an element of the crop plant with a bacterium comprising an edit in one or more loci of its genome is accomplished by coating said plant element with a substantially non-liquid formulation of the bacterium.

[0395] Aspect 31 : The method of Aspect 22:, wherein said plant element is a plant reproductive element.

[0396] Aspect 32: The method of Aspect 22:, wherein said plant element is a leaf.

[0397] Aspect 33: The method of Aspect 22:, wherein said plant element is a whole plant.

[0398] Aspect 34: A substantially pure composition comprising a genetically modified bacterium, wherein the genetically modified bacterium comprises one or more polynucleotide(s) encoding one or more polypeptide(s) involved in nitrogen fixation obtained from a second bacterium, wherein the second bacterium is from a different taxonomic genus than the genetically modified bacterium.

[0399] Aspect 35: A bacterial culture comprising the modified bacterium of Aspect 34:.

[0400] Aspect 36: A fermentation culture comprising the modified bacterium of Aspect 34:.

[0401] Aspect 37: An agricultural composition, comprising the modified bacterium of Aspect 34: and an agriculturally-acceptable carrier.

[0402] Aspect 38: The agricultural composition of Aspect 37:, further comprising a plant or plant element, wherein the modified bacterium is present in the agricultural composition in an amount effective for producing an improved phenotype in the plant.Docket # 24032-WO- PCT

[0403] Aspect 39: The agricultural composition of Aspect 38:, wherein the improved phenotype is an increase in the yield of the plant.

[0404] Aspect 40: Any of the methods or compositions of the preceding, wherein the first bacterium is an endophyte.

[0405] Aspect 41 : Any of the methods or compositions of the preceding, wherein the first bacterium is capable of colonizing plant root tissue.

[0406] Aspect 42: Any of the methods or compositions of the preceding, wherein the first bacterium is Gram-positive.

[0407] Aspect 43 : Any of the methods or compositions of the preceding, wherein the first bacterium is Gram-negative.

[0408] Aspect 44: Any of the methods or compositions of the preceding, wherein the second bacterium is Gram-positive.

[0409] Aspect 45: Any of the methods or compositions of the preceding, wherein the second bacterium is Gram-negative.

[0410] Aspect 46: Any of the methods or compositions of the preceding, wherein the first bacterium produces one or more composition that impact an insect, a fungus, and / or a nematode.

[0411] Aspect 47: Any of the methods or compositions of the preceding, wherein the first bacterium produces one or more composition that impact plant growth, health, yield, potassium solubilization, and phosphorous solubilization.

[0412] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. Various alterations, modifications, and improvements of the present disclosure that readily occur to those skilled in the art, including certain alterations, modifications, substitutions, and improvements are also part of this disclosure. For instance, while the particular examples below may illustrate the methods and embodiments described herein using a specific plant, the principles in these examples may be applied to any plant. Therefore, it will be appreciated that the scope of this invention is encompassed by the embodiments recited herein rather than solely by the specific examples that are exemplified below.Docket # 24032-WO- PCT

[0413] All cited patents and publications referred to in this application are herein incorporated by reference in their entirety, for all purposes, to the same extent as if each were individually and specifically incorporated by reference.Docket # 24032-WO- PCTEXAMPLES

[0414] The methods and compositions presented herein - based upon utilizing the disclosed isolated microbes, communities, consortia, and / or compositions comprising and / or produced by microbes or consortia or communities - improve one or more characteristics of plants, for example nitrogen fixation in agricultural crops. The abbreviation “uL” means “microliters”, “ug” means “micrograms”. Exemplary protocols are detailed herein; however, it is understood that alternative methods and / or variations may be employed.Example 1: Microbe Culture, Sequencing, and Target Selection

[0415] Wild-type and genetically modified strains were grown in culture media to obtain sufficient cellular growth. A subsample of each of the strains are then aseptically transferred to nitrogen-free growth media and incubated under microaerophilic conditions for 72 hours.

[0416] Isolates of interest were grown to mid-log phase in R2A media. DNA was extracted with the Qiagen Powersoil DNA extraction kit and sequencing libraries were constructed with the iGenomix RipTide kit as per manufacturer instructions. Sequencing was performed on an Illumina HiSeq with PE150. Quality control, trimming, assembly, and annotation were performed using the Bactopia 2 nextflow pipeline (Petit III RA, Read TD Bactopia- a flexible pipeline for complete analysis of bacterial genomes. mSystems 5 (2020)) Raw Illumina reads were trimmed to Q20 with BBDuk and any genome without a minimum of lOx coverage was discarded prior to assembly Assembly and annotation were carried out with Bactopia2 using default parameters. Checkm was used for purity with a contamination cutoff of < 5%. Taxonomy was assigned by GTDB-tk using default parameters with the April 2021 database (Pierre-Alain Chaumeil, Aaron J Mussig, Philip Hugenholtz, Donovan H Parks, GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database, Bioinformatics, Volume 36, Issue 6, 15 March 2020, Pages 1925-1927).

[0417] A selection of the strains were characterized and their nitrogen fixation-related genes identified. Editing targets of various polynucleotides in the genome were selected to increase Nitrogen fixation in the absence of exogenously-applied Nitrogen, in the presence of minimal added Nitrogen (e.g, ammonium), and in the presence of added Nitrogen.

[0418] The nitrogen fixation cluster (nif cluster) from two different Paenbacillus species (Strain 103408 P. durus. Strain 8619 P. potymyxa) were cloned into various species of Bacillus (Strain 105256 B. subtilis, Strain 14416 B. velezensis Strain 39400 B. thuririgiensisy' with a variety ofDocket # 24032-WO- PCT integration techniques, including replicative plasmid introduction, integration with transposons, and integration int the genome of the target (recipient) strains.Example 2: Editing of Strains

[0419] Edits of microbes to produce engineered strains that impart improved characteristics to plants with which they are associated may be accomplished by nucleotide insertion, nucleotide deletion, nucleotide replacement, and / or any combination or plurality of the preceding. The net effect may be one of upregulation, downregulation, knockout (of the target polynucleotide and / or the function of its encoded RNA or protein), and / or any combination or plurality of the preceding.

[0420] Any method known in the art may be used to accomplish editing of a bacterial strain. In some cases, a method described herein was used. In some cases, a method previously described was used, see, for example, WO2023039463A1 “Blind Editing of Polynucleotide Sequences” published 16 March 2023.

[0421] The Gram positive gene editing vector pMiniMad2 was obtained from the Bacillus Genetic Stock Center, and modified by the insertion of the TraJ origin of transfer, originally from vector pKVM4, between the Sall and BamHI restriction sites, yielding the mobilizable Gram positive gene editing vector pMMmob.Assembly of Editing Vectors

[0422] Polymerase Chain Reaction (PCR) was run using the appropriate primers with Q5 high- fidelity polymerase to amplify the upstream and downstream homology arms with the appropriate Gibson assembly overhangs from purified genomic DNA from the target strain. The PCR products were run on an agarose gel to confirm the appropriate size. The bands are excised and purified from the gel. The backbone vector pMMmob was digested with the restriction enzymes EcoRI and BamHI for at least 30 minutes at 37C in Cutsmart buffer. The digest was run on an agarose gel to confirm the appropriate size. The digested backbone was purified from the gel. Gibson assembly was performed by combining approximately lOOng of digested pMMmob with the insert fragments in a 1 :3:3 backbone:insert:insert molar ratio in a lOul volume, then adding lOul 2x Gibson Reagent. The reaction was incubated at 50C for 60 minutes then used for transformation into E. coli DH5a. l-5ul of the Gibson assembly mixture was added to 50ul of freshly thawed, chemically competent E. coli DH5ct cells and finger vortexed. The cell-plasmid mixture was incubated on ice for 30 minutes, heat shocked at 42C for 30 seconds, then movedDocket # 24032-WO- PCT back to ice for a further 5 minutes. ImL SOC (Super-Optimal Catabolism) medium was added, and the cells are incubated at 37C with 200RPM shaking for 60-90 minutes for recovery. Dilutions of the recovery culture were plated onto LB agar plates supplemented with lOOng / uL Ampicillin and incubated overnight at 37C. The plasmid region comprising the assembled inserts as amplified from several recovered colonies via colony PCR using GoTaq polymerase, and the PCR products were run on an agarose gel to confirm the expected sized product. Appropriately- sized PCR products are sent for Sanger sequencing to confirm proper assembly and lack of any off-target mutations in the editing cassette. Colonies confirmed to harbor the correct plasmid were inoculated into LB broth supplemented with lOOng / uL Ampicillin and grown overnight at 37C and 200RPM shaking. The plasmid was purified from the overnight culture and transformed into conjugation donor strain A. coli BW29472 via electroporation, lul of the purified plasmid was combined with 50ul of freshly thawed A. coli BW29472 electrocompetent cells and incubated for 5 minutes on ice. The cell-plasmid mixture was transferred into a pre-chilled 1mm electroporation cuvette on ice. Using an electroporator, a 1800V, 25uF, 200Q charge was applied to the cuvette, and the sample immediately resuspended in ImL SOC medium supplemented with 0.3mM 2,6-diaminopimelic acid (DAP). The resuspended cells were incubated at 37C with 200RPM shaking for 60-90 minutes for recovery. Dilutions of the recovery culture were plated onto LB agar plates supplemented with lOOng / uL Ampicillin and 0.3mM 2,6-diaminopimelic acid and incubated overnight at 37C. Recovered transformants were used as donor strains for conjugation.

[0423] For exogneous expression of the nif cluster on a replicative plasmid the replicative plasmid pBACOM, containing the colEl origin of replication and ampicillin resistance gene from pUC19, as well as the pUB origin of replication, traJ origin of transfer and the erythromycin and lincomycin resistance gene ermC, was used.

[0424] Using standard cloning techniques described above, the nif cluster from Paenibacillus durus 103408, comprised of the genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, orfl, hesA, and nifV was cloned into the pBACOM backbone, with the promoter PaprE cloned upstream of the nifB ribosome binding site, yielding the plasmid pBACOM_PaprE-CM103408nifB-nifV. Plasmids to be transformed into Bacillus strains via electroporation were first transformed via heat shock into chemically competent dam - / dem- E. coli. The transformed E. coli was grown overnight in liquid culture, then the plasmid was extracted for electroporation.Docket # 24032-WO- PCTConjugation - Used for transformation into Bacillus subtilis 39400

[0425] Recipient strains were inoculated into 5mL Tryptic Soy Broth (TSB) medium in 50mL conical tubes and grown overnight at 30C and 200RPM shaking. Donor E. colt BW29427 harboring the plasmid to be mobilized was inoculated into 5mL LB medium supplemented with lOOug / uL Ampicillin and 0.3mM 2,6-diaminopimelic acid (DAP) and grown overnight at 37C and 200RPM shaking. 1ml aliquots of the overnight donor and recipient cultures were spun down, washed in sterile water, combined and spotted onto the surface of LB agar plates supplemented with 0.3mM DAP for conjugative mating. Mating plates were incubated overnight at 25C, the permissive temperature for replication of pMMmob in Gram positive recipient strains. Other conditions required for Gram negative recipient strains were used as known in the art. Mating mixtures were resuspended by the addition of 1ml sterile water over the top of the spot and agitation with a sterile L-spreader. Resuspensions were collected in microcentrifuge tubes, washed, and resuspended in lOOul of sterile water. The concentrated cells were spread over TSA plates supplemented with MLS (25ug / ml Lincomycin, lug / ml Erythromycin) with no DAP added and incubated for 48-72 hours at 25C until the appearance of transconjugant colonies.Electroporation - used for transformation into Bacillus thuringiensis 39400 The dam- / dcm- prepared plasmids were electroporated into electrocompetent Bacillus thuringiensis 39400 and transformants were recovered on agar plates supplemented with selective antibiotics.Plasmid Integration

[0426] Recovered transconjugants were inoculated into 5mL TSB medium supplemented with MLS and grown for 48 hours at 25C with 200 RPM shaking, or until turbid. Dilutions of the culture were plated onto TSA + MLS plates and incubated overnight at 37C, the restrictive temperature for plasmid replication, until the appearance of integrated colonies.Plasmid Excision

[0427] Integrated colonies were re-inoculated into 5mL TSB medium supplemented with MLS and incubated overnight at 37C with 200RPM shaking. 5ul of the overnight culture was diluted into 5mL fresh TSB medium without antibiotics and grown overnight at 25C, 200RPM shaking. Subculturing of 5ul overnight culture into 5mL fresh TSB at 25C, 200RPM shaking was repeatedDocket # 24032-WO- PCT twice, for a total of three rounds of subculturing. Dilutions of the round three overnight culture were plated onto R2A plates lacking antibiotics and incubated at 30C overnight.

[0428] Excision of the plasmid was confirmed by picking individual colonies from the R2A plate and re-plating them in a grid format onto agar plates with and without MLS. Both plates were incubated at 30C for 24-48 hours, until the appearance of colonies. Colonies that grew when plated onto the plate without MLS, but not on the plate containing MLS, were confirmed to have excised and lost the plasmid.Confirmation of editing

[0429] The edit region was amplified from putative edited strains via colony PCR, and the presence of the proper edit is confirmed by the size of the band when run on an agarose gel (when possible), and / or by Sanger sequencing. The absence of the plasmid backbone was confirmed by PCR assaying of the MLS resistance cassette. Colonies yielding a band for the MLS cassette are confirmed to not be proper edits.

[0430] Sequence results ere analyzed to differentiate colonies that had excised to wild type from properly edited colonies, and the sequences were checked for off-target mutations.

[0431] Colonies that sequenced as proper edits without off-target mutations were given a modified strain designation, added to the Modified Strain Library, and made available to the project team for analysis.

[0432] Alternatively, any other method known in the art may be employed to effect any one or more of the polynucleotide edits described herein, for example but not limited to: targeting and / or homing nucleases, restriction endonucleases, zinc finger nucleases, meganucleases, Cas endonucleases, TAL effector nucleases, guided nucleases, random site mutations, blind editing, chemical mutagenesis, or radiation mutagenesis. Generally, a double-strand break is created at or near the target site to be edited, which is repaired by intracellular processes such as nonhom ologous end joining, homologous recombination, or homology -directed repair. The net effect can be any one or more of the following: insertion of at least one nucleotide, deletion of at least one nucleotide, replacement of at least one nucleotide, chemical alteration of at least one nucleotide. In some aspects, random, untargeted, stochastic, or other editing of the target polynucleotide (that which is to be edited) may be achieved, for example by radiation mutagenesis, chemical mutagenesis, blind editing (see for example WO2023039463A1 published 16 March 2023, or any other method known in the art. For the purposes of the editsDocket # 24032-WO- PCT described herein, any technique that is desired by the practitioner may be used to achieve the end result.

[0433] The edits were delivered to one or more parent strains and made available for in vitro and in planta evaluations.Replicative Plasmid Insertion

[0434] For delivery of replicative plasmids, the cloning, transformation, and confirmation steps were performed.

[0435] Additionally, a replicative empty plasmid pBACOM-empty with no introduced nif genes or promoter was transformed via electroporation as described above into 39400, yielding the strain PE39400-G279 to be used as an antibiotic resistant control. Summaries of the cloning processes are shown in Tables la - lb (Replicative plasmid insertion).Table lb: Replicative Plasmid Insertion Process SuccessPlasmid IDs include a “1” for 1-step transformation or “2” for 2-step transformation.Cl = Cloning; H = Host Strain; T = Transformation; I = Integration, E = Excision; Co = Confirmation.Docket # 24032-WO- PCT

[0436] Replicative plasmid insertion of genes from different species of Paenibacillus was successfully accomplished into different species of Bacillus, thus demonstrating intergeneric transformation. A representative strain transformed with an intergeneric replicative plasmid was selected for further testing, as described herein.Chromosomal Integration

[0437] For chromosomal integration of the Paenibacillus nif clusters, two steps of editing were required. During the first step, the genes between and including nifl) and w / I'were delivered along with the chloramphenicol resistance gene, catR. During the second step, the catR gene was removed simultaneously with the delivery of the PaprE promoter, nifB and nifH, yielding seamless delivery of the complete cluster.

[0438] For chromosomal integration in Bacillus thuringiensis 39400, the thuE gene locus was used as the integration location. For chromosomal integration in Bacillus subtilis 105256, the amyE gene locus was used as the integration location. Summaries of the cloning processes are shown in Table 1c (Integrative insertion).Table 1c: Summary of Intergeneric Chromosomal Integration of Paenibacillus nif clusters

[0439] Integration of genes from different species of Paenibacillus was successfully accomplished into different species of Bacillus, thus demonstrating intergeneric transformation.

[0440] A representative strain Bacillus thuringiensis Strain 39400 transformed with the nfix cluster from Paenibacillus durus Strain 103408 operably linked to the promoter from the aprE gene (plasmid insertion included PaprE-nifB-nifEI-nifD-nifK-nifE-nifP-nifX-orfl-hesA-nijV) was selected for further testing, as described herein.Docket # 24032-WO- PCTExample 3: Microbe Identification and Storage

[0441] Sequencing preparation for microbe identification, and long-term storage, is performed by the following method: Day 1 :

[0442] Use a 10 uL sterile tip to transfer a colony from a plate to a flask containing an appropriate liquid growth medium. Place the isolates on a shaker at room temperature and incubate for 2 days.Day 3:

[0443] Tubes may be cloudy after being on the shaker for 2 days. All samples are analyzed by PCR. Vortex each tube, collect a 50 uL sample from each vortexed tube, and dispense in a 96- well plate. Using a multichannel pipette, dispense 15 uL of the 50 uL samples into a new 96-well plate. The 96-well plate containing 35 uL of each sample will be used for phenotyping, and the 96-well plate containing 15 uL of each sample will be used for PCR analysis. 27F / 1492R primers are generally used for 16S PCR analysis, as they yield better results than PB36 / 38. Appropriate negative controls should be included with the plate and analyzed by PCR. The plate will be analyzed by PCR using an Eppendorf thermocycler. Once the PCR is finished, run a gel using standard gel electrophoresis techniques. This is important because most isolates are grown enough where they should ideally be put into long-term storage on day 3. The PCR and gel electrophoresis analysis are used to confirm that the isolates contain bacteria, rather than other microbes. For isolates that do not pass PCR or have clear broth, vortex tubes and use a loop to streak out onto a petri plate. Check after several days to see if anything grows, or if the tube is contaminated. For isolates that pass PCR, dispense 600 uL of 50% glycerol into a 2 ml screw cap tubes and add 1200 uL of the bacterial culture, such that the broth is stored in 20% glycerol. Store the glycerol stock at -80 °C and record an image of the gel of the PCR samples. Day 4:

[0444] Check the petri plates of the streaked isolates that failed PCR for growth. (During this time, the 2 ml broth tubes will remain on the shaker.) Once there is growth on the plate and the colonies appear to have been successfully isolated, dispense 600 uL of the broth-glycerol mixture in the small tube, and put both tubes in their respective -80 boxes. It is possible for isolates to fail the PCR check because of any of the following reasons: the primers may not work on all bacteria, the isolate is actually a fungus, the isolate is very adherent and therefore does notDocket # 24032-WO- PCT homogenize in the broth, the isolate produces too much EPS therefore needs dilution prior to PCR set-up, or the isolate is a slow grower. Over the next few days, continue checking the plate to confirm that only a single bacterial species was isolated. If contamination is observed, prepare a new isolate. Viability of the prepared glycerol stocks should be verified.Example 4: Formulation of Microbes

[0445] Microbes identified according to the previous examples may be formulated with additional components for application via methods such as, but not be limited to: seed treatment, root drench, root wash, seedling soak, foliar application, soil inocula, in-furrow application, sidedress application, soil pre-treatment, wound inoculation, drip tape irrigation, vectormediation via a pollinator, injection, osmopriming, hydroponics, aquaponics, aeroponics. The formulation comprising the microbes are prepared for agricultural application as a liquid, a solid, or a gas formulation. Application to the plant is achieved, for example, as a powder for surface deposition onto plant leaves, as a spray to the whole plant or selected plant element, as part of a drip to the soil or the roots, or as a coating onto the plant element prior to planting. Such examples are meant to be illustrative and not limiting to the scope of the invention.

[0446] Media components for an exemplary microbe preparation are shown below in Tables 2a- 2d. Add all contents with 50% of the final volume of water needed, and stir the solution at an elevated temperature until dissolved. After all contents have been dissolved, use sterile RO water to bring the solution to the final desired volume. Field trial preparations are typically performed using the 4x formulation.Table 2a: Exemplary media components and concentrations for microbe formulationTable 2b: Exemplary media components for microbe formulationDocket # 24032-WO- PCTTable 2c: Exemplary media components for m icrobe formulationTable 2d: Exemplary media components for microbe formulation

[0447] The procedure to mix TIX formulation is as follows: Measure all dry ingredients into a 50ml tube. Vortex the ingredients well to ensure xanthan gum is “separated” through the other carbon sources. Add about half of total sterile RO water to the mix, vortex. Use the long end of an L-spreader to break up chunks as much as you can. Heat some sterile RO water in the microwave to warm water bath temperature (45-50°C). Add the remaining sterile RO water to the mix, vortex. Repeat step 4 and vortex as needed until you have a clear solution with no lumps. Spin down the bubbles created in the process of mixing by using a centrifuge for 5-10 seconds on “fast spin”. Remember to have a balance to counter the formulation (TIX) tube. Allow formulation to cool to room temperature. 1. Mix in the microbial consortia. Vortex to ensure homogeneity. It is ideal to add microbes at a concentration of 10A9 CFU / ml to the formulation.

[0448] Apply the formulation to the plant or plant element for testing in field trial.Example 5: Application of Microbes to Plant Elements and Cultivation Thereof

[0449] A microbial composition (comprising one or more isolated microbes of a single strain, a consortium, a community, a combination, or any combination of the preceding) is preparedDocket # 24032-WO- PCT according to the previous Examples. The microbial composition comprises one or more microbes, optionally in combination with one or more additional microbes disclosed herein. Microbial Compositions for Application

[0450] In some methods, the microbial composition is dried and applied directly to a plant element.

[0451] In some methods, the microbial composition is suspended in a liquid formulation for application to a plant element.

[0452] In some methods, the microbial composition is combined with another composition, such as but not limited to: a carrier, a wetting agent, a stabilizer, a salt. In some methods, the other composition comprises a molecule that introduces additional agriculturally-beneficial outcomes to the plant to which the microbial composition is applied. The other composition includes, for example but not limited to: an herbicide, a fungicide, a bactericide, a pesticide, an insecticide, a nematicide, a biostimulant.Application Types

[0453] The microbial composition is applied to a plant element, at a time during development appropriate to the desired outcome, for example: in a formulation of a pre-planting soil drench / in-furrow application; as a seed or other reproductive element treatment; as a postplanting reproductive element application; as an in-furrow, drip, or drench application after planting; as a direct application to a plant element (e.g., root, leaf, stem); as an application to a harvested plant element (e.g., a fruit or a grain). Combinations of application types are also tested.Application Methods

[0454] The microbial composition is applied to (inoculating) a 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, which contains the microbial composition and which is configured to broadcast the same.

[0455] A seed coating of the microbial composition is applied to one or more seeds of a crop plant. Upon applying the isolated microbe as a seed coating, the seed is planted and cultivated according to practices established for that crop.Docket # 24032-WO- PCT

[0456] Alternatively, the microbial composition is applied to the soil for the benefit of a plant existing in that soil. Methods of soil application include in-furrow treatment, drench, and drip applications.

[0457] Alternatively, the microbial composition is applied to the surface of a plant or plant part after germination.

[0458] Alternatively, the microbial composition is applied to material obtained from the plant after harvest.

[0459] A control plot of plants, which did not have the isolated microbe applied, are also planted. Plants associated with the microbial composition exhibit improved characteristics of interest.

[0460] Application methods may be performed according to any protocol known in the art.

[0461] Plant elements, plants, or growth medium (e.g., soil) may further be inoculated with a disease or pest, according to the purpose of the test.

[0462] An exemplary, non-limiting protocol for drenching tomato plants is given below:1. Ten days after planting carefully separate plants out into 6 reps for each treatment. Plants are delicate and leaves can tear easily. Ensure that the size and overall appearance of plants is as uniform as possible (The purpose of thinning is continuing with an homogenous plant population). Transplant if there are not enough plants per reps. See step 3 for guidelines on transplanting.2. Begin thinning pots down to one plant per pot. Remove the smaller plant, one that is unhealthy or deformed in some way. If there are 2 or more healthy plants per pot, the extras can be transplanted into another pot. Use leftover soil prepped from initial planting or from pots where seeds did not germinate.3. To transplant: If some pots didn’t germinate, they can be fdled with a plant from another container. To do this simply scoop out the extra plant (trying to scoop out as much root mass as possible without disturbing the other plant) with a scoopula and place into a hole made in the empty pot. Firm the soil around the plant with slight finger pressure.4. Space out the pots into 6 pot lines (1 line of pots per treatment), will take 4 RL98 trays. Once done, have a look at all the treatments and consider making some pot switches to ensure some treatments don’t have all large plants and others have all large plants.Docket # 24032-WO- PCT5. Change gloves if necessary. Label each pot with your pre-prepared Avery Labels. Treatments should be labeled into rows of 6 replicates i.e., 1-1, 1-2, 1-3 to 1-6, etc. Makes it easier to find all replicates for each treatment6. Two weeks after planting (roughly 4 days after thinning and labeling), obtain treatments from the Microbiology team; set on the table with trays of prepped plants. Gather combitips, repeater, and RO water, (note: Plants should be watered lightly the day of treatment)7. Mix microbial solution by inverting tube / container (microbial treatment) 2-3 times or give a light shake. Set combitip to dispense 2ml. Collect treatment fluid into combitip, dispense first step back into the tube. Ensure the treatment you have corresponds to the row of plants to be treated. Once confirmed, gently dispense 2ml of treatment onto the surface soil of each pot, close to the stem but avoid direct contact with the stem and leaves.8. Dispose of combitip and repeat step 6 for all treatments. For the inoculated control (IC or InoCon) and untreated control (UTC), apply RO water in place of a treatment.Once all treatments have been applied, place plants back into growth chamber for (optional inoculation), growth, evaluation.Visualization of Microbes Associated with Plant Elements

[0463] Individual microbes can be tagged with a fluorescent protein according to methods known in the art. Microscopic image analysis demonstrated that the microbes disclosed herein were found associated with various plant tissues.Example 6: In vitro Testing

[0464] Wild Type and Edited strains were assessed for one or more of the following: root colonization, acetylene reduction activity, biofdm formation, turbidity (OD at 600 nm), oxygen tolerance, and gene expression.

[0465] NF11 media may be used. The recipe for NF 11 includes: Reagent Autoclavable (KH2PO4 5g / L 36.7mM; K2HPO4 5g / L 26.1mM; NaCl Ig / L 17.1mM; Yeast Extract O.lg / L; Mono-Sodium Glutamate (MSG) 0.86g / L 5.9mM), Filter Sterilized (Dextrose lOg / L; MgSO4 - 7H2O 0.2g / L 0.8mM; CaC12 - 2H2O 23mg / L 156uM; Trace Elements: FeSO4 5.4mg / L 35uM, MnSO4 - H2O 22.7mg / L 132uM, ZnSO4 0.0785mg / L 0.48mM, CuSO4 O.OOlmg / L 6.2nM, NaMoO4 0.004mg / L 19nM, NaNH4PHO4 - 4H2O OmM / 5mM).Docket # 24032-WO- PCTARA with GC-FID for Gram Positive Strains

[0466] Ensure all equipment and materials are sterilized. Wrap sealing equipment containers in foil prior to autoclaving so that they can be unwrapped in the Anaerobic chamber pass box and enter the Anaerobic chamber sterile. Seal vial openings with foil prior to sterilizing. Loose ‘seals’ are required to allow gas exchange in the pass box.1. Streak isolates from -80 C and incubate at 30 C or 25 C until colonies are observed.2. Spread one plate / isolate and incubate at 25 C or 30 C until a lawn is observed.3. Harvest plates and OD600 balance each isolate to approx. 0.3 to normalize the inoculant.4. Prepare the Anaerobic chamber by cleaning the surfaces and passing the sealing equipment through - ensure containers allow for gas exchange.5. Add 30 mL NF11 in 70mL vials - 3 reps / isolate.6. Add 150 uL inoculant / vial which was balanced to an OD600 of 0.3 using sterile water.7. Pass vials through Anaerobic chamber and seal under anaerobic conditions - include an empty vial (with foil ‘cap’) to add an anaerobic indicator for QC purposes.8. Place vials in 30 C, 200 rpm for 5 hours.9. After 5 hours, working in the fume hood, remove 10% (4mL) from the headspace of each vial and replace with the same volume of acetylene gas.10. Incubate at 30 C, 200 rpm for 48 hrs.11. At 48 hours take ImL headspace sample and place into a GC collection tube.12. Run samples in GC using instrument method for ethylene analysis ‘split 4’ which measures acetylene peak and ethylene peak.13. Amount of gas is quantified by peak area.14. Take OD600 readings of 200ul of the culture and TVCs of culture.15. Analyze ethylene gas as a percentage conversion of acetylene to ethylene. This produces an estimate of total conversion.

[0537] The volume of gas produced (ethylene) can either be quantified using calibration points in Chromeleon or by calculation from the % peak area. Acetylene + Ethylene peak area % must = 100% for this. From the knows amount of Acetylene added, the ethylene produced can be determined in mL. IM of gas = 24 dm3 or 24,000 ml. Therefore ImM of gas = 24 ml.

[0538] To calculate how many mM ethylene produced, divide amount by 24: mM ET= ml / 24

[0539] To calculate RATE: mM per hour per CFU, you need to calculate mM as described aboveDocket # 24032-WO- PCT

[0540] Rate = total ethylene mM / (time(h) x total CFU)ARA with Oxygen Tolerance Testing Protocol

[0541] Ensure all equipment and materials are sterilized. Wrap sealing equipment containers in foil prior to autoclaving so that they can be unwrapped in the Anaerobic chamber pass box and enter the Anaerobic chamber sterile. Seal vial openings with foil prior to sterilizing. Loose ‘seals’ are required to allow gas exchange in the pass box.1. Streak isolates from -80 C and incubate at 30 C or 25 C until colonies are observed.2. Spread one plate / isolate and incubate at 25 C or 30 C until a lawn is observed.3. Harvest plates and OD600 balance each isolate to approx. 0.3 to normalize the inoculant.4. Prepare the Anaerobic chamber by cleaning the surfaces and passing the sealing equipment through - ensure containers allow for gas exchange.5. Take NF11 media into the Anaerobic chamber after cleaning. Add agar at 20g / L to NF11 and place on hot plate. Briefly bring to boil to melt the agar. After melting agar, pour 30mL of the warmed agar into 70mL on their sides to maximize surface area to produce slants.6. Add 150 uL inoculant / vial which was balanced to an OD600 of 0.3 using sterile water. Do so trying to maximize the surface area exposed to the inoculate.7. Pass vials through Anaerobic chamber and seal under anaerobic conditions - include an empty vial (with foil ‘cap’) to add an anaerobic indicator for QC purposes.8. To adjust oxygen levels, after sealing the vial, take a thin needle syringe and remove the portion of anaerobic air from the via which will be replaced with 100% pure medical grade oxygen.9. The assay has been run at various oxygen conditions from 0% oxygen to 22% oxygen and can be increased to much higher oxygen conditions due to manual addition of oxygen. For example, to achieve 5% oxygen, remove 2.2mL anaerobic gas by hand and add 2mL of 100% pure oxygen back at this condition.10. Place vials in 30 C incubator for 5 hours.12. After 5 hours, working in the fume hood, remove 10% (4mL) from the headspace of each vial and replace with the same volume of acetylene gas. Incubate at 30 C for 48 hrs.13. At 48 hours take ImL headspace sample and place into a GC collection tube.Docket # 24032-WO- PCT14. Run samples in GC using instrument method for ethylene analysis ‘split 4’ which measures acetylene peak and ethylene people15. Amount of gas is quantified by peak area.16. Analyze ethylene gas as a percentage conversion of acetylene to ethylene. This produces an estimate of total conversion.

[0542] Volume of gas produced (ethylene) can either be quantified using calibration points in Chromeleon or by calculation from the % peak area. Acetylene + Ethylene peak area % must = 100% for this. From the known amount of Acetylene added, the ethylene produced can be determined in mb. IM of gas = 24 dm3 or 24,000 ml. Therefore, ImM of gas = 24 ml. To calculate how many mM ethylene produced, divide amount by 24: mM ET= ml / 24.

[0543] To calculate RATE: mM per hour per CFU, need to calculate mM as described above.

[0544] Rate = total ethylene mM / (time(h) x total CFU).

[0545] Bacillus Thuringiensis 39400(Strain 39400) was transformed to successfully introduce nifB-nijV from Paenibacillus durus (Strain 103408), as described above, to induce nitrogenase activity in a non-Nitrogen fixing strain.

[0546] Gas chromatography (GC) was performed on a wild-type Paenibacillus polymyxa strain (77155) as a positive control (known to be a nitrogen-fixing microorganism), a Bacillus thuringiensis strain transformed with an empty vector as a negative control (Strain 39400-G279), and experimental strains of Bacillus thuringiensis transformed with the nifB-nifV nfix cluster from Paenibacillus durus Strain 103408 operably linked to the aprE promoter (resulting Strain 39400-G280, comprising PaprE-nifB-nijV).

[0547] Among the isolates created, Strain 39400-G280 consistently showed increased activity over the negative control (Strain 39400-G279), as demonstrated by careful analysis and hand integration of the GC chromatogram.

[0548] Various media formulations and culture times were explored throughout the multiple assay that were run. Experiment CX27590 was cultured for the standard 48 hours prior to being harvested. Following the observations of CX27590, it was determined to extend the culture time to at least 72 hours to investigate if the extended time would allow for better signal detection. Experiment CX27704 was cultured for a total of six days with time points taken at 48h, 72h, and six days.Docket # 24032-WO- PCT

[0549] While the standard ARA methods require taking 1.0ml samples of replicate headspace for GC processing, 2.0ml samples were collected following the results of Experiment CX27572 (Investigation of GC LOD and Background Signal During C2H2 / C2H4 Assay) in effort to improve reliability of signal detection.Results

[0550] All intergeneric isolates were cultured with the addition MLS antibiotics (lug / ml Erythromicin and 25ug / ml Lincomycin) [l.Oul / ml culture fluid],

[0551] Strain 77155 (wild type Paenibacillus polymyxa ^EC 172, a known Nitrogen-fixing microorganism, as a positive control), Strain 39400-G279 (genetically modified Bacillus thuringiensis, transformed with an “empty” vector lacking the nfix cluster, as a negative control), and Strain 39400-G280 (genetically modified Bacillus thuringiensis, transformed with a vector comprising the nitrogen fixation cluster nifB-nifV from Strain 103408 Paenibacillus durus 103408) were assessed in three different assays, each in triplicate, in the acetylene reduction assay described above, under both OmM NH4 and 5mM NH4 conditions.

[0552] The positive control wild-type P. polymyxa strain 77155, naturally comprising a nitrogen fixation cluster comprising the genes nifB through nz / E, demonstrated ethylene production primarily under 0 mM NH4 conditions, as expected. The negative control Strain 39400-G279 did not demonstrate ethylene production under any condition, as expected. The experimental sample Strain 39400-G280 consistently demonstrated an ethylene production peak in one of the three replicates, in each of the three experiments under 5 mM NH4 conditions.

[0553] FIG. 1 shows that the B. thuringiensis strain 39400 that was transformed with a replicative plasmid comprising the nif cluster from P. durus strain 103408 (resulting Strain 39400-G280, lower bold chromatogram line) demonstrated nitrogen fixation capability, as demonstrated by the ethylene peak observed (shaded peak), as compared to the negative control strain 39400-G279 that did not produce an ethylene peak (upper thin chromatogram line).

[0554] The acetylene reduction to ethylene was only seen in the transformed strain under nitrogen-fortified conditions, not in nitrogen-limited media.

[0555] These results demonstrate that it is possible to impart nitrogen fixation capability to an organism not previously capable, including to an organism of a different genus than that of the source of the nitrogen fixation gene(s).Docket # 24032-WO- PCTOptimization

[0556] Further optimization of the gene expression, growth conditions, testing conditions, and / or other variables can be accomplished to further improve the results.

[0557] Possible optimizations that might increase the amplitude or consistency of ARA activity of Bacillus isolates transformed with a Paenibacillus nif expression plasmid include, but are not limited to, the following:• Using an alternate constitutive promoter for expression• Better optimizing the spacing of the constitutive promoter, ribosome binding site, and start codon for the expression cassette• Using an alternate replicate backbone with a different copy number• Extending the length of anaerobic growth over the course of the experiment• Optimizing the nitrogen free medium for better compatibility with diazotrophic growth in Bacillus strains• Testing alternate chassis Bacillus strains• Testing nif clusters from alternate Paenibacillus strains

[0558] Experiments that could shed light on the reasons for the observed inconsistency of ARA activity in 39400-G280 include:• Extracting and sequencing the replicative plasmid from the NF 11 cultures at the end of the experiment, to determine if there is any loss or modification of the plasmids.• Performing whole genome sequencing on the modified strains before and after each ARA experiment and comparing the sequences to see if there are spontaneous mutations to native DNA that are associated with absence or presence of activity.

[0559] Additional analysis, including RT-qPCR, RNA-Seq, Mass Spectrometry, or other assays, may help elucidate the genetic and transcriptional activity of the intergeneric nfix cluster.Root Colonization

[0560] Bacterial strains are prepared with the GFP gene integrated into its genome, using techniques known in the art. Seeds were treated with the strain(s), and using sterile technique, drop inoculated seeds into phytagel tubes. Tubes were placed in appropriate grow rooms and cover for 5 days to allow germination. The root tissue is separated from the seed and shoot, using an EtOH and flame sterilized tweezers and scalpels. The root tissue is cut to all be in the same focal plane and pressed at the same level on 0.8% water-agar in a square plate to image. TheDocket # 24032-WO- PCT same is performed for shoot tissue. The plant tissue is imaged for bacterial colonization using fluorescence microscopy.Biofdm Assay Protocol

[0561] This protocol was based on literature: “Effects of an EPS Biosynthesis Gene Cluster of Paenibacillus polymyxa WLY78 on Biofdm Formation and Nitrogen Fixation under Aerobic Conditions” (Chen 2021). Materials: Sterile 3 mL glass tubes, ‘Biofdm Broth (BFB)’ media, 0.1% Crystal Violet (aqueous) Solution, 40% Acetic acid. 7 days gave best overall biofdm results. Prepared using sterile technique. Results are shown in Table 3d.

[0562] The recipe for BFB includes: 5g / L KH2PO4, 5g / L K2HPO4, 0.86 g / L Mono sodium glutamate, 0.1 g / L yeast extract, Ig / L NH4C1 pH 7. Filter sterilizing after autoclaved: 36g / L glucose, 0.03g / L MgSO4.7H2O, 0.02g / L CaC12.2H2O, MnSO4-H2O 22.7 mg / L, ZnSO4 0.0785 mg / L, CuSO4 0.001 mg / L, Na2Mo04 0.004 mg / L, FeSO4 5.4mg / L.The method steps are:1. Streak isolates from -80 C.2. Make spread plates of each isolate.3. Autoclave 3 mL glass tubes in tube rack (x3 / isolate), use foil as a cover4. Harvest spread plates and OD600 balance to ~0.35. Fill each tube with 1 mL BFB.6. Inoculate with 10 uL / tube of spread plate harvest.7. Place foil cover back over tubes and incubate at 30 C, stationary for 7 days.8. After 7 days, start by removing the culture from tubes using long (1250 uL) pipette tips - collect culture in 2 mL snap cap tubes.9. Add water to the culture to reach final volume of 1 mL - Take OD600 reading.10. Wash glass tubes using RO water; fill approx, half-way, hold tube, sealing the top and shake to dislodge excess cellular material. Rinse several times.11. Remove excess water using long pipette tips.12. Add 1 mL / tube of 0.1% Crystal Violet solution and incubate for 10 minutes at room temperature.13. Remove Crystal Violet solution by pipette into a waste container (e. ., 50 mL falcon tube) and dispose in the incineration waste bin.14. Rinse glass tubes until water runs clear.Docket # 24032-WO- PCT15. Dry glass tubes (usually overnight).16. Add 1 mL 40% acetic acid solution to dissolve stained biofilm ring.17. Take OD570 reading.18. Normalize OD570 by OD600 (if appropriate).Strain Culture Production

[0549] Wild type and edited strains were assessed for CFU production capabilities at 48 hours. Gene Expression

[0550] Edited strains are evaluated for expression of the edited gene(s).Example 7: Plant testingNitrogen fixation capabilities of plants inoculated with edited strains

[0551] Plant tissue inoculated with edited strains are subjected to assay, as described above. Crop plant testing

[0552] The edited microbes described above are tested in various different types of monocot and / or dicot crop plants.

[0553] Plants are associated (physically contacted) with the wild-type and / or edited microbes described above, and tested in the greenhouse as well as in larger-scale field trials, according to standard protocols. Association may be accomplished by any one or more of the following: seed treatment, foliar treatment, in-furrow application, drench, side-dress.

[0554] Multiple replicates of plants are treated with the microbes described herein and grown. Data collected can include biomass, leaf area, plant height, root area, shoot Nitrogen, greenness, ND VI (capturing how much more near infrared light is reflected compared to visible red; a measure of the state of plant health based on how the plant reflects light at certain frequencies), NPCI (normalized pigment chlorophyll ratio index), PSRI (plant senescence reflectance index), and CCI (chlorophyll content index), and yield, and compared to an untreated control.

[0555] Taken together, these data demonstrate that although difficult to achieve, nitrogen fixation capabilities can be imparted to strains by the introduction of one or more nif genes from an intergeneric source, for the improvement of plants in agriculture.

Claims

Docket # 24032-WO- PCTTT IS CLAIMED:

1. A synthetic composition comprising:(a) a first microbe, wherein the first microbe lacks sufficient nitrogen fixation capability,(b) one or more polynucleotide(s) encoding one or more polypeptide(s) involved in nitrogen fixation obtained from a second bacterium; wherein the first microbe and the second microbe are from different taxonomic categories; wherein the first microbe comprises the one or more polynucleotides of (b); and wherein the first microbe comprising the one or more polynucleotides of (b) displays an improved phenotype as compared to a microbe not comprising the one or more polynucleotides of (b), wherein the improved phenotype is selected from the group consisting of: increased acetylene reduction capability, improved biofilm formation, increased turbidity in culture, greater nitrogen fixation, improved tolerance to oxygen levels, increased release of nitrogenous compounds, and any combination and / or plurality of the preceding.

2. The synthetic composition of Claim 1, wherein the different taxonomic categories are selected from the group consisting of: kingdom, phylum, class, order, family, and genus.

3. The synthetic composition of Claim 1, wherein the one or more polynucleotides of (b) comprises a gene(s) selected from the group consisting of: nifQ, nijB, nifA, nifL, nifF, nifM, nifV, iifS, nifU, nifX, nifN, nifE, nifY, nifK, nifE), nifH, niff nifB, nifH, nifHl nifD, nifK, nifE, nifV, nifX, orfl, hesA, hesA2, nifV, nifW, glnR, cueR, gin A, anfD, anfG, anfK vnfA, vnfD, vnfG, vnfK yggx, nifU, sox, and sod A.Docket # 24032-WO- PCT4. The synthetic composition of Claim 1, wherein the one or more polynucleotides comprise a nif cluster.

5. The synthetic composition of Claim 4, wherein the nif cluster is operably linked to a heterologous promoter.

6. The synthetic composition of Claim 1, wherein the first microbe and the second microbe are bacteria of different genera.

7. The synthetic composition of Claim 1, wherein the first microbe is of the genus Bacillus.

8. The synthetic composition of Claim 1, wherein the first microbe is Bacillus thuringiensis Bacillus subtilus, Bacillus cereus, or Bacillus velezensis.

9. The synthetic composition of Claim 1, wherein the second microbe is of the genus Paenbacillus.

10. The synthetic composition of Claim 1, further comprising a formulation component and / or an agricultural composition.

11. The synthetic composition of Claim 1, wherein the bacterium is present at a concentration of at least about 10A2 CFU / mL in a liquid formulation, or at least about 10A2 CFU / gram in a non-liquid formulation.

12. The synthetic composition of Claim 1, further comprising at least one additional microbe.

13. The synthetic composition of Claim 1, further comprising a plant or plant element.

14. The synthetic composition of Claim 13, wherein the plant or plant element comprises a transgene.

15. The synthetic composition of Claim 13, wherein the plant element is obtained from vegetative tissue.Docket # 24032-WO- PCT16. The synthetic composition of Claim 13, wherein the plant element is a plant reproductive element.

17. The synthetic composition of Claim 1, wherein the formulation component is selected from the group consisting of: a compound that improves the stability of the microbe, a preservative, a carrier, a surfactant, an anticomplex agent, and any plurality and / or combination thereof.

18. The synthetic composition of Claim 1, wherein the agricultural composition is selected from the group consisting of: a fungicide, a nematicide, a bactericide, an insecticide, an herbicide, a micronutrient, a macronutrient, Nitrogen, Phosphorous, Potassium, and any plurality and / or combination of the preceding.

19. A plurality of synthetic compositions of Claim 1, wherein said synthetic compositions are substantially confined within an object selected from the group consisting of: a tube, a petri dish, a bottle, ajar, an ampule, a package, a vessel, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and a case.

20. The synthetic composition of Claim 13, wherein the plant element is obtained from a monocot plant.

21. The synthetic composition of Claim 13, wherein the plant element is obtained from a dicot plant.

22. The synthetic composition of Claim 18, wherein the agricultural composition comprises a growth medium.

23. The synthetic composition of Claim 22, wherein the growth medium comprises soil.Docket # 24032-WO- PCT24. A plurality of synthetic compositions of Claim 23, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

25. A method of improving a characteristic of a plant, the method comprising:(a) associating an element of the plant with a synthetic composition, wherein the synthetic composition comprises: i. a first microbe, wherein the first microbe lacks sufficient nitrogen fixation capability; and ii. one or more polynucleotide(s) encoding one or more polypeptide(s) involved in nitrogen fixation obtained from a second microbe; wherein the first microbe and the second microbe are from different taxonomic categories;(a) placing the element of the crop plant in a medium that supports plant growth;(b) growing a plant from the element of the crop plant;(c) assessing one or more characteristics of the plant, wherein at least one of said characteristics is improved, as compared to the same characteristic of a plant not obtained from an element associated with the microbe of (a).

26. The method of Claim 25, wherein the different taxonomic categories are selected from the group consisting of: kingdom, phylum, class, order, family, and genus.

27. The method of Claim 25, wherein the one or more characteristics of (d) includes an improvement of nitrogen fixation, increase in biomass, increase in leaf area, increase in plant height, increase in root area, increase in shoot nitrogen composition, increase inDocket # 24032-WO- PCT greenness, increase in NDVT, increase in NPCI, increase in PSRI, increase in CCT, increase in yield, and any combination of the preceding.

28. The method of Claim 25, further comprising at least one additional microbe.

29. The method of Claim 25, wherein the associating an element of the crop plant with a bacterium comprising an edit in one or more loci of its genome is accomplished by a method selected from the group consisting of: in-furrow application, soil drench application, side-dress application, and any combination of the preceding.

30. The method of Claim 25, wherein the associating an element of the crop plant with a bacterium comprising an edit in one or more loci of its genome is accomplished by coating said plant element with a liquid formulation of the bacterium.

31. The method of Claim 25, wherein the associating an element of the crop plant with a bacterium comprising an edit in one or more loci of its genome is accomplished by coating said plant element with a substantially non-liquid formulation of the bacterium.

32. The method of Claim 25, wherein said plant element is a plant reproductive element.

33. The method of Claim 25, wherein said plant element is a leaf.

34. The method of Claim 25, wherein said plant element is a whole plant.

35. A substantially pure composition comprising a genetically modified bacterium, wherein the genetically modified bacterium comprises one or more polynucleotide(s) encoding one or more polypeptide(s) involved in nitrogen fixation obtained from a second bacterium, wherein the second bacterium is from a different taxonomic genus than the genetically modified bacterium.

36. A bacterial culture comprising the modified bacterium of Claim 35.

37. A fermentation culture comprising the modified bacterium of Claim 35.Docket # 24032-WO- PCT38. An agricultural composition, comprising the modified bacterium of Claim 35 and an agriculturally-acceptable carrier.

39. The agricultural composition of Claim 38, further comprising a plant or plant element, wherein the modified bacterium is present in the agricultural composition in an amount effective for producing an improved phenotype in the plant.

40. The agricultural composition of Claim 39, wherein the improved phenotype is an increase in the yield of the plant.

41. Any of the methods or compositions of the preceding, wherein the first and / or second bacterium is an endophyte.

42. Any of the methods or compositions of the preceding, wherein the first and / or second bacterium is capable of colonizing plant root tissue.

43. Any of the methods or compositions of the preceding, wherein the first and / or second bacterium is Gram-positive.

44. Any of the methods or compositions of the preceding, wherein the first and / or second first bacterium is Gram-negative.

45. Any of the methods or compositions of the preceding, wherein the first and / or second bacterium produces one or more composition that impact an insect, a fungus, and / or a nematode.

46. A synthetic composition, comprising a microbe of the genus Bacillus, further comprising one or more genes conferring the capability of nitrogen fixation, said one or more genes being obtained from a microbe of the genus Paenibacillus. wherein said one or more genes comprise nifB, nifH, nifD, nifK, nifE, nifV, nifX, hesA, and / or nijV.

47. The synthetic composition of Claim 46, further comprising a heterologous promoter.

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