WO2022261433A1 - Extender compositions and use thereof to increase on-seed adherence and stability of microbes - Google Patents

Extender compositions and use thereof to increase on-seed adherence and stability of microbes Download PDF

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Publication number
WO2022261433A1
WO2022261433A1 PCT/US2022/033002 US2022033002W WO2022261433A1 WO 2022261433 A1 WO2022261433 A1 WO 2022261433A1 US 2022033002 W US2022033002 W US 2022033002W WO 2022261433 A1 WO2022261433 A1 WO 2022261433A1
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Prior art keywords
composition
seed
microbes
gene
microbial
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PCT/US2022/033002
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French (fr)
Inventor
Richard Belcher
Farzaneh REZAEI
Scott Strobel
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Pivot Bio, Inc.
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Publication of WO2022261433A1 publication Critical patent/WO2022261433A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present disclosure relates to extender compositions to be used with microbes and microbial compositions, and methods of use thereof.
  • the compositions disclosed herein may
  • beneficial microbes can be cultured and transplanted to the soil near the root structure of the plant, or alternatively may be formulated in a seed coating.
  • compositions which increase the adherence of plant beneficial microbes on seed. Further, these compositions should maintain the viability of the microbes on-seed until use, and protect them from any pre-treatments already applied, thus increasing the stability of the microbes on the seed.
  • the disclosure provides a composition
  • a composition comprising: a) a sugar alcohol, at between about 5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at between about 10% and about 40% (w/v).
  • the disclosure further provides a composition
  • a composition comprising: a) a sugar alcohol, at between about 5% and about 70% (w/v); b) a chemical buffer wherein the buffer maintains the
  • composition at a neutral pH composition at a neutral pH
  • cultured microbes composition at a neutral pH
  • the disclosure further provides a composition
  • a composition comprising: a) a sugar alcohol, at between about 2.5% and about 30% (w/v); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH; c) cultured microbes; and d) a water-soluble polymer, at between about 5% and about 30% (w/v).
  • the disclosure further provides a seed coating kit comprising: a) an extender composition, comprising: i) a sugar alcohol; ii) a chemical buffer; and b) cultured microbes.
  • the extender composition of the kit comprises a water-soluble polymer.
  • the disclosure further provides a method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol, at between about 10% and about 80%
  • the disclosure further relates to seeds and plant tissues produced therefrom.
  • the disclosure further provides a method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol, at between about 2.5% and about 15%
  • the disclosure further relates to seeds and plant tissues produced therefrom.
  • the disclosure further provides a method of treating a seed or plant tissue, comprising
  • compositions comprising: a) a sugar alcohol, at between about 7.5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) a water-soluble polymer, at approximately between 5% and 40% (w/v); and d) cultured microbes to a seed or plant tissue.
  • a composition comprising: a) a sugar alcohol, at between about 7.5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) a water-soluble polymer, at approximately between 5% and 40% (w/v); and d) cultured microbes to a seed or plant tissue.
  • the disclosure further relates to seeds and plant tissues produced therefrom.
  • the disclosure further relates to a dried seed coating comprising: a) between about 50% and about 90% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) between about 5% to about 40% (w/w) cultured microbes.
  • the disclosure further relates to a dried seed coating comprising: a) between about 25% and about 50% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains
  • composition at a neutral pH; c) between about 17% and about 60% (w/w) water-soluble polymer; and d) between about 10% to about 40% (w/w) cultured microbes.
  • the disclosure further relates to a dried seed coating comprising: a) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) cultured microbes.
  • the disclosure further relates to a dried seed coating comprising: a) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) water- soluble polymer; and d) cultured microbes.
  • FIG. 1 is a bar graph showing the starting treatment titers of Kosakonia sacchari
  • FIG. 2 is a bar graph showing the application log loss of the treatments shown in FIG. 1 after application (seed coat) to two types of com seed (average of triplicated data).
  • FIG. 3 is a bar graph showing the on-seed stability (viability over time). The number of CPUs per seed were evaluated on day 0, day 7, and day 15 post seed coating (average of triplicated data). Seed was stored 21°C.
  • FIG. 4 is a line graph showing cell viability in CPUs per gram of powder (lyophilized) Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA-126740 over the
  • FIG. 5 is a line graph showing on-seed stability (viability over time) of microbes which were lyophilized and then reconstituted prior to mixture with extender composition PBX21 800 44L (“powder”, solid lines) compared to those that were in a broth solution prior to mixture with extender composition PBX21 800 44L (“broth”, dotted lines).
  • the number of CPUs per seed was evaluated over the course of 28 days post seed coating (average of triplicated data). Seed was stored 21 °C .
  • FIG. 6 is a line graph showing on-seed stability (viability over time) of microbes which were lyophilized and then reconstituted prior to mixture with extender composition
  • FIG. 7 is a line graph showing on-seed stability (viability over time) of liquid
  • FIG. 8 is a line graph showing on-seed stability (viability over time) of liquid
  • Plant tissues refers to any part of the plant during any aspect of the
  • Plant parts include leaves, roots, root hairs, rhizomes, stems, seed, ovules, pollen, flowers, fruit, cuttings, tubers, bulbs, etc.
  • An agricultural plant tissue “comprising” a dispersion of live microbes disclosed herein includes agricultural plant tissues to which the dispersion of live microbes has been applied by any of the means set forth herein, e.g., spraying, in-furrow application, seed treatment, etc.
  • Plant productivity refers generally to any aspect of growth or development of a plant that is a reason for which the plant is grown.
  • plant productivity can refer to the yield of grain or fruit harvested from a particular crop.
  • improved plant productivity refers broadly to improvements in yield of grain, fruit, flowers, or other plant parts harvested for various purposes, improvements in growth of plant
  • plant productivity is determined by comparing the productivity (e.g., yield) of a treated plant or seed (e.g., via a seed coating as described herein), vs. an untreated plant seed.
  • Microbes in and around food crops can influence the traits of those crops.
  • Plant traits that may be influenced by microbes include: yield (e.g., grain production, biomass generation, fruit development, flower set); nutrition (e.g., nitrogen, phosphorus, potassium, iron, micronutrient acquisition); abiotic stress management (e.g., drought tolerance, salt tolerance, heat tolerance); and biotic stress management (e.g., pest, weeds, insects, fungi, and bacteria).
  • yield e.g., grain production, biomass generation, fruit development, flower set
  • nutrition e.g., nitrogen, phosphorus, potassium, iron, micronutrient acquisition
  • abiotic stress management e.g., drought tolerance, salt tolerance, heat tolerance
  • biotic stress management e.g., pest, weeds, insects, fungi, and bacteria.
  • Strategies for altering crop traits include: increasing key metabolite concentrations; changing temporal dynamics of microbe influence on key metabolites; linking microbial metabolite
  • in planta refers to in the plant, on the plant, or intimately associated with the plant, depending upon context of usage (e.g. endophytic, epiphytic, or rhizospheric associations).
  • plant can include plant parts, tissue, leaves, roots, root
  • exogenous nitrogen refers to non-atmospheric nitrogen readily available in the soil, field, or growth medium that is present under non-nitrogen limiting conditions, including ammonia, ammonium, nitrate, nitrite, urea, uric acid, ammonium acids, etc.
  • non-nitrogen limiting conditions refers to non-atmospheric nitrogen available in the soil, field, media at concentrations greater than about 4 mM nitrogen, as disclosed by Kant et al. (2010. J. Exp. Biol. 62(4): 1499-1509), which is incoiporated herein by reference for all purposes.
  • a “wild type microbe,” e.g., a “wild type bacterium,” as used herein refers to a
  • Wild type microbes may be isolated and cultivated from a natural source. Wild type microbes may be selected for specific naturally occurring traits.
  • a “diazotroph” is a microbe that fixes atmospheric nitrogen gas into a more usable form, such as ammonia.
  • a diazotroph is a microorganism that is able to grow without external
  • All diazotrophs contain iron-molybdenum or -vanadium nitrogenase systems.
  • the increase of nitrogen fixation and/or the production of 1% or more of the nitrogen in the plant are measured relative to control plants, which have not been exposed to the bacteria of the present disclosure. All increases or decreases in bacteria are
  • water-soluble film package As used herein, a “water-soluble film package”, interchangeably used herein with “water-soluble package” refers to an encasement that is capable of disintegrating upon contact with a liquid, and is composed of a water-soluble film.
  • a “water-soluble film” refers to a film that is capable of disintegrating
  • the water-soluble film is “fully” soluble, meaning that all ingredients in the film are capable of fully dissolving in liquid such as water.
  • the water-soluble film further comprises one or more water-insoluble components, that still lose cohesion when exposed to a liquid, such as water.
  • the water-soluble film may contain granules, strips, netting, or other non-
  • a “non-intergeneric” remodeled microorganism is a microorganism that is formed by the deliberate combination of genetic material originally isolated from
  • a non-intergeneric remodeled microorganism can be used interchangeable with “intrageneric mutant” and “intrageneric microorganism”.
  • an “intragenic” microorganism is a microorganism that is engineered to comprise a genetic edit, or genetic modification, or genetic element, or genetic material (e.g. a nucleic acid sequence), that has been sourced from within the organism’s own species.
  • a “transgenic” microorganism is a microorganism that is engineered to comprise a genetic edit, or genetic modification, or genetic element, or genetic material (e.g. a nucleic acid sequence), that has been sourced from outside the organism’s taxonomic species.
  • 25 intergeneric remodeled microorganism has a synonymous meaning to “non-intergeneric engineered microorganism,” and will be utilized interchangeably.
  • applying,” “coating,” and “treating” agricultural plant seeds and tissues with the dispersion of microbes includes any means by which the plant seeds or tissues are made to come into contact (i.e. exposed) to a dispersion of microbes.
  • applying includes any means by which the plant seeds or tissues are made to come into contact (i.e. exposed) to a dispersion of microbes.
  • applying refers to placing or distributing the dispersion of microbes onto an area, volume, or quantity of agricultural plant seed or tissue (for example as a seed coat). Consequently, “applying” includes any of the following means of exposure to a dispersion of microbes: spraying, dripping, submerging, hand broadcast, machine spreading, brushing, machine broadcasting, and the like, onto agricultural plant seeds and tissues.
  • 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
  • cultured microorganism or “cultured microbes” refers to microbes that have been isolated and cultured. The culture can contain varying concentrations of said microbe.
  • 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
  • Microbes of the present disclosure may include spores and/or vegetative cells.
  • microbes of the present disclosure include microbes in a viable but non- culturable (VBNC) state.
  • stability refers to cell viability over time. In some embodiments, stability is reported as loss in cell viability (or percentage of remaining viability)
  • references to a percent microbial cell viability at a predetermined time is intended to describe the remaining percentage of viability from the original viability at the time the composition was first admixed and/or applied to a plant part.
  • discussion of a seed maintaining at least 50% microbial cell viability at 28 days means that the seed has 50% of the CPU at 28 days than it did at day 0 (when the seed was first coated).
  • a “seed treatment” refers to a substance that may be applied to
  • the seed treatment may provide one or more benefits to the seed and/or plant resulting from the seed.
  • seed treatments may include the dispersion of microbes disclosed herein, compositions disclosed herein, pesticides, herbicides, insecticides, nematicides, plant-growth promoting factors, fertilizers, and the like.
  • a seed treatment may also be a seed coating.
  • pre-treatmenf refers to the order of application, where “pre-treatments” are necessarily layered closer to the application locus (e.g., closer to the surface or center of a seed), with subsequent treatments covering over them.
  • Pre-treatment may refer to a single previous seed coating application (e.g. an herbicide), or may be used to collectively refer to all
  • colony forming unit or “CPU” as used herein is a unit used to estimate the number of viable microbial cells in a sample. Viable is defined as the ability to multiply under the controlled conditions. In some embodiments, counting colony-forming units involves culturing the microbes and counting only viable cells (e.g., cells capable of growing colonies),
  • a “chemical buffer,” “buffer solution,” “buffering agent,” or “buffer,” also known as a “pH buffer” or “hydrogen ion buffer,” consists of a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid.
  • neutral pH refers to a pH value of between 6 and 7.5.
  • a “dispersing agent” or “dispersant” is a substance that, when added to a solution or suspension of solid or liquid particles in a liquid, is capable of promoting the separation of the particles and thus, prevent clumping or settling of the particles.
  • a dispersing agent added to a suspension of microbes disclosed herein can improve and/or stabilize the suspension by promoting the separation of the microbes, and
  • a dispersing agent to a dispersion of microbes can promote rehydration, viability, and/or shelf- life of the microbes.
  • the dispersing agent is a biologically compatible dispersing agent, such as, for example, non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents.
  • polymer includes copolymers.
  • 5 soluble polymer refers to any synthetic, semisynthetic, or natural polymer that dissolves, disperses, or swells in water at least under some conditions, so as to be able to release ingredients admixed with the polymer and/or coated by the polymer into an aqueous solution.
  • CWT or “centum weight” is used in the context that is known in the art, as hundredweight for seed. Thus, an amount of treatment per CWT would refer to the
  • “extender” refers to compositions which prolong or maintain the viability of a microbe over time, thus increasing the overall stability of a composition comprising microbes and an extender. Extenders may also increase adherence of microbes to a seed or plant tissue.
  • a “plant enhancing agent” is any agent that provides a benefit, advantage, or protection to a seed or the plant resulting therefrom.
  • plant enhancing agents include fungicides, insecticides, biocides, herbicides, and nematicides.
  • seed coating refers to any coating on a seed or plant propagating material.
  • application log loss refers to a measurement of microbial adherence to seed or plant propagating material and is calculated by using the following equation: LOG((Day 0 treatment titers in CFU/ml) x (ml/seed application rate)) - LOG(Day 0 CFU/seed). Conceptionally this can be thought of as LOG(of theoretical microbial load per seed as determined by treatment titer and rate of application)- LOG(of actual microbial titer,
  • reconstituted refers to previously lyophilized microorganisms that have been formulated back to a liquid formulation, but which have not been permitted to grow/culture, since being formulated into the liquid formulation (e.g., through the addition of an aqueous solution).
  • reconstituted microbes are different from
  • Embodiments of the present disclosure define compositions based on their % content.
  • the % content is (v/v), which is calculated based on the volume of the recited ingredient divided by the volume of the composition (e.g., extender).
  • the % content is (w/v), which is calculated based on the weight (in grams) of the
  • the % content is (w/w), which is calculated based on the volume of the recited ingredient divided by the volume of the composition (e.g., extender). In some embodiments the % content is (w/w), which is calculated based on the weight of the recited ingredient divided by the weight of the composition (e.g., dry extender).
  • microbes coated on seed surfaces are often reduced by excess drying, environmental conditions, or mechanical stress during seed processing and/or storage. Furthermore, some microbial seed coatings may be incompatible with other types of seed treatments, including chemical pre-treatments. In addition, most seed treatment facilities are
  • the present disclosure provides extender compositions which prolong or maintain the viability of microbes on-seed, thus increasing the stability of microbes and shelf-life of coated seed.
  • the extender compositions are effective at improving stability of microbial
  • compositions disclosed herein further promote the adherence of microbes to seed during the seed coating process.
  • disclosure further teaches seed coating kits, methods of use, and seeds and plant propagating material produced therefrom which are coated with the compositions disclosed herein.
  • the present disclosure relates to a seed treatment composition
  • a seed treatment composition comprising: a) a sugar alcohol, b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH, c) a water-soluble polymer, and d) one or more cultured microbes.
  • the present disclosure relates to a seed treatment composition
  • the present disclosure relates to a liquid extender composition with cultured microbes, said extender comprising: a) a sugar alcohol, at approximately between 5% and 30% (w/v); b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at approximately between 5% and 30% (w/v).
  • the present disclosure relates to a liquid extender composition with cultured microbes, said extender comprising: a) a sugar alcohol, at approximately between 7.5% and 15% (w/v); b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at approximately between 5% and 20% (w/v).
  • the present disclosure relates to a liquid extender with cultured
  • said extender comprising: a) a sugar alcohol, at approximately between 20% and 40% (w/v) of the total volume; and b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH.
  • the extender compositions with microbes are allowed to dry on seeds.
  • the dried extender compositions with cultured microbes comprise
  • the dried extender compositions with cultured microbes comprise a sugar alcohol to polymer w/w ratio of about 4:3.
  • the present di sclosure teaches a dried seed coating compri sing: a) between about 50% and about 80% (w/w) sugar alcohol; b) a chemical buffer, wherein the
  • 20 buffer maintains the composition at a neutral pH; and c) between about 15% to about 40% (w/w) cultured microbes.
  • the dried seed coating comprises: a) about 72% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) about 24% (w/w) cultured microbes.
  • the present disclosure teaches a dried seed coating comprising: a) between about 25% and about 40% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) between about 17% and about 70% (w/w) water-soluble polymer; and d) between about 15% to about 40% (w/w) cultured microbes.
  • the dried seed coating comprises: a) about 35% (w/w) sugar
  • the extenders of the present disclosure are provided without the microbes, which are admixed with the extender prior to application on seeds.
  • the present disclosure relates to an extender composition comprising: a) a sugar alcohol, b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH,
  • the extender without the microbes comprises a) a sugar alcohol, and b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH.
  • the present disclosure relates to a liquid extender composition for increasing the stability of microbes, said extender comprising: a) a sugar
  • the present disclosure relates to a liquid extender composition for increasing the stability of microbes, said extender comprising: a) a sugar alcohol, at
  • the extender compositions without microbes are provided in dried form.
  • the dried extender compositions without cultured microbes comprise a sugar alcohol to polymer w/w ratio of between about 2:1 to 1:1.
  • the dried extender compositions without cultured microbes comprise a sugar alcohol to polymer w/w ratio of between 3 : 1 to 1 :3.
  • the extender compositions disclosed herein comprise a chemical buffer or buffering agent.
  • the buffering agent prevents fluctuations in the pH of the
  • the chemical buffer maintains the pH of the composition (extender with or without cultured microbes) in the pH range of pH 5-9, pH 5-8, pH 5-7, pH 5- 6, pH 6-9, pH 6-8, pH 6-7, pH 7-9, or pH 7-8. In some aspects, the chemical buffer maintains the composition at a neutral pH. In some aspects, the chemical buffer comprises potassium
  • the chemical buffer is a mixture of monopotassium phosphate (KH2PO4) and dipotassium phosphate (K2HPO4). In some aspects the chemical buffer is dipotassium phosphate at approximately between 0.5% and 3.0% of the total extender composition volume without the cultured microbes, and monopotassium phosphate at approximately between 0% and 2% of the total extender composition volume without the cultured microbes. In some aspects, the chemical buffer is dipotassium phosphate at
  • the chemical buffer is dipotassium phosphate at approximately between 0.25% and 1.5% of the total extender composition volume with the cultured microbes,
  • the chemical buffer is dipotassium phosphate at approximately 0.5-1% of the total extender composition volume with the cultured microbes, and monopotassium phosphate at approximately 0.25-0.5% of the total extender composition volume with the cultured microbes.
  • Non-limiting examples of buffering agents include potassium phosphates, sodium citrate, ascorbate, succinate, lactate, citric acid, boric acid, borax, hydrochloric acid, disodium
  • MES MES
  • PBS phosphate buffered saline
  • the chemical buffer is present in the extender composition with or without cultured microbes at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% (w/v), including all ranges and subranges therebetween.
  • the chemical buffer is present in the extender composition with or without cultured microbes at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,
  • compositions disclosed herein comprise a sugar.
  • the sugar is selected from the group consisting of monosaccharides, disaccharides,
  • the sugar includes one or more of trehalose, sucrose, or glycerol.
  • the sugar is a sugar alcohol or non-reducing sugar.
  • the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fiicitol, iditol, and inositol.
  • the sugar alcohol is sorbitol.
  • the sugar or sugar alcohol is present in the extender composition
  • the sugar alcohol is present in the extender composition without the cultured microbes at about 20% (w/v) with a polymer or about 60% (w/v) without a polymer. In some aspects, the sugar alcohol is present in the extender composition without the cultured microbes at about 10% (w/v) with a polymer or about 30% (w/v) without a polymer.
  • the sugar or sugar alcohol is present in the extender composition with or without cultured microbes at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
  • compositions disclosed herein comprise a polymer.
  • the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA), carboxymethyl cellulose (CMC),
  • the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
  • compositions and methods disclosed herein for example, synthetic polymers, naturally occurring polymers, copolymers, dry-phase polymers, wet-phase polymers, semi-dry
  • polymers 10 polymers, gel polymers, microporous polymers, emulsion polymers, film-forming polymers, allospheres (polymeric nanomaterials), electrospun polymers, cross-linked polymers, water- soluble polymers, and combinations thereof.
  • the polymer is a water-soluble polymer.
  • the polymer is a naturally occurring polymer. In some aspects, the
  • polymer is produced by a plant or plant part.
  • the polymer is derived from a plant, plant part, or substance therefrom.
  • the polymer is produced by an animal or animal part.
  • the polymer is derived from an animal, animal part, or substance therefrom.
  • the polymer is produced by a microbe such as an algae, protist, bacterium, or fungus.
  • the polymer is derived from a microbe or a
  • the polymer is an exopolymer. In some aspects, the polymer is an endopolymer.
  • the polymer contains only repeating units of one type of monomer. In some aspects, the polymer contains repeating units of more than one type of monomer (copolymer). In some aspects, the polymer structure is linear polymer - a linear polymer. In
  • the polymer structure is branched polymer - a branched polymer. In some aspects, the polymer structure is network polymer. In some aspects, the polymer is an interpenetrating network polymer.
  • the polymer is selected from: polyvinylpyrrolidone, polyvinylpyrrolidone-vinyl acetate copolymer (PVP-VA), 2-Pyrrolidinone, 1-
  • HPMC hydroxypropyl methylcellulose
  • HPMC hydroxypropyl methylcellulose
  • CMC carboxymethyl cellulose
  • Na- CMC sodium-carboxymethyl
  • polymethacrylic acid polymethacrylic acid, styrene-butadiene, acrylic, styrene-acrylic, vinyl acetate, tocopheryl polyethylene glycol succinate (TPGS)-based polymer, and poly(lactic-co-gly colic acid) (PLGA), etc.
  • TPGS polyethylene glycol succinate
  • PLGA poly(lactic-co-gly colic acid)
  • polymers that can be used with the compositions and methods disclosed herein include: polyvinyl acetates, polyvinyl acetate copolymers,
  • EVA ethylene vinyl acetate copolymers
  • polyvinyl alcohols polyvinyl alcohol copolymers
  • celluloses e.g., ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses, and carboxymethylcelluloses
  • polyvinylpyrolidones vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acylamide polymers and copolymers,
  • polyhydroxyethyl acrylate methylacrylamide monomers, polychloroprene, acrylamide homo- and copolymers, acrylic acid homo- and copolymer, cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose (sodium and other salts), carboxymethyl hydroxyethyl cellulose, hydroxyethyl cellulose, hydrophobically modified hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, water-soluble cellulose ethers, carboxy-vinyl
  • copolymers alginic acid, polyacrylic acid, sodium polyacrylate, partially and fully hydrolyzed polyvinyl alcohols, partially neutralized polyacrylic acid, polyalkylene glycol, polyvinylpyrrolidone and derivatives, starch and its derivatives, vinylpyrrolidone homo- and copolymers, polyacrylamide, attapulgite, montmorillonite, organically modified montmorillonite clays, alumina, precipitated silica, or any mixture thereof
  • the polymer is present in the extender compositions disclosed herein with or without cultured microbes at a % weight to volume of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,
  • the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA) and is present in the extender composition without the cultured microbes at approximately 15%. In some aspects, the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA) and is present in the extender composition with the cultured microbes at approximately 7.5%.
  • the polymer is present in the extender compositions disclosed herein
  • % (wt/wt) about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, including
  • the extender compositions disclosed herein are mixed with one or more cultured microbes and/or microbial compositions to be used as a seed coating. In some embodiments, the extender compositions disclosed herein are mixed with one or more
  • the microorganisms mixed with the compositions disclosed herein may be in a liquid or powder form, or may be reconstituted from a powder to a liquid form prior to mixing with the extender compositions disclosed herein.
  • the disclosed extender compositions exhibit higher protective effects on
  • the disclosed extender compositions can be directly mixed with dried microbes in powder form, without the need for a prior reconstitution step.
  • the microbial composition: extender composition ratio is between 1:1 and 1:4 by percent volume. In some aspects, the microbial composition is a liquid
  • composition and the ratio to the extender composition is 1 : 1 by percent volume.
  • the microbial composition is a powder and is reconstituted to a liquid prior to mixture with the extender composition at a powder: reconstituted liquid ratio of between 1:4 and 3:10 by weight to volume, including all ranges and subranges therebetween.
  • the powdered microbes are added directly to the liquid extender
  • the powdered microbes are added to the liquid extender composition at a ratio between 1 :3 and 1 :4. In some aspects, the powdered microbes are added to the liquid extender composition at a ratio of 3:10, 2.5:10, 2:10, or 1.5:10.
  • the extender compositions of the present disclosure comprise
  • the one or more cultured microbes comprise between 1.0 X 10 4 and 1.0 X 10 12 CFU/mL of the total volume of the mixed extender composition. In some aspects,
  • the microorganisms are at an initial concentration of 10 4 to 10 12 CFU/mL In some aspects, the microorganisms are at an initial concentration of 10 8 to 10 10 CFU/ml. In some aspects, the microorganisms are at an initial concentration of about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10°, or 10 12 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 10 8 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 10 9
  • the microorganisms are at an initial concentration of about 10 10 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 10 11 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 10 12 CFU/mL.
  • the cultured microbes are dried (e g., lyophilized) and present in the
  • 20 extender composition at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
  • the cultured microbes are at a concentration from about between 1.0 X 10 4 and 1.0 X 10 8 CFU per seed.
  • the cultured microbes are at an initial concentration of 10 4 , 10 5 , 10 6 , 10 7 , 10 8 per seed.
  • microbes can be obtained from any source, including environmental and
  • plant beneficial bacteria may be obtained from any general terrestrial environment, including its soils, plants, fungi, animals (including invertebrates) and other biota, including the sediments, water and biota of lakes and rivers; from the marine environment, its biota and sediments (for example, sea water, marine muds, marine plants, marine invertebrates (for example, sponges), marine vertebrates (for example, fish); the terrestrial and marine geosphere (regolith and rock, for example, crushed subterranean rocks,
  • cryosphere and its meltwater for example, filtered aerial dusts, cloud and rain droplets
  • atmosphere for example, filtered aerial dusts, cloud and rain droplets
  • urban, industrial and other man-made environments for example, accumulated organic and mineral matter on concrete, roadside gutters, roof surfaces, and road surfaces.
  • a plant having one or more desirable traits may be a plant having one or more desirable traits, for example a plant which naturally grows in a particular environment or under certain conditions of interest.
  • a certain plant may naturally grow in sandy soil or sand of high salinity, or under extreme temperatures, or with little water, or it may be resistant to certain pests or disease present in the environment, and it may be desirable for a commercial crop to be grown in such conditions,
  • the bacteria may be collected from commercial crops grown in such environments, or more specifically from individual crop plants best displaying a trait of interest amongst a crop grown in any specific environment: for example the fastest- growing plants amongst a crop grown in saline-limiting soils, or the least damaged plants in
  • the bacteria may be collected from a plant of interest or any material occurring in the environment of interest, including fungi and other animal and plant biota, soil, water, sediments, and other elements of the environment as
  • the bacteria may be isolated from plant tissue. This isolation can occur from any appropriate tissue in the plant, including for example root, stem and leaves, and plant reproductive tissues. Non-limiting examples of plant tissues include a seed, seedling, leaf, cutting, plant, bulb, tuber, root, and rhizomes. In some
  • microorganisms are isolated from a seed. In ssoommee embodiments, microorganisms are isolated from a root. [00103] Persons having skill in the art will be familiar with techniques for recovering microbes from various environmental sources. For example, microbes useful in the compositions and methods disclosed herein can be obtained by extracting microbes from surfaces or tissues of native plants; grinding seeds to isolate microbes; planting seeds in diverse
  • the parameters for processing samples may be varied to isolate different types of associative microbes, such as rhizospheric, epiphytes, or endophytes.
  • associative microbes such as rhizospheric, epiphytes, or endophytes.
  • some methods for isolation from plants include the sterile excision of the plant material of interest (e.g. root or stem lengths,
  • the surface-sterilized plant material can be crushed in a sterile liquid (usually water) and the liquid suspension, including small pieces of the crushed plant material spread over the surface of a suitable solid agar medium, or media, which may or may not be selective (e.g. contain only
  • the plant root or foliage samples may not be surface sterilized but only washed gently thus including surfacedwelling epiphytic microorganisms in the isolation process, or the epiphytic microbes can be
  • the roots may be processed without washing off small quantities of soil attached to the roots, thus including microbes that colonize the plant rhizosphere. Otherwise, soil adhering to the roots can be removed, diluted and spread
  • Microbes may also be sourced from a repository, such as environmental strain collections, instead of initially isolating from a first plant.
  • the microbes can be genotyped and phenotyped, via sequencing the genomes of isolated microbes; profiling the composition of
  • Selected candidate strains or populations can be obtained via sequence data; phenotype data; plant data (e.g., genome, phenotype, and/or yield data); soil data (e.g., pH, N/P/K content, and/or bulk soil biotic communities); or any combination of these
  • the one or more cultured microbes are selected from species
  • the one or more microbes comprise Kosakonia sacchari. In some aspects, the one or more cultured microbes is Kosakonia sacchari
  • the one or more cultured microbes is Klebsiella variicola. In some aspects, the one or more cultured microbes is Klebsiella variicola PTA-126740, as described in International Patent Publication No. WO2021222567.
  • the one or more cultured microorganisms of the disclosure are those from Table 2. In other aspects, the one or more cultured microorganisms of the disclosure are derived from a microorganism of Table 2. For example, a strain, child, mutant, or derivative,
  • microbes from Table 2 are provided herein.
  • the disclosure contemplates all possible combinations of microbes listed in Table 2, said combinations sometimes forming a microbial consortia.
  • the microbes from Table 2 can be combined with any plant, active molecule (synthetic, organic, etc.), adjuvant, carrier, supplement, biofilm, or biological in a microbial composition.
  • the one or more cultured microbes are a microbial composition comprising at least one of a polymer, sugar, biofilm, and isolated biofilm compositions.
  • the microbes of this disclosure are nitrogen fixing microbes
  • microbes usefill in the compositions and methods disclosed herein are spore forming microbes, for example spore forming bacteria.
  • bacteria useful in the compositions and methods disclosed herein are Gram positive bacteria or Gram negative bacteria.
  • the bacteria are endospore forming bacteria of the Firmicute phylum. In some embodiments, the bacteria are diazotrophs. In some embodiments, the bacteria are not diazotrophs.
  • compositions and methods of the disclosure are used with an archaea, such as, for example, Methanothermobacter thermoautotrophicus, Methanosarcina
  • the one or more cultured microbes include, but are not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus
  • Bacillus aminovorans Bacillus amylolyticus (also known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus coagulans, Bacillus endoparasiticus Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus lacticola,
  • Bacillus lactimorbus Bacillus lactis.
  • Bacillus laterosporus also known as Brevibacillus laterosporus
  • Bacillus lautus Bacillus lentimorbus
  • Bacillus lentus Bacillus licheniformis
  • Bacillus maroccamis Bacillus megaterium, Bacillus metiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus.
  • Bacillus papillae Bacillus psychrosaccharolyticus
  • Bacillus pumilus Bacillus siamensis
  • Bacillus smithii Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis. Bacillus uniflagellatus, Bradyrhizobium japonicum, Brevibacillus brevis, Brevibacillus laterosporus (formerly Bacillus laterosporus), Chromobacterium subtsugae, Delftia acidovorans, Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly Bacillus popilliae), Pantoea agglomerans, Pasteuria penetrans (formerly Bacillus penetrans), Pasteuria usgae, Pectobacterium carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa,
  • Pseudomonas aureofaciens Pseudomonas cepacia (formerly known as Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas fluoresce ns, Pseudomonas proradix, Pseudomonas putida, Pseudomonas syringae, Serratia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saraceticus, Streptomyces
  • the bacterium is Azotobacter chroococcum, Methanosarcina barkeri,
  • the bacterium is a species of Clostridium, for example Clostridium pasteuriamim, Clostridium beijerinckii, Clostridium perfringens, Clostridium
  • the one or more cultured microbes used with the compositions and methods of the present disclosure are cyanobacteria.
  • cyanobacteria examples include Anabaena (for example Anagaena sp. PCC7120), Nostoc (for example Nostoc punctiforme), or Synechocystis (for example Synechocystis sp. PCC6803).
  • the one or more cultured microbes used with the compositions and methods of the present disclosure belong to the phylum Chlorobi, for example Chlorobium tepidum.
  • microbes used with the compositions and methods of the present disclosure comprise a gene homologous to a known NifH gene. Sequences of known
  • NifH genes may be found in, for example, the Zehr lab NifH database, (wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, April 4, 2014), or the Buckley lab NifH database (www.css.comell.edu/faculty/buckley/nifh.htm, and Gaby, John Christian, and Daniel H. Buckley. "A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria.” Database 2014 (2014): bauOOL).
  • microbes used with the compositions and methods of the present disclosure comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%,
  • microbes used with the compositions and methods of the present disclosure comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more than 99% sequence identity to a sequence from the Buckley lab NifH
  • compositions and methods described herein make use of bacteria that are able to self-propagate efficiently on the leaf surface, root surface, or inside
  • the bacteria described herein are isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen.
  • the one or more cultured microbes is an endophyte or an epiphyte or a bacterium inhabiting the plant rhizosphere (rhizospheric bacteria). Endophytes
  • the bacteria can be a seed-borne endophyte.
  • Seed-borne endophytes include bacteria associated with or derived from the seed of a grass or plant, such as a seed-borne bacterial endophyte found in
  • the seed-borne bacterial endophyte can be associated with or derived from the surface of the seed; alternatively, or in addition, it can be associated with or derived from the interior seed compartment (e.g., of a surface-sterilized seed). In some aspects, a seed-borne bacterial endophyte is capable of replicating within the plant tissue, for example, the interior of the seed.
  • the seed-borne bacterial endophyte is capable of surviving desiccation.
  • microbes for example those that exhibit complementary colonization (different nutrient utilization, temporal occupation, oxygen adaptability, and/or spatial occupation), and/or different benefits to the seed or plant (nitrogen fixation, pest and/or pathogen control, etc.) can be used with the compositions and methods disclosed herein to increase on-seed adherence and stability of the microbe(s).
  • the one or more cultured microbes used in the compositions and methods of the disclosure can comprise a plurality of different microorganism taxa in combination.
  • the bacteria may include Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Deljtia, Bradyrhizobiun, Sinorhizobium
  • Proteobacteria such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Deljtia, Bradyrhizobiun, Sinorhizobium
  • the bacteria used in compositions and methods of this disclosure may include nitrogen fixing bacterial consortia of two or more species.
  • one or more bacterial species of the bacterial consortia may be capable of fixing nitrogen.
  • one or more species of the bacterial consortia facilitate or enhance the ability of other bacteria to fix nitrogen.
  • the bacteria which fix nitrogen and the bacteria which enhance the ability of other bacteria to fix nitrogen may be the same or different.
  • a bacterial strain is able to fix nitrogen when in combination with a different bacterial strain, or in a certain bacterial consortia, but may be unable to fix nitrogen in a monoculture. Examples
  • bacterial genera which may be found in a nitrogen fixing bacterial consortia include, but are not limited to, Herbaspirillum, Azospirillum, Enterobacter, and Bacillus.
  • Bacteria that can be used in the compositions and methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp.
  • the bacteria are selected from the group consisting of: Azotobacter vinelandii, Bradyrhizobium
  • the bacteria are of the genus Enterobacter or Rahnella. In some aspects, the bacteria are of the genus Frankia, or Clostridium. Examples of bacteria of the genus Clostridium include, but are not limited to, Clostridium acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perjringens, and Clostridium tetani. In some aspects, the bacteria are of the genus
  • Paenibacillus for example Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus, Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chibensis, Paenibacillus glucanolyticus, Paenibacillus illinoisensis, Paenibacillus larvae subsp. Larvae, Paenibacillus larvae subsp.
  • Pulvifaciens Paenibacillus lautus, Paenibacillus macerans, Paenibacillus macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli, Paenibacillus peoriae, or Paenibacillus polymyxa.
  • bacteria for use in the present compositions and methods can be a member of one or more of the following taxa: Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Ancylobacter, Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus, Brevundimonas,
  • Sediminibacterium Serratia, Shigella, Shinella, Sinorhizobium, Sinosporangium,
  • Sphingobacterium Sphingomonas, Sphingopyxis, Sphingosinicella, Staphylococcus,
  • the bacteria are Gram-negative bacteria of a genus selected
  • Acetobacter Acetobacter, Achromobacter, Aerobacter, Anabaena, Azoarcus, Azomonas, Azorhizobium, Azospirillum, Azotobacter, Beijemickia, Bradyrhizobium, Burkholderia, Citrobacter, Derxia, Enterobacter, Herbaspirillum, Klebsiella, Kluyvera, Kosakonia, Nostoc, Mesorhizobium, Rahnella, Rhizobium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Serratia Sinorhizobium, Spirillum, Trichodesmium, and Xanthomonas.
  • a bacterial species selected from at least one of the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella. In some aspects, a
  • the species utilized can be one or more of: Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari, and Rahnella aquatilis.
  • a Gram positive microbe may have a Molybdenum-Iron nitrogenase system comprising: nifH, niJD, niJK, nijB, niJE, nifN, nijX, hesA, nifV, nifW, nifU,
  • a Gram positive microbe may have a vanadium nitrogenase system comprising: vnfDG, vnjK, vnfE, vnfN, vupC, vupB, vupA, vnjV, vnJRl, vnfH, vnfR2, vnfA (transcriptional regulator).
  • a Gram positive microbe may have an iron-only nitrogenase system comprising: anfK, anjG, anfD, anfH, anfA (transcriptional regulator).
  • a Gram positive microbe may have a nitrogenase system
  • glnB comprising glnB, an&glnK (nitrogen signaling proteins).
  • enzymes involved in nitrogen metabolism in Gram positive microbes include glnA (glutamine synthetase), gdh (glutamate dehydrogenase), bdh (3 -hydroxy butyrate dehydrogenase), glutaminase, gltAB/gltB/gltS (glutamate synthase), asnA/asnB (aspartate- ammonia ligase/asparagine synthetase), and ansA/ansZ (asparaginase).
  • proteins involved in nitrogen include glnA (glutamine synthetase), gdh (glutamate dehydrogenase), bdh (3 -hydroxy butyrate dehydrogenase), glutaminase, gltAB/gltB/gltS (glutamate syntha
  • 20 transport in Gram positive microbes include amtB (ammonium transporter), glnK (regulator of ammonium transport), glnPHQ/ glnQHMP (ATP-dependent glutamine/glutamate transporters), glnT/alsT/yrbD/yflA (glutamine-like proton symport transporters), and ⁇ P/gltT/yhcl/nqt (glutamate-like proton symport transporters).
  • amtB ammonium transporter
  • glnK regulatory of ammonium transport
  • glnPHQ/ glnQHMP ATP-dependent glutamine/glutamate transporters
  • glnT/alsT/yrbD/yflA glutamine-like proton symport transporters
  • ⁇ P/gltT/yhcl/nqt glutamate-like proton symport transporters
  • Gram positive microbes for use within the present compositions include
  • Paenibacillus polymixa Paenibacillus riograndensis, Paenibacillus sp., Frankia sp., Heliobacterium sp., Heliobacterium chlorum, Heliobacillus sp., Heliophilum sp., Heliorestis sp., Clostridium acetobidylicum, Clostridium sp., Methanobacterium sp., Micrococcus sp., Mycobacterium flavum, Mycobacterium sp., Arthrobacter s spp..,, Agromyces sp., Corynebacterium autitrophicum, Corynebacterium sp., Micromonospora sp.,
  • the microorganism which is combined with the compositions disclosed herein is genetically modified to have improved nitrogen fixation capabilities.
  • the microbes comprise one or more genetic variations introduced into one or
  • the genetic variation may be introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ.
  • the genetic variation may be a variation in a gene encoding a protein with functionality selected from the group consisting of: glutamine
  • the genetic variation may be a mutation that results in one or more of: increased expression or activity of nifA or glutaminase; decreased expression or activity of nifL, ntrB, glutamine synthetase, glnB, glnK, draT, amtB; decreased expression or activity of nifA or glutaminase; decreased expression or activity of nifL, ntrB, glutamine synthetase, glnB, glnK, draT, amtB; decreased
  • the genetic variation may be a variation in a gene selected from the group consisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and combinations thereof.
  • the microbe has a disrupted (e.g., deleted or partially deleted)
  • the microbe has a nifL gene that has been disrupted with the introduction of a promoter sequence that acts on the nifA gene.
  • the promoter is a K. variicola PinflC promoter.
  • the promoter is a K. sacchari Prm5 promoter.
  • the microbe has a glnE gene that has been altered to remove the
  • the microbe has a deletion of the glnD gene.
  • the genetic variation introduced into one or more microorganisms may be a knockout mutation or it may abolish a regulatory sequence of a target gene, or it may comprise
  • a heterologous regulatory sequence for example, insertion of a regulatory sequence found within the genome of the same bacterial species or genus.
  • the regulatory sequence can be chosen based on the expression level of a gene in a bacterial culture or within plant tissue. The genetic variation may be produced by chemical mutagenesis. The plants grown may be exposed to biotic or abiotic stressors.
  • the one or more cultured microbes for use with the compositions and methods disclosed herein also envision altering the impact of ATP or O2 on the circuitry, or replacing the circuitry with other regulatory cascades
  • Gene clusters can be reengineered to generate functional products under the control of a heterologous regulatory system. By eliminating native regulatory elements outside of, and within, coding sequences of gene clusters, and replacing them with alternative regulatory systems, the functional products of complex genetic operons and other gene clusters can be controlled and/or moved to
  • the synthetic gene clusters can be controlled by genetic circuits or other inducible regulatory systems, thereby controlling the products’ expression as desired.
  • the expression cassettes can be designed to act as logic gates, pulse generators, oscillators, switches, or memory devices.
  • the controlling expression cassette can be designed to act as logic gates, pulse generators, oscillators, switches, or memory devices.
  • the expression cassette 15 be linked to a promoter such that the expression cassette functions as an environmental sensor, such as an oxygen, temperature, touch, osmotic stress, membrane stress, or redox sensor.
  • an environmental sensor such as an oxygen, temperature, touch, osmotic stress, membrane stress, or redox sensor.
  • nifL, nifA, nijT, and nijX genes can be eliminated from the nif gene cluster.
  • Synthetic genes can be designed by codon randomizing the DNA encoding each amino acid sequence. Codon selection is performed, specifying that codon usage be as
  • Proposed sequences are scanned for any undesired features, such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, sigma 54 and sigma 70 promoters, cryptic ribosome binding sites, and rho independent terminators.
  • Synthetic ribosome binding sites are chosen to match the strength of each corresponding native ribosome binding site, such as by constructing a
  • a fluorescent reporter plasmid in which the 150 bp surrounding a gene's start codon (from -60 to +90) is fused to a fluorescent gene.
  • This chimera can be expressed under control of the Ptac promoter, and fluorescence measured via flow cytometiy.
  • a library of reporter plasmids using 150 bp (-60 to +90) of a synthetic expression cassette is generated.
  • a synthetic expression cassette can consist of a random DNA
  • Some examples of genetic alterations which may be made in Gram positive microbes include: deleting glnR to remove negative regulation of BNF in the presence of environmental
  • GlnR is the main regulator of N metabolism and fixation in, e.g., Paenibacillus species.
  • the genome of a Paenibacillus species does not contain a gene to produce glnR.
  • the genome of a Paenibacillus species does not contain a gene to produce glnE or glnD.
  • the genome of a Paenibacillus species does contain a gene to produce glnB or glnK.
  • Paenibacillus sp. WLY78 doesn’t contain a gene
  • Paenibacillus polymixa E681 lacks glnK and gdh, has several nitrogen compound transporters, but only amtB appears to be controlled by GlnR.
  • Paenibacillus sp. JDR2 has glnK, gdh and most other central nitrogen metabolism genes, has many fewer nitrogen compound
  • Paenibacillus riograndensis SBR5 contains a standard glnRA operon, an fdx gene, a main nif operon, a secondary nif operon, and an anf operon (encoding iron-only nitrogenase). Putative glnR/tnrA sites were found upstream of each of these operons. GlnR may regulate all of the above operons, except the anf operon. GlnR may bind to each of these regulatoiy sequences as a dimer.
  • Paenibacillus N-fixing strains may fall into two subgroups: Subgroup I, which contains only a minimal nif gene cluster and subgroup n, which contains a minimal cluster, plus an uncharacterized gene between nifX and hesA, and often other clusters duplicating some of the nif genes, such as nifH, nifHDK, nifBEN, or clusters encoding vanadaium nitrogenase (ynf) or iron-only nitrogenase (anf) genes.
  • Subgroup I which contains only a minimal nif gene cluster
  • subgroup n which contains a minimal cluster, plus an uncharacterized gene between nifX and hesA, and often other clusters duplicating some of the nif genes, such as nifH, nifHDK, nifBEN, or clusters encoding vanadaium nitrogenase (ynf) or iron-only nitrogenase (anf) genes.
  • the genome of a Paenibacillus species may not contain a gene to produce glnB or glnK.
  • the genome of a Paenibacillus species may contain a minimal nif cluster with 9 genes transcribed from a sigma-70 promoter.
  • a Paenibacillus nif cluster is negatively regulated by nitrogen or oxygen.
  • the genome of a Paenibacillus species does not contain a gene to produce sigma-54.
  • Paenibacillus sp. WLY78 does not contain a gene for sigma-54.
  • a nif cluster is regulated by glnR, and/or TnrA.
  • activity of a nif cluster is altered by altering
  • GlnR glutamine synthetase
  • TnrA TnrA
  • GlnR binds and represses gene expression in the presence of excess intracellular glutamine and AMP.
  • GlnR may be to prevent the influx and intracellular production of glutamine and ammonium under conditions of high nitrogen availability.
  • TnrA may bind and/or activate (or repress) gene expression in the presence of limiting intracellular glutamine, and/or in the presence of FBI-GS.
  • the activity of a Bacilli nif cluster is altered by altering the activity of GlnR.
  • FBI-GS Feedback-inhibited glutamine synthetase
  • Several bacterial species have a GlnR/TnrA binding site upstream of the nif cluster. Altering the binding of FBI-GS and GlnR may alter the activity of the nif pathway.
  • the microbes are non-intergeneric remodeled microbes.
  • non-intergeneric indicates that the genetic variations introduced into the host do not contain nucleic acid sequences from outside the host genus.
  • the microbes are intragenic. Therefore, in some embodiments, the microbes are not transgenic.
  • promoters for promoter swapping are selected from within the microbe’s genome, or genus.
  • Exemplary non-intergeneric genetic variations include a mutation in the gene of interest that may improve the function of the protein encoded by the gene; a constitutionally active promoter that can replace the endogenous promoter of the gene of interest to increase the expression of the gene; a mutation that will inactivate the gene of interest; the insertion of a promoter from within the host’s genome into a heterologous location, e.g. insertion of the
  • a genetic variation may comprise an inactivating mutation of the nifL gene (negative regulator of nitrogen fixation pathway) and/or comprise replacing the endogenous promoter of the nifA and/or nifH gene (nitrogenase iron protein that catalyzes a key reaction to fix atmospheric nitrogen) with a constitutionally active promoter that will
  • the one or more cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, ni/J, nifH, nifD, nifK, nifY, nifE, nifN, nifU,
  • nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nijQ a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii,yjbE,fhaB, pehA, otsB, treZ, glsA2, or combinations thereof.
  • the one or more cultured microbes are non-intergeneric remodeled bacteria capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
  • One or more of the microorganisms described above may be mixed with the compositions described herein and used as a treatment seed coating for a seed or plant propagating material.
  • Conventional or otherwise suitable coating equipment or techniques may be used to coat the seeds or plant propagating material with the seed coating treatments described above. Suitable equipment is deemed to include drum coaters, fluidized beds, rotary
  • the extender compositions disclosed herein increase microbial adherence to seed or plant propagating material. In some embodiments, the extender
  • compositions disclosed herein increase adherence of live microbes to seed or plant propagating material.
  • the extender compositions disclosed herein increase stability of microbes during treatment of a seed or plant propagating material.
  • the microbes exhibit a log loss of less than 1.5 on seed. In some aspects, the microbes exhibit a log loss of less than 1 on seed.
  • the extender compositions disclosed herein increase microbial growth factor
  • microbes mixed with the extender compositions disclosed herein maintain at least 50% viability on seed after 28 days storage at room temperature. In some aspects, microbes mixed with the extender compositions disclosed herein maintain at least 60% viability on seed after 28 days storage at room temperature. In some aspects, microbes mixed with the extender compositions disclosed herein
  • microbes mixed with the extender compositions disclosed herein maintain at least 70% viability on seed after 28 days storage at room temperature.
  • microbes mixed with the extender compositions disclosed herein maintain at least 80% viability on seed after 28 days storage at room temperature.
  • microbes mixed with the extender compositions disclosed herein maintain at least 90% viability on seed after 28 days storage at room temperature.
  • the composition is a seed coat present on a plant seed or other plant propagation material. In some aspects, the composition is a seed coat present on a plant seed or other plant propagation material that has at least one pre-treatment. In some aspects, the pre-treatment is a plant enhancing agent. In some embodiments, the pre-treatment is an insecticide, herbicide, fungicide, biocide, or nematicide.
  • compositions of the present disclosure allow for downstream treatment of seed or plant propagation material.
  • the seed or plant propagation material to be coated with the compositions disclosed herein may have any number of pre-treatments, such as, plant enhancing agents, for example, insecticides, fungicides, herbicides, nematicides, and the like.
  • Insecticides Al) the class of carbamates consisting of aldicarb, alanycarb, benfuracarb, carbaryl, carbofuran, carbosulfan, methiocarb, methomyl, oxamyl, pirimicarb, propoxur and thiodicarb; A2) the class of organophosphates consisting of acephate, azinphos- ethyl, azinphos-methyl, chlorfenvinphos, chlorpyrifos, chlorpyrifos-methyl, demeton-S-
  • pyrafluprole and pyriprole 5 pyrafluprole and pyriprole; A5) the class of neonicotinoids consisting of acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam; A6) the class of spinosyns such as spinosad and spinetoram; A7) chloride channel activators from the class of mectins consisting of abamectin, emamectin benzoate, ivermectin, lepimectin and milbemectin; A8) juvenile hormone mimics such as hydroprene, kinoprene, methoprene,
  • 20 receptor agonists such as amitraz; Al 8) mitochondrial complex electron transport inhibitors pyridaben, tebufenpyrad, tolfenpyrad, flufenerim, cyenopyrafen, cyflumetofen, hydramethylnon, acequinocyl or fluacrypyrim;A19) voltage-dependent sodium channel blockers such as indoxacarb and metaflumizone; A20) inhibitors of the lipid synthesis such as spirodicl ofen, spiromesifen and spirotetramat; A21) ryanodine receptor-modulators from the
  • azadirachtin such as azadirachtin, amidoflumet, bifenazate, fluensulfone, piperonyl butoxide, pyridalyl, sulfoxaflor; or A23) sodium channel modulators from the class of pyrethroids consisting of acrinathrin, allethrin, bifenthrin, cyfluthrin, gamma-cyhalothrin, lambda- cyhalothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta-cypermethrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, tau- fluvalinate, permethrin, silafluofen, tefluthrin and tralomethr
  • Fungicides Bl) azoles selected from the group consisting of bitertanol,
  • carboxamides selected from the group consisting of carboxin, benalaxyl, benalaxyl-M, fenhexamid, flutolanil, furametpyr, mepronil, metalaxyl, mefenoxam, ofurace, oxadixyl, oxy carboxin, penthiopyrad, isopyrazam, thifluzamide, tiadinil, 3,4-dichloro- N-(2-cyanophenyl)isothiazole-5-carboxamide, dimethomorph, flumorph, flumetover, fluopicolide (picobenzamid), zoxamide, carpropamid, diclocymet, mandipropamid, N-(2-(443-
  • heterocyclic compounds selected from the group consisting of fluazinam, pyrifenox, bupirimate, cyprodinil, fenarimol, ferimzone, mepanipyrim, nuarimol, pyrimethanil, triforine, fenpiclonil, fludioxonil, aldimorph, dodemorph, fenpropimorph, tridemorph, fenpropidin, iprodione, procymidone, vinclozolin, famoxadone, fenamidone, octhilinone, proben-azole, 5- chloro-7-(4-methyl-piperidin-l-yl)-6-(2,4,6-trifluorophenyl)41,2,4]triazolo[l,5-a]pyrimidine,
  • 25 derivatives binapacryl, dinocap, dinobuton, sulfur-containing heterocyclyl compounds: dithianon, isoprothiolane, organometallic compounds: fentin salts, organophosphorus compounds: edifenphos, iprobenfos, fosetyl, fosetyl-aluminum, phosphorous acid and its salts, pyrazophos, tolclofos-methyl, organochlorine compounds: dichlofluanid, flusulfamide, hexachloro-benzene, phthalide, pencycuron, quintozene, thiophanate-methyl, tolylfluanid,
  • Herbicides Cl) acetyl-CoA carboxylase inhibitors (ACC), for example
  • cyclohexenone oxime ethers such as alloxydim, clethodim, cloproxydim, cycloxydim, sethoxydim, tralkoxydim, butroxydim, clefoxydim or tepraloxydim; phenoxyphenoxypropionic esters, such as clodinafop-propargyl, cyhalofop-butyl, diclofop- methyl, fenoxaprop-ethyl, fenoxaprop-P-ethyl, fenthiapropethyl, fluazifop-butyl, fluazifop-P- butyl, haloxyfop-ethoxyethyl, haloxyfop-methyl, haloxyfop-P-methyl, isoxapyrifop,
  • arylaminopropionic acids such as flamprop-methyl or flamprop-isopropyl
  • C2 acetolactate synthase inhibitors ALS
  • imidazolinones such as imazapyr, imazaquin, imazamethabenz-methyl (imazame), imazamox, imazapic or imazethapyr
  • pyrimidyl ethers such as pyrithiobac-acid, pyrithiobac-sodium, bispyribac-sodium. KIH-6127 or pyribenzoxym;
  • sulfonamides such as florasulam, flumetsulam or metosulam; or sulfonylureas, such as amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, halosulfuron-methyl, imazosulfuron, metsulfuron-methyl, nicosulfuron, primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, thifensulfuron-methyl,
  • amides for example allidochlor (CDAA), benzoylprop- ethyl, bromobutide, chiorthiamid. diphenamid, etobenzanidibenzchlomet), fluthiamide, fosamin or monalide;
  • auxin herbicides for example pyridinecarboxylic acids, such as clopyralid or picloram; or 2,4-D or benazolin;
  • auxin transport inhibitors for example
  • C6 carotenoid biosynthesis inhibitors, for example benzofenap, clomazone (dimethazone), diflufenican, fluorochloridone, fluridone, pyrazolynate, pyrazoxyfen, isoxaflutole, isoxachlortole, mesotrione, sulcotrione (chlormesulone), ketospiradox, flurtamone, norflurazon or amitrol; C7) enolpyruvylshikimate-3-phosphate synthase inhibitors (EPSPS), for example glyphosate or sulfosate; C8) glutamine synthetase inhibitors, for example bilanafos (bialaphos) or glufosinate-ammonium; C9) lipid biosynthesis inhibitors, for example anilides, such as anilofos or mefenacet; chloroacetanilides, such as
  • esprocarb molinate, pebulate, prosulfocarb, thiobencarb (benthiocarb), tri-allate or ve olate; or benfuresate or perfluidone; CIO) mitosis inhibitors, for example
  • carbamates such as asulam, carbetamid, chlorpropham, orbencarb, pronamid (propyzamid), propham or tiocarbazil; dinitroanilines, such as benefin, butralin, dinitramin, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine or trifluralin; pyridines, such as dithiopyr or thiazopyr; or butamifos, chlorthal-dimethyl (DCPA) or maleic hydrazide; Cl l) protoporphyrinogen IX oxidase inhibitors, for example diphenyl ethers, such as acifluorfen,
  • nipyraclofen 20 nipyraclofen; Cl 2) photosynthesis inhibitors, for example propanil, pyridate or pyridafol; benzothiadiazinones, such as bentazone; dinitrophenols, for example bromofenoxim, dinoseb, dinoseb-acetate, dinoterb or DNOC; dipyridylenes, such as cyperquat-chloride, difenzoquat- methyl sulfate, diquat or paraquat-dichloride; ureas, such as chlorbromuron, chlorotoluron, difenoxuron, dimefuron, diuron, ethidimuron, fenuron, fluometuron, isoproturonisouron,
  • Nematicides Benomyl, cloethocarb, aldoxycarb, tirpate, diamidafos, fenamiphos,
  • Biocides may be a chemical substance or a microorganism.
  • a biocide may be a bacteria, such as Pseudomonas, Enterobacter, Stenotrophomonas,
  • Plant Growth Regulators or Hormones such as clofibric acid, 2,3,5- triiodobenzoic acid; D2) Auxins such as 4-CPA, 2,4-D, 2,4-DB, 2,4-DEP, dichlorprop, fenoprop, IAA, IB A, naphthaleneacetamide, a-naphthaleneacetic acids, 1 -naphthol, naphthoxyacetic acids, potassium naphthenate, sodium naphthenate, 2,4,5-T; D3) cytokinins, such as 21P, benzyl adenine, 4-hydroxyphenethyl alcohol, kinetin, zeatin; D4) defoliants, such as
  • 25 as calcium cyanamide, dimethipin, endothal, ethephon, merphos, metoxuron, pentachlorophenol, thidiazuron, tribufos; D5) ethylene inhibitors, such as aviglycine, 1- methylcyclopropene; D6) ethylene releasers, such as ACC, et messagingl, ethephon, glyoxime; D7) gametocides, such as fenridazon, maleic hydrazide; D8) gibberellins, such as gibberellins, gibberellic acid; D9) growth inhibitors, such as abscisic acid, ancymidol, butralin, carbaryl,
  • the seed coating comprises microbes at a concentration of about 1 x 10 4 to about 1 x 10 11 CPU per seed at the time of planting, when planted within 28 days of application.
  • the microbes are at a concentration of about 1 x 10 5 to about 1 x io 7 CPU per seed at the time of planting.
  • the microbes are at a concentration of about 1 x io 6 CPU per seed.
  • Table 3 below utilizes various CPU concentrations per seed in a contemplated seed treatment embodiment (rows across) and various seed acreage planting densities (1 st column: 15K-41K) to calculate the total amount of CPU per acre, which would be utilized in various
  • compositions and methods to increase the adherence and on- seed stability of plant beneficial microbes can improve a
  • traits that may be introduced or improved include: root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size, photosynthesis rate, tolerance to drought, heat tolerance, salt tolerance, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, level of a metabolite, and
  • the desirable traits including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the improved traits)
  • compositions and methods described herein can improve plant traits, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, and increasing fruit or seed unit weight.
  • the plant grown from the treated seed or plant material has improved health, yield, stress resistance, growth, or agronomic characteristics relative to a control plant.
  • Traits that may be improved by the compositions and methods disclosed herein include any observable characteristic of the seed or the plant resulting therefrom, including, for example, growth rate, height, weight, color, taste, smell, changes in the production of one or more compounds by the plant (including for example, metabolites, proteins, drugs,
  • compositions and methods disclosed herein may result in a change in genotypic information (for example, a change in the pattern of plant gene expression such as those associated with increased nitrogen fixation, in response to the microbes).
  • the plants show the absence, suppression or inhibition of a certain feature or trait (such as an undesirable feature or trait) as opposed to the
  • the trait improved may be nitrogen fixation, including in a plant not previously capable of nitrogen fixation.
  • enhanced levels of nitrogen fixation are achieved in the presence of fertilizer supplemented with glutamine, ammonia, or other chemical source of nitrogen. Methods for assessing degree of nitrogen fixation are known and
  • ingredients/compositions disclosed herein may be packaged together as a kit for seed coating.
  • a dry microbial powder from one or more species of cultured microbes could be encased in commercial-grade water-soluble packaging
  • the extender composition may be provided in a kit with an aqueous solution of cultured microbes. The end-user would then mix the components of the kit for use with any seed coating equipment.
  • the extender compositions disclosed herein are a part of a seed
  • the ingredients for the extender composition are provided in a dry form, with the end-user combining the sugar alcohol, chemical buffer, and optionally water- soluble polymer with water to create a liquid extender composition.
  • the extender composition is provided premixed in a liquid form.
  • the one or more cultured microbes is provided in a liquid form.
  • the one or more cultured microbes have been lyophilized and are provided in a powder formulation.
  • the kit further comprises a buffer for reconstitution of the powdered microbes.
  • the ingredients for the buffer are provided in a dry form to be mixed by the end user.
  • the ingredients for the buffer are provided pre-mixed in a liquid form.
  • Dry microbial formulations typically have a longer shelf compared to liquid microbial formulations, therefore are useful if the microbes need to be transported or stored prior to use. Dry microbial formulations however, have several disadvantages, as described below.
  • the seed coating kit described herein may comprise powder forms of microbes and or dried extender ingredients encased in a safe, convenient and eco-friendly water-soluble package.
  • the disclosure provides the water-soluble packages described in any one of: US 7,357,891, US 8,617,589, WO 2014/202412, WO 2014/202412, WO
  • the water-soluble packages disclosed herein improve the shelf stability of the microbes contained therein. In some embodiments, the microbes in the water- soluble packages disclosed herein have improved shelf stability relative to comparable liquid
  • the water-soluble packages disclosed herein When the water-soluble packages disclosed herein are brought into contact with a liquid (such as, water or an aqueous solution), the package disintegrates, releasing the powder microbes and or extender ingredients contained therein into the liquid, thereby forming a liquid that can be applied to seeds or plant propagating material and/or mixed with the extender
  • a liquid such as, water or an aqueous solution
  • compositions disclosed herein to generate a seed treatment coating are disclosed herein to generate a seed treatment coating.
  • water-soluble packages has several advantages.
  • the use of the water- soluble packages disclosed herein obviates the need for direct handling of the dry microbial powder by the end-user, such as a seed treater or a farmer, thus eliminating any real or perceived safety concerns due to the potential inhalation or contact of the microbial powder
  • the water-soluble packages disclosed herein can be designed to contain a standardized unit of microbes for simplified dosing.
  • the use of the water- soluble packages disclosed herein can promote uniform dispersion of the powdered microbe.
  • the components of the water-soluble packages help stabilize the microbes during dry storage and improve dispersion of the microbe in liquids.
  • the water-soluble packages disclosed herein enhance the shelf life of the microbes contained therein, since they provide an effective barrier between the microbial powder, and moisture and/or oxygen.
  • the water-soluble packages are environmentfriendly and reduce packaging waste, while having the potential to be aesthetically pleasing.
  • the water-soluble packages disclosed herein enable the co-administration of components (such as, microbes and one or more additives) that may not be amenable to being in contact with each other prior to the time of administration, during storage, and/or for long periods of time. Furthermore, when the disclosed agricultural components are brought in
  • composition and/or buffer is prevented until use by placing, for example, the sugar, chemical buffer, and optionally water-soluble polymer in separate compartments of the disclosed packages.
  • the water-soluble package comprises two or more compartment(s). In some embodiments, the two or more compartment(s) comprise different
  • sugars, buffering salts, and water-soluble polymers are provided in one compartment and the cultured microbes in another.
  • the water-soluble polymer is present in the water- soluble package itself (e.g., forms the film, which is later dissolved into the liquid extender solution when placed in water).
  • more than one species of cultured microbes are provided in the seed coating kit.
  • contact between the different species of powdered microbes is prevented until use by placing them in separate compartments of the water-soluble package.
  • the seed coating kits disclosed herein comprise one or more
  • microbes selected from species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enter obacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof.
  • the one or more microbes comprise Kosakonia sacchari.
  • the one or more microbes comprise Kosakonia sacchari.
  • the one or more microbes is Kosakonia sacchari PTA- 126743.
  • the one or more microbes is Klebsiella variicola.
  • the one or more microbes is Klebsiella variicola PTA- 126740.
  • the seed coating kits disclosed herein comprise microbes having at least one genetic variation introduced into a member selected from the group
  • nifA, nifL, ntrB, ntrC polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, niJH, nifD, nijK vtijY, nijE, nifN, nifU, nrfS, nijV, nifW, nijZ, nijM, nifF, nifB, nijQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, faaB, pehA, otsB, treZ, gbsA2, or combinations
  • the seed coating kits disclosed herein comprise non-
  • the one or more cultured microbes are provided as a microbial composition.
  • the microbial composition has been lyophilized and is provided
  • the diy powder has been agglomerated to produce granules.
  • the microbial composition is provided in a liquid form.
  • the microbial composition comprises at least one of a polymer, sugar, biofilm, and isolated biofilm compositions
  • the microbial composition comprises a stabilizer, bulking agent, anticaking agent, dispersant, or any combination thereof.
  • a microbial stabilizer is an agent that acts to stabilize the microorganism population within the agricultural composition. In some embodiments, the microbial stabilizer decreases or slows the decay rate of the microbial population. In some embodiments, the microbial stabilizer accomplishes this change in the decay rate by maintaining the microorganisms in a
  • the microbial stabilizer improves microbial survival rate, decreases microbial decay, improves microbial metabolic activity, improves microbial catabolic gene expression, improves the microbial colonization rate, or decreases toxin
  • the microbial stabilizer increases the survival rate of microbial cells after storage, e.g., after 1, 2, 3, 4, 5, or 6 months of storage.
  • the log loss of CFU/mL of microbes after the storage period is less than 1.
  • the log loss is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2.
  • the microbial stabilizer improves the metabolic activity and/or catabolic gene expression of the microorganisms comprised by the agricultural composition after the storage period.
  • the microbial stabilizer improves the colonization rate of the
  • the microbial stabilizer decreases toxin accumulation.
  • the toxin is a direct product or byproduct of nitrogen fixation.
  • the toxin is ammonia or ammonium.
  • the toxin is produced during cell growth/division.
  • the microbial stabilizer is a sugar. In some embodiments, the microbial stabilizer is a non-reducing sugar.
  • Sugars suitable for use include, but are not limited to, sucrose, oligofructose, glucose and fructose.
  • Monosaccharides suitable for use include, but are not limited to, trehalose, sucrose, lactose, melibiose, and lactulose.
  • the microbial stabilizer is trehalose. In some embodiments, the microbial stabilizer is a
  • Polysaccharides suitable for use include, but are not limited to, maltodextrin, microcrystalline cellulose, and dextran. Additional carbohydrates suitable for use as microbial stabilizers include, but are not limited to, pentoses (e.g., ribose, xylose), hexoses (e.g., mannose, sorbose), oligosaccharides (e.g., raffinose), and oligofructoses.
  • the microbial stabilizer is a sugar alcohol.
  • glycerol glycerol
  • mannitol glycerol
  • sorbitol glycerol
  • the microbial stabilizer is an amino acid. In some embodiments, the microbial stabilizer is glycine, proline, glutamate, or cysteine. In some embodiments, the microbial stabilizer is a protein or protein hydrolysate. Proteins or protein hydrolysates suitable for use as microbial stabilizers within the agricultural composition of the
  • the microbial stabilizer is skimmed milk, starch, humic acid, chitosan, CMC, com steep liquor, molasses, paraffin, pinolene, NFSM, MgSCh, liquid growth medium, horse serum, or Ficoll.
  • the microbial stabilizer is a desiccant.
  • a desiccant As used herein, a
  • desiccant can include any compound or mixture of compounds that can be classified as a desiccant regardless of whether the compound or compounds are used in such concentrations that they in fact have a desiccating effect on the liquid inoculant.
  • desiccants are ideally compatible with the microbial population used, and should promote the ability of the microbial population to survive application on the agricultural plant tissues or the environs thereof and to survive desiccation.
  • suitable desiccants include one or more of trehalose,
  • sucrose glycerol
  • methylene glycol e.g., glycerol
  • suitable desiccants include, but are not limited to, non-reducing sugars and sugar alcohols (e.g., mannitol or sorbitol).
  • the microbial stabilizer also acts as a physical stabilizer.
  • the substance acting as a microbial stabilizer has properties of a thickening agent and therefore also acts as a physical stabilizer.
  • composition of the present disclosure comprising both a physical and a microbial stabilizer does so by comprising the same agent that has characteristics of both types of stabilizer.
  • the concentration of microbial stabilizer in powdered microbes is in the range from about 0.1% w/v to about 30% w/v.
  • the microbial composition comprises a physical stabilizer.
  • a “physical stabilizer” refers to a substance that improves the homogeneity of the composition, such that the microbial cells are at a similar density throughout the liquid composition. By increasing homogeneity, the physical stabilizer prevents high concentrations of cells and/or toxins from accumulating in any one sub-volume of the dispersion of live microbes.
  • the physical stabilizer increases the viscosity of the dispersion of live microbes.
  • the physical stabilizer is a thickening agent.
  • the physical stabilizer is an anti-settling agent.
  • the physical stabilizer is a suspension aid.
  • the physical stabilizer acts to maintain microbial cells in suspension, improving the cell’s resistance to settle statically and
  • a physical stabilizer may also have properties of a microbial stabilizer and vice versa.
  • the physical stabilizer is a polysaccharide.
  • Polysaccharides suitable for use as physical stabilizers include, but are not limited to, polyethylene glycol (PEG), xanthan gum, pectin, and alginates.
  • PEG polyethylene glycol
  • xanthan gum xanthan gum
  • pectin pectin
  • alginates alginates
  • the physical stabilizer is a protein or protein hydrolysate. Proteins or protein hydrolysates suitable for use as physical stabilizers include, but are not limited to, gluten, collagen, gelatin, elastin, keratin, and albumin. In some embodiments, the physical stabilizer is a polymer. Polymers suitable for use as physical stabilizers include, but are not limited to, Carbopol® (CBP) polymers, methylene glycol, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poyacrylate, hydroxyethyl cellulose, or hydroxypropyl
  • CBP Carbopol®
  • PVA polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • poyacrylate hydroxyethyl cellulose, or hydroxypropyl
  • the physical stabilizer is a gum or its derivative. Gums and their derivatives suitable for use as physical stabilizers include, but are not limited to, guar gum, gum Arabic, gum tragacanth, xanthan gum, derivitized guar, hydroxypropyl guar, and polysaccharide gums. In some embodiments, the physical stabilizer is a CBP polymer.
  • compositions/seed coating kits disclosed herein
  • compositions or seed coating kits disclosed herein may comprise additional components.
  • additional components may include protectants and beneficial ingredients including but not limited to animal and bird repellants, attractants, baits, herbicides, herbicide safeners, antidessicants, antitranspirants, frost prevention aids, inoculants, dyes, brighteners, markers, synergists, pigments, UV protectants, antioxidants, leaf
  • polish 15 polish, pigmentation stimulants and inhibitors, surfactants, moisture retention aids, humic acids and humates, lignins and lignates, bitter flavors, irritants, malodorous ingredients, molluscicides (e.g., slugs and snails), nematicides, rodenticides, defoliants, desiccants, sticky traps, IPM (integrated pest management) lures, chemosterilants, plant defense boosters (harpin protein and chitosan), and other beneficial or detrimental agents applied to the surface of the
  • multiple active agents are readily formulated within a given agricultural composition, for example, multiple active agents may include two or more of any of the following fungicides, fertilizers, pesticides, herbicides, and any type of active ingredient or class of active ingredient.
  • the seed coating kits disclosed herein further comprise one or
  • Fertilizers include anhydrous ammonia, urea, ammonium nitrate, and urea-ammonium nitrate (UAN) compositions, among many others.
  • pop-up fertilization and/or starter fertilization is used in combination with the methods and bacteria of the present disclosure.
  • nitrogen stabilizers are used in combination with the methods
  • Nitrogen stabilizers include nitrapyrin, 2-chloro-6- (trichloromethyl) pyridine, N-SERVE 24, INSTINCT, dicyandiamide (BCD).
  • Urease inhibitors include N-(n-butyl)-thiophosphoric triamide (NBPT), AGROTAIN, AGROTAIN PLUS, and AGROTAIN PLUS SC. Further, the disclosure contemplates utilization of AGROTAIN ADVANCED 1.0, AGROTAIN DRI-MAXX, and AGROTAIN ULTRA.
  • stabilized forms of fertilizer can be used.
  • a stabilized form of fertilizer is SUPER U, containing 46% nitrogen in a stabilized, urea-based
  • SUPERU contains urease and nitrification inhibitors to guard from denitrification, leaching, and volatilization.
  • Stabilized and targeted foliar fertilizer such as NIT AMIN may also be used herein.
  • Pop-up fertilizers are commonly used in com fields. Pop-up fertilization comprises applying a few pounds of nutrients with the seed at planting. Pop-up fertilization is used to
  • Slow- or controlled-release fertilizer that may be used herein entails: A fertilizer containing a plant nutrient in a form which delays its availability for plant uptake and use after application, or which extends its availability to the plant significantly longer than a reference ‘rapidly available nutrient fertilizer’ such as ammonium nitrate or urea, ammonium phosphate
  • Such delay of initial availability or extended time of continued availability may occur by a variety of mechanisms. These include controlled water solubility of the material by semi-permeable coatings, occlusion, protein materials, or other chemical forms, by slow hydrolysis of water-soluble low molecular weight compounds, or by other unknown means.
  • Stabilized nitrogen fertilizer that may be used herein entails: A fertilizer to which a nitrogen stabilizer has been added.
  • a nitrogen stabilizer is a substance added to a fertilizer which extends the time the nitrogen component of the fertilizer remains in the soil in the urea- N or ammoniacal-N form.
  • Nitrification inhibitor that may be used herein entails: A substance that inhibits the
  • Some examples include: (1) 2-chloro-6- (trichloromethyl-pyridine), common name Nitrapyrin, manufactured by Dow Chemical; (2) 4- amino-l,2,4-6-triazole-HCl, common name ATC, manufactured by Ishihada Industries; (3) 2,4-diamino-6-trichloro-methyltriazine, common name CI-1580, manufactured by American Cyanamid; (4) Dicyandiamide, common name DCD, manufactured by Showa Denko; (5)
  • Urease inhibitor that may be used herein entails: A substance that inhibits hydrolytic action on urea by the enzyme urease. Thousands of chemicals have been evaluated as soil urease inhibitors (Kiss and Simihaian, 2002). However, only a few of the many compounds tested meet the necessary requirements of being nontoxic, effective at low concentration,
  • urea solid and solutions
  • urease inhibitors Four main classes of urease inhibitors have been proposed: (a) reagents which interact with the sulphydryl groups (sulphydryl reagents), (b) hydroxamates, (c) agricultural crop protection chemicals, and (d) structural analogues of urea
  • N-(n-Butyl) thiophosphoric triamide (NBPT), phenylphosphorodiamidate (PPD/ PPDA), and hydroquinone are probably the most thoroughly studied urease inhibitors (Kiss and Simihaian, 2002). Research and practical testing has also been carried out with N-(2-nitrophenyl) phosphoric acid triamide (2-NPT) and ammonium thiosulphate (ATS).
  • N-N-N-N-NPT N-(2-nitrophenyl) phosphoric acid triamide
  • ATS ammonium thiosulphate
  • the oigano-phosphorus compounds are structural analogues of urea and
  • compositions or seed coating kits disclosed herein may comprise trace metal ions, such as molybdenum ions, iron ions, manganese ions, or combinations of these ions.
  • trace metal ions such as molybdenum ions, iron ions, manganese ions, or combinations of these ions.
  • compositions or seed coating kits disclosed herein may comprise additional carriers, besides those which may be included in the microbial compositions.
  • Additional carriers may include beta-glucan, carboxylmethyl cellulose (CMC), bacterial extracellular polymeric substance (EPS), sugar, trehalose, maltose, animal milk, milk powder, or other suitable carriers.
  • encapsulating refers to enclosing the compositions of the present
  • Encapsulation can be done by any method known in the art for the purpose, or any method that can be conceived to result in the components of the seed coating kit being encapsulated within the package.
  • compositions and methods disclosed herein can be applied to a number of seeds
  • the seed or plant part is an agricultural crop. In some embodiments the seed or plant part is a monocot. In some embodiments, the seed or plant part is a dicot. In some embodiments, the seed is a com seed. In some embodiments, the com seed comprises a pre-treatment.
  • compositions and methods disclosed herein including but not limited to, rice, sorghum, canola, tomato, strawberry, and barley.
  • application rate may vary depending on the microbe(s) and type of seed or plant material coated. In some aspects, the application rate
  • the seed 20 may be between 1.0 oz/CWT and 20.0 oz/CWT, including all ranges and subranges therebetween. Additionally, the seed may be naked (untreated) or pre-treated with any number of plant-enhancing agents, including but not limited to, fungicides, insecticides, biocides, herbicides, and nematicides. Additional examples of agriculturally important crops are listed below.
  • the plant belongs to the genera Hordeum, Oryza, Zea, and Triticeae.
  • crop plants include maize, rice, wheat, barley, sorghum, millet, oats, rye triticale, buckwheat, sweet com, sugar cane, onions, tomatoes, strawberries, asparagus, canola, soybean, potato, vegetables, cereals, and oilseeds.
  • the plant is a genetically modified organism (GMO), non-GMO, organic, or conventional plant.
  • compositions and methods described herein are suitable for plant tissues from any of a variety of transgenic plants, non-transgenic plants, and hybrid plants thereof.
  • the plants are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and/or
  • Some examples of these plants may include pineapple, banana, coconut, lily, grasspeas and grass; and dicotyledonous plants, such as, for example, peas, alfalfa, tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, grape, cotton, sunflower, thale cress, canola, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron, and pomelo),
  • plant tissues or plant parts e.g., seeds, from a
  • Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncal es, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales.
  • Plants belonging to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales. In some embodiments, the
  • 15 monocotyledonous plant can be selected from the group consisting of a maize, rice, wheat, barley, and sugarcane.
  • plant tissues or plant parts, e.g., seeds, from a dicotyledonous plant are treated, including those belonging to the orders of the Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Comales,
  • the dicotyledonous plant can be selected from the group consisting of cotton, soybean, pepper, and tomato.
  • compositions and methods described herein are suitable for any of a variety of non-genetically modified maize plants or parts thereof.
  • the com is organic.
  • compositions and methods described herein are suitable for any non-genetically modified hybrids, varieties, lineages, etc. Corn varieties generally fall under six categories:
  • Yellow su varieties include Earlivee, Early Sunglow, Sundance, Early Golden Bantam, lochief, Merit, Jubilee, and Golden Cross Bantam.
  • White su varieties include True Platinum, Country Gentleman, Silver Queen, and Stowell’s Evergreen. Bicolor su varieties
  • Multicolor su varieties include Hookers, Triple Play, Painted Hill, Black Mexican/Aztec.
  • Yellow se varieties include Buttergold, Precocious, Spring Treat, Sugar Buns, Colorow, Kandy King, Bodacious R/M, Tuxedo, Incredible, Merlin, Miracle, and Kandy Korn
  • White se varieties include Spring Snow, Sugar Pearl, Whiteout, Cloud Nine, Alpine, Silver King, and Argent.
  • Bicolor se varieties include Sugar Baby, Fleet, Bon Jour, Trinity, Bi-Licious, Temptation, Luscious, Ambrosia, Accord, Brocade, Lancelot, Precious Gem, Peaches and Cream Mid EH, and Delectable R/M.
  • Multicolor se varieties include Ruby Queen.
  • Yellow sh2 varieties include Extra Early Super Sweet, Takeoff, Early Xtra Sweet,
  • White sh2 varieties include Summer Sweet White, Tahoe, Aspen, Treasure, How Sweet It Is, and Camelot.
  • Bicolor sh2 varieties include Summer Sweet Bicolor, Radiance, Honey ‘N Pearl, Aloha, Dazzle, Hudson, and Phenomenal.
  • Yellow sy varieties include Applause, Infe o, Honeytreat, and Honey Select.
  • White sy varieties include Silver Duchess, Cinderella, Mattapoisett, Avalon, and Captivate.
  • Bicolor sy varieties include Pay Dirt, Revelation, Renaissance, Charisma, Synergy, Montauk, Kristine, Serendipity/Providence, and Cameo.
  • Yellow augmented supersweet varieties include Xtra-Tender IddA, Xtra-Tender
  • White augmented supersweet varieties include Xtra-Tender 3dda, Xtra-Tender 31 dd, Mirai 421W, XTH 3673, and Devotion.
  • Bicolor augmented supersweet varieties include Xtra-Tender 2dda, Xtra-Tender 21dd, Kickoff XR, Mirai 308BC, Anthem XR, Mirai 336BC, Fantastic XR, Triumph, Mirai 301BC, Stellar, American Dream, Mirai 350BC, and Obsession.
  • Flint com varieties include Bronze-Orange, Candy Red Flint, Floriani Red Flint, Glass Gem, Indian Ornamental (Rainbow), Mandan Red Flour, Painted Mountain, Petmecky, Cherokee White Flour,
  • Popco varieties include Monarch Butterfly, Y ellow Butterfly, Midnight Blue, Ruby Red, Mixed Baby Rice, Queen Mauve, Mushroom Flake, Japanese Hull-less, Strawberry, Blue Shaman, Miniature Colored, Miniature Pink, Pennsylvania Dutch Butter Flavor, and Red Strawberry.
  • Dent com varieties include Bloody Butcher, Blue Clarage, Ohio Blue Clarage, Cherokee White Eagle, Hickory Cane, Hickory King, Jellicorse Twin, Kentucky Rainbow, Daymon Morgan’s Knt. Butcher, Learning, Learning’s Yellow, McCormack’s Blue Giant, Neal Paymaster, Pungo Creek Butcher, Reid’s Yellow Dent, Rotten Clarage, and Tennessee
  • com varieties include P1618W, P1306W, P1345, Pl 151, Pl 197, P0574, P0589, and P0157.
  • W white com.
  • compositions and methods described herein are suitable for any hybrid of the maize varieties set forth herein.
  • compositions and methods described herein are suitable for any of a hybrid, variety, lineage, etc. of genetically modified maize plants or part thereof. Furthermore, the compositions and methods described herein are suitable for any of the following genetically modified maize events, which have been approved in one or more countries, or any new genetically modified com event, which may include Bt traits, glufosinate resistance, glyphosate
  • 32138 32138 SPT Maintainer
  • 3272 ENOGEN
  • 3272 x Btll 3272 x btl l x GA21, 3272 x Btl 1 x MIR604, 3272 x Btl 1 x MIR604 x GA21, 3272 x Btl 1 x MIR604 x TC1507 x 5307 x GA21, 3272 x GA21, 3272 xMIR604, 3272 x MIR604 x GA21, 4114, 5307 (AGRISURE Duracade), 5307 x GA21, 5307 x MIR604 x Bill x TC1507 x GA21 (AGRISURE Duracade 5122), 5307 x MIR604 x Bil l x TC1507 x GA21 x MIR162
  • the present disclosure relates to a seed or plant tissue treated with the kits and compositions disclosed herein.
  • the seed is a com seed.
  • microbes of the present disclosure are derived from two wild-type strains.
  • Strain CI006 is a bacterial species previously classified in the genus Enterobacter (see
  • Strain CI019 is a bacterial species classified in the genus Rahnella.
  • the deposit information for the CI006 Kosakonia wild type (WT) and CI019 Rahnella WT are found in Table 2.
  • NCMA National Center for Marine Algae and Microbiota
  • a biologically pure culture of Klebsiella variicola was deposited on August 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA, and assigned NCMA Patent Deposit Designation number 201708001.
  • NCMA National Center for Marine Algae and Microbiota
  • NCMA National Center for Marine Algae and Microbiota
  • a biologically pure culture of a Paenibacilhis polymyxa (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PT A-126581.
  • a biologically pure culture of a Paenibacilhis polymyxa (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PT A-126581.
  • a Paraburkholderia tropica (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126582.
  • a biologically pure culture of a Herbaspirillwn aquaticum (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801
  • a biologically pure culture of a Metakosakonia intestini (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126585.
  • a biologically pure culture of a Klebsiella variicola variant/remodeled strain was deposited on March 25, 2020 with the
  • ATCC American Type Culture Collection
  • a biologically pure culture of a Kosakonia sacchari variant/remodeled strain was deposited on March 25, 2020 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126743.
  • the applicable deposit information is found in Table 2.
  • This example describes the preparation of microbial culture to be tested in combination with extenders of the present disclosure.
  • Kosakonia sacchari strain PTA- 126743 and Klebsiella variicola strain PTA- 126740 were separately grown in fermenters to saturation to create microbial culture broths.
  • Freeze dried powders were reconstituted at 1 g per 10 mL of IX PBS.
  • Cell viability of the dried microbial powder was tested by plating samples and measuring colony forming units per gram of powder.
  • Initial cell viability assays showed viability between 1 x 10 11 and 1 x 10 12 CFU/gram of powder.
  • Example 2 Extender compositions increase adherence of microbes to seed
  • This microbial 1 : 1 blend was then mixed with the extender compositions or buffer control of Table 4 at a 1 : 1 ratio to form final seed treatment compositions.
  • Table 5 below shows the final % by volume contents of the seed treatment compositions created in this example.
  • test compositions based on Liquid Extender PBX21 800 50L were created with each of the test extender and control liquids as follows: 1) test compositions based on Liquid Extender PBX21 800 50L, 2) test compositions based on Liquid Extender PBX21 800 44L, and 3)
  • Seed 1 contained 3 fungicide pretreatments, 1 insecticide pretreatment, and 1 biological pretreatment.
  • Seed 2 contained 6 fungicide pretreatments, 2 insecticide pretreatments, and 3 gram-positive sporulating biological s.
  • Example 3 Extender compositions increase stability of microbes on-seed
  • the seed treatment compositions with the extenders of the present disclosure maintained a greater percentage of viable cells, overall resulting in high stability over time.
  • extender composition PBX21 800 44L approximately 22.5% of cells on-seed were still
  • Example 2 demonstrated that extenders of the present disclosure reduce on-seed application loss
  • Example 3 demonstrated that extenders further improve the stability of microbes that make it onto the seed. Together, these two properties help produce higher and
  • Example 4 Storage stability of dried microbial powder microbes
  • Example 1 generated two microbial delivery forms that could be used to produce the seed treatment compositions of the present disclosure.
  • the first was microbial culture broths.
  • the second was dried microbial powder, produced by lyophilizing and (optionally) milling the
  • Example 5 Reconstituted dried microbial powder microbes produce highest microbial titers during on-seed storage when used with extender compositions
  • Two seed treatment compositions were prepared. The first was prepared by mixing the microbial culture broths of example 1 (undiluted, unconcentrated, unformulated) directly with the extender formulations of example 2, in a 1 : 1 ratio (Broth Seed Treatment). The second
  • Example 10 was prepared by resuspending dried microbial powders from example 1 in buffer at 10% wt/v PTA-126743 and 10% wt/v PTA-126740. This reconstituted microbe liquid was then mixed with extender composition PBX21 800 44L at a 1:1 ratio as described in Example 2 (Reconstituted Microbe Seed Treatment). Controls using the microbial culture broth and reconstituted microbe liquid were also prepared using buffer control A. Table 6 below shows
  • Microbe + Buffer Control A were applied to the same hybrid com seed at 5.4 fl oz/CWT. Seed was stored at 21 °C and viability in CFU/seed was measured over the course of 28 days (FIG. 5) to evaluate microbe stability.
  • PBX21 800 44L can increase microbial viability when added to a Microbial Culture Broth Seed Treatment.
  • Example 6 Extender compositions maintain on-seed viability of microbial powders above le+4 CFU/seed 90 days after application on treated corn seed
  • Example 5 showed that the use of an extender composition could increase on-seed
  • Dry microbial powder was prepared as described above in Example 4 was stored in sealed mylar bags at 21°C and opened at time of seed treatment (aged 0, 1, 2, 3, months). This
  • Example 30 example tested a 1 :1 ratio blend of Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA-126740, as described in Example 1. 8.6% weight powder dry microbe was combined with 96.5% volume of either extender PBX21 800 77L (see Example 2, Table 4) or control solution B; mixed well and treated within 4 hours of mixing. In contrast with Example 5, microbes in this example were directly added to the extender in powdered (e.g., lyophilized) form, without first being resuspended in buffer.
  • Seed treatment was applied at 2.4 fl oz/unit application rate on a Hege 11 seed treater in 50 g batches of seed with a spin time of 35 sec.
  • Seed 1 contained 6 fungicide pretreatments, 2 insecticide pretreatment, and 3 biological pretreatments.
  • Seed 2 contained 3 fungicide pretreatments and 1 insecticide pretreatments.
  • Seeds were extracted in 1 X PBS buffer (5 seeds/ 5 mL PBS in 50 ml conical tube) and shaken at 1600 rpm for 30 min. Serial dilutions (1:10) were performed in 1XPBS and spread plated on TSA+10 ug/ml Erythromycin plates. Plates were incubated at 30°C for ⁇ 24h prior to colony forming unit (CFU) counts. CFU/seed was calculated as (CFU/mL) x (mL/seed).
  • Example 7 Extender compositions maintain on-seed viability of liquid microbes above le+4 CFU/seed 30 days after application on treated seed
  • Example 5 showed that the use of an extender composition could increase on-seed viability when added to either a microbial culture broth or powder (reconstituted lyophilized
  • liquid microbes were stored in Scholle bladders and stored at 21 °C prior to seed treatment (bladders only opened up to 3 times to reduce air exchange). Two volume to volume ratios were evaluated, and data from both were compiled:
  • compositions were mixed well and treated within 4 hours of mixing. Seed treatment was applied at 2.4 fl oz/unit application rate on a Hege 11 seed treater in 50 g batches
  • Seed contained either 6 fungicides, 2 insecticides, and 3 biologicals, or 3 fungicides, 1 insecticide, and 1 biological. Data was compiled as an average between both. All seed was stored at 21°C for viability studies.
  • liquid microbes were stored as described above, except only one volume ratio was evaluated: 75% volume liquid microbe + 25% volume of either extender PBX21 800 44L or control solution A; mixed well and treated within 4 hours of mixing.
  • Seed treatment was applied at 10 fl oz/CWT of wheat application rate on a Hege 11 seed treater in 50 g batches of seed with a spin time of 35 sec. Seed contained 6 fungicides, 2
  • On-seed viability was determined overtime using the following seed extraction and plating methods: Seeds were extracted in IX PBS buffer (25 seeds/ 25 mL PBS in 50 ml conical tube) and shaken at 1600 rpm for 30 min. Serial dilutions (1:10) were performed in IXPBS and spread plated on TSA+10 ug/ml Erythromycin plates. Plates were incubated at 30°C for ⁇ 24h
  • CFU/seed was calculated as (CFU/mL) x (mL/seed).
  • a composition comprising: a) a sugar alcohol or sugar, at between about 5% and about 30% (w/v);
  • composition of embodiment 1, wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-
  • CMC carboxymethyl cellulose
  • hydroxypropyl methylcellulose alginate, and combinations thereof.
  • composition of embodiment 2, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
  • composition of embodiment 3 wherein the polyvinylpyrrolidone-vinyl acetate
  • PVP-VA is present in the composition at between about 15% and about 20% (w/v).
  • PVP-VA poly vinylpyrrolidone- vinyl acetate
  • PVP-VA polyvinylpyrrolidone-vinyl acetate
  • a composition comprising: a) a sugar alcohol or sugar, at between about 5% and about 70%(wA); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH; and
  • composition of embodiment 8, wherein the sugar alcohol or sugar is present in
  • composition at about 60% (w/v).
  • a composition comprising: a) a sugar alcohol or sugar, at between about 2.5% and about 30% (wA); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH;
  • composition of embodiment 10, wherein the sugar alcohol or sugar is between about 7.5% and about 15% (wA).
  • composition of embodiment 10 or 10.1, wherein the water-soluble polymer is between about 10% and about 20% (wA).
  • PVP polyvinylpyrrolidone
  • PVP-VA polyvinylpyrrolidone-vinyl acetate
  • CMC carboxymethyl cellulose
  • alginate hydroxypropyl methylcellulose
  • composition of embodiment 11, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
  • PVP-VA polyvinylpyrrolidone-vinyl acetate
  • PVP-VA polyvinylpyrrolidone-vinyl acetate
  • PVP-VA is present in the composition at about 11% (w/v).
  • PVP-VA polyvinylpyrrolidone-vinyl acetate
  • composition of any one of embodiments 1-14, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fucitol, iditol, and inositol.
  • composition of any one of embodiments 1-16, wherein the chemical buffer comprises dipotassium phosphate at approximately between 0.5% and 4% of the total volume, and monopotassium phosphate at approximately between 0% and 2% of the
  • composition of embodiment 17 or 17.1 wherein the chemical buffer is dipotassium phosphate at approximately 1% of the total volume, and monopotassium phosphate at approximately 0.5% of the total volume.
  • the chemical buffer is di potassium phosphate at approximately between 0.5% and 2% of the total volume, and monopotassium phosphate at approximately between 0% and 0.5% of the total volume.
  • composition of embodiment 19, wherein the chemical buffer is dipotassium
  • composition of any one of embodiments 8-20, wherein the cultured microbes are selected from a species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium,
  • composition of embodiment 21, wherein the one or more microbes comprise Kosakonia sacchari.
  • composition of embodiment 22, wherein the cultured microbes comprise Kosakonia sacchari PTA- 126743.
  • composition of embodiment 21, wherein the cultured microbes comprise Klebsiella variicola.
  • composition of embodiment 24, wherein the cultured microbes comprise Klebsiella
  • composition of any one of embodiments 8-25, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA- 126743.
  • composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species that is a transgenic microbial species.
  • composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species having a non-intergeneric genomic modification.
  • GlnD 25 glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
  • the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
  • microbial species that is a non intergeneric remodeled microbial species comprising at least one of: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated
  • composition of any one of embodiments 8-26.8, wherein the cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine
  • a microbial composition comprising at least one of a polymer, sugar, biofilm, and isolated biofilm compositions.
  • composition of any one of embodiments 8-29, wherein the cultured microbes to extender composition ratio is between 1 : 1 and 1 :4 by percent volume.
  • composition of embodiment 32, wherein the cultured microbes comprise between
  • composition of embodiment 36, wherein the pre-treatment is an insecticide, herbicide, fungicide, biocide, or nematicide.
  • composition of any one of embodiments 1-37 wherein the composition maintains at least 40% microbial cell viability at 14 days post seed treatment.
  • composition of any one of embodiments 1-37 wherein the composition maintains at least 50% microbial cell viability at 14 days post seed treatment.
  • a plant seed or plant propagation material comprising the composition of any one of
  • a seed coating kit comprising: a) an extender composition, comprising:
  • the seed coating kit of embodiment 51 wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP- VA), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
  • PVP polyvinylpyrrolidone
  • PVP- VA polyvinylpyrrolidone-vinyl acetate
  • CMC carboxymethyl cellulose
  • alginate alginate
  • microbes are encapsulated within a water-soluble package.
  • the seed coating kit of any one of embodiments 50-64, wherein the cultured microbes are selected from species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and
  • the seed coating kit of embodiment 68, wherein the cultured microbes comprise Klebsiella variicola PT A-l 26740.
  • the seed coating kit of any one of embodiments 50-64, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA-126743.
  • the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or noncoding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
  • the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
  • microbial species 30 comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
  • the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
  • the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
  • microbial species 20 comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated amtB gene that results in the lack of expression of said amtB gene.
  • 25 species comprising at least one of: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said m/Z, gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated glnD gene that results in a truncated GlnD protein lacking a uridyl-transferase domain
  • the cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD,
  • nijJ 5 glnE, nijJ, nifH, nifD, nifK, nijY, nifE, niJN, nifU, nifS, nifl', niJW, niJZ, nijM, nifF, nifB, nijQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, JhaB,pehA, otsB, treZ, glsA2, or combinations thereof.
  • a method of treating a seed or plant tissue comprising applying a composition comprising: a) a sugar alcohol or sugar, at between about 10% and about 80% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral
  • a method of treating a seed or plant tissue comprising applying a composition comprising:
  • a sugar alcohol or sugar at between about 2.5% and about 15% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) a water-soluble polymer, at approximately between 5% and 20% (w/v); and d) cultured microbes
  • a method of treating a seed or plant tissue comprising applying a composition comprising: a) a sugar alcohol or sugar, at between about 7.5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral
  • a dried seed coating comprising: a) between about 50% and about 90% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) between about 5% to about 40% (w/w) cultured microbes.
  • the dried seed coating of embodiment 97 comprising: a) about 72 or 84% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and
  • a dried seed coating comprising: a) between about 25% and about 50% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH;
  • the dried seed coating of embodiment 99 comprising: a) about 35, 44, or 30% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral
  • a dried seed coating comprising: a) sugar alcohol or sugar;
  • a dried seed coating comprising: a) sugar alcohol or sugar;
  • polymer 30 polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA), carboxymethyl cellulose (CMC), hydroxypropyl methyl cellulose, alginate, and combinations thereof.
  • PVP polyvinylpyrrolidone
  • PVP-VA polyvinylpyrrolidone-vinyl acetate
  • CMC carboxymethyl cellulose
  • alginate alginate
  • 25 microbes comprise Klebsiella variicola PTA-126740.
  • microbes comprise at least one microbial species that is a remodeled microbe.
  • 25 microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
  • 30 microbial species having at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE;
  • the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
  • 15 microbial species is a non intergeneric remodeled microbial species comprising a mutated amtB gene that results in the lack of expression of said amtB gene.
  • a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated glnD gene that results in a truncated GlnD protein lacking a uridyl-transferase domain or lack of expression of said glnD gene, and combinations thereof.
  • any one of embodiments 97-113, wherein the cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nijH, nijD, nijK, nifY, nifE, nifN, nifU, nifS, ni/K, nifW, nijZ, nijM, nifF, nifB,

Abstract

The present disclosure provides extender compositions which prolong or maintain the viability of microbes on-seed, thus increasing the stability and shelf-life of the microbes on coated seed, even when the seed has been pre-treated with other chemicals and/or biologicals (plant enhancing agents). The compositions disclosed herein further promote the adherence of microbes to seed during the seed coating process. The disclosure further teaches seed coating kits, methods of use, and seeds and plant propagating material produced therefrom which are coated with the compositions disclosed herein.

Description

EXTENDER COMPOSITIONS AND USE THEREOF TO INCREASE ON-SEED
ADHERENCE AND STABILITY OF MICROBES
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of International Patent Application No.
5 PCT/US2021/036863, filed on June 10, 2021, which is hereby incorporated by reference in its entirety for all purposes.
FIELD
[002] The present disclosure relates to extender compositions to be used with microbes and microbial compositions, and methods of use thereof. The compositions disclosed herein may
10 be used to improve microbial stability on a seed which in turn can improve yield and productivity, and have other beneficial effects on agricultural crops.
BACKGROUND
[003] Application of plant beneficial microbes, such as nitrogen-fixing bacteria, to crops can increase agricultural yield, while potentially decreasing the use of fertilizers. These
15 beneficial microbes can be cultured and transplanted to the soil near the root structure of the plant, or alternatively may be formulated in a seed coating. However, it is often cumbersome and inefficient to apply microbes directly to a seed, and once applied, the microbial viability declines. Seed that has already been pre-treated can also interfere with microbial viability. Seed coatings with low seed adherence rates also raise safety concerns, as unused coating
20 increases the chances of contact with farm hands.
[004] Thus, there continues to be a need for compositions which increase the adherence of plant beneficial microbes on seed. Further, these compositions should maintain the viability of the microbes on-seed until use, and protect them from any pre-treatments already applied, thus increasing the stability of the microbes on the seed.
25 SUMMARY
[005] The disclosure provides a composition comprising: a) a sugar alcohol, at between about 5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at between about 10% and about 40% (w/v).
[006] The disclosure further provides a composition comprising: a) a sugar alcohol, at between about 5% and about 70% (w/v); b) a chemical buffer wherein the buffer maintains the
5 composition at a neutral pH; and c) cultured microbes.
[007] The disclosure further provides a composition comprising: a) a sugar alcohol, at between about 2.5% and about 30% (w/v); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH; c) cultured microbes; and d) a water-soluble polymer, at between about 5% and about 30% (w/v).
10 [008] The disclosure further provides a seed coating kit comprising: a) an extender composition, comprising: i) a sugar alcohol; ii) a chemical buffer; and b) cultured microbes. In some aspects, the extender composition of the kit comprises a water-soluble polymer.
[009] The disclosure further provides a method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol, at between about 10% and about 80%
15 (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) cultured microbes to a seed or plant tissue. The disclosure further relates to seeds and plant tissues produced therefrom.
[0010] The disclosure further provides a method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol, at between about 2.5% and about 15%
20 (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) a water-soluble polymer, at approximately between 5% and 20% (w/v); and d) cultured microbes to a seed or plant tissue. The disclosure further relates to seeds and plant tissues produced therefrom.
[0011] The disclosure further provides a method of treating a seed or plant tissue, comprising
25 applying a composition comprising: a) a sugar alcohol, at between about 7.5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) a water-soluble polymer, at approximately between 5% and 40% (w/v); and d) cultured microbes to a seed or plant tissue. The disclosure further relates to seeds and plant tissues produced therefrom.
30 [0012] The disclosure further relates to a dried seed coating comprising: a) between about 50% and about 90% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) between about 5% to about 40% (w/w) cultured microbes.
[0013] The disclosure further relates to a dried seed coating comprising: a) between about 25% and about 50% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains
5 the composition at a neutral pH; c) between about 17% and about 60% (w/w) water-soluble polymer; and d) between about 10% to about 40% (w/w) cultured microbes.
[0014] The disclosure further relates to a dried seed coating comprising: a) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) cultured microbes.
10 [0015] The disclosure further relates to a dried seed coating comprising: a) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) water- soluble polymer; and d) cultured microbes.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 is a bar graph showing the starting treatment titers of Kosakonia sacchari
15 strain PTA-126743 and Klebsiella variicola strain PTA-126740 mixed with buffer control, extender composition PBX21 800 44L, and extender composition PBX21 800 50L, as the number of colony forming units (CPUs) per mL (average of triplicated data).
[0017] FIG. 2 is a bar graph showing the application log loss of the treatments shown in FIG. 1 after application (seed coat) to two types of com seed (average of triplicated data).
20 [0018] FIG. 3 is a bar graph showing the on-seed stability (viability over time). The number of CPUs per seed were evaluated on day 0, day 7, and day 15 post seed coating (average of triplicated data). Seed was stored 21°C.
[0019] FIG. 4 is a line graph showing cell viability in CPUs per gram of powder (lyophilized) Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA-126740 over the
25 course of three months. Powders were stored at 20-25°C in opaque containers.
[0020] FIG. 5 is a line graph showing on-seed stability (viability over time) of microbes which were lyophilized and then reconstituted prior to mixture with extender composition PBX21 800 44L (“powder”, solid lines) compared to those that were in a broth solution prior to mixture with extender composition PBX21 800 44L (“broth”, dotted lines). The number of CPUs per seed was evaluated over the course of 28 days post seed coating (average of triplicated data). Seed was stored 21 °C .
[0021] FIG. 6 is a line graph showing on-seed stability (viability over time) of microbes which were lyophilized and then reconstituted prior to mixture with extender composition
5 PBX21 800 77L compared to those that were reconstituted prior to mixture with a control solution. The number of CPUs per seed was evaluated over the course of time as (CFU/mL) x (mL/seed). Seed was stored 21 °C. Error bars are 95% confidence intervals around the mean CFU/seed for each timepoint.
[0022] FIG. 7 is a line graph showing on-seed stability (viability over time) of liquid
10 microbes which were mixed with extender composition PBX21 800 44L compared to those that were mixed with a control solution. The solutions were applied to chemically treated com seeds. The number of CPUs per seed was evaluated over the course of time as (CFU/mL) x (mL/seed). Seed was stored 21 °C.
[0023] FIG. 8 is a line graph showing on-seed stability (viability over time) of liquid
15 microbes which were mixed with extender composition PBX21 800 44L compared to those that were mixed with a control solution. The solutions were applied to chemically treated wheat seeds. The number of CPUs per seed was evaluated over the course of time as (CFU/mL) x (mL/seed). Seed was stored 21°C.
DETAILED DESCRIPTION
20 Definitions
[0024] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be constmed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are
25 to be constmed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11,
30 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.
5 [0025] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value
10 or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.
[0026] “Plant tissues,” as used herein, refers to any part of the plant during any aspect of the
15 growing cycle, including seeds, seedlings, plants, or plant parts. Plant parts include leaves, roots, root hairs, rhizomes, stems, seed, ovules, pollen, flowers, fruit, cuttings, tubers, bulbs, etc. An agricultural plant tissue “comprising” a dispersion of live microbes disclosed herein includes agricultural plant tissues to which the dispersion of live microbes has been applied by any of the means set forth herein, e.g., spraying, in-furrow application, seed treatment, etc.
20 [0027] “Plant productivity” refers generally to any aspect of growth or development of a plant that is a reason for which the plant is grown. For food crops, such as grains or vegetables, “plant productivity” can refer to the yield of grain or fruit harvested from a particular crop. As used herein, improved plant productivity refers broadly to improvements in yield of grain, fruit, flowers, or other plant parts harvested for various purposes, improvements in growth of plant
25 parts, including stems, leaves and roots, promotion of plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight, and similar improvements of the growth and development of plants. In some embodiments, plant productivity is determined by comparing the productivity (e.g., yield) of a treated plant or seed (e.g., via a seed coating as described herein), vs. an untreated plant seed.
30 [0028] Microbes in and around food crops can influence the traits of those crops. Plant traits that may be influenced by microbes include: yield (e.g., grain production, biomass generation, fruit development, flower set); nutrition (e.g., nitrogen, phosphorus, potassium, iron, micronutrient acquisition); abiotic stress management (e.g., drought tolerance, salt tolerance, heat tolerance); and biotic stress management (e.g., pest, weeds, insects, fungi, and bacteria). Strategies for altering crop traits include: increasing key metabolite concentrations; changing temporal dynamics of microbe influence on key metabolites; linking microbial metabolite
5 production/degradation to new environmental cues; reducing negative metabolites; and improving the balance of metabolites or underlying proteins.
[0029] As used herein, “in planta" refers to in the plant, on the plant, or intimately associated with the plant, depending upon context of usage (e.g. endophytic, epiphytic, or rhizospheric associations). As used herein, the term “plant” can include plant parts, tissue, leaves, roots, root
10 hairs, rhizomes, stems, seeds, ovules, pollen, flowers, fruit, etc.
[0030] As used herein, “exogenous nitrogen” refers to non-atmospheric nitrogen readily available in the soil, field, or growth medium that is present under non-nitrogen limiting conditions, including ammonia, ammonium, nitrate, nitrite, urea, uric acid, ammonium acids, etc.
15 [0031J As used herein, “non-nitrogen limiting conditions” refers to non-atmospheric nitrogen available in the soil, field, media at concentrations greater than about 4 mM nitrogen, as disclosed by Kant et al. (2010. J. Exp. Biol. 62(4): 1499-1509), which is incoiporated herein by reference for all purposes.
[0032] A “wild type microbe,” e.g., a “wild type bacterium,” as used herein refers to a
20 microbe that has not been genetically modified. Wild type microbes may be isolated and cultivated from a natural source. Wild type microbes may be selected for specific naturally occurring traits.
[0033] A “diazotroph” is a microbe that fixes atmospheric nitrogen gas into a more usable form, such as ammonia. A diazotroph is a microorganism that is able to grow without external
25 sources of fixed nitrogen. All diazotrophs contain iron-molybdenum or -vanadium nitrogenase systems.
[0034] In some embodiments, the increase of nitrogen fixation and/or the production of 1% or more of the nitrogen in the plant are measured relative to control plants, which have not been exposed to the bacteria of the present disclosure. All increases or decreases in bacteria are
30 measured relative to control bacteria. All increases or decreases in plants are measured relative to control plants. [0035] As used herein, a “water-soluble film package”, interchangeably used herein with “water-soluble package” refers to an encasement that is capable of disintegrating upon contact with a liquid, and is composed of a water-soluble film.
[0036] As used herein, a “water-soluble film” refers to a film that is capable of disintegrating
5 upon contact with a liquid. In some embodiments, the water-soluble film is “fully” soluble, meaning that all ingredients in the film are capable of fully dissolving in liquid such as water. In some embodiments, the water-soluble film further comprises one or more water-insoluble components, that still lose cohesion when exposed to a liquid, such as water. For example, in some embodiments, the water-soluble film may contain granules, strips, netting, or other non-
10 water-soluble ingredients that are held together to form a pouch with water-soluble ingredients, such that exposure of the film to water or another liquid still causes the film to lose integrity and dissipate, even if some components are not fully solvated in the liquid.
[0037] As used herein, a “non-intergeneric” remodeled microorganism is a microorganism that is formed by the deliberate combination of genetic material originally isolated from
15 organisms of the same taxonomic genera. A non-intergeneric remodeled microorganism can be used interchangeable with “intrageneric mutant" and “intrageneric microorganism”.
[0038] As used herein, an “intragenic" microorganism, is a microorganism that is engineered to comprise a genetic edit, or genetic modification, or genetic element, or genetic material (e.g. a nucleic acid sequence), that has been sourced from within the organism’s own species.
20 [0039] As used herein, a “transgenic” microorganism, is a microorganism that is engineered to comprise a genetic edit, or genetic modification, or genetic element, or genetic material (e.g. a nucleic acid sequence), that has been sourced from outside the organism’s taxonomic species.
[0040] As used herein, in the context of non-intergeneric microorganisms, the term “remodeled" is used synonymously with the term “engineered”. Consequently, a “non-
25 intergeneric remodeled microorganism” has a synonymous meaning to “non-intergeneric engineered microorganism,” and will be utilized interchangeably.
[0041] As used herein, “applying,” “coating,” and “treating” agricultural plant seeds and tissues with the dispersion of microbes includes any means by which the plant seeds or tissues are made to come into contact (i.e. exposed) to a dispersion of microbes. In some embodiments,
30 “applying” refers to placing or distributing the dispersion of microbes onto an area, volume, or quantity of agricultural plant seed or tissue (for example as a seed coat). Consequently, “applying” includes any of the following means of exposure to a dispersion of microbes: spraying, dripping, submerging, hand broadcast, machine spreading, brushing, machine broadcasting, and the like, onto agricultural plant seeds and tissues.
[0042] As used herein, when the disclosure discuses a particular microbial deposit by
5 accession number, it is understood that the disclosure also contemplates a microbial strain having all of the identifying characteristics of said deposited microbe, and/or a mutant thereof.
[0043] 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
10 of other living organisms and contains only the individual microbe in question. As used herein, “cultured microorganism” or “cultured microbes” refers to microbes that have been isolated and cultured. 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
15 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), afFd in part, rev’d 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,
20 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 B12 produced by microbes), incorporated herein by reference.
25 [0044] Microbes of the present disclosure may include spores and/or vegetative cells. In some embodiments, microbes of the present disclosure include microbes in a viable but non- culturable (VBNC) state.
[0045] As used herein, the term “stability” refers to cell viability over time. In some embodiments, stability is reported as loss in cell viability (or percentage of remaining viability)
30 after a preset period of time. References to a percent microbial cell viability at a predetermined time (e.g., 28 days) is intended to describe the remaining percentage of viability from the original viability at the time the composition was first admixed and/or applied to a plant part. Thus, discussion of a seed maintaining at least 50% microbial cell viability at 28 days means that the seed has 50% of the CPU at 28 days than it did at day 0 (when the seed was first coated).
[0046] As used herein, a “seed treatment” refers to a substance that may be applied to
5 agricultural seeds. The seed treatment may provide one or more benefits to the seed and/or plant resulting from the seed. Without limitation, seed treatments may include the dispersion of microbes disclosed herein, compositions disclosed herein, pesticides, herbicides, insecticides, nematicides, plant-growth promoting factors, fertilizers, and the like. A seed treatment may also be a seed coating.
10 [0047] Certain embodiments of the disclosure refer to seed “pre-treatment.” As used herein the term “pre-treatmenf’ refers to the order of application, where “pre-treatments” are necessarily layered closer to the application locus (e.g., closer to the surface or center of a seed), with subsequent treatments covering over them. Pre-treatment may refer to a single previous seed coating application (e.g. an herbicide), or may be used to collectively refer to all
15 previous treatments adhering to the locus of application (e.g. a seed).
[0048] The term “colony forming unit” or “CPU” as used herein is a unit used to estimate the number of viable microbial cells in a sample. Viable is defined as the ability to multiply under the controlled conditions. In some embodiments, counting colony-forming units involves culturing the microbes and counting only viable cells (e.g., cells capable of growing colonies),
20 in contrast with microscopic examination which counts all cells, living or dead.
[0049] As used herein, a “chemical buffer,” “buffer solution,” “buffering agent,” or “buffer,” also known as a “pH buffer” or “hydrogen ion buffer,” consists of a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid.
[0050] As used herein, “neutral pH” refers to a pH value of between 6 and 7.5.
25 [0051] As used herein, a “dispersing agent” or “dispersant” is a substance that, when added to a solution or suspension of solid or liquid particles in a liquid, is capable of promoting the separation of the particles and thus, prevent clumping or settling of the particles. In some embodiments, a dispersing agent added to a suspension of microbes disclosed herein can improve and/or stabilize the suspension by promoting the separation of the microbes, and
30 preventing the clumping or settling of the microbes. In some embodiments, addition of a dispersing agent to a dispersion of microbes can promote rehydration, viability, and/or shelf- life of the microbes. In some embodiments, the dispersing agent is a biologically compatible dispersing agent, such as, for example, non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents.
[0052] As used herein, the term “polymer” includes copolymers. As used herein, “water-
5 soluble polymer” refers to any synthetic, semisynthetic, or natural polymer that dissolves, disperses, or swells in water at least under some conditions, so as to be able to release ingredients admixed with the polymer and/or coated by the polymer into an aqueous solution.
[0053] As used herein, “CWT” or “centum weight” is used in the context that is known in the art, as hundredweight for seed. Thus, an amount of treatment per CWT would refer to the
10 amount used to treat 100 pounds of seed.
[0054] As used herein, “extender” refers to compositions which prolong or maintain the viability of a microbe over time, thus increasing the overall stability of a composition comprising microbes and an extender. Extenders may also increase adherence of microbes to a seed or plant tissue.
15 [0055] As used herein, a “plant enhancing agent” is any agent that provides a benefit, advantage, or protection to a seed or the plant resulting therefrom. Non-limiting examples of plant enhancing agents include fungicides, insecticides, biocides, herbicides, and nematicides.
[0056] As used herein, “seed coating” refers to any coating on a seed or plant propagating material.
20 [0057] As used herein, “application log loss” refers to a measurement of microbial adherence to seed or plant propagating material and is calculated by using the following equation: LOG((Day 0 treatment titers in CFU/ml) x (ml/seed application rate)) - LOG(Day 0 CFU/seed). Conceptionally this can be thought of as LOG(of theoretical microbial load per seed as determined by treatment titer and rate of application)- LOG(of actual microbial titer,
25 empirically measured). Lower log loss values indicate higher microbial adherence
[0058] As used herein, “reconstituted” refers to previously lyophilized microorganisms that have been formulated back to a liquid formulation, but which have not been permitted to grow/culture, since being formulated into the liquid formulation (e.g., through the addition of an aqueous solution). In some embodiments, reconstituted microbes are different from
30 microbes that have not been lyophilized, or which have been allowed to substantially grow/divide (e.g., cultured) since being formulated into liquid suspension. [0059] Embodiments of the present disclosure define compositions based on their % content. In some embodiments the % content is (v/v), which is calculated based on the volume of the recited ingredient divided by the volume of the composition (e.g., extender). In some embodiments the % content is (w/v), which is calculated based on the weight (in grams) of the
5 recited ingredient divided by the volume (in liter) of the composition (e.g., extender). In some embodiments the % content is (w/w), which is calculated based on the volume of the recited ingredient divided by the volume of the composition (e.g., extender). In some embodiments the % content is (w/w), which is calculated based on the weight of the recited ingredient divided by the weight of the composition (e.g., dry extender).
10 Overview
[0060] The stability of microbes coated on seed surfaces is often reduced by excess drying, environmental conditions, or mechanical stress during seed processing and/or storage. Furthermore, some microbial seed coatings may be incompatible with other types of seed treatments, including chemical pre-treatments. In addition, most seed treatment facilities are
15 not equipped to apply dry microbial powder to seeds, and there are health concerns associated with microbial powders, which could be inhaled during the treatment process. The present disclosure provides extender compositions which prolong or maintain the viability of microbes on-seed, thus increasing the stability of microbes and shelf-life of coated seed. In some embodiments, the extender compositions are effective at improving stability of microbial
20 treatments, even when the seed has been pre-treated with other chemicals and/or biologicals, such as plant enhancing agents. The compositions disclosed herein further promote the adherence of microbes to seed during the seed coating process. The disclosure further teaches seed coating kits, methods of use, and seeds and plant propagating material produced therefrom which are coated with the compositions disclosed herein.
25 Extender compositions
[0061] In some embodiments, the present disclosure relates to a seed treatment composition comprising: a) a sugar alcohol, b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH, c) a water-soluble polymer, and d) one or more cultured microbes.
[0062] In some embodiments, the present disclosure relates to a seed treatment composition
30 comprising: a) a sugar alcohol, b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH, and c) one or more cultured microbes. [0063] In some embodiments, the present disclosure relates to a liquid extender composition with cultured microbes, said extender comprising: a) a sugar alcohol, at approximately between 5% and 30% (w/v); b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at approximately between 5% and 30% (w/v).
5 [0064] In some embodiments, the present disclosure relates to a liquid extender composition with cultured microbes, said extender comprising: a) a sugar alcohol, at approximately between 7.5% and 15% (w/v); b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at approximately between 5% and 20% (w/v).
[0065] In some embodiments, the present disclosure relates to a liquid extender with cultured
10 microbes, said extender comprising: a) a sugar alcohol, at approximately between 20% and 40% (w/v) of the total volume; and b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH.
[0066] In some embodiments the extender compositions with microbes are allowed to dry on seeds. In some embodiments, the dried extender compositions with cultured microbes comprise
15 a sugar alcohol to polymer w/w ratio of between about 2:1 to 1 : 1. In some embodiments, the dried extender compositions with cultured microbes comprise a sugar alcohol to polymer w/w ratio of about 4:3.
[0067] In some embodiments, the present di sclosure teaches a dried seed coating compri sing: a) between about 50% and about 80% (w/w) sugar alcohol; b) a chemical buffer, wherein the
20 buffer maintains the composition at a neutral pH; and c) between about 15% to about 40% (w/w) cultured microbes.
[0068] In some embodiments, the dried seed coating comprises: a) about 72% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) about 24% (w/w) cultured microbes.
25 [0069] In some embodiments, the present disclosure teaches a dried seed coating comprising: a) between about 25% and about 40% (w/w) sugar alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) between about 17% and about 70% (w/w) water-soluble polymer; and d) between about 15% to about 40% (w/w) cultured microbes.
[0070] In some embodiments, the dried seed coating comprises: a) about 35% (w/w) sugar
30 alcohol; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) about 26% water-soluble polymer; and d) about 35% (w/w) cultured microbes. [0071] In some embodiments, the extenders of the present disclosure are provided without the microbes, which are admixed with the extender prior to application on seeds. Thus, in some embodiments, the present disclosure relates to an extender composition comprising: a) a sugar alcohol, b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH,
5 and c) a water-soluble polymer. In some embodiments the extender without the microbes comprises a) a sugar alcohol, and b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH.
[0072] In some embodiments, the present disclosure relates to a liquid extender composition for increasing the stability of microbes, said extender comprising: a) a sugar
10 alcohol, at approximately between 15% and 30% (w/v); b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at approximately between 10% and 40% (w/v).
[0073] In some embodiments, the present disclosure relates to a liquid extender composition for increasing the stability of microbes, said extender comprising: a) a sugar alcohol, at
15 approximately between 40% and 80% (w/v) of the total volume; and b) a chemical buffer, wherein said buffer maintains the composition at a neutral pH.
[0074] In some embodiments the extender compositions without microbes are provided in dried form. Thus in some embodiments, the dried extender compositions without cultured microbes comprise a sugar alcohol to polymer w/w ratio of between about 2:1 to 1:1. In
20 some embodiments, the dried extender compositions without cultured microbes comprise a sugar alcohol to polymer w/w ratio of between 3 : 1 to 1 :3.
Buffering agents
[0075] In some embodiments, the extender compositions disclosed herein comprise a chemical buffer or buffering agent. The buffering agent prevents fluctuations in the pH of the
25 composition, and thus prevents toxic levels of acidity or basicity for microorganisms.
[0076] In some embodiments, the chemical buffer maintains the pH of the composition (extender with or without cultured microbes) in the pH range of pH 5-9, pH 5-8, pH 5-7, pH 5- 6, pH 6-9, pH 6-8, pH 6-7, pH 7-9, or pH 7-8. In some aspects, the chemical buffer maintains the composition at a neutral pH. In some aspects, the chemical buffer comprises potassium
30 phosphate. In some aspects, the chemical buffer is a mixture of monopotassium phosphate (KH2PO4) and dipotassium phosphate (K2HPO4). In some aspects the chemical buffer is dipotassium phosphate at approximately between 0.5% and 3.0% of the total extender composition volume without the cultured microbes, and monopotassium phosphate at approximately between 0% and 2% of the total extender composition volume without the cultured microbes. In some aspects, the chemical buffer is dipotassium phosphate at
5 approximately 1-2% of the total extender composition volume without the cultured microbes, and monopotassium phosphate at approximately 0.5-1% of the total extender composition volume without the cultured microbes.
[0077] In some aspects the chemical buffer is dipotassium phosphate at approximately between 0.25% and 1.5% of the total extender composition volume with the cultured microbes,
10 and monopotassium phosphate at approximately between 0% and 1% of the total extender composition volume with the cultured microbes. In some aspects, the chemical buffer is dipotassium phosphate at approximately 0.5-1% of the total extender composition volume with the cultured microbes, and monopotassium phosphate at approximately 0.25-0.5% of the total extender composition volume with the cultured microbes. While K2HPO4 and KH2PO4 were
15 used as the buffering salts in the example compositions disclosed herein, one skilled in the art will appreciate that, in some embodiments, other chemical buffers capable of maintaining a neutral or approximately neutral pH may be used.
[0078] Non-limiting examples of buffering agents include potassium phosphates, sodium citrate, ascorbate, succinate, lactate, citric acid, boric acid, borax, hydrochloric acid, disodium
20 hydrogen phosphate, acetic acid, formic acid, glycine, bicarbonate, phosphate, tartaric acid, Tris-glycine, Tris-NaCl, Tris-ethylenediamine tetraacetic acid (“EDTA”), Tris-borate, Tris- borate-EDTA, Tris-acteate-EDTA (“TAB”), Tris-buffered saline, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (“HEPES”), 3-(N-morpholino) propanesulfonic acid (“MOPS”), piperazine-l,4-bis(2-ethanesulfonic acid) (“PIPES”), 2-(N-morpholino)ethanesulfonic acid
25 (“MES”), and phosphate buffered saline (“PBS”). Table 1 also provides exemplary buffering agents as well as their pKa values and useful pH ranges.
Table 1: Exemplary buffering agents
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
[0079] Additional buffers and instructions on how to prepare them can be found in, e.g., “Common Buffers and Stock Solutions” (2011) Current Protocols in Nucleic Acid Chemistry, A.2A.1-A.2A.14 and in the Sigma Aldrich “Buffer Reference Center” available on the world
5 wide web at sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/leaming- center/buffer-reference-center.html, the contents of each of which are incorporated herein in their entirety. Persons having skill in the art will appreciate that the amount of buffer needed to maintain the desired pH will depend on the buffer used, and the total volume of extender solution.
10 [0080] In some aspects, the chemical buffer is present in the extender composition with or without cultured microbes at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% (w/v), including all ranges and subranges therebetween. [0081] In some aspects, the chemical buffer is present in the extender composition with or without cultured microbes at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,
5 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% (w/w), including all ranges and subranges therebetween (e.g., in seed coatings).
Sugars
[0082] In some embodiments, the compositions disclosed herein comprise a sugar. In some aspects, the sugar is selected from the group consisting of monosaccharides, disaccharides,
10 tri saccharides, and polysaccharides. In some aspects, the sugar includes one or more of trehalose, sucrose, or glycerol. In some aspects, the sugar is a sugar alcohol or non-reducing sugar. In some aspects, the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fiicitol, iditol, and inositol. In some aspects, the sugar alcohol is sorbitol.
[0083] In some aspects, the sugar or sugar alcohol is present in the extender composition
15 with or without cultured microbes at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% (w/v), including
20 all ranges and subranges therebetween. In some aspects, the sugar alcohol is present in the extender composition without the cultured microbes at about 20% (w/v) with a polymer or about 60% (w/v) without a polymer. In some aspects, the sugar alcohol is present in the extender composition without the cultured microbes at about 10% (w/v) with a polymer or about 30% (w/v) without a polymer.
25 [0084] In some aspects, the sugar or sugar alcohol is present in the extender composition with or without cultured microbes at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
30 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or 83% (w/w), including all ranges and subranges therebetween (e.g., in seed coatings). Polymers
[0085] In some embodiments, the compositions disclosed herein comprise a polymer. In some aspects, the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA), carboxymethyl cellulose (CMC),
5 hydroxypropyl methylcellulose, alginate, and combinations thereof. In some aspects, the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
[0086] One skilled in the art will appreciate that a large variety of polymers may be used with the compositions and methods disclosed herein, for example, synthetic polymers, naturally occurring polymers, copolymers, dry-phase polymers, wet-phase polymers, semi-dry
10 polymers, gel polymers, microporous polymers, emulsion polymers, film-forming polymers, allospheres (polymeric nanomaterials), electrospun polymers, cross-linked polymers, water- soluble polymers, and combinations thereof. In some aspects, the polymer is a water-soluble polymer.
[0087] In some aspects, the polymer is a naturally occurring polymer. In some aspects, the
15 polymer is produced by a plant or plant part. In some aspects, the polymer is derived from a plant, plant part, or substance therefrom. In some aspects, the polymer is produced by an animal or animal part. In some aspects, the polymer is derived from an animal, animal part, or substance therefrom. In some aspects, the polymer is produced by a microbe such as an algae, protist, bacterium, or fungus. In some aspects, the polymer is derived from a microbe or a
20 substance therefrom. In some aspects, the polymer is an exopolymer. In some aspects, the polymer is an endopolymer.
[0088] In some aspects, the polymer contains only repeating units of one type of monomer. In some aspects, the polymer contains repeating units of more than one type of monomer (copolymer). In some aspects, the polymer structure is linear polymer - a linear polymer. In
25 some aspects, the polymer structure is branched polymer - a branched polymer. In some aspects, the polymer structure is network polymer. In some aspects, the polymer is an interpenetrating network polymer.
[0089] In some aspects, the polymer is selected from: polyvinylpyrrolidone, polyvinylpyrrolidone-vinyl acetate copolymer (PVP-VA), 2-Pyrrolidinone, 1-
30 ethenylhexadecyl-, homopolymer, carrageenan, sodium alginate, hydroxypropyl methylcellulose (HPMC), polyethylene glycol, gum arabic, maltodextrin, sodium alginate, alginate, xanthan gum, carboxymethyl cellulose (CMC), sodium-carboxymethyl cellulose (Na- CMC), starch BR-07, starch BR-08, starch, and starch-derivatives, pullulan, chitosan, glycosaminoglycans (GAGs), keratin sulfate GAG, hyaluronic acid GAG, heparin sulfate GAG, chondroitin sulfate GAG, polymerized fibrin, polymethylcrylate, polyacrylic acid,
5 polymethacrylic acid, styrene-butadiene, acrylic, styrene-acrylic, vinyl acetate, tocopheryl polyethylene glycol succinate (TPGS)-based polymer, and poly(lactic-co-gly colic acid) (PLGA), etc.
[0090] Additional non-limiting examples of polymers that can be used with the compositions and methods disclosed herein include: polyvinyl acetates, polyvinyl acetate copolymers,
10 ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses (e.g., ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses, and carboxymethylcelluloses), polyvinylpyrolidones, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acylamide polymers and copolymers,
15 polyhydroxyethyl acrylate, methylacrylamide monomers, polychloroprene, acrylamide homo- and copolymers, acrylic acid homo- and copolymer, cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose (sodium and other salts), carboxymethyl hydroxyethyl cellulose, hydroxyethyl cellulose, hydrophobically modified hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, water-soluble cellulose ethers, carboxy-vinyl
20 copolymers, alginic acid, polyacrylic acid, sodium polyacrylate, partially and fully hydrolyzed polyvinyl alcohols, partially neutralized polyacrylic acid, polyalkylene glycol, polyvinylpyrrolidone and derivatives, starch and its derivatives, vinylpyrrolidone homo- and copolymers, polyacrylamide, attapulgite, montmorillonite, organically modified montmorillonite clays, alumina, precipitated silica, or any mixture thereof
25 [0091] In some aspects, the polymer is present in the extender compositions disclosed herein with or without cultured microbes at a % weight to volume of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,
30 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, including all ranges and subranges therebetween. In some aspects, the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA) and is present in the extender composition without the cultured microbes at approximately 15%. In some aspects, the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA) and is present in the extender composition with the cultured microbes at approximately 7.5%.
[0092] In some aspects, the polymer is present in the extender compositions disclosed herein
5 with or without cultured microbes at a % (wt/wt) of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, including
10 all ranges and subranges therebetween (e.g., in dried seed coatings).
Example microbes and microbial compositions for use with disclosed compositions
[0093] In some embodiments, the extender compositions disclosed herein are mixed with one or more cultured microbes and/or microbial compositions to be used as a seed coating. In some embodiments, the extender compositions disclosed herein are mixed with one or more
15 cultured microbes and/or microbial compositions for storage or use as a liquid (e.g., direct application to soil or other surface). The microorganisms mixed with the compositions disclosed herein may be in a liquid or powder form, or may be reconstituted from a powder to a liquid form prior to mixing with the extender compositions disclosed herein. In some embodiments, the disclosed extender compositions exhibit higher protective effects on
20 microorganisms that are not lyophilized and resuspended in buffer (e.g., broth samples). In some embodiments, the disclosed extender compositions can be directly mixed with dried microbes in powder form, without the need for a prior reconstitution step.
[0094] In some aspects, the microbial composition: extender composition ratio is between 1:1 and 1:4 by percent volume. In some aspects, the microbial composition is a liquid
25 composition and the ratio to the extender composition is 1 : 1 by percent volume.
[0095] In some aspects, the microbial composition is a powder and is reconstituted to a liquid prior to mixture with the extender composition at a powder: reconstituted liquid ratio of between 1:4 and 3:10 by weight to volume, including all ranges and subranges therebetween. In some embodiments, the powdered microbes are added directly to the liquid extender
30 composition at ratio of 1:1, 1 :2, 1:3, 1 :4, 1:5, 1 :6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30 by weight to volume (grams to mL). In some embodiments, the powdered microbes are added to the liquid extender composition at a ratio between 1 :3 and 1 :4. In some aspects, the powdered microbes are added to the liquid extender composition at a ratio of 3:10, 2.5:10, 2:10, or 1.5:10.
[0096] In some embodiments, the extender compositions of the present disclosure comprise
5 powdered microbes at a % weight (grams to mL) to volume of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, including all ranges and subranges therebetween.
[0097] In some aspects the one or more cultured microbes comprise between 1.0 X 104 and 1.0 X 1012 CFU/mL of the total volume of the mixed extender composition. In some aspects,
10 the microorganisms are at an initial concentration of 104 to 1012 CFU/mL In some aspects, the microorganisms are at an initial concentration of 108 to 1010 CFU/ml. In some aspects, the microorganisms are at an initial concentration of about 104, 105, 106, 107, 108, 109, 1010, 10°, or 1012 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 108 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 109
15 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 1010 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 1011 CFU/mL. In some aspects, the microorganisms are at an initial concentration of about 1012 CFU/mL.
[0098] In some aspects, the cultured microbes are dried (e g., lyophilized) and present in the
20 extender composition at approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,
44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,
60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% (w/w), including all ranges
25 and subranges therebetween (e.g. in a dried seed coating).
[0099] In some embodiments, the cultured microbes are at a concentration from about between 1.0 X 104 and 1.0 X 108 CFU per seed. Thus, in some embodiments, the cultured microbes are at an initial concentration of 104, 105, 106, 107, 108 per seed.
[00100] These microbes can be obtained from any source, including environmental and
30 commercial sources. For example, plant beneficial bacteria may be obtained from any general terrestrial environment, including its soils, plants, fungi, animals (including invertebrates) and other biota, including the sediments, water and biota of lakes and rivers; from the marine environment, its biota and sediments (for example, sea water, marine muds, marine plants, marine invertebrates (for example, sponges), marine vertebrates (for example, fish); the terrestrial and marine geosphere (regolith and rock, for example, crushed subterranean rocks,
5 sand and clays); the cryosphere and its meltwater; the atmosphere (for example, filtered aerial dusts, cloud and rain droplets); urban, industrial and other man-made environments (for example, accumulated organic and mineral matter on concrete, roadside gutters, roof surfaces, and road surfaces).
[00101] The plants from which the bacteria (or any microbe according to the disclosure) are
10 obtained may be a plant having one or more desirable traits, for example a plant which naturally grows in a particular environment or under certain conditions of interest. By way of example, a certain plant may naturally grow in sandy soil or sand of high salinity, or under extreme temperatures, or with little water, or it may be resistant to certain pests or disease present in the environment, and it may be desirable for a commercial crop to be grown in such conditions,
15 particularly if they are, for example, the only conditions available in a particular geographic location. By way of further example, the bacteria may be collected from commercial crops grown in such environments, or more specifically from individual crop plants best displaying a trait of interest amongst a crop grown in any specific environment: for example the fastest- growing plants amongst a crop grown in saline-limiting soils, or the least damaged plants in
20 crops exposed to severe insect damage or disease epidemic, or plants having desired quantities of certain metabolites and other compounds, including fiber content, oil content, and the like, or plants displaying desirable colors, taste or smell. The bacteria may be collected from a plant of interest or any material occurring in the environment of interest, including fungi and other animal and plant biota, soil, water, sediments, and other elements of the environment as
25 referred to previously.
[00102] The bacteria (or any microbe according to the disclosure) may be isolated from plant tissue. This isolation can occur from any appropriate tissue in the plant, including for example root, stem and leaves, and plant reproductive tissues. Non-limiting examples of plant tissues include a seed, seedling, leaf, cutting, plant, bulb, tuber, root, and rhizomes. In some
30 embodiments, microorganisms are isolated from a seed. In ssoommee embodiments, microorganisms are isolated from a root. [00103] Persons having skill in the art will be familiar with techniques for recovering microbes from various environmental sources. For example, microbes useful in the compositions and methods disclosed herein can be obtained by extracting microbes from surfaces or tissues of native plants; grinding seeds to isolate microbes; planting seeds in diverse
5 soil samples and recovering microbes from tissues; or inoculating plants with exogenous microbes and determining which microbes appear in plant tissues. The parameters for processing samples may be varied to isolate different types of associative microbes, such as rhizospheric, epiphytes, or endophytes. By way of example, some methods for isolation from plants include the sterile excision of the plant material of interest (e.g. root or stem lengths,
10 leaves), surface sterilization with an appropriate solution (e.g. 2% sodium hypochlorite), after which the plant material is placed on nutrient medium for microbial growth. Alternatively, the surface-sterilized plant material can be crushed in a sterile liquid (usually water) and the liquid suspension, including small pieces of the crushed plant material spread over the surface of a suitable solid agar medium, or media, which may or may not be selective (e.g. contain only
15 phytic acid as a source of phosphorus). This approach is especially useful for bacteria which form isolated colonies and can be picked off individually to separate plates of nutrient medium, and further purified to a single species by well-known methods. Alternatively, the plant root or foliage samples may not be surface sterilized but only washed gently thus including surfacedwelling epiphytic microorganisms in the isolation process, or the epiphytic microbes can be
20 isolated separately, by imprinting and lifting off pieces of plant roots, stem or leaves onto the surface of an agar medium and then isolating individual colonies as above. This approach is especially useful for bacteria, for example. Alternatively, the roots may be processed without washing off small quantities of soil attached to the roots, thus including microbes that colonize the plant rhizosphere. Otherwise, soil adhering to the roots can be removed, diluted and spread
25 out onto agar of suitable selective and non-selective media to isolate individual colonies of rhizospheric bacteria.
[00104] Microbes may also be sourced from a repository, such as environmental strain collections, instead of initially isolating from a first plant. The microbes can be genotyped and phenotyped, via sequencing the genomes of isolated microbes; profiling the composition of
30 communities in planta, characterizing the transcriptomic functionality of communities or isolated microbes; or screening microbial features using selective or phenotypic media (e.g., nitrogen fixation or phosphate solubilization phenotypes). Selected candidate strains or populations can be obtained via sequence data; phenotype data; plant data (e.g., genome, phenotype, and/or yield data); soil data (e.g., pH, N/P/K content, and/or bulk soil biotic communities); or any combination of these
[00105] In some embodiments, the one or more cultured microbes are selected from species
5 of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof. In some aspects, the one or more microbes comprise Kosakonia sacchari. In some aspects, the one or more cultured microbes is Kosakonia sacchari
10 PTA-126743, as described in International Patent Publication No. WO2021222567. In some aspects, the one or more cultured microbes is Klebsiella variicola. In some aspects, the one or more cultured microbes is Klebsiella variicola PTA-126740, as described in International Patent Publication No. WO2021222567.
[00106] While Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA-
15 126740 were used in the experiments disclosed herein, it will be understood by one skilled in the art that any type of beneficial microbe(s) may be used with the compositions and methods disclosed herein to increase on-seed adherence and stability of the microbe(s). Non-limiting examples of other beneficial microbes are provided below in Table 2, for example.
Table 2: Microorganisms Deposited under the Budapest Treaty
Figure imgf000026_0001
Figure imgf000027_0001
[00107] In some aspects, the one or more cultured microorganisms of the disclosure are those from Table 2. In other aspects, the one or more cultured microorganisms of the disclosure are derived from a microorganism of Table 2. For example, a strain, child, mutant, or derivative,
5 of a microorganism from Table 2 are provided herein. The disclosure contemplates all possible combinations of microbes listed in Table 2, said combinations sometimes forming a microbial consortia. The microbes from Table 2, either individually or in any combination, can be combined with any plant, active molecule (synthetic, organic, etc.), adjuvant, carrier, supplement, biofilm, or biological in a microbial composition. In some aspects, the one or more cultured microbes are a microbial composition comprising at least one of a polymer, sugar, biofilm, and isolated biofilm compositions.
[00108] In some embodiments, the microbes of this disclosure are nitrogen fixing microbes,
5 for example nitrogen fixing bacteria, nitrogen fixing archaea, nitrogen fixing fungi, nitrogen fixing yeast, nitrogen fixing algae, or nitrogen fixing protozoa. In some aspects, microbes usefill in the compositions and methods disclosed herein are spore forming microbes, for example spore forming bacteria. In some aspects, bacteria useful in the compositions and methods disclosed herein are Gram positive bacteria or Gram negative bacteria. In some
10 embodiments, the bacteria are endospore forming bacteria of the Firmicute phylum. In some embodiments, the bacteria are diazotrophs. In some embodiments, the bacteria are not diazotrophs.
[00109] In some embodiments, the compositions and methods of the disclosure are used with an archaea, such as, for example, Methanothermobacter thermoautotrophicus, Methanosarcina
15 barkeri, Methanospirillum hungatei, Methanobacterium bryantii, Methanococcus thermolithotrophicus, and Methanococcus maripaludis.
[00110] In some embodiments, the one or more cultured microbes include, but are not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus
20 aminoglucosidicus, Bacillus aminovorans, Bacillus amylolyticus (also known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus coagulans, Bacillus endoparasiticus Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus lacticola,
25 Bacillus lactimorbus, Bacillus lactis. Bacillus laterosporus (also known as Brevibacillus laterosporus), Bacillus lautus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus maroccamis, Bacillus megaterium, Bacillus metiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus. Bacillus papillae, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus siamensis,
30 Bacillus smithii, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis. Bacillus uniflagellatus, Bradyrhizobium japonicum, Brevibacillus brevis, Brevibacillus laterosporus (formerly Bacillus laterosporus), Chromobacterium subtsugae, Delftia acidovorans, Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly Bacillus popilliae), Pantoea agglomerans, Pasteuria penetrans (formerly Bacillus penetrans), Pasteuria usgae, Pectobacterium carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa,
5 Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known as Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas fluoresce ns, Pseudomonas proradix, Pseudomonas putida, Pseudomonas syringae, Serratia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saraceticus, Streptomyces
10 venezuelae, Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus nematophila, Rhodococcus globerulus AQ719 (NRR.L Accession No. B-21663), Bacillus sp. AQ175 (ATCC Accession No. 55608), Bacillus ^. AQ 177 (ATCC Accession No. 55609), Bacillus sp. AQ178 (ATCC Accession No. 53522), and Streptomyces sp. strain NRRL Accession No. B-30145. In some embodiments, the bacterium is Azotobacter chroococcum, Methanosarcina barkeri,
15 Klesiella pneumoniae, Azotobacter vinelandii, Rhodobacter spharoides, Rhodobacter capsulatus, Rhodobcter palustris, Rhodosporillum rubrum, Rhizobium leguminosarum or Rhizobium etli.
[00111] In some embodiments, the bacterium is a species of Clostridium, for example Clostridium pasteuriamim, Clostridium beijerinckii, Clostridium perfringens, Clostridium
20 tetani, Clostridium acetobutylicum.
[00112] In some embodiments, the one or more cultured microbes used with the compositions and methods of the present disclosure are cyanobacteria. Examples of cyanobacterial genera include Anabaena (for example Anagaena sp. PCC7120), Nostoc (for example Nostoc punctiforme), or Synechocystis (for example Synechocystis sp. PCC6803).
25 [00113] In some embodiments, the one or more cultured microbes used with the compositions and methods of the present disclosure belong to the phylum Chlorobi, for example Chlorobium tepidum.
[00114] In some embodiments, microbes used with the compositions and methods of the present disclosure comprise a gene homologous to a known NifH gene. Sequences of known
30 NifH genes may be found in, for example, the Zehr lab NifH database, (wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, April 4, 2014), or the Buckley lab NifH database (www.css.comell.edu/faculty/buckley/nifh.htm, and Gaby, John Christian, and Daniel H. Buckley. "A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria." Database 2014 (2014): bauOOL). In some aspects, microbes used with the compositions and methods of the present disclosure comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%,
5 96%, 98%, 99% or more than 99% sequence identity to a sequence from the Zehr lab NifH database, (wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, April 4, 2014). In some aspects, microbes used with the compositions and methods of the present disclosure comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more than 99% sequence identity to a sequence from the Buckley lab NifH
10 database, (Gaby, John Christian, and Daniel H. Buckley. "A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria." Database 2014 (2014): bauOOL).
[00115] In some embodiments, the compositions and methods described herein make use of bacteria that are able to self-propagate efficiently on the leaf surface, root surface, or inside
15 plant tissues without inducing a damaging plant defense reaction, or bacteria that are resistant to plant defense responses. In some embodiments, the bacteria described herein are isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen.
[00116] In some embodiments, the one or more cultured microbes is an endophyte or an epiphyte or a bacterium inhabiting the plant rhizosphere (rhizospheric bacteria). Endophytes
20 are organisms that enter the interior of plants without causing disease symptoms or eliciting the formation of symbiotic structures, and are of agronomic interest because they can enhance plant growth and improve the nutrition of plants (e.g., through nitrogen fixation). The bacteria can be a seed-borne endophyte. Seed-borne endophytes include bacteria associated with or derived from the seed of a grass or plant, such as a seed-borne bacterial endophyte found in
25 mature, dry, undamaged (e.g., no cracks, visible fungal infection, or prematurely germinated) seeds. The seed-borne bacterial endophyte can be associated with or derived from the surface of the seed; alternatively, or in addition, it can be associated with or derived from the interior seed compartment (e.g., of a surface-sterilized seed). In some aspects, a seed-borne bacterial endophyte is capable of replicating within the plant tissue, for example, the interior of the seed.
30 Also, In some aspects, the seed-borne bacterial endophyte is capable of surviving desiccation.
[00117] Further, one skilled in the art will understand that assemblages of microbes, for example those that exhibit complementary colonization (different nutrient utilization, temporal occupation, oxygen adaptability, and/or spatial occupation), and/or different benefits to the seed or plant (nitrogen fixation, pest and/or pathogen control, etc.) can be used with the compositions and methods disclosed herein to increase on-seed adherence and stability of the microbe(s).
5 [00118] Thus, the one or more cultured microbes used in the compositions and methods of the disclosure, can comprise a plurality of different microorganism taxa in combination. By way of example, the bacteria may include Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Deljtia, Bradyrhizobiun, Sinorhizobium
10 anAHalomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium),and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium). The bacteria used in compositions and methods of this disclosure may include nitrogen fixing bacterial consortia of two or more species. In some embodiments, one or more bacterial species of the bacterial consortia may be capable of fixing nitrogen. In some
15 embodiments, one or more species of the bacterial consortia facilitate or enhance the ability of other bacteria to fix nitrogen. The bacteria which fix nitrogen and the bacteria which enhance the ability of other bacteria to fix nitrogen may be the same or different. In some aspects, a bacterial strain is able to fix nitrogen when in combination with a different bacterial strain, or in a certain bacterial consortia, but may be unable to fix nitrogen in a monoculture. Examples
20 of bacterial genera which may be found in a nitrogen fixing bacterial consortia include, but are not limited to, Herbaspirillum, Azospirillum, Enterobacter, and Bacillus.
[00119] Bacteria that can be used in the compositions and methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp. In some aspects, the bacteria are selected from the group consisting of: Azotobacter vinelandii, Bradyrhizobium
25 japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti. In some aspects, the bacteria are of the genus Enterobacter or Rahnella. In some aspects, the bacteria are of the genus Frankia, or Clostridium. Examples of bacteria of the genus Clostridium include, but are not limited to, Clostridium acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perjringens, and Clostridium tetani. In some aspects, the bacteria are of the genus
30 Paenibacillus, for example Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus, Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chibensis, Paenibacillus glucanolyticus, Paenibacillus illinoisensis, Paenibacillus larvae subsp. Larvae, Paenibacillus larvae subsp. Pulvifaciens, Paenibacillus lautus, Paenibacillus macerans, Paenibacillus macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli, Paenibacillus peoriae, or Paenibacillus polymyxa.
5 [00120] In some embodiments, bacteria for use in the present compositions and methods can be a member of one or more of the following taxa: Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Ancylobacter, Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus, Brevundimonas,
10 Burkholderia, Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio, Chryseobacterium, Citrobacter, Clostridium, Coraliomargarita, Corynebacterium, Cupriavidus, Curtobacterium, Curvibacter, Deinococcus, Delftia, Desemzia, Devosia, Dokdonella, Dyella, Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia, Escherichia/Shigella, Exiguobacterium, Ferroglobus, Filimonas, Finegoldia, Flavisolibacter,
15 Flavobacterium, Frigoribacterium, Gluconacetobacter, Hafhia, Halobaculum, Halomonas, Halosimplex, Herbaspirillum, Hymenobacter, Klebsiella, Kocuria, Kosakonia, Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas, Massilia, Mesorhizobium, Methylobacterium, Microbacterium, Micrococcus, Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum, Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus, Oxalophagus,
20 Paenibacillus, Panteoa, Pantoea, Pelomonas, Perlucidibaca, Plantibacter , Polynucleobacter, Propionibacterium, Propioniciclava, Pseudoclavibacter, Pseudomonas, Pseudonocardia, Pseudoxanthomonas, Psychrobacter, Rahnella, Ralstonia, Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas, Roseateles, Ruminococcus, Sebaldella, Sediminibacillus,
Sediminibacterium, Serratia, Shigella, Shinella, Sinorhizobium, Sinosporangium,
25 Sphingobacterium, Sphingomonas, Sphingopyxis, Sphingosinicella, Staphylococcus,
Stenotrophomonas, Strenotrophomonas, Streptococcus, Streptomyces, Stygiolobus,
Suljurisphaera, Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Variovorax, WPS-2 genera incertae sedis, Xanthomonas, and Zimmermannella.
[00121] In some embodiments, the bacteria are Gram-negative bacteria of a genus selected
30 from the following list: Acetobacter, Achromobacter, Aerobacter, Anabaena, Azoarcus, Azomonas, Azorhizobium, Azospirillum, Azotobacter, Beijemickia, Bradyrhizobium, Burkholderia, Citrobacter, Derxia, Enterobacter, Herbaspirillum, Klebsiella, Kluyvera, Kosakonia, Nostoc, Mesorhizobium, Rahnella, Rhizobium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Serratia Sinorhizobium, Spirillum, Trichodesmium, and Xanthomonas.
[00122] In some embodiments, a bacterial species selected from at least one of the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella. In some aspects, a
5 combination of bacterial species from the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella. In some aspects, the species utilized can be one or more of: Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari, and Rahnella aquatilis.
[00123] In some embodiments, a Gram positive microbe may have a Molybdenum-Iron nitrogenase system comprising: nifH, niJD, niJK, nijB, niJE, nifN, nijX, hesA, nifV, nifW, nifU,
10 niJS, nifll, and nijI2. In some aspects, a Gram positive microbe may have a vanadium nitrogenase system comprising: vnfDG, vnjK, vnfE, vnfN, vupC, vupB, vupA, vnjV, vnJRl, vnfH, vnfR2, vnfA (transcriptional regulator). In some aspects, a Gram positive microbe may have an iron-only nitrogenase system comprising: anfK, anjG, anfD, anfH, anfA (transcriptional regulator). In some aspects, a Gram positive microbe may have a nitrogenase system
15 comprising glnB, an&glnK (nitrogen signaling proteins). Some examples of enzymes involved in nitrogen metabolism in Gram positive microbes include glnA (glutamine synthetase), gdh (glutamate dehydrogenase), bdh (3 -hydroxy butyrate dehydrogenase), glutaminase, gltAB/gltB/gltS (glutamate synthase), asnA/asnB (aspartate- ammonia ligase/asparagine synthetase), and ansA/ansZ (asparaginase). Some examples of proteins involved in nitrogen
20 transport in Gram positive microbes include amtB (ammonium transporter), glnK (regulator of ammonium transport), glnPHQ/ glnQHMP (ATP-dependent glutamine/glutamate transporters), glnT/alsT/yrbD/yflA (glutamine-like proton symport transporters), and ^P/gltT/yhcl/nqt (glutamate-like proton symport transporters).
[00124] Examples of Gram positive microbes for use within the present compositions include
25 Paenibacillus polymixa, Paenibacillus riograndensis, Paenibacillus sp., Frankia sp., Heliobacterium sp., Heliobacterium chlorum, Heliobacillus sp., Heliophilum sp., Heliorestis sp., Clostridium acetobidylicum, Clostridium sp., Methanobacterium sp., Micrococcus sp., Mycobacterium flavum, Mycobacterium sp., Arthrobacter s spp..,, Agromyces sp., Corynebacterium autitrophicum, Corynebacterium sp., Micromonospora sp.,
30 Propionibacteria sp., Streptomyces sp., and Microbacterium sp. Genetic alterations to microbes
[00125] In some embodiments, the microorganism which is combined with the compositions disclosed herein is genetically modified to have improved nitrogen fixation capabilities. Thus, in some aspects, the microbes comprise one or more genetic variations introduced into one or
5 more genes regulating nitrogen fixation. The genetic variation may be introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The genetic variation may be a variation in a gene encoding a protein with functionality selected from the group consisting of: glutamine
10 synthetase, glutaminase, glutamine synthetase adenylyltransferase, transcriptional activator, anti-transcriptional activator, pyruvate flavodoxin oxidoreductase, flavodoxin, and NAD+- dinitrogen-reductase aDP-D-ribosyltransferase. The genetic variation may be a mutation that results in one or more of: increased expression or activity of nifA or glutaminase; decreased expression or activity of nifL, ntrB, glutamine synthetase, glnB, glnK, draT, amtB; decreased
15 adenylyl-removing activity of GlnE; decreased expression of GlnD; or decreased uridylyl- removing activity of GlnD. The genetic variation may be a variation in a gene selected from the group consisting of: bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and combinations thereof.
[00126] In some embodiments, the microbe has a disrupted (e.g., deleted or partially deleted)
20 nifL gene. In some aspects, the microbe has a nifL gene that has been disrupted with the introduction of a promoter sequence that acts on the nifA gene. In some aspects, e.g., when the microbe is a strain of K. variicola, the promoter is a K. variicola PinflC promoter. In some aspects, e.g., when the microbe is a strain of K. sacchari, the promoter is a K. sacchari Prm5 promoter. In some aspects, the microbe has a glnE gene that has been altered to remove the
25 adenylyl-removing (AR) domain, while leaving the coding region for the adenyltransferase (AT) domain, which is functionally expressed. In some aspects, the microbe has a deletion of the glnD gene.
[00127] The genetic variation introduced into one or more microorganisms may be a knockout mutation or it may abolish a regulatory sequence of a target gene, or it may comprise
30 insertion of a heterologous regulatory sequence, for example, insertion of a regulatory sequence found within the genome of the same bacterial species or genus. The regulatory sequence can be chosen based on the expression level of a gene in a bacterial culture or within plant tissue. The genetic variation may be produced by chemical mutagenesis. The plants grown may be exposed to biotic or abiotic stressors. However, in some aspects, the one or more cultured microbes for use with the compositions and methods disclosed herein also envision altering the impact of ATP or O2 on the circuitry, or replacing the circuitry with other regulatory cascades
5 in the cell, or altering genetic circuits other than nitrogen fixation. Gene clusters can be reengineered to generate functional products under the control of a heterologous regulatory system. By eliminating native regulatory elements outside of, and within, coding sequences of gene clusters, and replacing them with alternative regulatory systems, the functional products of complex genetic operons and other gene clusters can be controlled and/or moved to
10 heterologous cells, including cells of different species other than the species from which the native genes were derived. Once re-engineered, the synthetic gene clusters can be controlled by genetic circuits or other inducible regulatory systems, thereby controlling the products’ expression as desired. The expression cassettes can be designed to act as logic gates, pulse generators, oscillators, switches, or memory devices. The controlling expression cassette can
15 be linked to a promoter such that the expression cassette functions as an environmental sensor, such as an oxygen, temperature, touch, osmotic stress, membrane stress, or redox sensor.
[00128] As an example, the nifL, nifA, nijT, and nijX genes can be eliminated from the nif gene cluster. Synthetic genes can be designed by codon randomizing the DNA encoding each amino acid sequence. Codon selection is performed, specifying that codon usage be as
20 divergent as possible from the codon usage in the native gene. Proposed sequences are scanned for any undesired features, such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, sigma 54 and sigma 70 promoters, cryptic ribosome binding sites, and rho independent terminators. Synthetic ribosome binding sites are chosen to match the strength of each corresponding native ribosome binding site, such as by constructing a
25 fluorescent reporter plasmid in which the 150 bp surrounding a gene's start codon (from -60 to +90) is fused to a fluorescent gene. This chimera can be expressed under control of the Ptac promoter, and fluorescence measured via flow cytometiy. To generate synthetic ribosome binding sites, a library of reporter plasmids using 150 bp (-60 to +90) of a synthetic expression cassette is generated. Briefly, a synthetic expression cassette can consist of a random DNA
30 spacer, a degenerate sequence encoding an RBS library, and the coding sequence for each synthetic gene. Multiple clones are screened to identify the synthetic ribosome binding site that best matched the native ribosome binding site. Synthetic operons that consist of the same genes as the native operons are thus constructed and tested for functional complementation. A further exemplary description of synthetic operons is provided in US20140329326.
[00129] Some examples of genetic alterations which may be made in Gram positive microbes include: deleting glnR to remove negative regulation of BNF in the presence of environmental
5 nitrogen, inserting different promoters directly upstream of the nif cluster to eliminate regulation by GlnR in response to environmental nitrogen, mutating glnA to reduce the rate of ammonium assimilation by the GS-GOGAT pathway, deleting amtB to reduce uptake of ammonium from the media, mutating glnA so it is constitutively in the feedback-inhibited (FBI- GS) state, to reduce ammonium assimilation by the GS-GOGAT pathway.
10 [00130] GlnR is the main regulator of N metabolism and fixation in, e.g., Paenibacillus species. In some aspects, the genome of a Paenibacillus species does not contain a gene to produce glnR. In some aspects, the genome of a Paenibacillus species does not contain a gene to produce glnE or glnD. In some aspects, the genome of a Paenibacillus species does contain a gene to produce glnB or glnK. For example, Paenibacillus sp. WLY78 doesn’t contain a gene
15 for glnB, or its homologs found in the archaeon Methanococcus maripaludis, nifll and nifI2. In some aspects, the genomes of Paenibacillus species are variable. For example, Paenibacillus polymixa E681 lacks glnK and gdh, has several nitrogen compound transporters, but only amtB appears to be controlled by GlnR. In another example, Paenibacillus sp. JDR2 has glnK, gdh and most other central nitrogen metabolism genes, has many fewer nitrogen compound
20 transporters, but does have glnPHQ controlled by GlnR. Paenibacillus riograndensis SBR5 contains a standard glnRA operon, an fdx gene, a main nif operon, a secondary nif operon, and an anf operon (encoding iron-only nitrogenase). Putative glnR/tnrA sites were found upstream of each of these operons. GlnR may regulate all of the above operons, except the anf operon. GlnR may bind to each of these regulatoiy sequences as a dimer.
25 [00131] Paenibacillus N-fixing strains may fall into two subgroups: Subgroup I, which contains only a minimal nif gene cluster and subgroup n, which contains a minimal cluster, plus an uncharacterized gene between nifX and hesA, and often other clusters duplicating some of the nif genes, such as nifH, nifHDK, nifBEN, or clusters encoding vanadaium nitrogenase (ynf) or iron-only nitrogenase (anf) genes.
30 [00132] In some embodiments, the genome of a Paenibacillus species may not contain a gene to produce glnB or glnK. In some aspects, the genome of a Paenibacillus species may contain a minimal nif cluster with 9 genes transcribed from a sigma-70 promoter. In some aspects, a Paenibacillus nif cluster is negatively regulated by nitrogen or oxygen. In some aspects, the genome of a Paenibacillus species does not contain a gene to produce sigma-54. For example, Paenibacillus sp. WLY78 does not contain a gene for sigma-54. In some aspects, a nif cluster is regulated by glnR, and/or TnrA. In some aspects, activity of a nif cluster is altered by altering
5 activity of glnR, and/or TnrA.
[00133] In Bacilli, glutamine synthetase (GS) is feedback-inhibited by high concentrations of intracellular glutamine, causing a shift in confirmation (referred to as FBI-GS). Nif clusters contain distinct binding sites for the regulators GlnR and TnrA in several Bacilli species. GlnR binds and represses gene expression in the presence of excess intracellular glutamine and AMP.
10 A role of GlnR may be to prevent the influx and intracellular production of glutamine and ammonium under conditions of high nitrogen availability. TnrA may bind and/or activate (or repress) gene expression in the presence of limiting intracellular glutamine, and/or in the presence of FBI-GS. In some embodiments, the activity of a Bacilli nif cluster is altered by altering the activity of GlnR.
15 [00134] Feedback-inhibited glutamine synthetase (FBI-GS) may bind GlnR and stabilize binding of GlnR to recognition sequences. Several bacterial species have a GlnR/TnrA binding site upstream of the nif cluster. Altering the binding of FBI-GS and GlnR may alter the activity of the nif pathway.
[00135] In some embodiments, the microbes are non-intergeneric remodeled microbes. The
20 term “non-intergeneric” indicates that the genetic variations introduced into the host do not contain nucleic acid sequences from outside the host genus. In some embodiments, the microbes are intragenic. Therefore, in some embodiments, the microbes are not transgenic. For example, for non-transgenic microbes with varied promoters, promoters for promoter swapping are selected from within the microbe’s genome, or genus.
25 [00136] Exemplary non-intergeneric genetic variations include a mutation in the gene of interest that may improve the function of the protein encoded by the gene; a constitutionally active promoter that can replace the endogenous promoter of the gene of interest to increase the expression of the gene; a mutation that will inactivate the gene of interest; the insertion of a promoter from within the host’s genome into a heterologous location, e.g. insertion of the
30 promoter into a gene that results in inactivation of said gene and upregulation of a downstream gene; and the like. The mutations can be point mutations, insertions, and/or deletions (full or partial deletion of the gene). For example, in some embodiments, to improve the nitrogen fixation activity of the host microbe, a genetic variation may comprise an inactivating mutation of the nifL gene (negative regulator of nitrogen fixation pathway) and/or comprise replacing the endogenous promoter of the nifA and/or nifH gene (nitrogenase iron protein that catalyzes a key reaction to fix atmospheric nitrogen) with a constitutionally active promoter that will
5 drive the expression of the nifA and/or nifH gene constitutively.
[00137] In some embodiments, the one or more cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, ni/J, nifH, nifD, nifK, nifY, nifE, nifN, nifU,
10 nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nijQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii,yjbE,fhaB, pehA, otsB, treZ, glsA2, or combinations thereof.
[00138] In some aspects, the one or more cultured microbes are non-intergeneric remodeled bacteria capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
[00139] Additional examples of microorganisms and genetic modifications suitable for the
15 microorganisms for use with the compositions and methods of the present disclosure may be found in International Patent publication Nos. WO/2020/006246A1, WO/2020/118111A1, WO/2021/146209A1, WO/2021/222643A1, and W02020/014498, the contents of which are herein incorporated by reference in their entirety.
Seed Coating Treatments
20 [00140] One or more of the microorganisms described above may be mixed with the compositions described herein and used as a treatment seed coating for a seed or plant propagating material. Conventional or otherwise suitable coating equipment or techniques may be used to coat the seeds or plant propagating material with the seed coating treatments described above. Suitable equipment is deemed to include drum coaters, fluidized beds, rotary
25 coaters, side vended pan, tumble mixers and spouted beds, but any suitable equipment or technique may be used. Additionally, various coating machines are available to a person skilled in the art.
[00141] In some embodiments, the extender compositions disclosed herein increase microbial adherence to seed or plant propagating material. In some embodiments, the extender
30 compositions disclosed herein increase adherence of live microbes to seed or plant propagating material. In some embodiments, the extender compositions disclosed herein increase stability of microbes during treatment of a seed or plant propagating material. In some aspects, the microbes exhibit a log loss of less than 1.5 on seed. In some aspects, the microbes exhibit a log loss of less than 1 on seed.
[00142] In some embodiments, the extender compositions disclosed herein increase microbial
5 stability on seed or plant propagating material. In some aspects, microbes mixed with the extender compositions disclosed herein maintain at least 50% viability on seed after 28 days storage at room temperature. In some aspects, microbes mixed with the extender compositions disclosed herein maintain at least 60% viability on seed after 28 days storage at room temperature. In some aspects, microbes mixed with the extender compositions disclosed herein
10 maintain at least 70% viability on seed after 28 days storage at room temperature. In some aspects, microbes mixed with the extender compositions disclosed herein maintain at least 80% viability on seed after 28 days storage at room temperature. In some aspects, microbes mixed with the extender compositions disclosed herein maintain at least 90% viability on seed after 28 days storage at room temperature.
15 [00143] In some aspects, the composition is a seed coat present on a plant seed or other plant propagation material. In some aspects, the composition is a seed coat present on a plant seed or other plant propagation material that has at least one pre-treatment. In some aspects, the pre-treatment is a plant enhancing agent. In some embodiments, the pre-treatment is an insecticide, herbicide, fungicide, biocide, or nematicide.
20 Pre-treatments
[00144] The compositions of the present disclosure allow for downstream treatment of seed or plant propagation material. For example, the seed or plant propagation material to be coated with the compositions disclosed herein may have any number of pre-treatments, such as, plant enhancing agents, for example, insecticides, fungicides, herbicides, nematicides, and the like.
25 Examples of various pre-treatments are listed in the paragraphs below.
[00145] Insecticides: Al) the class of carbamates consisting of aldicarb, alanycarb, benfuracarb, carbaryl, carbofuran, carbosulfan, methiocarb, methomyl, oxamyl, pirimicarb, propoxur and thiodicarb; A2) the class of organophosphates consisting of acephate, azinphos- ethyl, azinphos-methyl, chlorfenvinphos, chlorpyrifos, chlorpyrifos-methyl, demeton-S-
30 methyl, diazinon, dichlorvos/DDVP, dicrotophos, dimethoate, disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion, methamidaphos, methidathion, mevinphos, monocrotophos, oxymethoate, oxydemeton-methyl, parathion, parathion-methyl, phenthoate, phorate, phosalone, phosmet, phosphamidon, pirimiphos-methyl, quinalphos, terbufos, tetrachlorvinphos, triazophos and trichlorfon; A3) the class of cyclodiene organochlorine compounds such as endosulfan; A4) the class of fiproles consisting of ethiprole, fipronil,
5 pyrafluprole and pyriprole; A5) the class of neonicotinoids consisting of acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam; A6) the class of spinosyns such as spinosad and spinetoram; A7) chloride channel activators from the class of mectins consisting of abamectin, emamectin benzoate, ivermectin, lepimectin and milbemectin; A8) juvenile hormone mimics such as hydroprene, kinoprene, methoprene,
10 fenoxycarb and pyriproxyfen; A9) selective homopteran feeding blockers such as pymetrozine, flonicamid and pyrifluquinazon; A10) mite growth inhibitors such as clofentezine, hexythiazox and etoxazole; All) inhibitors of mitochondrial ATP synthase such as diafenthiuron, fenbutatin oxide and propargite; uncouplers of oxidative phosphorylation such as chlorfenapyr; Al 2) nicotinic acetylcholine receptor channel blockers such as bensultap, cartap hydrochloride,
15 thiocyclam and thiosultap sodium; Al 3) inhibitors of the chitin biosynthesis type 0 from the benzoylurea class consisting of bistrifluron, diflubenzuron, flufenoxuron, hexaflumuron, lufenuron, novaluron and teflubenzuron; A14) inhibitors of the chitin biosynthesis type 1 such as buprofezin; A15) moulting disruptors such as cyromazine; A16) ecdyson receptor agonists such as methoxyfenozide, tebufenozide, halofenozide and chromafenozide; Al 7) octopamin
20 receptor agonists such as amitraz; Al 8) mitochondrial complex electron transport inhibitors pyridaben, tebufenpyrad, tolfenpyrad, flufenerim, cyenopyrafen, cyflumetofen, hydramethylnon, acequinocyl or fluacrypyrim;A19) voltage-dependent sodium channel blockers such as indoxacarb and metaflumizone; A20) inhibitors of the lipid synthesis such as spirodicl ofen, spiromesifen and spirotetramat; A21) ryanodine receptor-modulators from the
25 class of diamides consisting of flubendiamide, the phthalamide compounds (R)-3-Chlor-Nl- { 2-methyl-4-[ 1 ,2,2,2-tetrafluor- 1 -(trifluormethyl)ethyl]phenyl } -N2-( 1 -methyl -2-methyl sulfonylethyl)phthalamid and (S)-3-Chlor-Nl-{2-methyl-441,2,2,2-tetrafluor-l- (trifluormethyl)ethyl]phenyl } -N2-( 1 -methyl-2-methylsulfonylethyl)phthalamid, chloranthraniliprole and cyantraniliprole; A22) compounds of unknown or uncertain mode of
30 action such as azadirachtin, amidoflumet, bifenazate, fluensulfone, piperonyl butoxide, pyridalyl, sulfoxaflor; or A23) sodium channel modulators from the class of pyrethroids consisting of acrinathrin, allethrin, bifenthrin, cyfluthrin, gamma-cyhalothrin, lambda- cyhalothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta-cypermethrin, deltamethrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, tau- fluvalinate, permethrin, silafluofen, tefluthrin and tralomethrin and any suitable combinations thereof.
[00146] Fungicides: Bl) azoles selected from the group consisting of bitertanol,
5 bromuconazole, cyproconazole, difenoconazole, diniconazole, enilconazole, epoxi conazole, fluquinconazole, fenbuconazole, flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, penconazole, propiconazole, prothioconazole, simeconazole, triadimefon, triadimenol, tebuconazole, tetraconazole, triticonazole, prochloraz, pefurazoate, imazalil, triflumizole, cyazofamid, benomyl, carbendazim, thia-bendazole,
10 fuberidazole, ethaboxam, etridiazole and hymexazole, azaconazole, diniconazole-M, oxpoconazol, paclobutrazol, uni con azol, l-(4-chloro-phenyl)-2-([l,2,4]triazol-l-yl)- cycloheptanol and imazalilsulfphate; B2) strobilurins selected from the group consisting of azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl, methominostrobin, orysastrobin, picoxystrobin, pyraclostrobin, trifloxystrobin, methyl (2-
15 chloro-541 -(3 -methylbenzyloxyimino)ethyl]benzy l)carbamate, methyl (2-chloro-5 -[ 1 -(6- methylpyridin-2-ylmethoxyimino)ethyl]benzyl)carbamate and methyl 2-(ortho-(2,5- dimethylphenyloxymethylene)-phenyl)-3-methoxyacrylate, 2-(2-(6-(3-chloro-2-methyl- phenoxy)-5-fluoro-pyrimidin-4-yloxy)-phenyl)-2-methoxyimino-N-methyl-acetamide and 3- methoxy-2-(2-(N-(4-methoxy-phenyl)-cyclopropanecarboximidoylsulfanylmethyl)-phenyl)-
20 acrylic acid methyl ester; B3) carboxamides selected from the group consisting of carboxin, benalaxyl, benalaxyl-M, fenhexamid, flutolanil, furametpyr, mepronil, metalaxyl, mefenoxam, ofurace, oxadixyl, oxy carboxin, penthiopyrad, isopyrazam, thifluzamide, tiadinil, 3,4-dichloro- N-(2-cyanophenyl)isothiazole-5-carboxamide, dimethomorph, flumorph, flumetover, fluopicolide (picobenzamid), zoxamide, carpropamid, diclocymet, mandipropamid, N-(2-(443-
25 (4-chlorophenyl)prop-2-ynyloxy]-3-methoxyphenyl)ethyl)-2-methanesulfonyl-amino-3- methylbutyramide,N-(2-(4-[3-(4-chloro-phenyl)prop-2-ynyloxy]-3-methoxy-phenyl)ethyl)-2- ethanesulfonylamino-3-methylbutyramide, methyl 3-(4-chlorophenyl)-3-(2- isopropoxycarbonyl-amino-3-methyl-butyrylamino)propionate, N-(4'-bromobiphenyl-2-yl)-4- difluoromethylA-methylthiazole-6-carboxamide, N-(4'-trifluoromethyl-biphenyl-2-yl)-4-
30 difluoromethyl-2-methylthiazole-5-carboxamide, N-(4'-chloro-3'-fluorobiphenyl-2-yl)-4- difluoromethyl-2-methyl-thiazole-5-carboxamide, N-(3',4’-dichloro-4-fluorobiphenyl-2-yl)-3- difluoro-methyl-l-methyl-pyrazole-4-carboxamide, N-(3',4'-dichloro-5-fluorobiphenyl-2-yl)- 3 -difluoromethyl- 1 -methylpyrazole-4-carboxamide, N-(2-cyano-phenyl)-3 ,4- dichloroisothiazole-5-carboxamide, 2-amino-4-methyl-thiazole-5-carboxanilide, 2-chloro-N- (1 , 1 ,3-trimethyl-indan-4-yl)-ni cotinamide, N-(2-( 1 ,3-dimethylbutyl)-phenyl)- 1 ,3-dimethyl-5- fluoro-lH-pyrazole-4-carboxamide, N-(4'-chloro-3',5-difluoro-biphenyl-2-yl)-3-
5 difluoromethyl-l-methyl-lH-pyrazole-4-carboxamide,N-(4'-chloro-3', 5-difluoro-biphenyl-2- yl)-3-trifluoromethyl-l-methyl- 1 H-pyrazole-4-carboxamide, N-(3 4'-dichloro-5-fluoro- biphenyl-2-yl)-3-trifluoromethyl- 1 -methyl- lH-pyrazole-4-carboxamide, N-(3 5-difluoro-4'- methyl-biphenyl-2-yl)-3-difluoromethyl-l-methyl-lH-pyrazole-4-carboxamide, N-(3', 5- difluoro-4'-methyl-biphenyl-2-yl)-3-trifluoromethyl-l-methyl-lH-pyrazole-4-carboxamide,
10 N-(cis-2-bicyclopropyl-2-yl-phenyl)-3-difluoromethyl- 1 -methyl- lH-pyrazole-4-carboxamide, N-(trans-2-bicyclopropyl-2-yl-phenyl)-3-difluoro-methyl-l-methyl-lH-pyrazole-4- carboxamide, fluopyram, N-(3-ethyl-3,5-5-trimethyl-cyclohexyl)-3-formylamino-2-hydroxy- benzamide, oxytetracyclin, silthiofam, N-(6-methoxy-pyridin-3-yl) cyclopropanecarboxamide, 2-iodo-N-phenyl -benzamide, N-(2-bicyclo-propyl-2-yl-phenyl)-3 -
15 difluormethyl-l-methylpyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-l,3- dimethylpyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-l,3-dimethyl-5- fluoropyrazol-4-yl-carboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-5-chloro-l,3-dimethyl- pyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-3-fluoromethyl-l- methylpyrazol-4-ylcarboxamide,N-(3',4’,5’-trifluorobiphenyl-2-yl)-3-(chlorofluoromethyl)-l-
20 methylpyrazol-4-ylcarboxamide,N-(3',4',5'-trifluorobiphenyl-2-yl)-3-difluoromethyl-l- methylpyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-3-difluoromethyl-5- fluoro-l-methylpyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-5-chloro-3- difluoromethyl-l-methylpyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-3- (chlorodifluoromethyl)-l-methylpyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-
25 l-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide, N-(3',4',5'-trifluorobiphenyl-2-yl)-5- fluoro- 1 -methyl-3 -trifluoromethylpyrazol-4-ylcarboxamide, N-(3 ',4', 5 '-trifluorobiphenyl-2- yl)-5-chloro-l-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide, N-(2',4',5'- trifluorobiphenyl-2-yl)-l,3-dimethylpyrazol-4-ylcarboxamide, N-(2',4',5'-trifluorobiphenyl-2- yl)- 1 ,3-dimethyl-5-fluoropyrazol-4-ylcarboxamide, N-(2',4',5'-trifluorobiphenyl-2-yl)-5-
30 chloro-l,3-dimethylpyrazol-4-ylcarb oxami de, N-(2',4',5'-trifluorobiphenyl-2-yl)-3- fluoromethyl- 1 -methylpyrazol-4-ylcarboxamide, N-(2',4',5 '-trifluorobiphenyl-2-yl)-3 -
(chlorofluoromethyl)- 1 -methylpyrazol-4-yl carboxamide, N-(2 ',4', 5 '-trifluorobiphenyl-2-yl)-3 - difluoromethyl- 1 -methyl pyrazol -4-yl carboxamide, N-(2 ',4', 5 '-trifluorobiphenyl-2-yl)-3 - difluoromethyl-5-fluoro- 1 -methylpyrazol-4-ylcarboxamide, N-(2',4',5 '-trifluorobiphenyl-2- yl)-5-chloro-3-difluoromethyl-l-methylpyrazol-4-ylcarboxamide, N-(2',4',5'- trifluorobiphenyl-2-yl)-3 -(chlorodifluoromethyl)- 1 -methylpyrazol-4-ylcarboxamide, N- (2',4',5'-trifluorobiphenyl-2-yl)-l-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide, N-
5 (2',4',5'-trifluorobiphenyl-2-yl)-5-fluoro-l-methyl-3-trifluoromethylpyrazol-4- yl carboxamide, N-(2',4',5'-trifluorobiphenyl-2-yl)-5-chloro-l -methyl-3- trifluoromethylpyrazol-4-ylcarboxamide, N-(3',4'-dichloro-3-fluorobiphenyl-2-yl)-l-methyl- 3-trifluoromethyl- lH-pyrazole-4-carboxamide, N-(3 ',4'-dichloro-3 -fluorobiphenyl-2-yl)- 1 - methyl-3-difluoromethyl-lH-pyrazole-4-carboxamide, N-(3',4'-difluoro-3-fluorobiphenyl-2-
10 yl)- l-methyl-3 -trifluoromethyl- lH-pyrazole-4-carboxamide, N-(3 ',4'-difluoro-3- fluorobiphenyl-2-yl)-l-methyl-S-difluoromethyl-lH-pyrazole-4-carboxamide, N-(3'-chloro- 4 '-fluoro-3 -fluorobiphenyl-2-yl)- 1 -methyl -3 -difluoromethyl- lH-pyrazole-4-carboxamide, N- (3 ',4'-dichloro-4-fluorobiphenyl-2-yl)-l -methyl-3-trifluoromethyl- lH-pyrazole-4- carboxamide, N-(3',4'-difluoro-4-fluorobiphenyl-2-yl)-l-methyl-S-trifluoromethyl-lH-
15 pyrazole-4-carboxamide, N-(3 ',4'-dichloro-4-fluorobiphenyl-2-yl)- 1 -methyl-3- difluoromethyl-lH-pyrazole-4-carboxamide, N-(3',4'-difluoro-4-fluorobiphenyl-2-yl)-l- methyl-3-difluoromethyl-lH-pyrazole-4-carboxamide, N-(3 '-chloro-4'-fluoro-4- fluorobiphenyl-2-yl)-l-methyl-S-difluoromethyl-lH-pyrazole-4-caiboxamide, N-(3',4'- dichloro-5-fluorobiphenyl-2-yl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carboxamide, N-
20 (3',4'-difluoro-5-fluorobiphenyl-2-yl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4- carboxamide, N-(3 ',4'-dichloro-5-fluorobiphenyl-2-yl)- 1 -methyl-S-difluoromethyl- 1H- pyrazole-carboxamide, N-(3 ',4'-difluoro-5-fluorobiphenyl-2-yl)- 1 -methyl-3-difluoromethyl- lH-pyrazole-4-carboxamide, N-(3',4'-dichloro-5-fluorobiphenyl-2-yl)-l,3-dimethyl-lH- pyrazole-4-carboxamide, N-(3'-chloro-4'-fluoro-5-fluorobiphenyl-2-yl)-l-methyl-3-
25 difluoromethyl-lH-pyrazole-4-carboxamide, N-(4'-fluoro-4-fluorobiphenyl-2-yl)-l-methyl-3- trifluoromethyl-lH-pyrazole-4-carboxamide,N-(4'-fluoro-5-fluorobiphenyl-2-yl)-l-methyl-3- trifluoromethyl-lH-pyrazole-4-carboxamide, N-(4'-chloro-5-fluorobiphenyl-2-yl)-l-methyl-
3 -trifluoromethyl- lH-pyrazole-4-carboxamide, N-(4'-methyl-5-fluorobiphenyl-2-yl)- 1 - methyl-3-trifluoromethyl-lH-pyrazole-4-carboxamide, N-(4'-fluoro-5-fluorobiphenyl-2-yl)-
30 l,3-dimethyl-lH-pyrazole-4-carboxamide, N-(4'-chloro-5-fluorobiphenyl-2-yl)-l,3-dimethyl- lH-pyrazole-4-carboxamide, N-(4'-methyl-5-fluorobiphenyl-2-yl)-l,3-dimethyl-lH-pyrazole-
4-carboxamide,N-(4'-fluoro-6-fluorobiphenyl-2-yl)-l-methyl-3-trifluoromethyl-lH-pyrazole- 4-carboxamide, N-(4'-chloro-6-fluorobiphenyl-2-yl)-l-methyl-3-trifluoromethyl-lH- pyrazole-4-carboxamide, N-[2-( 1 , 1 ,2,3 , 3 ,3 -hexafluoropropoxy )-phenyl]-3 -difluoromethyl- 1 - methyl-lH-pyrazole-4-carboxamide, N-[4'-(trifluoromethylthio)-biphenyl-2-yl]-3- difluoromethyl-1 -methyl- lH-pyrazole-4-carboxamide aanndd N44'-(trifluoromethylthio)- biphenyl-2-yl]-l-methyl-3-trifluoromethyl-l-methyl-lH-pyrazole-4-carboxamide; B4)
5 heterocyclic compounds selected from the group consisting of fluazinam, pyrifenox, bupirimate, cyprodinil, fenarimol, ferimzone, mepanipyrim, nuarimol, pyrimethanil, triforine, fenpiclonil, fludioxonil, aldimorph, dodemorph, fenpropimorph, tridemorph, fenpropidin, iprodione, procymidone, vinclozolin, famoxadone, fenamidone, octhilinone, proben-azole, 5- chloro-7-(4-methyl-piperidin-l-yl)-6-(2,4,6-trifluorophenyl)41,2,4]triazolo[l,5-a]pyrimidine,
10 anilazine, diclomezine, pyroquilon, proquinazid, tricyclazole, 2-butoxy-6-iodo-3- propylchromen-4-one, acibenzolar-S-methyl, captafol, captan, dazomet, folpet, fenoxanil, quinoxyfen, N,N-dimethyl-3 -(3 -bromo-6-fluoro-2-methylindole- 1 -sulfonyl)-[ 1 ,2,4]triazole- 1 - sulfonamide, 5-ethyl-6-octyl41 ,2,4]triazolo[l 2,3,5,6-tetrachloro-4-methanesulfonyl-pyridine, 3,4,5-trichloro-pyridine-2,6-di-carbonitrile, N-(l-(5-bromo-3-chloro-pyridin-2-yl)-ethyl)-2,4-
15 di chloro-nicotinamide, N-((5-bromo-3-chloro pyridin-2-yl)-methyl)-2,4-dichloro- nicotinamide, diflumetorim, nitrapyrin, dodemorphacetate, fluoroimid, blasticidin-S, chinomethionat, debacarb, difenzoquat, difenzoquat-methylsulphat, oxolinic acid and piperalin; B5) carbamates selected from the group consisting of mancozeb, maneb, metam, methasulphocarb, metiram, ferbam, propineb, thiram, zineb, ziram, diethofencarb,
20 iprovalicarb, benthiavalicarb, propamocarb, propamocarb hydrochlorid, 4-fluorophenyl N-(l- (1 -(4-cyanophenyl)-ethanesulfonyl)but-2-yl)carbamate, methyl 3-(4-chloro-phenyl)-3-(2- isopropoxycarbonylamino-3-methyl-butyrylamino)propanoate; or B6) other fungicides selected from the group consisting of guanidine, dodine, dodine free base, iminoctadine, guazatine, antibiotics: kasugamycin, streptomycin, polyoxin, validamycin A, nitrophenyl
25 derivatives: binapacryl, dinocap, dinobuton, sulfur-containing heterocyclyl compounds: dithianon, isoprothiolane, organometallic compounds: fentin salts, organophosphorus compounds: edifenphos, iprobenfos, fosetyl, fosetyl-aluminum, phosphorous acid and its salts, pyrazophos, tolclofos-methyl, organochlorine compounds: dichlofluanid, flusulfamide, hexachloro-benzene, phthalide, pencycuron, quintozene, thiophanate-methyl, tolylfluanid,
30 others: cyflufenamid, cymoxanil, dimethirimol, ethirimol, furalaxyl, metrafenone and spiroxamine, guazatine-acetate, iminoc-tadine-triacetate, iminoctadine-tris(albesilate), kasugamycin hydrochloride hydrate, dichlorophen, pentachlorophenol and its salts, N-(4- chloro-2-nitro-phenyl)-N-ethyl-4-methyl-benzenesulfonamide, did nitrothal-isopropyl, tecnazen, biphenyl, bronopol, diphenylamine, mildiomycin, oxincopper, prohexadione calcium, N-(cyclopropylmethoxyimino-(6-difluoromethoxy-2,3-difluoro-phenyl)-methyl)-2- phenyl acetamide, N'-(4-(4-chloro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl- N-methyl formamidine, N'-(4-(4-fluoro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-
5 ethyl-N-methyl formamidine, N'-(2-methyl-5-trifluormethyl-4-(3-trimethylsilanyl-propoxy)- phenyl)-N-ethyl-N-methylformamidine and N'-(5-difluormethyl-2-methyl-4-(3- trimethylsilanyl-propoxy)-phenyl)-N-ethyl-N-methyl formamidine, and any combinations thereof
[00147] Herbicides: Cl) acetyl-CoA carboxylase inhibitors (ACC), for example
10 cyclohexenone oxime ethers, such as alloxydim, clethodim, cloproxydim, cycloxydim, sethoxydim, tralkoxydim, butroxydim, clefoxydim or tepraloxydim; phenoxyphenoxypropionic esters, such as clodinafop-propargyl, cyhalofop-butyl, diclofop- methyl, fenoxaprop-ethyl, fenoxaprop-P-ethyl, fenthiapropethyl, fluazifop-butyl, fluazifop-P- butyl, haloxyfop-ethoxyethyl, haloxyfop-methyl, haloxyfop-P-methyl, isoxapyrifop,
15 propaquizafop, quizalofop-ethyl, quizalofop-P-ethyl or quizalofop-tefuiyl; or arylaminopropionic acids, such as flamprop-methyl or flamprop-isopropyl; C2 acetolactate synthase inhibitors (ALS), for example imidazolinones, such as imazapyr, imazaquin, imazamethabenz-methyl (imazame), imazamox, imazapic or imazethapyr; pyrimidyl ethers, such as pyrithiobac-acid, pyrithiobac-sodium, bispyribac-sodium. KIH-6127 or pyribenzoxym;
20 sulfonamides, such as florasulam, flumetsulam or metosulam; or sulfonylureas, such as amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, halosulfuron-methyl, imazosulfuron, metsulfuron-methyl, nicosulfuron, primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, thifensulfuron-methyl,
25 triasulfuron, tribenuron-methyl, triflusulfuron-methyl, tritosulfuron, sulfosulfuron, foramsulfuron or iodosulfuron; C3) amides, for example allidochlor (CDAA), benzoylprop- ethyl, bromobutide, chiorthiamid. diphenamid, etobenzanidibenzchlomet), fluthiamide, fosamin or monalide; C4) auxin herbicides, for example pyridinecarboxylic acids, such as clopyralid or picloram; or 2,4-D or benazolin; C5) auxin transport inhibitors, for example
30 naptalame or diflufenzopyr; C6) carotenoid biosynthesis inhibitors, for example benzofenap, clomazone (dimethazone), diflufenican, fluorochloridone, fluridone, pyrazolynate, pyrazoxyfen, isoxaflutole, isoxachlortole, mesotrione, sulcotrione (chlormesulone), ketospiradox, flurtamone, norflurazon or amitrol; C7) enolpyruvylshikimate-3-phosphate synthase inhibitors (EPSPS), for example glyphosate or sulfosate; C8) glutamine synthetase inhibitors, for example bilanafos (bialaphos) or glufosinate-ammonium; C9) lipid biosynthesis inhibitors, for example anilides, such as anilofos or mefenacet; chloroacetanilides, such as
5 dimethenamid, S-dimethenamid, acetochlor, alachlor, butachlor, butenachlor, diethatyl-ethyl, dimethachlor, metazachlor, metolachlor, S-metolachlor, pretilachlor, propachlor, prynachlor, terbuchlor, thenylchlor or xylachlor; thioureas, such as butylate, cycloate, di-allate, dimepiperate, EPTC. esprocarb, molinate, pebulate, prosulfocarb, thiobencarb (benthiocarb), tri-allate or ve olate; or benfuresate or perfluidone; CIO) mitosis inhibitors, for example
10 carbamates, such as asulam, carbetamid, chlorpropham, orbencarb, pronamid (propyzamid), propham or tiocarbazil; dinitroanilines, such as benefin, butralin, dinitramin, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine or trifluralin; pyridines, such as dithiopyr or thiazopyr; or butamifos, chlorthal-dimethyl (DCPA) or maleic hydrazide; Cl l) protoporphyrinogen IX oxidase inhibitors, for example diphenyl ethers, such as acifluorfen,
15 acifluorfen-sodium, aclonifen, bifenox, chlomitrofen (CNP), ethoxyfen, fluorodifen, fluoroglycofen-ethyl, fomesafen, furyloxyfen, lactofen, nitrofen, nitrofluorfen or oxyfluorfen; oxadiazoles, such as oxadiargyl or oxadiazon; cyclic imides, such as azafenidin, butafenacil, carfentrazone-ethyl, cinidon-ethyl, flumiclorac-pentyl, flumioxazin, flumipropyn, flupropacil, fluthiacet-methyl, sulfentrazone or thidiazimin; or pyrazoles, such as ET-751.JV 485 or
20 nipyraclofen; Cl 2) photosynthesis inhibitors, for example propanil, pyridate or pyridafol; benzothiadiazinones, such as bentazone; dinitrophenols, for example bromofenoxim, dinoseb, dinoseb-acetate, dinoterb or DNOC; dipyridylenes, such as cyperquat-chloride, difenzoquat- methyl sulfate, diquat or paraquat-dichloride; ureas, such as chlorbromuron, chlorotoluron, difenoxuron, dimefuron, diuron, ethidimuron, fenuron, fluometuron, isoproturonisouron,
25 linuron, methabenzthiazuron, methazole, metobenzuron, metoxuron, monolinuron, neburon, siduron or tebuthiuron; phenols, such as bromoxynil or ioxynil; chloridazon; triazines, such as ametryn, atrazine, cyanazine, desmein, dimethamethryn, hexazinone, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbutryn, terbutylazine or trietazine; triazinones, such as metamitron or metribuzin; uracils, such as bromacil, lenacil or terbacil; or
30 biscarbamates, such as desmedipham or phenmedipham; C13) synergists, for example oxiranes, such as tridiphane; C14) CIS cell wall synthesis inhibitors, for example isoxaben or dichlobenil; Cl 6) various other herbicides, for example dichloropropionic acids, such as dalapon; dihydrobenzofurans, such as ethofumesate; phenylacetic acids, such as chlorfenac (fenac); or aziprotryn, barban, bensulide, benzthiazuron, benzofluor, buminafos, buthidazole, buturon, cafenstrole, chlorbufam, chlorfenprop-methyl, chloroxuron, cinmethylin, cumyluron, cycluron, cyprazine, cyprazole, dibenzyluron, dipropetryn, dymron, eglinazin-ethyl, endothall, ethiozin, flucabazone, fluorbentranil, flupoxam, isocarb amid, isopropalin, karbutilate,
5 mefluidide, monuron, napropamide, napropanilide, nitralin, oxaciclomefone, phenisopham, piperophos, procyazine, profluralin, pyributicarb, secbumeton, sulfallate (CDEC), terbucarb, triaziflam, triazofenamid or trimeturon; or their environmentally compatible salts or combinations thereof.
[00148] Nematicides: Benomyl, cloethocarb, aldoxycarb, tirpate, diamidafos, fenamiphos,
10 cadusafos, dichlofenthion, ethoprophos, fensulfothion, fosthiazate, heterophos, isamidofof, isazofos, phosphocarb, thionazin, imicyafos, mecarphon, acetoprole, benclothiaz, chloropicrin, dazomet, fluensulfone, oxamyl, terbufos and suitable combinations thereof.
[00149] Biocides: Biocides may be a chemical substance or a microorganism. By way of example, a biocide may be a bacteria, such as Pseudomonas, Enterobacter, Stenotrophomonas,
15 Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium, Halomonas, Bacillus (for example Bacillus amyloliquefaciens, Bacillus firmus), Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium, Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium.
20 [00150] Plant Growth Regulators or Hormones: DI) Antiauxins, such as clofibric acid, 2,3,5- triiodobenzoic acid; D2) Auxins such as 4-CPA, 2,4-D, 2,4-DB, 2,4-DEP, dichlorprop, fenoprop, IAA, IB A, naphthaleneacetamide, a-naphthaleneacetic acids, 1 -naphthol, naphthoxyacetic acids, potassium naphthenate, sodium naphthenate, 2,4,5-T; D3) cytokinins, such as 21P, benzyl adenine, 4-hydroxyphenethyl alcohol, kinetin, zeatin; D4) defoliants, such
25 as calcium cyanamide, dimethipin, endothal, ethephon, merphos, metoxuron, pentachlorophenol, thidiazuron, tribufos; D5) ethylene inhibitors, such as aviglycine, 1- methylcyclopropene; D6) ethylene releasers, such as ACC, etacelasil, ethephon, glyoxime; D7) gametocides, such as fenridazon, maleic hydrazide; D8) gibberellins, such as gibberellins, gibberellic acid; D9) growth inhibitors, such as abscisic acid, ancymidol, butralin, carbaryl,
30 chlorphonium, chlorpropham, dikegulac, flumetralin, fluoridamid, fosamine, glyphosine, isopyrimol, jasmonic acid, maleic hydrazide, mepiquat, piproctanyl, prohydrojasmon, propham, tiaojiean, 2,3,5-tri-iodobenzoic acid; D10) morphactins, such as chlorfluren, chlorflurenol, dichlorflurenol, flurenol; Dl l) growth retardants, such as chlormequat, daminozide, flurprimidol, mefluidide, paclobutrazol, tetcyclacis, uniconazole; D12) growth stimulators, such as brassinolide, brassinolide-ethyl, DCPTA, forchlorfenuron, hymexazol, prosuler, triacontanol; D13) unclassified plant growth regulators, such as bachmedesh,
5 benzofluor, buminafos, carvone, choline chloride, ciobutide, clofencet, cyanamide, cyclanilide, cycloheximide, cyprosulfamide, epocholeone, ethychlozate, ethylene, fuphenthiourea, furalane, heptopargil, holosulf, inabenfide, karetazan, lead arsenate, methasulfocarb, prohexadione, pydanon, sintofen, triapenthenol, trinexapac.
Concentrations and Rates of Application of Microbes
10 [00151] In some embodiments, the seed coating comprises microbes at a concentration of about 1 x 104 to about 1 x 1011 CPU per seed at the time of planting, when planted within 28 days of application. In some aspects, the microbes are at a concentration of about 1 x 105 to about 1 x io7 CPU per seed at the time of planting. In some aspects, the microbes are at a concentration of about 1 x io6 CPU per seed.
15 [00152] Using com as an example, in the United States, about 10% of com acreage is planted at a seed density of above about 36,000 seeds per acre; 1/3 of the com acreage is planted at a seed density of between about 33,000 to 36,000 seeds per acre; 1/3 of the com acreage is planted at a seed density of between about 30,000 to 33,000 seeds per acre, and the remainder of the acreage is variable. See, “Com Seeding Rate Considerations,” written by Steve Butzen,
20 available on the world wide web at pioneer.com/home/site/us/agronomy/library/com-seeding- rate-considerations/.
[00153] Table 3 below utilizes various CPU concentrations per seed in a contemplated seed treatment embodiment (rows across) and various seed acreage planting densities (1st column: 15K-41K) to calculate the total amount of CPU per acre, which would be utilized in various
25 agricultural scenarios (i.e. seed treatment concentration per seed x seed density planted per acre). Thus, if one were to utilize a seed treatment with 1 x io6 CPU per seed and plant 30,000 seeds per acre, then the total CPU content per acre would be 3 x 1O10 (i.e. 30K * 1 x 106). Table 3: Total CFU Per Acre Calculation for Corn Seed Treatment Embodiments
Figure imgf000049_0001
Figure imgf000050_0001
Improvement of Plant Traits
[00154] The disclosure provides compositions and methods to increase the adherence and on- seed stability of plant beneficial microbes. These plant beneficial microbes can improve a
5 variety of desirable traits in a plant. Examples of traits that may be introduced or improved include: root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size, photosynthesis rate, tolerance to drought, heat tolerance, salt tolerance, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, level of a metabolite, and
10 proteome expression. The desirable traits, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the improved traits)
15 grown under identical conditions. In some aspects, the compositions and methods described herein can improve plant traits, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, and increasing fruit or seed unit weight. In some aspects, the plant grown from the treated seed or plant material has improved health, yield, stress resistance, growth, or agronomic characteristics relative to a control plant. [00155] Traits that may be improved by the compositions and methods disclosed herein include any observable characteristic of the seed or the plant resulting therefrom, including, for example, growth rate, height, weight, color, taste, smell, changes in the production of one or more compounds by the plant (including for example, metabolites, proteins, drugs,
5 carbohydrates, oils, and any other compounds). In some aspects, the compositions and methods disclosed herein may result in a change in genotypic information (for example, a change in the pattern of plant gene expression such as those associated with increased nitrogen fixation, in response to the microbes). In some aspects, the plants show the absence, suppression or inhibition of a certain feature or trait (such as an undesirable feature or trait) as opposed to the
10 presence of a certain feature or trait (such as a desirable feature or trait).
[00156] The trait improved may be nitrogen fixation, including in a plant not previously capable of nitrogen fixation. In some embodiments, enhanced levels of nitrogen fixation are achieved in the presence of fertilizer supplemented with glutamine, ammonia, or other chemical source of nitrogen. Methods for assessing degree of nitrogen fixation are known and
15 may be employed to assess the methods described herein.
Seed coating kits
[00157] The ingredients/compositions disclosed herein may be packaged together as a kit for seed coating. For example, in some embodiments a dry microbial powder from one or more species of cultured microbes could be encased in commercial-grade water-soluble packaging
20 (commonly seen in the detergent industry) and provided in a kit with an extender composition. In some embodiments, the extender composition may be provided in a kit with an aqueous solution of cultured microbes. The end-user would then mix the components of the kit for use with any seed coating equipment.
[00158] In some embodiments, the extender compositions disclosed herein are a part of a seed
25 coating kit comprising the ingredients for the extender composition and one or more cultured microbes. In some aspects, the ingredients for the extender composition are provided in a dry form, with the end-user combining the sugar alcohol, chemical buffer, and optionally water- soluble polymer with water to create a liquid extender composition. In some aspects, the extender composition is provided premixed in a liquid form.
30 [00159] In some aspects, the one or more cultured microbes is provided in a liquid form. In some aspects, the one or more cultured microbes have been lyophilized and are provided in a powder formulation. In some aspects, and the kit further comprises a buffer for reconstitution of the powdered microbes. In some aspects, the ingredients for the buffer are provided in a dry form to be mixed by the end user. In some aspects, the ingredients for the buffer are provided pre-mixed in a liquid form.
5 [00160] Dry microbial formulations (such as, powders or granules) typically have a longer shelf compared to liquid microbial formulations, therefore are useful if the microbes need to be transported or stored prior to use. Dry microbial formulations however, have several disadvantages, as described below.
[00161] There may be real and perceived safety concerns over the direct handling of dry
10 powders containing microbes. For instance, farmers and treatment applicators may be concerned about potential inhalation of the microbial powder or contact of the microbial powder with skin or eye. This concern may manifest either in the transport/mixing of the extenders, or in post seed application steps, where non-adhered microbes must be disposed or reused. By increasing microbial adherence, the extenders of the present disclosure reduce
15 percentage of unattached microbes that must be dealt with later.
[00162] It may also be challenging for end-users to apply accurate and reproducible doses of diy formulations, at least in part, because of potential weight variability due to differing moisture content of the products in different climates, or volume fluctuations with particle aggregation. Moreover, the packaging of microbial diy powder for individual field doses can
20 result in excess plastic and non-degradable waste.
[00163] Thus, the seed coating kit described herein may comprise powder forms of microbes and or dried extender ingredients encased in a safe, convenient and eco-friendly water-soluble package. In some embodiments, the disclosure provides the water-soluble packages described in any one of: US 7,357,891, US 8,617,589, WO 2014/202412, WO 2014/202412, WO
25 2010/0088112, EP 1375637, EP 1394065, and US 2001/0033883, the packages comprising any one or more of the dehydrated microbes disclosed herein.
[00164] In some embodiments, the water-soluble packages disclosed herein improve the shelf stability of the microbes contained therein. In some embodiments, the microbes in the water- soluble packages disclosed herein have improved shelf stability relative to comparable liquid
30 formulations, dry powders or granules comprising the same microbes which are not encapsulated by the water-soluble packages disclosed herein. [00165] When the water-soluble packages disclosed herein are brought into contact with a liquid (such as, water or an aqueous solution), the package disintegrates, releasing the powder microbes and or extender ingredients contained therein into the liquid, thereby forming a liquid that can be applied to seeds or plant propagating material and/or mixed with the extender
5 compositions disclosed herein to generate a seed treatment coating.
[00166] The use of water-soluble packages has several advantages. First, the use of the water- soluble packages disclosed herein obviates the need for direct handling of the dry microbial powder by the end-user, such as a seed treater or a farmer, thus eliminating any real or perceived safety concerns due to the potential inhalation or contact of the microbial powder
10 with skin or eye. Second, the water-soluble packages disclosed herein can be designed to contain a standardized unit of microbes for simplified dosing. Third, the use of the water- soluble packages disclosed herein can promote uniform dispersion of the powdered microbe. For instance, in some embodiments, the components of the water-soluble packages help stabilize the microbes during dry storage and improve dispersion of the microbe in liquids.
15 Therefore, the use of the water-soluble packages promotes consistency in application results.
[00167] Fourth, the water-soluble packages disclosed herein enhance the shelf life of the microbes contained therein, since they provide an effective barrier between the microbial powder, and moisture and/or oxygen. Fifth, the water-soluble packages are environmentfriendly and reduce packaging waste, while having the potential to be aesthetically pleasing.
20 Sixth, they allow for single dose administration of multiple components that may not be amenable to comingling during storage, or which benefit from different administration timings. Finally, one or more components of the water-soluble packages disclosed herein (such as, polyvinyl alcohols) are released into the dispersion of live microbes upon contact with the liquid. Such components may enhance the survival of the microbes in the dispersion and/or on
25 seed, when the dispersion is applied as a seed treatment.
[00168] The water-soluble packages disclosed herein enable the co-administration of components (such as, microbes and one or more additives) that may not be amenable to being in contact with each other prior to the time of administration, during storage, and/or for long periods of time. Furthermore, when the disclosed agricultural components are brought in
30 contact with a liquid to form a dispersion that can be applied to plants or plant parts, it may be desirable to bring different components in contact with each other in a timed and/or ordered fashion. This kind of regulation of the contact (as well as the timing and/or the order of the contact) between the components can be achieved using the compartmentalization of the disclosed water-soluble packages, as described below.
Compartmentalization of water-soluble packages
[00169] In some embodiments, contact among the separate ingredients of the extender
5 composition and/or buffer is prevented until use by placing, for example, the sugar, chemical buffer, and optionally water-soluble polymer in separate compartments of the disclosed packages.
[00170] In some embodiments, the water-soluble package comprises two or more compartment(s). In some embodiments, the two or more compartment(s) comprise different
10 sugars, buffering salts, and water-soluble polymers. In some embodiments, the sugar alcohol, chemical buffer and water-soluble polymer are provided in one compartment and the cultured microbes in another. In some embodiments, the water-soluble polymer is present in the water- soluble package itself (e.g., forms the film, which is later dissolved into the liquid extender solution when placed in water).
15 [00171] In some embodiments, more than one species of cultured microbes are provided in the seed coating kit. In some embodiments, contact between the different species of powdered microbes is prevented until use by placing them in separate compartments of the water-soluble package.
[00172] In some embodiments, the seed coating kits disclosed herein comprise one or more
20 microbes selected from species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enter obacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof. In some embodiments, the one or more microbes comprise Kosakonia sacchari. In some embodiments,
25 the one or more microbes is Kosakonia sacchari PTA- 126743. In some embodiments, the one or more microbes is Klebsiella variicola. In some embodiments, the one or more microbes is Klebsiella variicola PTA- 126740.
[00173] In some embodiments, the seed coating kits disclosed herein comprise microbes having at least one genetic variation introduced into a member selected from the group
30 consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, niJH, nifD, nijK vtijY, nijE, nifN, nifU, nrfS, nijV, nifW, nijZ, nijM, nifF, nifB, nijQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, faaB, pehA, otsB, treZ, gbsA2, or combinations thereof.
[00174] In some embodiments, the seed coating kits disclosed herein comprise non-
5 intergeneric remodeled bacteria capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
Microbial compositions
[00175] In some embodiments, the one or more cultured microbes are provided as a microbial composition. In some aspects, the microbial composition has been lyophilized and is provided
10 in a dry powder form. In some aspects, the diy powder has been agglomerated to produce granules. In some aspects, the microbial composition is provided in a liquid form. In some embodiments, the microbial composition comprises at least one of a polymer, sugar, biofilm, and isolated biofilm compositions In some embodiments, the microbial composition comprises a stabilizer, bulking agent, anticaking agent, dispersant, or any combination thereof.
15 Stabilizers
[00176] A microbial stabilizer is an agent that acts to stabilize the microorganism population within the agricultural composition. In some embodiments, the microbial stabilizer decreases or slows the decay rate of the microbial population. In some embodiments, the microbial stabilizer accomplishes this change in the decay rate by maintaining the microorganisms in a
20 semi-dormant state. In a semi-dormant state, microorganisms do not respond to environmental conditions as rapidly as they would in an active state.
[00177] In some embodiments, the microbial stabilizer improves microbial survival rate, decreases microbial decay, improves microbial metabolic activity, improves microbial catabolic gene expression, improves the microbial colonization rate, or decreases toxin
25 accumulation.
[00178] In some embodiments, the microbial stabilizer increases the survival rate of microbial cells after storage, e.g., after 1, 2, 3, 4, 5, or 6 months of storage. In some embodiments, the log loss of CFU/mL of microbes after the storage period is less than 1. In some embodiments, the log loss is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2. [00179] In some embodiments, the microbial stabilizer improves the metabolic activity and/or catabolic gene expression of the microorganisms comprised by the agricultural composition after the storage period.
[00180] In some embodiments, the microbial stabilizer improves the colonization rate of the
5 microorganisms in the agricultural plant.
[00181] In some embodiments, the microbial stabilizer decreases toxin accumulation. In some embodiments, the toxin is a direct product or byproduct of nitrogen fixation. In some embodiments, the toxin is ammonia or ammonium. In some embodiments, the toxin is produced during cell growth/division.
10 [00182] In some embodiments, the microbial stabilizer is a sugar. In some embodiments, the microbial stabilizer is a non-reducing sugar. Sugars suitable for use include, but are not limited to, sucrose, oligofructose, glucose and fructose. Monosaccharides suitable for use include, but are not limited to, trehalose, sucrose, lactose, melibiose, and lactulose. In some embodiments, the microbial stabilizer is trehalose. In some embodiments, the microbial stabilizer is a
15 polysaccharide. Polysaccharides suitable for use include, but are not limited to, maltodextrin, microcrystalline cellulose, and dextran. Additional carbohydrates suitable for use as microbial stabilizers include, but are not limited to, pentoses (e.g., ribose, xylose), hexoses (e.g., mannose, sorbose), oligosaccharides (e.g., raffinose), and oligofructoses. In some embodiments, the microbial stabilizer is a sugar alcohol. Sugar alcohols suitable for use
20 include, but are not limited to, glycerol, mannitol, and sorbitol.
[00183] In some embodiments, the microbial stabilizer is an amino acid. In some embodiments, the microbial stabilizer is glycine, proline, glutamate, or cysteine. In some embodiments, the microbial stabilizer is a protein or protein hydrolysate. Proteins or protein hydrolysates suitable for use as microbial stabilizers within the agricultural composition of the
25 present disclosure include, but are not limited to, malt extract, milk powder, casein, whey powder, and yeast extract. In some embodiments, the microbial stabilizer is skimmed milk, starch, humic acid, chitosan, CMC, com steep liquor, molasses, paraffin, pinolene, NFSM, MgSCh, liquid growth medium, horse serum, or Ficoll.
[00184] In some embodiments, the microbial stabilizer is a desiccant. As used herein, a
30 “desiccant” can include any compound or mixture of compounds that can be classified as a desiccant regardless of whether the compound or compounds are used in such concentrations that they in fact have a desiccating effect on the liquid inoculant. Such desiccants are ideally compatible with the microbial population used, and should promote the ability of the microbial population to survive application on the agricultural plant tissues or the environs thereof and to survive desiccation. Examples of suitable desiccants include one or more of trehalose,
5 sucrose, glycerol, and methylene glycol. Other suitable desiccants include, but are not limited to, non-reducing sugars and sugar alcohols (e.g., mannitol or sorbitol).
[00185] In some embodiments, the microbial stabilizer also acts as a physical stabilizer. In some embodiments, the substance acting as a microbial stabilizer has properties of a thickening agent and therefore also acts as a physical stabilizer. In some embodiments, a microbial
10 composition of the present disclosure comprising both a physical and a microbial stabilizer does so by comprising the same agent that has characteristics of both types of stabilizer.
[00186] In some embodiments, the concentration of microbial stabilizer in powdered microbes is in the range from about 0.1% w/v to about 30% w/v.
[00187] In some embodiments, the microbial composition comprises a physical stabilizer. As
15 used herein, a “physical stabilizer” refers to a substance that improves the homogeneity of the composition, such that the microbial cells are at a similar density throughout the liquid composition. By increasing homogeneity, the physical stabilizer prevents high concentrations of cells and/or toxins from accumulating in any one sub-volume of the dispersion of live microbes.
20 [00188] In some embodiments, the physical stabilizer increases the viscosity of the dispersion of live microbes. In some embodiments, the physical stabilizer is a thickening agent. In some embodiments, the physical stabilizer is an anti-settling agent. In some embodiments, the physical stabilizer is a suspension aid. In some embodiments, the physical stabilizer acts to maintain microbial cells in suspension, improving the cell’s resistance to settle statically and
25 flow under shear or rheological shear-thinning. In some embodiments, a physical stabilizer may also have properties of a microbial stabilizer and vice versa.
[00189] In some embodiments, the physical stabilizer is a polysaccharide. Polysaccharides suitable for use as physical stabilizers include, but are not limited to, polyethylene glycol (PEG), xanthan gum, pectin, and alginates. In some embodiments, the physical stabilizer is
30 xanthan gum. In some embodiments, the physical stabilizer is a protein or protein hydrolysate. Proteins or protein hydrolysates suitable for use as physical stabilizers include, but are not limited to, gluten, collagen, gelatin, elastin, keratin, and albumin. In some embodiments, the physical stabilizer is a polymer. Polymers suitable for use as physical stabilizers include, but are not limited to, Carbopol® (CBP) polymers, methylene glycol, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poyacrylate, hydroxyethyl cellulose, or hydroxypropyl
5 methylcellulose. In some embodiments, the physical stabilizer is a gum or its derivative. Gums and their derivatives suitable for use as physical stabilizers include, but are not limited to, guar gum, gum Arabic, gum tragacanth, xanthan gum, derivitized guar, hydroxypropyl guar, and polysaccharide gums. In some embodiments, the physical stabilizer is a CBP polymer.
Additional components of the compositions/seed coating kits disclosed herein
10 [00190] In some embodiments, compositions or seed coating kits disclosed herein may comprise additional components. These additional components may include protectants and beneficial ingredients including but not limited to animal and bird repellants, attractants, baits, herbicides, herbicide safeners, antidessicants, antitranspirants, frost prevention aids, inoculants, dyes, brighteners, markers, synergists, pigments, UV protectants, antioxidants, leaf
15 polish, pigmentation stimulants and inhibitors, surfactants, moisture retention aids, humic acids and humates, lignins and lignates, bitter flavors, irritants, malodorous ingredients, molluscicides (e.g., slugs and snails), nematicides, rodenticides, defoliants, desiccants, sticky traps, IPM (integrated pest management) lures, chemosterilants, plant defense boosters (harpin protein and chitosan), and other beneficial or detrimental agents applied to the surface of the
20 plant seed or tissue . In some embodiments, multiple active agents are readily formulated within a given agricultural composition, for example, multiple active agents may include two or more of any of the following fungicides, fertilizers, pesticides, herbicides, and any type of active ingredient or class of active ingredient.
[00191] In some embodiments, the seed coating kits disclosed herein further comprise one or
25 more of a fertilizer, nitrogen stabilizer, or urease inhibitor. Fertilizers include anhydrous ammonia, urea, ammonium nitrate, and urea-ammonium nitrate (UAN) compositions, among many others. In some embodiments, pop-up fertilization and/or starter fertilization is used in combination with the methods and bacteria of the present disclosure.
[00192] In some embodiments, nitrogen stabilizers are used in combination with the methods
30 and bacteria of the present disclosure. Nitrogen stabilizers include nitrapyrin, 2-chloro-6- (trichloromethyl) pyridine, N-SERVE 24, INSTINCT, dicyandiamide (BCD). Urease inhibitors include N-(n-butyl)-thiophosphoric triamide (NBPT), AGROTAIN, AGROTAIN PLUS, and AGROTAIN PLUS SC. Further, the disclosure contemplates utilization of AGROTAIN ADVANCED 1.0, AGROTAIN DRI-MAXX, and AGROTAIN ULTRA.
[00193] In some embodiments, stabilized forms of fertilizer can be used. For example, a stabilized form of fertilizer is SUPER U, containing 46% nitrogen in a stabilized, urea-based
5 granule, SUPERU contains urease and nitrification inhibitors to guard from denitrification, leaching, and volatilization. Stabilized and targeted foliar fertilizer such as NIT AMIN may also be used herein.
[00194] Pop-up fertilizers are commonly used in com fields. Pop-up fertilization comprises applying a few pounds of nutrients with the seed at planting. Pop-up fertilization is used to
10 increase seedling vigor.
[00195] Slow- or controlled-release fertilizer that may be used herein entails: A fertilizer containing a plant nutrient in a form which delays its availability for plant uptake and use after application, or which extends its availability to the plant significantly longer than a reference ‘rapidly available nutrient fertilizer’ such as ammonium nitrate or urea, ammonium phosphate
15 or potassium chloride. Such delay of initial availability or extended time of continued availability may occur by a variety of mechanisms. These include controlled water solubility of the material by semi-permeable coatings, occlusion, protein materials, or other chemical forms, by slow hydrolysis of water-soluble low molecular weight compounds, or by other unknown means.
20 [00196] Stabilized nitrogen fertilizer that may be used herein entails: A fertilizer to which a nitrogen stabilizer has been added. A nitrogen stabilizer is a substance added to a fertilizer which extends the time the nitrogen component of the fertilizer remains in the soil in the urea- N or ammoniacal-N form.
[00197] Nitrification inhibitor that may be used herein entails: A substance that inhibits the
25 biological oxidation of ammoniacal-N to nitrate-N. Some examples include: (1) 2-chloro-6- (trichloromethyl-pyridine), common name Nitrapyrin, manufactured by Dow Chemical; (2) 4- amino-l,2,4-6-triazole-HCl, common name ATC, manufactured by Ishihada Industries; (3) 2,4-diamino-6-trichloro-methyltriazine, common name CI-1580, manufactured by American Cyanamid; (4) Dicyandiamide, common name DCD, manufactured by Showa Denko; (5)
30 Thiourea, common name TU, manufactured by Nitto Ryuso; (6) 1 -mercapto- 1, 2, 4-triazole, common name MT, manufactured by Nippon; (7) 2-amino-4-chloro-6-methyl-pyramidine, common name AM, manufactured by Mitsui Toatsu; (8) 3,4-dimethylpyrazole phosphate (DMPP), from BASF; (9) l-amide-2-thiourea (ASU), from Nitto Chemical Ind.; (10) Ammoniumthiosulphate (ATS); (11) lH-l,2,4-triazole (HPLC); (12) 5-ethylene oxide-3- trichloro-methlyl,2,4-thiodiazole (Terrazole), from Olin Mathieson; (13) 3-methylpyrazole (3-
5 MP); (14) l-carbamoyle-3-methyl-pyrazole (CMP); (15) Neem; and (16) DMPP.
[00198] Urease inhibitor that may be used herein entails: A substance that inhibits hydrolytic action on urea by the enzyme urease. Thousands of chemicals have been evaluated as soil urease inhibitors (Kiss and Simihaian, 2002). However, only a few of the many compounds tested meet the necessary requirements of being nontoxic, effective at low concentration,
10 stable, and compatible with urea (solid and solutions), degradable in the soil and inexpensive. They can be classified according to their structures and their assumed interaction with the enzyme urease (Watson, 2000, 2005). Four main classes of urease inhibitors have been proposed: (a) reagents which interact with the sulphydryl groups (sulphydryl reagents), (b) hydroxamates, (c) agricultural crop protection chemicals, and (d) structural analogues of urea
15 and related compounds. N-(n-Butyl) thiophosphoric triamide (NBPT), phenylphosphorodiamidate (PPD/ PPDA), and hydroquinone are probably the most thoroughly studied urease inhibitors (Kiss and Simihaian, 2002). Research and practical testing has also been carried out with N-(2-nitrophenyl) phosphoric acid triamide (2-NPT) and ammonium thiosulphate (ATS). The oigano-phosphorus compounds are structural analogues of urea and
20 are some of the most effective inhibitors of urease activity, blocking the active site of the enzyme (Watson, 2005).
[00199] In some embodiments, the compositions or seed coating kits disclosed herein may comprise trace metal ions, such as molybdenum ions, iron ions, manganese ions, or combinations of these ions. The concentration of ions in examples of compositions as described
25 herein may between about 0.1 mM and about 50 mM. In some embodiments, the compositions or seed coating kits disclosed herein may comprise additional carriers, besides those which may be included in the microbial compositions. Additional carriers may include beta-glucan, carboxylmethyl cellulose (CMC), bacterial extracellular polymeric substance (EPS), sugar, trehalose, maltose, animal milk, milk powder, or other suitable carriers. In some embodiments,
30 peat or planting materials can be used as a carrier, or biopolymers in which a composition is entrapped in the biopolymer can be used as a carrier. [00200] Additional components for inclusion in the seed coating kits disclosed herein may be found in International Patent Publication No. WO/2020/006064A3, the contents of which are herein incorporated by reference in their entirety for all purposes.
[00201] As used herein, “encapsulating” refers to enclosing the compositions of the present
5 disclosure within water-soluble film package. Encapsulation can be done by any method known in the art for the purpose, or any method that can be conceived to result in the components of the seed coating kit being encapsulated within the package.
Plant species for use with the disclosed compositions, kits, and methods
[00202] The compositions and methods disclosed herein can be applied to a number of seeds
10 and plant parts. In some embodiments, the seed or plant part is an agricultural crop. In some embodiments the seed or plant part is a monocot. In some embodiments, the seed or plant part is a dicot. In some embodiments, the seed is a com seed. In some embodiments, the com seed comprises a pre-treatment.
[00203] While pre-treated com and wheat seed was used in the examples disclosed herein, it
15 will be understood by one skilled in the art that seed or plant propagating material of any agriculturally important crop could be used with the compositions and methods disclosed herein, including but not limited to, rice, sorghum, canola, tomato, strawberry, and barley. Similarly, one skilled in the art will appreciate that the application rate may vary depending on the microbe(s) and type of seed or plant material coated. In some aspects, the application rate
20 may be between 1.0 oz/CWT and 20.0 oz/CWT, including all ranges and subranges therebetween. Additionally, the seed may be naked (untreated) or pre-treated with any number of plant-enhancing agents, including but not limited to, fungicides, insecticides, biocides, herbicides, and nematicides. Additional examples of agriculturally important crops are listed below.
25 [00204] In some embodiments, the plant belongs to the genera Hordeum, Oryza, Zea, and Triticeae. Non-limiting examples of crop plants include maize, rice, wheat, barley, sorghum, millet, oats, rye triticale, buckwheat, sweet com, sugar cane, onions, tomatoes, strawberries, asparagus, canola, soybean, potato, vegetables, cereals, and oilseeds. In some embodiments, the plant is a genetically modified organism (GMO), non-GMO, organic, or conventional plant.
30 In some embodiments, the compositions and methods described herein are suitable for plant tissues from any of a variety of transgenic plants, non-transgenic plants, and hybrid plants thereof.
[00205] In some embodiments, the plants are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and/or
5 forestry. Some examples of these plants may include pineapple, banana, coconut, lily, grasspeas and grass; and dicotyledonous plants, such as, for example, peas, alfalfa, tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, grape, cotton, sunflower, thale cress, canola, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron, and pomelo),
10 pepper, bean, lettuce, Panicum virgatum (switch), Sorghum bicolor (sorghum, Sudan), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (com), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet),
15 Panicum spp. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum- 25 wheat X rye), Bamboo, Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricin us communis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix curminghamiana (king palm), Syagrus romanzoffiana (queen palm),
20 Linum usitatissimum (flax), Brassica j uncea, Manihot esculenta (cassaya), Ly cop er si con esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brussel sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium
25 cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona
30 officinalis, Coichicum autumnale, Veratrum califomica, Digitalis lanata, Digitalis purpurea, Dioscorea 5 spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus womorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita
5 (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare (barley), and Lolium spp. (rye).
[00206] In some embodiments, plant tissues or plant parts, e.g., seeds, from a
10 monocotyledonous plant are treated. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncal es, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales. In some embodiments, the
15 monocotyledonous plant can be selected from the group consisting of a maize, rice, wheat, barley, and sugarcane.
[00207] In some embodiments, plant tissues or plant parts, e.g., seeds, from a dicotyledonous plant are treated, including those belonging to the orders of the Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Comales,
20 Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphoibiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales,
25 Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales, and Violates. In some embodiments, the dicotyledonous plant can be selected from the group consisting of cotton, soybean, pepper, and tomato.
[00208] Additional plants and seeds acceptable for use within the methods and compositions
30 of the present disclosure may be found in Interational Publication Nos. WG/2020/006246A1 and WO/2020/006064A3, the contents of each of which are herein incorporated by reference in their entirety. [00209] The compositions and methods described herein are suitable for any of a variety of non-genetically modified maize plants or parts thereof. In some embodiments, the com is organic. The compositions and methods described herein are suitable for any non-genetically modified hybrids, varieties, lineages, etc. Corn varieties generally fall under six categories:
5 sweet com, flint corn, popcorn, dent com, pod com, and flour com.
Sweet Com
[00210] Yellow su varieties include Earlivee, Early Sunglow, Sundance, Early Golden Bantam, lochief, Merit, Jubilee, and Golden Cross Bantam. White su varieties include True Platinum, Country Gentleman, Silver Queen, and Stowell’s Evergreen. Bicolor su varieties
10 include Sugar & Gold, Quickie, Double Standard, Butter & Sugar, Sugar Dots, Honey & Cream. Multicolor su varieties include Hookers, Triple Play, Painted Hill, Black Mexican/Aztec.
[00211] Yellow se varieties include Buttergold, Precocious, Spring Treat, Sugar Buns, Colorow, Kandy King, Bodacious R/M, Tuxedo, Incredible, Merlin, Miracle, and Kandy Korn
15 EH. White se varieties include Spring Snow, Sugar Pearl, Whiteout, Cloud Nine, Alpine, Silver King, and Argent. Bicolor se varieties include Sugar Baby, Fleet, Bon Jour, Trinity, Bi-Licious, Temptation, Luscious, Ambrosia, Accord, Brocade, Lancelot, Precious Gem, Peaches and Cream Mid EH, and Delectable R/M. Multicolor se varieties include Ruby Queen.
[00212] Yellow sh2 varieties include Extra Early Super Sweet, Takeoff, Early Xtra Sweet,
20 Raveline, Summer Sweet Yellow, Krispy King, Garrison, Illini Gold, Challenger, Passion, Excel, Jubilee SuperSweet, mini Xtra Sweet, and Crisp ‘N Sweet. White sh2 varieties include Summer Sweet White, Tahoe, Aspen, Treasure, How Sweet It Is, and Camelot. Bicolor sh2 varieties include Summer Sweet Bicolor, Radiance, Honey ‘N Pearl, Aloha, Dazzle, Hudson, and Phenomenal.
25 [00213] Yellow sy varieties include Applause, Infe o, Honeytreat, and Honey Select. White sy varieties include Silver Duchess, Cinderella, Mattapoisett, Avalon, and Captivate. Bicolor sy varieties include Pay Dirt, Revelation, Renaissance, Charisma, Synergy, Montauk, Kristine, Serendipity/Providence, and Cameo.
[00214] Yellow augmented supersweet varieties include Xtra-Tender IddA, Xtra-Tender
30 lldd, Mirai 131Y, Mirai 130Y, Vision, and Mirai 002. White augmented supersweet varieties include Xtra-Tender 3dda, Xtra-Tender 31 dd, Mirai 421W, XTH 3673, and Devotion. Bicolor augmented supersweet varieties include Xtra-Tender 2dda, Xtra-Tender 21dd, Kickoff XR, Mirai 308BC, Anthem XR, Mirai 336BC, Fantastic XR, Triumph, Mirai 301BC, Stellar, American Dream, Mirai 350BC, and Obsession.
5
Flint Com
[00215] Flint com varieties include Bronze-Orange, Candy Red Flint, Floriani Red Flint, Glass Gem, Indian Ornamental (Rainbow), Mandan Red Flour, Painted Mountain, Petmecky, Cherokee White Flour,
10 Popcorn
[00216] Popco varieties include Monarch Butterfly, Y ellow Butterfly, Midnight Blue, Ruby Red, Mixed Baby Rice, Queen Mauve, Mushroom Flake, Japanese Hull-less, Strawberry, Blue Shaman, Miniature Colored, Miniature Pink, Pennsylvania Dutch Butter Flavor, and Red Strawberry.
15 Dent Com
[00217] Dent com varieties include Bloody Butcher, Blue Clarage, Ohio Blue Clarage, Cherokee White Eagle, Hickory Cane, Hickory King, Jellicorse Twin, Kentucky Rainbow, Daymon Morgan’s Knt. Butcher, Learning, Learning’s Yellow, McCormack’s Blue Giant, Neal Paymaster, Pungo Creek Butcher, Reid’s Yellow Dent, Rotten Clarage, and Tennessee
20 Red Cob.
[00218] In some embodiments, com varieties include P1618W, P1306W, P1345, Pl 151, Pl 197, P0574, P0589, and P0157. W = white com.
[00219] In some embodiments, the compositions and methods described herein are suitable for any hybrid of the maize varieties set forth herein.
25 [00220] The compositions and methods described herein are suitable for any of a hybrid, variety, lineage, etc. of genetically modified maize plants or part thereof. Furthermore, the compositions and methods described herein are suitable for any of the following genetically modified maize events, which have been approved in one or more countries, or any new genetically modified com event, which may include Bt traits, glufosinate resistance, glyphosate
30 resistance, etc.: 32138 (32138 SPT Maintainer), 3272 (ENOGEN), 3272 x Btll, 3272 x btl l x GA21, 3272 x Btl 1 x MIR604, 3272 x Btl 1 x MIR604 x GA21, 3272 x Btl 1 x MIR604 x TC1507 x 5307 x GA21, 3272 x GA21, 3272 xMIR604, 3272 x MIR604 x GA21, 4114, 5307 (AGRISURE Duracade), 5307 x GA21, 5307 x MIR604 x Bill x TC1507 x GA21 (AGRISURE Duracade 5122), 5307 x MIR604 x Bil l x TC1507 x GA21 x MIR162
5 (AGRISURE Duracade 5222), 59122 (Herculex RW), 59122 x DAS40278, 59122 x GA21, 59122 x MIR604, 59122 x MIR604 x GA21, 59122 x MIR604 x TC1507, 59122 x MIR604 x TC1507 x GA21, 59122 x MON810, 59122 x MON810 x MIR604, 59122 x MON810 x NK603, 59122 x MON810 x NK603 x MIR604, 59122 x MON88017, 59122 x MON88017 x DAS40278, 59122 x NK603 (Herculex RW ROUNDUP READY 2), 59122 x NK603 x
10 MIR604, 59122 x TC1507 x GA21, 676, 678, 680, 3751 IR, 98140, 98140 x 59122, 98140 x TC1507, 98140 x TC1507x 59122, BtlO (BtlO), Btl 1 [X4334CBR, X4734CBR] (AGRISURE CB/LL), Btl 1 x 5307, Btl 1 x 5307 x GA21, Btl 1 x 59122 x MIR604, Brl 1 x 59122 x MIR604 x GA21, Btll x 59122 x MIR604 x TC1507, M53, M56, DAS-59122-7, Btll x 59122 x MIR604 x TC1507 x GA21, Btll x 59122 x TC1507, TC1507 x DAS-59122-7, Btll x 59122
15 x TC1507 x GA21, Btl l x GA21 (AGRISURE GT/CB/LL), Btll x MIR162 (AGRISURE Viptera 2100), Bill x MIR162 x 5307, Btll x MIR162 x 5307 x GA21, Btll x MIR162 x GA21 (AGRISURE Viptera 3110), Btll x MIR162 x MIR604 (AGRISURE Viptera 3100), Btl 1 x MIR162 x MIR604 x 5307, Btl 1 x MIR162 x MIR604 x 5307 x GA21 , Btl 1 x MIR162 x MIR604 x GA21 (AGRISURE Viptera 3111 / AGRISURE Viptera 4), Btll, MIR162 x
20 MIR604 x MON89034 x 5307 x GA21, Btl 1 x MIR162 x MIR604 x TC1507, Btl 1 x MIR162 x MIR604 x TC1507 x 5307, Btl 1 x MIR162 x MIR604 x TC1507 x GA21, Btl 1 x MIR162 x MON89034, Btll x MIR162 x MON89034 x GA21, Btl l x MIR162 x TC1507, Btll x MIR162 x TC1507 x 5307, Btll x MIR162 x TC1507 x 5307 x GA21, Btll x MR162 x TCI 507 x GA21 (AGRISURE Viptera 3220), BT11 x MIR604 (Agrisure BC/LL/RW), Btl 1
25 x MIR604 x 5307, Btl 1 x MIR604 x 5307 x GA21, Btl 1 x MIR604 x GA21 , Btl 1 x MIR604 x TC1507, Btll x MIR604 x TC1507 x 5307, Btll x MIR604 x TC1507 x GA21, Btll x MON89034 x GA21, Btl 1 x TCI 507, Btl 1 x TC1507 x 5307, Btl 1 x TCI 507 x GA21, Btl 76 [176] (NaturGard KnockOut / Maximizer), BVLA430101, CBH-351 (STARLINK Maize), DAS40278 (ENLIST Maize), DAS40278 x NK603, DBT418 (Bt Xtra Maize), DLL25 [B 16],
30 GA21 (ROUNDUP READY Maize / AGRISURE GT), GA21 x MON810 (ROUNDUP READY Yieldgard Maize), GA21 x T25, HCEM485, LY038 (MAVERA Maize), LY038 x MON810 (MAVERA Yieldgard Maize), MIR162 (AGRISURE Viptera), MIR162 x 5307, MIR162 x 5307 x GA21, MIR162 x GA21, MIR162 x MIR604, MIR162 x MIR604 x 5307, MIR162 x MIR604 x 5307 x GA21, MIR162 x MIR604 x GA21, MIR162 x MIR604 x TC1507 x 5307, MIR162 x MIR604 x TCI 507 x 5307 x GA21, MIR162 x MIR604 x TC1507 x GA21, MIR162 x MON89034, MIR162 x NK603, MIR162 x TC1507, MIR162 x TC1507 x 5307, MIR162 x TC1507 x 5307 x GA21, MIR162 x TC1507 x GA21, MIR604 (AGRISURE RW),
5 MIR604 x 5307, MIR604 x 5307 x GA21, MIR604 x GA21 (AGRISURE GT/RW), MIR604 x NK603, MIR604 x TC1507, MIR604 x TC1507 x 5307, MIR604 x TC1507 x 5307 xGA21, MIR604 x TC1507 x GA21, MON801 [MON80100], MON802, MON809, MON810 (YIELDGARD, MAIZEGARD), MON810 x MIR162, MON810 x MIR162 x NK603, MON810 x MIR604, MON810 x MON88017 (YIELDGARD VT Triple), MON810 x NK603
10 x MIR604, MON832 (ROUNDUP READY Maize), MON863 (YIELDGARD Rootworm RW, MAXGARD), MON863 x MON810 (YIELDGARD Plus), MON863 x MON810 x NK603 (YIELDGARD Plus with RR), MON863 x NK603 (YIELDGARD RW + RR), MON87403, MON87411, MON87419, MON87427 (ROUNDUP READY Maize), MON87427 x 59122, MON87427 x MON88017, MON87427 x MON88017 x 59122, MON87427 x MON89034,
15 MON87427 x MON89034 x 59122, MON87427 x MON89034 x MIR162 x MON87411, MON87427 x MON89034 x MON88017, MON87427 x MON89034 x MON88017 x 59122, MON87427 x MON89034 x NK603, MON87427 x MON89034 x TCI 507, MON87427 x MON89034 x TC1507 x 59122, MON87427 x MON89034 x TC1507 x MON87411 x 59122, MON87427 x MON89034 x TC1507 x MON87411 x 59122 x DAS40278, MON87427 x
20 MON89034 x TCI 507 x MON88017 , MON87427 x MON89034 x MIR162 x NK603, MON87427 x MON89034 x TC1507 x MON88017 x 59122, MON87427 x TC1507, MON87427 x TC1507 x 59122, MON87427 x TC1507 x MON88017, MON87427 x TC1507 x MON88017 x 59122, MON87460 (GENUITY DROUGHTGARD), MON87460 x MON88017, MON87460 x MON89034 x MON88017, MON87460 x MON89034 x NK603,
25 MON87460 x NK603, MON88017, MON88017 x DAS40278, MON89034, MON89034 x 59122, MON89034 x 59122 x DAS40278, MON89034 x 59122 x MON88017, MON89034 x 59122 x MON88017 x DAS40278, MON89034 x DAS40278, MON89034 x MON87460, MON89034 x MON88017 (GENUITY VT Triple Pro), MON89034 x MON88017 x DAS40278, MON89034 x NK603 (GENUITY VT Double Pro), MON89034 x NK603 x
30 DAS40278, MON89034 x TC1507, MON89034 x TC1507 x 59122, MON89034 x TC1507 x 59122 x DAS40278, MON89034 x TC1507 x DAS40278, MON89034 x TC1507 x MON88017, MON89034 x TC1507 x MON88017 x 59122 (GENUITY SMARTSTAX), MON89034 x TC1507 x MON88017 x 59122 x DAS40278, MON89034 x TC1507 x MON88017 x DAS40278, MON89034 x TCI 507 x NK603 (POWER CORE), MON89034 x TCI 507 x NK603 x DAS40278, MON89034 x TCI 507 x NK603 x MIR162, MON89034 x TC1507 x NK603 x MIR162 x DAS40278, MON89034 x GA21, MS3 (INVIGOR Maize), MS6 (INVIGOR Maize), MZHG0JG, MZIR098, NK603 (ROUNDUP READY 2 Maize),
5 NK603 x MON810 x 4114 x MIR604, NK603 x MON810 (YIELDGARD CB + RR), NK603 x T25 (ROUNDUP READY LIBERTY LINK Maize), T14 (LIBERTY LINK Maize), T25 (LIBERTY LINK Maize), T25 x MON810 (LIBERTY LINK YIELDGARD Maize), TC1507 (HERCULEX I, HERCULEX CB), TC1507 x 59122 x MON810 x MIR604 x NK603 (OPTIMUM INTRASECT XTREME), TC1507 x MON810 x MIR604 x NK603, TCI 507 x
10 5307, TC1507 x 5307 x GA21, TC1507 x 59122 (HERCULEX XTRA), TC1507 x 59122 x DAS40278, TC1507 x 59122 x MON810, TC1507 x 59122 x MON810 x MIR604, TC1507 x 59122 x MON810 x NK603 (OPTIMUM INTRASECT XTRA), TC1507 x 59122 x MON88017, TC1507 x 59122 x MON88017 x DAS40278, TC1507 x 59122 x NK603 (HERCULEX XTRA RR), TC1507 x 59122 x NK603 x MIR604, TC1507 x DAS40278,
15 TC1507 x GA21, TC1507 x MIR162 x NK603, TC1507 x MIR604 x NK603 (OPTIMUM TRISECT), TC1507 x MON810, TC1507 x MON810 x MIR162, TC1507 x MON810 x MIR162 xNK603, TC1507 x MON810 x MIR604, TC1507 x MON810 x NK603 (OPTIMUM INTRASECT), TC1507 x MON810 x NK603 x MIR604, TC1507 x MON88017, TC1507 x MON88017 x DAS40278, TCI 507 x NK603 (HERCULEX I RR), TCI 507 x NK603 x
20 DAS40278, TC6275, and VCO-01981-5.
[00221] In some embodiments, the present disclosure relates to a seed or plant tissue treated with the kits and compositions disclosed herein. In some embodiments, the seed is a com seed.
BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE
DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT
25 PROCEDURES
[00222] The microbial deposits of the present disclosure were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Puipose of Patent Procedure (Budapest Treaty).
[00223] Applicants state that pursuant to 37 C.F.R. § 1.808(a)(2) “all restrictions imposed by
30 the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent.” This statement is subject to paragraph (b) of this section (z.e. 37 C.F.R. § 1.808(b)). [00224] The Enterobacter sacchari has now been reclassified as Kosakonia sacchari, the name for the organism may be used interchangeably throughout the present disclosure.
[00225] Some microbes of the present disclosure are derived from two wild-type strains. Strain CI006 is a bacterial species previously classified in the genus Enterobacter (see
5 aforementioned reclassification into Kosakonia). Strain CI019 is a bacterial species classified in the genus Rahnella. The deposit information for the CI006 Kosakonia wild type (WT) and CI019 Rahnella WT are found in Table 2.
[00226] Some microorganisms described in this application were deposited on January 06, 2017 or August 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota
10 (NCMA), located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA. As aforementioned, all deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The Bigelow National Center for Marine Algae and Microbiota accession numbers and dates of deposit for the aforementioned Budapest Treaty deposits are provided in Table 2.
15 [00227] Biologically pure cultures of Kosakonia sacchari (WT), Rahnella aquatilis (WT), and a variant/remodeled Kosakonia sacchari strain were deposited on January 06, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201701001, 201701003, and 201701002, respectively. The applicable deposit
20 information is found in Table 2.
[00228] Biologically pure cultures of variant/remodeled Kosakonia sacchari strains were deposited on August 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201708004, 201708003, and
25 201708002, respectively. The applicable deposit information is found in Table 2.
[00229] A biologically pure culture of Klebsiella variicola (WT) was deposited on August 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA, and assigned NCMA Patent Deposit Designation number 201708001. Biologically pure cultures of two Klebsiella variicola
30 variants/remodeled strains were deposited on December 20, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201712001 and 201712002, respectively. The applicable deposit information is found in Table 2.
[00230] Biologically pure cultures of two Kosakonia sacchari variants/remodeled strains were
5 deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Numbers PTA-126575 and PTA-126576. Biologically pure cultures of four Klebsiella variicola variants/remodeled strains were deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas,
10 Virginia 20110-2209, USA and assigned ATCC Patent Deposit Numbers PTA-126577, PTA- 126578, PTA-126579 and PTA-126580. A biologically pure culture of a Paenibacilhis polymyxa (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PT A-126581. A biologically pure culture of
15 a Paraburkholderia tropica (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126582. A biologically pure culture of a Herbaspirillwn aquaticum (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801
20 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126583. Biologically pure cultures of four Metakosakonia intestini variants/remodeled strains were deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Numbers PTA-126584, PTA-126586,
25 PTA-126587 and PTA-126588. A biologically pure culture of a Metakosakonia intestini (WT) strain was deposited on December 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126585. A biologically pure culture of a Klebsiella variicola variant/remodeled strain was deposited on March 25, 2020 with the
30 American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126740. A biologically pure culture of a Kosakonia sacchari variant/remodeled strain was deposited on March 25, 2020 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126743. The applicable deposit information is found in Table 2.
EXAMPLES
5 Example 1: Preparation of microbial cultures
[00231] This example describes the preparation of microbial culture to be tested in combination with extenders of the present disclosure.
[00232] Kosakonia sacchari strain PTA- 126743 and Klebsiella variicola strain PTA- 126740 were separately grown in fermenters to saturation to create microbial culture broths.
10 [00233] Portions of these microbial culture broths were subsequently concentrated 20X by tangential flow filtration. The cell concentrates were each blended 1 : 1 v/v with a formulation concentrate solution comprising 30% w/v sucrose and 15% w/v oligofructose to create liquid microbial stocks.
[00234] These liquid microbial stocks/microbial concentrates were subsequently freeze dried
15 in trays in a laboratory scale lyophilizer. After the completion of the lyophilization program, the dry material was milled to pass through a 500 micron mesh. Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA-126740 freeze dried powders were both aged 2 weeks at 21 °C prior to being reconstituted and mixed with the extender compositions for seed treatment.
20 [00235] Freeze dried powders were reconstituted at 1 g per 10 mL of IX PBS. Cell viability of the dried microbial powder was tested by plating samples and measuring colony forming units per gram of powder. Initial cell viability assays showed viability between 1 x 1011 and 1 x 1012 CFU/gram of powder.
Example 2: Extender compositions increase adherence of microbes to seed
25 [00236] To evaluate the adherence of microbes to seed, multiple extender compositions were compared to a control composition composed of only buffering salts. As buffering salts may provide some benefit to biologicals, this comparison evaluated the effects of extender compositions comprising combinations of i) sugars and buffering salts, or ii) sugars, polymers, and buffering salts. The compositions tested are shown below in Table 4. All values are
30 presented as a % (w/v). Table 4 - Extender and buffer control compositions.
Figure imgf000072_0001
[00237] The PBX21 800 50L and PBX21 800 44L extenders of Table 4 were mixed with microbes and applied to seeds to test adherence. This example tested a 1 : 1 ratio: blend of the
5 microbial culture broths prepared from Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA- 126740, as described in Example 1.
[00238] This microbial 1 : 1 blend was then mixed with the extender compositions or buffer control of Table 4 at a 1 : 1 ratio to form final seed treatment compositions. Table 5 below shows the final % by volume contents of the seed treatment compositions created in this example.
10 Table 5 - Percent content of microbes and extender compositions tested (% by volume)
Figure imgf000072_0002
[00239] In total three seed treatment compositions were created with each of the test extender and control liquids as follows: 1) test compositions based on Liquid Extender PBX21 800 50L, 2) test compositions based on Liquid Extender PBX21 800 44L, and 3)
15 Buffer Control. Viability tests were conducted on all three of the freshly mixed seed treatment compositions to establish a time 0 CFU/ml (i.e., original or starting titer). As shown in the bar graph of FIG. 1, all three seed treatment compositions had similar starting titers of microbes, between 1.0 X 109and 1.0 X 10wCFU/mL.
[00240] The three treatments were applied to two types of com seed at 5.4 fl oz/CWT using a
20 Hege 11 liquid seed treater. Seed 1 contained 3 fungicide pretreatments, 1 insecticide pretreatment, and 1 biological pretreatment. Seed 2 contained 6 fungicide pretreatments, 2 insecticide pretreatments, and 3 gram-positive sporulating biological s.
[00241] To determine efficacy of microbial attachment to seed, application log loss was calculated (FIG. 2). This takes into consideration the starting titer going onto seed (FIG. 1),
5 the seed per pound of a given seed type, volume applied per pound of seed, and how much is measured from the extracted seed. Specifically, application log loss was calculated by using the equation: LOG((Day 0 treatment titers in CFU/ml) x (ml/seed application rate)) - LOG(Day 0 CFU/seed). All treatments were triplicated, using 3 biological reps each microbial blend/seed combination. Controls were also tested in triplicate; all were autoclave sterilized prior to
10 application to the seed. The gram-positive biologicals in seed 2 were screened out during plate counting using antibiotic treated media that did not interfere with the growth of Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA-126740. Thus, all CFU measurements represent the microbial attachment of the microbes in the seed treatment compositions tested in this example.
15 [00242] There are inherent limitations, considering extraction efficiencies, plating accuracies, stressors associated with seed treatment, and coverage efficiency onto seed during the treating procedure. Therefore, one skilled in the art expects an application log loss of at least 0.5 for these inherent limitations alone. The data gathered for this experiment (FIG. 2) indicates that both extender compositions PBX21 800 44L and PBX21 800 50L statistically improve
20 microbial adherence to seed, compared to a Buffer Control. As shown in the bar graph of FIG. 2, extender compositions PBX21 800 44L and PBX21 800 50L on seed 1 had log losses of less than 1 and approximately 1.25, respectively, whereas the buffer control on seed 1 exhibited a log loss of approximately 1.75. Similar results were achieved on seed 2.
[00243] This data demonstrates that both tested extender compositions promote the adherence
25 of microbes to seeds over buffer controls alone.
Example 3: Extender compositions increase stability of microbes on-seed
[00244] To determine the effect of the extender compositions on microbial stability on-seed, the viability of the strains on the seeds was measured over time. Treated seed prepared in Example 2 was stored at 21°C and analyzed for CFU/seed as done in Example 2. Measurements
30 were taken at Day 0, 7, and 15 post application to track viability loss over time (FIG. 3). [00245] The results show that extender compositions PBX21 800 44L and PBX21 800 50L statistically improve the on-seed stability of microbes compared to Buffer Control A. Buffer controls exhibit significant viability reductions in the first 7 days before leveling off. For the buffer controls, approximately 3.36% of cells on-seed were still viable at day 15 for seed 1
5 (2,776 remaining viable from 82,666), and approximately 5.2% of cells on-seed were still viable at day 15 for seed 2 (3,200 remaining viable from 61,333).
[00246] The seed treatment compositions with the extenders of the present disclosure maintained a greater percentage of viable cells, overall resulting in high stability over time. For extender composition PBX21 800 44L, approximately 22.5% of cells on-seed were still
10 viable at day 15 for seed 1 (102,000 remaining viable from 453,333), and approximately 55% of cells on-seed were still viable at day 15 for seed 2 (172,333 remaining viable from 313,333). For extender composition PBX21 800 50L, approximately 21% of cells were still viable at day 15 for seed 1 (34,700 remaining viable from 165,000), and approximately 23.8% of cells on-seed were still viable at day 15 for seed 2 (68,333 remaining viable from 286,666). Thus,
15 application of the extender compositions disclosed herein increased on-seed stability of microbes from 3-5% (without extender) to greater than 20% (with extender).
[00247] Example 2 demonstrated that extenders of the present disclosure reduce on-seed application loss, and Example 3 demonstrated that extenders further improve the stability of microbes that make it onto the seed. Together, these two properties help produce higher and
20 more predictable target titers of biologicals prior to planting.
Example 4: Storage stability of dried microbial powder microbes
[00248] Example 1 generated two microbial delivery forms that could be used to produce the seed treatment compositions of the present disclosure. The first was microbial culture broths. The second was dried microbial powder, produced by lyophilizing and (optionally) milling the
25 liquid microbial stock.
[00249] In order to test how the lyophilization step would affect stability of the two different species of microbes, the cell viability of the mixed powder was monitored by CFU plating on a monthly basis (FIG. 4). The two species of microbes were lyophilized separately and then mixed after the drying process. The results showed no obvious differences between Kosakonia
30 sacchari strain PT A-l 26743 and Klebsiella variicola strain PTA-126740. The cell titers of both strains declined by less than 1 log CFU/g over the course of 3 months. The strains also maintained their 1 : 1 ratio while in storage.
Example 5: Reconstituted dried microbial powder microbes produce highest microbial titers during on-seed storage when used with extender compositions
5 [00250] Next, Applicant sought to test the on-seed stability of seed treatment compositions made directly with liquid microbial stocks versus reconstituted dried microbial powders.
[00251] Two seed treatment compositions were prepared. The first was prepared by mixing the microbial culture broths of example 1 (undiluted, unconcentrated, unformulated) directly with the extender formulations of example 2, in a 1 : 1 ratio (Broth Seed Treatment). The second
10 was prepared by resuspending dried microbial powders from example 1 in buffer at 10% wt/v PTA-126743 and 10% wt/v PTA-126740. This reconstituted microbe liquid was then mixed with extender composition PBX21 800 44L at a 1:1 ratio as described in Example 2 (Reconstituted Microbe Seed Treatment). Controls using the microbial culture broth and reconstituted microbe liquid were also prepared using buffer control A. Table 6 below shows
15 the final % by volume of the Reconstitute Microbe Seed Treatment.
Table 6 - Ratio of powdered microbes to extender and buffer compositions
Figure imgf000075_0001
[00252] The four compositions (1- Microbial Culture Broth Seed Treatment; 2- Reconstituted Microbe Seed Treatment; 3- Microbial Culture Broth+ Buffer Control A; and 4- Reconstituted
20 Microbe + Buffer Control A) were applied to the same hybrid com seed at 5.4 fl oz/CWT. Seed was stored at 21 °C and viability in CFU/seed was measured over the course of 28 days (FIG. 5) to evaluate microbe stability.
[00253] Due to the concentration process employed with tangential flow filtration, there was a higher starting titer with the Reconstituted Microbe Seed Treatment compared to Microbial
25 Culture Broth Seed Treatment. At 14 days post seed treatment, the microbial viability on-seed of powdered microbes reconstituted and mixed with the extender PBX21 800 44L was greater than 57% of the starting on-seed viability (9.2 X 105 of 1.61 X 106). At 28 days, Reconstituted Microbe Seed Treatment mixed with extender PBX21 800 44L was still greater than 1.0 X 105 CFU/seed, which was 23.8% of the starting viability.
[00254] At 14 days post seed treatment, the microbial viability on-seed of liquid microbes mixed with the extender PBX21 800 44L was approximately 17% of the starting on-seed
5 viability (4.93 X 104 of 2.9 X 105). At 28 days, the Microbial Culture Broth Seed Treatment mixed with extender PBX21 800 44L was less than 1.0 X 105 CFU/seed, however compared to starting titers on-seed, it also maintained 13.4% viability. This data demonstrates that extender composition PBX21 800 44L can prolong viability of either powder (reconstituted) or Broth forms of microbial seed treatments, and that the highest microbial titers on-seed are
10 achieved with the combination of the Reconstituted Microbes plus an extender composition. Without wishing to be bound by any one theory, the present inventors believe that the reconstituted microorganisms exhibit improved on-seed stability properties than nonreconstituted microorganisms.
[00255] Additionally, a difference was observed between the Microbial Culture Broth Seed
15 Treatment mixed with Buffer Control and the Reconstituted Microbial Seed Treatment mixed with PBX21 800 44L (compare points B and D on FIG. 5). Without the extender composition, the Microbial Culture Broth treatment did not have any viable cells by day 28. However, with the addition of extender composition PBX21 800 44L, approximately 13.4% of cells were still viable at day 28. This data demonstrates that the addition of an extender composition, such
20 as PBX21 800 44L can increase microbial viability when added to a Microbial Culture Broth Seed Treatment.
Example 6: Extender compositions maintain on-seed viability of microbial powders above le+4 CFU/seed 90 days after application on treated corn seed
[00256] Example 5 showed that the use of an extender composition could increase on-seed
25 viability when added to either a microbial culture broth or powder (reconstituted lyophilized microbes). A similar experiment was carried out treated com seed using powdered microbes and the viability tested over 90 days.
[00257] Dry microbial powder was prepared as described above in Example 4 was stored in sealed mylar bags at 21°C and opened at time of seed treatment (aged 0, 1, 2, 3, months). This
30 example tested a 1 :1 ratio blend of Kosakonia sacchari strain PTA-126743 and Klebsiella variicola strain PTA-126740, as described in Example 1. 8.6% weight powder dry microbe was combined with 96.5% volume of either extender PBX21 800 77L (see Example 2, Table 4) or control solution B; mixed well and treated within 4 hours of mixing. In contrast with Example 5, microbes in this example were directly added to the extender in powdered (e.g., lyophilized) form, without first being resuspended in buffer.
5 [00258] Seed treatment was applied at 2.4 fl oz/unit application rate on a Hege 11 seed treater in 50 g batches of seed with a spin time of 35 sec. Seed 1 contained 6 fungicide pretreatments, 2 insecticide pretreatment, and 3 biological pretreatments. Seed 2 contained 3 fungicide pretreatments and 1 insecticide pretreatments.
[00259] All seed was stored at 21 °C for viability studies. On-seed viability was determined
10 over time using the following seed extraction and plating methods: Seeds were extracted in 1 X PBS buffer (5 seeds/ 5 mL PBS in 50 ml conical tube) and shaken at 1600 rpm for 30 min. Serial dilutions (1:10) were performed in 1XPBS and spread plated on TSA+10 ug/ml Erythromycin plates. Plates were incubated at 30°C for ~24h prior to colony forming unit (CFU) counts. CFU/seed was calculated as (CFU/mL) x (mL/seed).
15 [00260] As shown in FIG. 6, at 90 days post seed treatment, the microbial viability on-seed of powdered microbes reconstituted and mixed with the extender PBX21 800 77L was still above le+4 CFU/seed, compared with the control, which dropped below le+4 CFU/seed after 30 days. Thus, the Dry Microbes + Control resulted in faster decay rate on seed compared with Dry microbe + extender. This data suggests that the extender provides protection for gram¬
20 negative Nitrogen Fixing bacteria applied as an overtreatment on chemically treated seeds.
Example 7: Extender compositions maintain on-seed viability of liquid microbes above le+4 CFU/seed 30 days after application on treated seed
[00261] Example 5 showed that the use of an extender composition could increase on-seed viability when added to either a microbial culture broth or powder (reconstituted lyophilized
25 microbes). A similar experiment was carried out treated com and wheat seed using liquid microbes and the viability tested.
[00262] For the com seed, liquid microbes were stored in Scholle bladders and stored at 21 °C prior to seed treatment (bladders only opened up to 3 times to reduce air exchange). Two volume to volume ratios were evaluated, and data from both were compiled:
30 1. 75% volume liquid microbe + 25% volume of either extender PBX21 800 44L or control solution A 2. 50% volume liquid microbe + 50% volume of either extender PBX21 800 44L or control solution A
[00263] Both compositions were mixed well and treated within 4 hours of mixing. Seed treatment was applied at 2.4 fl oz/unit application rate on a Hege 11 seed treater in 50 g batches
5 of seed with a spin time of 35 sec. Seed contained either 6 fungicides, 2 insecticides, and 3 biologicals, or 3 fungicides, 1 insecticide, and 1 biological. Data was compiled as an average between both. All seed was stored at 21°C for viability studies.
[00264] On-seed viability was determined overtime using procedure described in Example 6. Data for both 75% and 50% volume liquid compositions was combined. As shown in FIG. 7,
10 the viability of the microbes + extender on-seed after 25 days was still approximately le+4 CFU/seed, whereas the viability of microbes + control was less than le+2 CFU/seed. Thus, the data suggests that liquid microbes + control results in faster decay rate on treated seed compared with liquid microbe + extender, indicating the extender provides protection for gramnegative Nitrogen Fixing bacteria applied as an overtreatment on chemically treated seeds.
15 [00265] For the wheat seeds, liquid microbes were stored as described above, except only one volume ratio was evaluated: 75% volume liquid microbe + 25% volume of either extender PBX21 800 44L or control solution A; mixed well and treated within 4 hours of mixing.
[00266] Seed treatment was applied at 10 fl oz/CWT of wheat application rate on a Hege 11 seed treater in 50 g batches of seed with a spin time of 35 sec. Seed contained 6 fungicides, 2
20 insecticides, and 3 biologicals. All seed was stored at 21 °C for viability studies.
[00267] On-seed viability was determined overtime using the following seed extraction and plating methods: Seeds were extracted in IX PBS buffer (25 seeds/ 25 mL PBS in 50 ml conical tube) and shaken at 1600 rpm for 30 min. Serial dilutions (1:10) were performed in IXPBS and spread plated on TSA+10 ug/ml Erythromycin plates. Plates were incubated at 30°C for ~24h
25 prior to colony forming unit (CFU) counts. CFU/seed was calculated as (CFU/mL) x (mL/seed).
[00268] As shown in FIG. 8, after 14 days wheat seeds treated with liquid microbe + extender had approximately le+5 CFU/seed, compared with the control, which had less than le+4 CFU/seed.
30 INCORPORATION BY REFERENCE
[00270] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. PCT published application WO2021222567 is also hereby incorporated by reference in its entirety
5 for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
NUMBERED EMBODIMENTS
1. A composition comprising: a) a sugar alcohol or sugar, at between about 5% and about 30% (w/v);
5 b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at between about 10% and about 40% (w/v).
2. The composition of embodiment 1, wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-
10 VA), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
3. The composition of embodiment 2, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
4. The composition of embodiment 3, wherein the polyvinylpyrrolidone-vinyl acetate
15 (PVP-VA) is present in the composition at between about 15% and about 20% (w/v).
4.1 The composition of embodiment 3, wherein the poly vinylpyrrolidone- vinyl acetate (PVP-VA) is present in the composition at about 15% (w/v).
4.2 The composition of embodiment 3, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA) is present in the composition at about 17% (w/v).
20 5. The composition of any one of embodiments 1-4.2, wherein the sugar or sugar alcohol is present in the composition at between about 10% and about 20% (w/v).
5.1 The composition of any one of embodiments 1-4.2, wherein the sugar or sugar alcohol is present in the composition at about 11% (w/v).
5.2 The composition of any one of embodiments 1-4.2, wherein the sugar or sugar alcohol
25 is present in the composition at about 20% (w/v).
6. The composition of any one of embodiments 1-5.2, wherein the composition is mixed with cultured microbes to create a liquid seed treatment.
7. The liquid seed treatment of embodiment 6, wherein the sugar or sugar alcohol comprises between 2.5% and 30% (w/v) and the water-soluble polymer comprises
30 between 5% and 30% (w/v). 8. A composition comprising: a) a sugar alcohol or sugar, at between about 5% and about 70%(wA); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH; and
5 c) cultured microbes.
8.1 The composition of embodiment 8, wherein the sugar alcohol or sugar is present in the composition between about 5% and about 20% (wA).
8.2 The composition of embodiment 8, wherein the sugar alcohol or sugar is present in the composition between about 20% and about 30% (wA).
10 9. The composition of embodiment 8, wherein the sugar alcohol or sugar is present in the composition at about 30% (wA).
9.1 The composition of embodiment 8, wherein the sugar alcohol or sugar is present in the composition at about 50% (wA).
9.2 The composition of embodiment 8, wherein the sugar alcohol or sugar is present in
15 the composition at about 60% (w/v).
10. A composition comprising: a) a sugar alcohol or sugar, at between about 2.5% and about 30% (wA); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH;
20 c) cultured microbes; and d) a water-soluble polymer, at between about 5% and about 30% (wA).
10.1 The composition of embodiment 10, wherein the sugar alcohol or sugar is between about 7.5% and about 15% (wA).
10.2 The composition of embodiment 10 or 10.1, wherein the water-soluble polymer is
25 between about 5% and about 20% (wA).
10.3 The composition of embodiment 10 or 10.1, wherein the water-soluble polymer is between about 10% and about 20% (wA).
11. The composition of any one of embodiments 10-10.3, wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
12. The composition of embodiment 11, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
5 13. The composition of embodiment 12, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA) is present in the composition at about 7.5% (w/v).
13.1 The composition of embodiment 12, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA) is present in the composition at about 15% (w/v).
13.2 The composition of embodiment 12, wherein the polyvinylpyrrolidone-vinyl acetate
10 (PVP-VA) is present in the composition at about 11% (w/v).
13.3 The composition of embodiment 12, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA) is present in the composition at about 8.5% (w/v).
14. The composition of any one of embodiments 1-13.3, wherein the chemical buffer comprises potassium phosphate.
15 15. The composition of any one of embodiments 1-14, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fucitol, iditol, and inositol.
16. The composition of any one of embodiments 1-15, wherein the sugar alcohol is sorbitol.
17. The composition of any one of embodiments 1-16, wherein the chemical buffer comprises dipotassium phosphate at approximately between 0.5% and 1.5% of the total
20 volume, and monopotassium phosphate at approximately between 0% and 1% of the total volume.
17.1 The composition of any one of embodiments 1-16, wherein the chemical buffer comprises dipotassium phosphate at approximately between 0.5% and 4% of the total volume, and monopotassium phosphate at approximately between 0% and 2% of the
25 total volume.
18. The composition of embodiment 17 or 17.1 , wherein the chemical buffer is dipotassium phosphate at approximately 1% of the total volume, and monopotassium phosphate at approximately 0.5% of the total volume. 19. The composition of any one of embodiments 8-16, wherein the chemical buffer is di potassium phosphate at approximately between 0.5% and 2% of the total volume, and monopotassium phosphate at approximately between 0% and 0.5% of the total volume.
20. The composition of embodiment 19, wherein the chemical buffer is dipotassium
5 phosphate at approximately 1% of the total volume, and monopotassium phosphate at approximately 0.5% of the total volume.
21. The composition of any one of embodiments 8-20, wherein the cultured microbes are selected from a species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium,
10 Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof.
22. The composition of embodiment 21, wherein the one or more microbes comprise Kosakonia sacchari.
15 23. The composition of embodiment 22, wherein the cultured microbes comprise Kosakonia sacchari PTA- 126743.
24. The composition of embodiment 21, wherein the cultured microbes comprise Klebsiella variicola.
25. The composition of embodiment 24, wherein the cultured microbes comprise Klebsiella
20 variicola PTA-126740.
26. The composition of any one of embodiments 8-25, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA- 126743.
26.1 The composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species genetically engineered to fix atmospheric
25 nitrogen and provide such to a host plant.
26.2 The composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species that is a remodeled microbe.
26.3 The composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species that is a transgenic microbial species. 26.4 The composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species having a non-intergeneric genomic modification.
26.5 The composition of any one of embodiments 8-26, wherein the cultured microbes
5 comprise at least one microbial species that is a non intergeneric remodeled microbial species capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
26.6 The composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or non¬
10 coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
26.7 The composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising an introduced control sequence operably linked to at least one
15 gene of the nitrogen fixation or assimilation genetic regulatory network.
26.8 The composition of any one of embodiments 8-26, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
20 27. The composition of any one of embodiments 8-26.8, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or noncoding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or
25 glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
27.1 The composition of any one of embodiments 8-26.8, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial
30 species having a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene. 27.2 The composition of any one of embodiments 8-26.8, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
5 27.3 The composition of any one of embodiments 8-26.8, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated amtB gene that results in the lack of expression of said amtB gene.
27.4 The composition of any one of embodiments 8-26.8, wherein the cultured microbes
10 comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising at least one of: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated
15 glnD gene that results in a truncated GlnD protein lacking a uridyl-transferase domain or lack of expression of saidg/zzZ) gene, and combinations thereof.
28. The composition of any one of embodiments 8-26.8, wherein the cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine
20 synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, ni/K, nifY, niJE, nifN, nxfU, nifS, niJV, ni/W, nifZ, niJM, nifF, niJB, niJQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, fhaB,pehA, otsB, treZ, glsA2, or combinations thereof.
29. The composition of any one of embodiments 8-28, wherein the cultured microbes are
25 in a microbial composition comprising at least one of a polymer, sugar, biofilm, and isolated biofilm compositions.
30. The composition of any one of embodiments 8-29, wherein the cultured microbes to extender composition ratio is between 1 : 1 and 1 :4 by percent volume.
31. The composition of any one of embodiments 8-29, wherein the cultured microbes are
30 at a concentration of between about 1.0 X 104 and about 1.0 X 1012 CFU/mL of the total volume. 32. The composition of any one of embodiments 8-31, wherein the cultured microbes are reconstituted microbes.
33. The composition of embodiment 32, wherein the cultured microbes are at a dry weight to extender volume ratio between 1:10 and 1 :30 w/v.
5 33.1 The composition of embodiment 32, wherein the cultured microbes are at a dry weight to extender volume ratio between 1 :5 and 1 :30 w/v.
33.2 The composition of embodiment 32, wherein the cultured microbes are at a dry weight to extender volume ratio between 1 : 100 and 100: 1 w/v.
34. The composition of embodiment 32, wherein the cultured microbes comprise between
10 1% and 15% dry weight by volume of the composition.
35. The composition of any one of embodiments 1-34, wherein the composition is a seed coat present on a plant seed or other plant propagation material.
36. The composition of any one of embodiments 1-34, wherein the composition is a seed coat present on a plant seed or other plant propagation material with a pre-treatment.
15 37. The composition of embodiment 36, wherein the pre-treatment is an insecticide, herbicide, fungicide, biocide, or nematicide.
38. The composition of any one of embodiments 8-37, wherein the microbes exhibit an application log loss of less than 1.5 when applied to a seed.
39. The composition of any one of embodiments 8-37, wherein the microbes exhibit an
20 application log loss of less than 1 when applied to a seed.
40. The composition of any one of embodiments 1-37, wherein the composition maintains at least 10% microbial cell viability at 14 days post seed treatment.
41. The composition of any one of embodiments 1-37, wherein the composition maintains at least 20% microbial cell viability at 14 days post seed treatment.
25 42. The composition of any one of embodiments 1-37, wherein the composition maintains at least 30% microbial cell viability at 14 days post seed treatment.
43. The composition of any one of embodiments 1-37, wherein the composition maintains at least 40% microbial cell viability at 14 days post seed treatment. 44. The composition of any one of embodiments 1-37, wherein the composition maintains at least 50% microbial cell viability at 14 days post seed treatment.
45. The composition of any one of embodiments 1-37, wherein the composition maintains at least 10% microbial cell viability at 28 days post seed treatment.
5 46. The composition of any one of embodiments 1-37, wherein the composition maintains at least 20% microbial cell viability at 28 days post seed treatment.
47. The composition of any one of embodiments 1-37, wherein the composition maintains at least 30% microbial cell viability at 28 days post seed treatment.
48. A plant seed or plant propagation material comprising the composition of any one of
10 embodiments 1-47.
49. The plant seed of embodiment 48, wherein the plant seed is a com seed.
49.1 The plant seed of embodiment 48, wherein the plant seed is a wheat seed.
50. A seed coating kit comprising: a) an extender composition, comprising:
15 i) a sugar alcohol or sugar; ii) a chemical buffer; and b) cultured microbes.
51. The seed coating kit of embodiment 50, wherein the extender composition comprises iii) a water-soluble polymer.
20 52. The seed coating kit of embodiment 51 , wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP- VA), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
53. The seed coating kit of embodiment 52, wherein the polymer is polyvinylpyrrolidone¬
25 vinyl acetate (PVP-VA).
54. The seed coating kit of embodiment 53, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA) is present in the extender composition between about 5% and about 30% (w/v).
55. The seed coating kit of embodiment 51, wherein the sugar alcohol or sugar is between
30 about 5% and about 30% (w/v) of the extender composition. 56. The seed coating kit of embodiment 50, wherein the sugar alcohol or sugar is between about 10% and about 70% (w/v) of the extender composition.
57. The seed coating kit of embodiment 51, wherein the sugar alcohol or sugar to water- soluble polymer ratio is between 2: 1 and 1 : 1 w/w.
5 58. The seed coating kit of any one of embodiments 50-57, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fiicitol, iditol, and inositol.
59. The seed coating kit of any one of embodiments 50-58, wherein the sugar alcohol is sorbitol.
10 60. The seed coating kit of any one of embodiments 50-59, wherein the cultured microbes are in a liquid formulation.
61. The seed coating kit of any one of embodiments 50-59, wherein the cultured microbes are a powder formulation of lyophilized microbes.
62. The seed coating kit of embodiment 61, wherein the powder formulation of lyophilized
15 microbes are encapsulated within a water-soluble package.
63. The seed coating kit of embodiment 62, wherein the water-soluble package comprises two or more compartments.
64. The seed coating kit of embodiment 63, wherein the two or more compartments each comprise a different species of microbe.
20 65. The seed coating kit of any one of embodiments 50-64, wherein the cultured microbes are selected from species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and
25 combinations thereof.
66. The seed coating kit of embodiment 65, wherein the cultured microbes comprise Kosakonia sacchari.
67. The seed coating kit of embodiment 66, wherein the cultured microbes comprise Kosakonia sacchari PTA-126743. 68. The seed coating kit of embodiment 65, wherein the cultured microbes comprise Klebsiella variicola.
69. The seed coating kit of embodiment 68, wherein the cultured microbes comprise Klebsiella variicola PT A-l 26740.
5 70. The seed coating kit of any one of embodiments 50-64, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA-126743.
70.1 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is genetically engineered to fix atmospheric nitrogen and provide such to a host plant.
10 70.2 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a remodeled microbe.
70.3 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a transgenic microbial species.
70.4 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes
15 comprise at least one microbial species having a non-intergeneric genomic modification.
70.5 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
20 70.6 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or noncoding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
25 70.7 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
70.8 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes
30 comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
70.9 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial
5 species having at least one genetic variation introduced into at least one gene, or noncoding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or
10 decreased uridylyl-removing activity of GlnD.
71.1 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
15 71.2 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
71.3 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes
20 comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated amtB gene that results in the lack of expression of said amtB gene.
71.4 The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial
25 species comprising at least one of: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said m/Z, gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated glnD gene that results in a truncated GlnD protein lacking a uridyl-transferase domain
30 or lack of expression of saidgZnD gene, and combinations thereof. 72. The seed coating kit of any one of embodiments 50-70, wherein the cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD,
5 glnE, nijJ, nifH, nifD, nifK, nijY, nifE, niJN, nifU, nifS, nifl', niJW, niJZ, nijM, nifF, nifB, nijQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, JhaB,pehA, otsB, treZ, glsA2, or combinations thereof.
73. The seed coating kit of any one of embodiments 50-72, wherein the cultured microbes are in a microbial composition comprising at least one of a stabilizer, polymer, sugar,
10 bulking agent, anticaking agent, dispersant, biofilm, and isolated biofilm compositions.
74. The seed coating kit of any one of embodiments 50-73, wherein the sugar alcohol/sugar and/or chemical buffer is provided in a dry form.
75. The seed coating kit of any one of embodiments 51-74, wherein the water-soluble polymer is provided in a dry form.
15 76. The seed coating kit of embodiment 74, wherein the sugar alcohol/sugar and/or chemical buffer are encapsulated within a water-soluble package.
77. The seed coating kit of embodiment 75, wherein the water-soluble polymer is encapsulated within a water-soluble package.
78. The seed coating kit of embodiment 75, wherein the water-soluble polymer is the water-
20 soluble package.
79. A method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol or sugar, at between about 10% and about 80% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral
25 pH; and c) cultured microbes to a seed or plant tissue.
79.1 The method of embodiment 79, wherein the sugar alcohol or sugar, at between about 20% and about 30% (w/v). 79.2 The method of embodiment 79, wherein the sugar alcohol or sugar, at between about 40% and about 80% (w/v).
80. A method of treating a seed or plant tissue, comprising applying a composition comprising:
5 a) a sugar alcohol or sugar, at between about 2.5% and about 15% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) a water-soluble polymer, at approximately between 5% and 20% (w/v); and d) cultured microbes
10 to a seed or plant tissue.
80.1 A method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol or sugar, at between about 7.5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral
15 pH; c) a water-soluble polymer, at approximately between 5% and 40% (w/v); and d) cultured microbes to a seed or plant tissue.
81. The method any one of embodiments 79-80.1, wherein the composition has a microbial
20 concentration of about 1.0 X 104 to 1.0 X 1012 CPU per mL.
82. The method of any one of embodiments 79-81, wherein the composition is applied at a rate of between 1.0 fl oz/CWT and 20.0 fl oz/CWT.
83. The method of embodiment 82, wherein the composition is applied at an approximate rate of 5.4 fl oz/CWT.
25 83.1 The method of embodiment 82, wherein the composition is applied at an approximate rate of 2.4 fl oz/unit, and adjusted based on seed/lb to fl oz/CWT.
83.2 The method of embodiment 82, wherein the composition is applied at an approximate rate of 2.4 fl oz/CWT.
84. A treated seed or plant tissue produced by the method of any one of embodiments 79-
30 83.2. 85. The treated seed of embodiment 84, wherein the seed or plant tissue has a microbial concentration of about 1.0 X 103 to 1.0 X 1011 CPU per seed.
86. The treated seed of embodiment 85, wherein the seed or plant tissue has a microbial concentration of about 1.0 X 103 to 1.0 X 107 CPU per seed.
5 87. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least 10% microbial cell viability at 14 days post seed treatment.
88. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least 20% microbial cell viability at 14 days post seed treatment.
89. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least
10 30% microbial cell viability at 14 days post seed treatment.
90. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least 40% microbial cell viability at 14 days post seed treatment.
91. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least 50% microbial cell viability at 14 days post seed treatment.
15 92. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least 10% microbial cell viability at 28 days post seed treatment.
93. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least 20% microbial cell viability at 28 days post seed treatment.
94. The treated seed of any one of embodiments 84-86, wherein the seed maintains at least
20 30% microbial cell viability at 28 days post seed treatment.
95. The treated seed of any one of embodiments 84-94, wherein the seed comprises a pretreatment.
96. The treated seed of embodiment 95, wherein the pre-treatment is selected from the group consisting of an insecticide, herbicide, fungicide, or nematicide.
25 97. A dried seed coating comprising: a) between about 50% and about 90% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) between about 5% to about 40% (w/w) cultured microbes. 98. The dried seed coating of embodiment 97, comprising: a) about 72 or 84% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and
5 c) about 24 or 11% (w/w) cultured microbes.
99. A dried seed coating comprising: a) between about 25% and about 50% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH;
10 c) between about 17% and about 60% (w/w) water-soluble polymer; and d) between about 10% to about 40% (w/w) cultured microbes.
100. The dried seed coating of embodiment 99, comprising: a) about 35, 44, or 30% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral
15 pH; c) about 26%, 45, or 33 water-soluble polymer; and d) about 35. 20, or 21% (w/w) cultured microbes.
101. A dried seed coating comprising: a) sugar alcohol or sugar;
20 b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) cultured microbes.
102. A dried seed coating comprising: a) sugar alcohol or sugar;
25 b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) water-soluble polymer; and d) cultured microbes.
103. The dried seed coating of any one of embodiments 99-100, and 102, wherein the
30 polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA), carboxymethyl cellulose (CMC), hydroxypropyl methyl cellulose, alginate, and combinations thereof.
104. The dried seed coating of embodiment 103, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
5 105. The dried seed coating of any one of embodiments 97-104, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fucitol, iditol, and inositol.
106. The dried seed coating of any one of embodiments 97-104, wherein the sugar alcohol is sorbitol.
10 107. The dried seed coating of any one of embodiments 97-106, wherein the cultured microbes are a powder formulation of lyophilized microbes.
108. The dried seed coating of any one of embodiments 97-107, wherein the cultured microbes are selected from species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus,
15 Bradyrhizobium, Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof.
109. The dried seed coating of any one of embodiments 97-108, wherein the cultured microbes comprise Kosakonia sacchari.
20 110. The dried seed coating of any one of embodiments 97-108, wherein the cultured microbes comprise Kosakonia sacchari PTA-126743.
111. The dried seed coating of any one of embodiments 97-108, wherein the cultured microbes comprise Klebsiella variicola.
112. The dried seed coating of any one of embodiments 97-108, wherein the cultured
25 microbes comprise Klebsiella variicola PTA-126740.
113. The dried seed coating of any one of embodiments 97-108, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA- 126743. 114. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is genetically engineered to fix atmospheric nitrogen and provide such to a host plant.
115. The dried seed coating of any one of embodiments 97-113, wherein the cultured
5 microbes comprise at least one microbial species that is a remodeled microbe.
116. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a transgenic microbial species.
117. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species having a non-intergeneric genomic
10 modification.
118. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
15 119. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
20 120. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
121. The dried seed coating of any one of embodiments 97-113, wherein the cultured
25 microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
122. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled
30 microbial species having at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE;
5 or decreased uridylyl-removing activity of GlnD.
123. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated nz/t, gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
10 124. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
125. The dried seed coating of any one of embodiments 97-113, wherein at least one
15 microbial species is a non intergeneric remodeled microbial species comprising a mutated amtB gene that results in the lack of expression of said amtB gene.
126. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising at least one of: a mutated nifL gene that has been altered
20 to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated glnD gene that results in a truncated GlnD protein lacking a uridyl-transferase domain or lack of expression of said glnD gene, and combinations thereof.
25 127. The dried seed coating of any one of embodiments 97-113, wherein the cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nijH, nijD, nijK, nifY, nifE, nifN, nifU, nifS, ni/K, nifW, nijZ, nijM, nifF, nifB,
30 nijQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, or combinations thereof. 128. The dried seed coating of any one of embodiments 97-127, wherein the cultured microbes are in a microbial composition comprising at least one of a stabilizer, polymer, sugar, bulking agent, anticaking agent, dispersant, biofilm, and isolated biofilm compositions.
5

Claims

CLAIMS What is claimed is:
1. A composition comprising: a) a sugar alcohol or sugar, at between about 5% and about 30% (w/v);
5 b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) a water-soluble polymer, at between about 10% and about 40% (w/v).
2. The composition of claim 1, wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA),
10 carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
3. The composition of claim 2, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
4. The composition of claim 3, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA)
15 is present in the composition at between about 15% and about 20% (w/v).
5. The composition of claim 3, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA) is present in the composition at about 15% (w/v).
6. The composition of claim 3, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-VA) is present in the composition at about 17% (w/v).
20 7. The composition of claim 1, wherein the sugar or sugar alcohol is present in the composition at between about 10% and about 20% (w/v).
8. The composition of claim 1, wherein the sugar or sugar alcohol is present in the composition at about 11% (w/v).
9. The composition of claim 1, wherein the sugar or sugar alcohol is present in the
25 composition at about 20% (w/v).
10. The composition of claim 1, wherein the composition is mixed with cultured microbes to create a liquid seed treatment.
11. The liquid seed treatment of claim 10, wherein the sugar or sugar alcohol comprises between 2.5% and 30% (w/v) and the water-soluble polymer comprises between 5%
30 and 30% (w/v).
12. A composition comprising: a) a sugar alcohol or sugar, at between about 5% and about 70%(w/v); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH; and
5 c) cultured microbes.
13. The composition of claim 12, wherein the sugar alcohol or sugar is present in the composition between about 5% and about 20% (wA).
14. The composition of claim 12, wherein the sugar alcohol or sugar is present in the composition between about 20% and about 30% (w/v).
10 15. The composition of claim 12, wherein the sugar alcohol or sugar is present in the composition at about 30% (w/v).
16. The composition of claim 12, wherein the sugar alcohol or sugar is present in the composition at about 50% (w/v).
17. The composition of claim 12, wherein the sugar alcohol or sugar is present in the
15 composition at about 60% (w/v).
18. A composition comprising: a) a sugar alcohol or sugar, at between about 2.5% and about 30% (wA); b) a chemical buffer wherein the buffer maintains the composition at a neutral pH;
20 c) cultured microbes; and d) a water-soluble polymer, at between about 5% and about 30% (w/v).
19. The composition of claim 18, wherein the sugar alcohol or sugar is between about 7.5% and about 15% (w/v).
20. The composition of claim 18, wherein the water-soluble polymer is between about 5%
25 and about 20% (w/v).
21. The composition of claim 18, wherein the water-soluble polymer is between about 10% and about 20% (w/v).
22. The composition of claim 18, wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA),
30 carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
23. The composition of claim 22, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
24. The composition of claim 23, wherein the polyvinylpyrrolidone-vinyl acetate (PVP- VA) is present in the composition at about 7.5% (w/v).
5 25. The composition of claim 23, wherein the polyvinylpyrrolidone-vinyl acetate (PVP- VA) is present in the composition at about 15% (w/v).
26. The composition of claim 23, wherein the poly vinylpyrrolidone- vinyl acetate (PVP- VA) is present in the composition at about 11% (w/v).
27. The composition of claim 23, wherein the polyvinylpyrrolidone-vinyl acetate (PVP-
10 VA) is present in the composition at about 8.5% (w/v).
28. The composition of claim 1, 12, or 18, wherein the chemical buffer comprises potassium phosphate.
29. The composition of claim 1, 12, or 18, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fucitol, iditol, and inositol.
15 30. The composition of claim 1, 12, or 18, wherein the sugar alcohol is sorbitol.
31. The composition of claim 1, 12, or 18, wherein the chemical buffer comprises dipotassium phosphate at approximately between 0.5% and 1.5% of the total volume, and monopotassium phosphate at approximately between 0% and 1% of the total volume.
20 32. The composition of claim 1, 12, or 18, wherein the chemical buffer comprises di potassium phosphate at approximately between 0.5% and 4% of the total volume, and monopotassium phosphate at approximately between 0% and 2% of the total volume.
33. The composition of claim 1, 12, or 18, wherein the chemical buffer is dipotassium phosphate at approximately 1% of the total volume, and monopotassium phosphate at
25 approximately 0.5% of the total volume.
34. The composition of claim 1, 12, or 18, wherein the chemical buffer is dipotassium phosphate at approximately between 0.5% and 2% of the total volume, and monopotassium phosphate at approximately between 0% and 0.5% of the total volume.
35. The composition of claim 1, 12, or 18, wherein the chemical buffer is dipotassium phosphate at approximately 1% of the total volume, and monopotassium phosphate at approximately 0.5% of the total volume.
36. The composition of claim 12 or 18, wherein the cultured microbes are selected from a
5 species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof.
37. The composition of claim 12 or 18, wherein the one or more microbes comprise
10 Kosakonia sacchari.
38. The composition of claim 12 or 18, wherein the cultured microbes comprise Kosakonia sacchari PTA- 126743.
39. The composition of claim 12 or 18, wherein the cultured microbes comprise Klebsiella variicola.
15 40. The composition of claim 12 or 18, wherein the cultured microbes comprise Klebsiella variicola PT
41. The composition of claim 12 or 18, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA-126743.
42. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one
20 microbial species genetically engineered to fix atmospheric nitrogen and provide such to a host plant.
43. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a remodeled microbe.
44. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one
25 microbial species that is a transgenic microbial species.
45. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species having a non-intergeneric genomic modification.
46. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species capable of
30 fixing atmospheric nitrogen in the presence of exogenous nitrogen.
47. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
5 48. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
49. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one
10 microbial species that is a non intergeneric remodeled microbial species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
50. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least
15 one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
20 51. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
52. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one
25 microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl- removing (AR) domain.
53. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a
30 mutated amtB gene that results in the lack of expression of said amtB gene.
54. The composition of claim 12 or 18, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising at least one of: a mutated nijL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated
5 GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated glnD gene that results in a truncated GlnD protein lacking a uridyl-transferase domain or lack of expression of said glnD gene, and combinations thereof.
55. The composition of claim 12 or 18, wherein the cultured microbes comprises at least
10 one genetic variation introduced into a member selected from the group consisting of: nifA, nijL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, gbiA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nijD, nifK, nifY, nijE, nijN, nifU, nifS, nijV, nifW, nijZ, nifM, nijF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE,fhaB,pehA, otsB, treZ,
15 glsA2, or combinations thereof.
56. The composition of claim 12 or 18, wherein the cultured microbes are in a microbial composition comprising at least one of a polymer, sugar, biofilm, and isolated biofilm compositions.
57. The composition of claim 12 or 18, wherein the cultured microbes to extender
20 composition ratio is between 1 : 1 and 1 :4 by percent volume.
58. The composition of claim 12 or 18, wherein the cultured microbes are at a concentration of between about 1.0 X 104 and about 1.0 X 1012 CFU/mL of the total volume.
59. The composition of claim 12 or 18, wherein the cultured microbes are reconstituted microbes.
25 60. The composition of claim 12 or 18, wherein the cultured microbes are at a dry weight to extender volume ratio between 1:10 and 1 :30 w/v.
61. The composition of claim 12 or 18, wherein the cultured microbes are at a dry weight to extender volume ratio between 1:5 and 1 :30 w/v.
62. The composition of claim 12 or 18, wherein the cultured microbes are at a dry weight
30 to extender volume ratio between 1 : 100 and 100: 1 w/v.
63. The composition of claim 12 or 18, wherein the cultured microbes comprise between 1% and 15% dry weight by volume of the composition.
64. The composition of claim 1, 12, or 18, wherein the composition is a seed coat present on a plant seed or other plant propagation material.
5 65. The composition of claim 1, wherein the composition is a seed coat present on a plant seed or other plant propagation material with a pre-treatment.
66. The composition of claim 12, wherein the composition is a seed coat present on a plant seed or other plant propagation material with a pre-treatment.
67. The composition of claim 18, wherein the composition is a seed coat present on a plant
10 seed or other plant propagation material with a pre-treatment.
68. The composition of claim 65, 66, or 67, wherein the pre-treatment is an insecticide, herbicide, fungicide, biocide, or nematicide.
69. The composition of claim 12 or 18, wherein the microbes exhibit an application log loss of less than 1.5 when applied to a seed.
15 70. The composition of claim 12 or 18, wherein the microbes exhibit an application log loss of less than 1 when applied to a seed.
71. The composition of claim 1, 12, or 18, wherein the composition maintains at least 10% microbial cell viability at 14 days post seed treatment.
72. The composition of claim 1, 12, or 18, wherein the composition maintains at least 20%
20 microbial cell viability at 14 days post seed treatment.
73. The composition of claim 1, 12, or 18, wherein the composition maintains at least 30% microbial cell viability at 14 days post seed treatment.
74. The composition of claim 1, 12, or 18, wherein the composition maintains at least 40% microbial cell viability at 14 days post seed treatment.
25 75. The composition of claim 1, 12, or 18, wherein the composition maintains at least 50% microbial cell viability at 14 days post seed treatment.
76. The composition of claim 1, 12, or 18, wherein the composition maintains at least 10% microbial cell viability at 28 days post seed treatment.
77. The composition of claim 1, 12, or 18, wherein the composition maintains at least 20% microbial cell viability at 28 days post seed treatment.
78. The composition of claim 1, 12, or 18, wherein the composition maintains at least 30% microbial cell viability at 28 days post seed treatment.
5 79. A plant seed or plant propagation material comprising the composition of claim 1, 12, or 18.
80. A com seed comprising the composition of claim 1, 12, or 18.
81. A wheat seed comprising the composition of claim 1, 12, or 18.
82. A seed coating kit comprising:
10 a) an extender composition, comprising: i) a sugar alcohol or sugar; ii) a chemical buffer; and b) cultured microbes.
83. The seed coating kit of claim 82, wherein the extender composition comprises iii) a
15 water-soluble polymer.
84. The seed coating kit of claim 83, wherein the polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP- VA), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
20 85. The seed coating kit of claim 83, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
86. The seed coating kit of claim 83, wherein the polyvinylpyrrolidone-vinyl acetate (PVP- VA) is present in the extender composition between about 5% and about 30% (w/v).
87. The seed coating kit of claim 82, wherein the sugar alcohol or sugar is between about
25 5% and about 30% (w/v) of the extender composition.
88. The seed coating kit of claim 82, wherein the sugar alcohol or sugar is between about 10% and about 70% (w/v) of the extender composition.
89. The seed coating kit of claim 83, wherein the sugar alcohol or sugar to water-soluble polymer ratio is between 2: 1 and 1 : 1 w/w.
90. The seed coating kit of claim 82, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fucitol, iditol, and inositol.
91. The seed coating kit of claim 82, wherein the sugar alcohol is sorbitol.
92. The seed coating kit of claim 82, wherein the cultured microbes are in a liquid
5 formulation.
93. The seed coating kit of claim 82, wherein the cultured microbes are a powder formulation of lyophilized microbes.
94. The seed coating kit of claim 93, wherein the powder formulation of lyophilized microbes are encapsulated within a water-soluble package.
10 95. The seed coating kit of claim 94, wherein the water-soluble package comprises two or more compartments.
96. The seed coating kit of claim 95, wherein the two or more compartments each comprise a different species of microbe.
97. The seed coating kit of claim 82, wherein the cultured microbes are selected from
15 species of the following genera: Achromobacter, Agrobacterium, Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof.
98. The seed coating kit of claim 97, wherein the cultured microbes comprise Kosakonia
20 sacchari.
99. The seed coating kit of claim 98, wherein the cultured microbes comprise Kosakonia sacchari PTA-126743.
100. The seed coating kit of claim 97, wherein the cultured microbes comprise Klebsiella variicola.
25 101. The seed coating kit of claim 100, wherein the cultured microbes comprise Klebsiella variicola PTA-126740.
102. The seed coating kit of claim 82, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA-126743.
103. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is genetically engineered to fix atmospheric nitrogen and provide such to a host plant.
104. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one
5 microbial species that is a remodeled microbe.
105. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a transgenic microbial species.
106. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species having a non-intergeneric genomic modification.
10 107. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
108. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least
15 one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
109. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising an introduced control sequence operably linked to at least one gene of the nitrogen fixation
20 or assimilation genetic regulatory network.
110. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
25 111. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
112. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a
5 mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
113. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-
10 removing (AR) domain.
114. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated amtB gene that results in the lack of expression of said amtB gene.
115. The seed coating kit of claim 82, wherein the cultured microbes comprise at least one
15 microbial species that is a non intergeneric remodeled microbial species comprising at least one of: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated glnD gene that results in
20 a truncated GlnD protein lacking a uridyl-transferase domain or lack of expression of said glnD gene, and combinations thereof.
116. The seed coating kit of claim 82, wherein the cultured microbes comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat,
25 amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifll, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii,yjbE,fhaB,pehA, otsB, treZ, glsA2, or combinations thereof.
117. The seed coating kit of claim 82, wherein the cultured microbes are in a microbial
30 composition comprising at least one of a stabilizer, polymer, sugar, bulking agent, anticaking agent, dispersant, biofilm, and isolated biofilm compositions.
118. The seed coating kit of claim 82, wherein the sugar alcohol/sugar and/or chemical buffer is provided in a dry form.
119. The seed coating kit of claim 83, wherein the water-soluble polymer is provided in a diy form.
5 120. The seed coating kit of claim 118, wherein the sugar alcohol/sugar and/or chemical buffer are encapsulated within a water-soluble package.
121. The seed coating kit of claim 119, wherein the water-soluble polymer is encapsulated within a water-soluble package.
122. The seed coating kit of claim 121, wherein the water-soluble polymer is the water-
10 soluble package.
123. A method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol or sugar, at between about 10% and about 80% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral
15 pH; and c) cultured microbes to a seed or plant tissue.
124. The method of claim 123, wherein the sugar alcohol or sugar, at between about 20% and about 30% (w/v).
20 125. The method of claim 123, wherein the sugar alcohol or sugar, at between about 40% and about 80% (w/v).
126. A method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol or sugar, at between about 2.5% and about 15% (w/v);
25 b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) a water-soluble polymer, at approximately between 5% and 20% (w/v); and d) cultured microbes to a seed or plant tissue.
30
127. A method of treating a seed or plant tissue, comprising applying a composition comprising: a) a sugar alcohol or sugar, at between about 7.5% and about 30% (w/v); b) a chemical buffer, wherein the buffer maintains the composition at a neutral
5 pH; c) a water-soluble polymer, at approximately between 5% and 40% (w/v); and d) cultured microbes to a seed or plant tissue.
128. The method of claim 123, 126, or 127, wherein the composition has a microbial
10 concentration of about 1.0 X 104 to 1.0 X 1012 CPU per mL.
129. The method of claim 123, 126, or 127, wherein the composition is applied at a rate of between 1.0 fl oz/CWT and 20.0 fl oz/CWT.
130. The method of claim 123, 126, or 127, wherein the composition is applied at an approximate rate of 5.4 fl oz/CWT.
15 131. The method of claim 123, 126, or 127, wherein the composition is applied at an approximate rate of 2.4 fl oz/unit, and adjusted based on seed/lb to fl oz/CWT.
132. The method of claim 123, 126, or 127, wherein the composition is applied at an approximate rate of 2.4 fl oz/CWT.
133. A treated seed or plant tissue produced by the method of claim 123.
20 134. A treated seed or plant tissue produced by the method of claim 126.
135. A treated seed or plant tissue produced by the method of claim 127.
136. The treated seed of claim 133, 134, or 135, wherein the seed or plant tissue has a microbial concentration of about 1.0 X 103 to 1.0 X 1011 CPU per seed.
137. The treated seed of claim 133, 134, or 135, wherein the seed or plant tissue has a
25 microbial concentration of about 1.0 X 103 to 1.0 X 107 CPU per seed.
138. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 10% microbial cell viability at 14 days post seed treatment.
139. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 20% microbial cell viability at 14 days post seed treatment.
140. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 30% microbial cell viability at 14 days post seed treatment.
141. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 40% microbial cell viability at 14 days post seed treatment.
5 142. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 50% microbial cell viability at 14 days post seed treatment.
143. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 10% microbial cell viability at 28 days post seed treatment.
144. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 20%
10 microbial cell viability at 28 days post seed treatment.
145. The treated seed of claim 133, 134, or 135, wherein the seed maintains at least 30% microbial cell viability at 28 days post seed treatment.
146. The treated seed of claim 133, 134, or 135, wherein the seed comprises a pre-treatment.
147. The treated seed of claim 146, wherein the pre-treatment is selected from the group
15 consisting of an insecticide, herbicide, fungicide, or nematicide.
148. A dried seed coating comprising: a) between about 50% and about 90% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and
20 c) between about 5% to about 40% (w/w) cultured microbes.
149. The dried seed coating of claim 148, comprising: a) about 72 or 84% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and
25 c) about 24 or 11% (w/w) cultured microbes.
150. A dried seed coating comprising: a) between about 25% and about 50% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH;
30 c) between about 17% and about 60% (w/w) water-soluble polymer; and d) between about 10% to about 40% (w/w) cultured microbes.
151. The dried seed coating of claim 150, comprising: a) about 35, 44, or 30% (w/w) sugar alcohol or sugar; b) a chemical buffer, wherein the buffer maintains the composition at a neutral
5 pH; c) about 26%, 45, or 33 water-soluble polymer; and d) about 35. 20, or 21% (w/w) cultured microbes.
152. A dried seed coating comprising: a) sugar alcohol or sugar;
10 b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; and c) cultured microbes.
153. A dried seed coating comprising: a) sugar alcohol or sugar;
15 b) a chemical buffer, wherein the buffer maintains the composition at a neutral pH; c) water-soluble polymer; and d) cultured microbes.
154. The dried seed coating of claim 150, 152, or 153, wherein the polymer is selected from
20 the group consisting of polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate (PVP-VA), carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose, alginate, and combinations thereof.
155. The dried seed coating of claim 150, 152, or 153, wherein the polymer is polyvinylpyrrolidone-vinyl acetate (PVP-VA).
25 156. The dried seed coating of claim 148, 150, 152, or 153, wherein the sugar alcohol is selected from the group consisting of sorbitol, mannitol, galactitol, fucitol, iditol, and inositol.
157. The dried seed coating of claim 148, 150, 152, or 153, wherein the sugar alcohol is sorbitol.
158. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes are a powder formulation of lyophilized microbes.
159. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes are selected from species of the following genera: Achromobacter, Agrobacterium,
5 Anabaena, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Clostridium, Enterobacter, Klebsiella, Kluyvera, Kosakonia, Mesorhizobium, Microbacterium, Pseudomonas, Rahnella, Rhizobium, Sinorhizobium, and combinations thereof.
160. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes
10 comprise Kosakonia sacchari.
161. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise Kosakonia sacchari PTA-126743.
162. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise Klebsiella variicola.
15 163. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise Klebsiella variicola PTA-126740.
164. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise Klebsiella variicola PTA-126740 and Kosakonia sacchari PTA-126743.
165. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes
20 comprise at least one microbial species that is genetically engineered to fix atmospheric nitrogen and provide such to a host plant.
166. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise at least one microbial species that is a remodeled microbe.
167. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes
25 comprise at least one microbial species that is a transgenic microbial species.
168. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise at least one microbial species having a non-intergeneric genomic modification.
169. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
170. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes
5 comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or noncoding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
171. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes
10 comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
172. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial
15 species comprising a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
173. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species having at least one genetic variation introduced into at least one gene, or non¬
20 coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
25 174. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
175. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes
30 comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
176. The dried seed coating of claim 148, 150, 152, or 153, wherein at least one microbial species is a non intergeneric remodeled microbial species comprising a mutated amtB
5 gene that results in the lack of expression of said amtB gene.
177. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes comprise at least one microbial species that is a non intergeneric remodeled microbial species comprising at least one of: a mutated nijL gene that has been altered to comprise a heterologous promoter inserted into said nijL gene; a mutated glnE gene that results
10 in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; a mutated glnD gene that results in a truncated GlnD protein lacking a uridyl-transferase domain or lack of expression of said glnD gene, and combinations thereof.
178. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes
15 comprises at least one genetic variation introduced into a member selected from the group consisting of: niJA, nijL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnfi, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, niJJ, nijH, nijD, nijK, nijY, nijE, nijN, nijU, nijS, nijV, nijW, nijZ, nijM, nijF, nijB, nijQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE,
20 jhaB,pehA, otsB, treZ, glsA2, or combinations thereof.
179. The dried seed coating of claim 148, 150, 152, or 153, wherein the cultured microbes are in a microbial composition comprising at least one of a stabilizer, polymer, sugar, bulking agent, anticaking agent, dispersant, biofilm, and isolated biofilm compositions.
25
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