WO2022125639A1 - Bactéries modifiées associées à une plante et leurs procédés d'utilisation - Google Patents

Bactéries modifiées associées à une plante et leurs procédés d'utilisation Download PDF

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Publication number
WO2022125639A1
WO2022125639A1 PCT/US2021/062372 US2021062372W WO2022125639A1 WO 2022125639 A1 WO2022125639 A1 WO 2022125639A1 US 2021062372 W US2021062372 W US 2021062372W WO 2022125639 A1 WO2022125639 A1 WO 2022125639A1
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plant
bacillus
strain
modified
bacillus strain
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PCT/US2021/062372
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English (en)
Inventor
James A. Baum
Michael J. Crawford
Larry A. Gilbertson
Xiangqian Li
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Monsanto Technology Llc
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Publication of WO2022125639A1 publication Critical patent/WO2022125639A1/fr

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    • 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
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • 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
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)

Definitions

  • the present disclosure relates to modified plant-associated bacteria expressing insecticidal proteins, and methods of making and using modified bacteria to protect an agricultural plant, seed, or seedling from insects.
  • Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most well-known species expressing insecticidal proteins, but certain strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus also have insecticidal properties. These bacteria have been formulated as insecticides and commercial products for application to agricultural crops.
  • Bacterial insecticides can offer effective control of many insect pests, and are generally non-toxic and non-pathogenic to animals and humans.
  • these bacterial strains are often vulnerable to environmental stresses, such as heat, desiccation and ultraviolet radiation, and are not typically adapted to proliferate and survive within various plant tissues or on the exterior surfaces (e.g., phyllosphere or rhizosphere) of a plant.
  • these insecticidal bacteria are often short-lived following their application to a plant, and require repeated applications for successful insect control.
  • a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein.
  • the DNA molecule may further comprise a Bacillus promoter operably linked to the sequence encoding the insecticidal protein.
  • the Bacillus promoter may comprise a DNA sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6.
  • the modified plant-associated Bacillus strain may be a Bacillus amyloliquefaciens, Bacillus butanolivorans , Bacillus megaterium Bni), Bacillus pumilus, Bacillus simplex, Bacillus velezensis, or Bacillus subtilis strain.
  • the insecticidal protein may be Cry 1 Ac, TIC2463, or a combination thereof.
  • the insecticidal protein may comprise or consist of a protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or similar to SEQ ID NO: 2 or SEQ ID NO: 4, or the sequence encoding the insecticidal protein may comprise or consist of a DNA sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or similar to SEQ ID NO: 1 or SEQ ID NO: 3.
  • a modified plant-associated Bacillus strain may provide insecticidal activity against one or more insects within the Lepidopteran order or the Coleopteran order, or a combination thereof.
  • a modified plant-associated Bacillus strain may provide insecticidal activity against one or more of the following insects: Black cutworm (BCW), Colorado Potato Beetle (CPB), Diamondback moth (DBM), and Western com rootworm (WCR).
  • a modified plant-associated Bacillus strain may confer a positive insecticidal trait or benefit to a crop plant when the crop plant is treated or associated with the modified plant-associated Bacillus strain.
  • a bacterial population or culture comprising a modified plant- associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein.
  • the bacterial population or culture may comprise two or more modified plant-associated Bacillus species or strains.
  • a pure or substantially pure population or culture of a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein.
  • a composition comprising a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein, or a bacterial population or culture of a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein, and an agriculturally acceptable carrier.
  • the modified plant-associated Bacillus strain may be present in the composition at a concentration of at least 10 3 cfu per milliliter or gram.
  • a plant, plant part or plant seed having applied or coated on at least a portion of its outer surface a composition comprising a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein, or a bacterial population or culture of a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein.
  • the composition may further comprise an agriculturally acceptable carrier.
  • the plant, plant part or plant seed may be transgenic.
  • the plant, plant part or plant seed may be a monocotyledonous plant, plant part or plant seed, such as a corn, wheat, rice, barley, or cereal plant, plant part or plant seed.
  • the plant, plant part or plant seed may be a dicotyledonous plant, plant part or plant seed, such as a soybean, alfalfa, sunflower, cotton, canola, sugar beet or vegetable plant, plant part or plant seed.
  • a bag or container comprising plant seed having applied or coated on at least a portion of its outer surface a composition comprising a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein, or a bacterial population or culture of a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein.
  • a plant is provided that is grown or developed from a plant seed having applied or coated on at least a portion of its outer surface a composition comprising a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein, or a bacterial population or culture of a modified plant-associated Bacillus strain comprising a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein.
  • the plant grown or developed from the plant seed may comprise or be associated with the modified plant-associated Bacillus strain or a progeny thereof.
  • a plant part or plant seed from the grown or developed plant is provided, wherein the plant part or plant seed may comprise or be associated with the modified plant-associated Bacillus strain or a progeny thereof.
  • a method for making a modified plant- associated Bacillus strain comprising: contacting or mixing a plant-associated Bacillus strain or bacterial cell with a recombinant and/or heterologous DNA molecule comprising a sequence encoding an insecticidal protein; and isolating or selecting a modified plant-associated Bacillus strain having the recombinant and/or heterologous DNA molecule.
  • the plant-associated Bacillus strain may be a Bacillus amyloliquefaciens, Bacillus butanolivorans, Bacillus megaterium, Bacillus pumilus, Bacillus simplex, Bacillus velezensis, or Bacillus subtilis strain.
  • the DNA molecule may be introduced or transferred into the Bacillus strain or bacterial cell via a horizontal gene transfer technique, such as via transformation.
  • the DNA molecule may further comprise a Bacillus promoter operably linked to the sequence encoding the insecticidal protein.
  • the DNA molecule may be a plasmid.
  • the DNA molecule may be recombinant and/or comprise a Bacillus promoter that is heterologous with respect to the sequence encoding the insecticidal protein.
  • a method for making a modified plant- associated Bacillus strain comprising: contacting or mixing a plant-associated Bacillus strain or bacterial cell with a bacterial donor cell comprising a DNA molecule comprising a sequence encoding an insecticidal protein; and isolating or selecting a modified plant-associated Bacillus strain or bacterial cell having the DNA molecule, wherein the DNA molecule is heterologous with respect to the plant-associated Bacillus strain or bacterial cell.
  • the plant- associated Bacillus strain may be a Bacillus amyloliquefaciens, Bacillus butanolivorans, Bacillus megaterium, Bacillus pumilus, Bacillus simplex, Bacillus velezensis, or Bacillus subtilis strain.
  • the DNA molecule may be introduced or transferred into the Bacillus strain or bacterial cell via a horizontal gene transfer technique, such as via conjugation.
  • the DNA molecule may further comprise a Bacillus promoter operably linked to the sequence encoding the insecticidal protein.
  • the DNA molecule may be a plasmid.
  • the DNA molecule may be recombinant and/or comprise a Bacillus promoter that is heterologous with respect to the sequence encoding the insecticidal protein.
  • a method for making a modified plant- associated Bacillus strain comprising: contacting or mixing a plant-associated Bacillus strain or bacterial cell with a bacterial virus or bacteriophage comprising a DNA molecule comprising a sequence encoding an insecticidal protein; and isolating or selecting a modified plant-associated Bacillus strain or bacterial cell having the DNA molecule, wherein the DNA molecule is heterologous with respect to the plant-associated Bacillus strain or bacterial cell.
  • the plant-associated Bacillus strain may be a Bacillus amyloliquefaciens, Bacillus butanolivorans, Bacillus megaterium, Bacillus pumilus, Bacillus simplex, Bacillus velezensis, or Bacillus subtilis strain.
  • the DNA molecule may be introduced or transferred into the Bacillus strain or bacterial cell via a horizontal gene transfer technique, such as via transduction.
  • the DNA molecule may further comprise a Bacillus promoter operably linked to the sequence encoding the insecticidal protein.
  • the DNA molecule may be a recombinant and/or comprise a Bacillus promoter that is heterologous with respect to the sequence encoding the insecticidal protein.
  • a method for treating a plant, plant part or plant seed comprising: applying or coating a modified plant-associated Bacillus strain as provided herein, or a bacterial population or culture of a modified plant-associated Bacillus strain as provided herein, or a composition of any of the foregoing, on or to a plant, plant part or plant seed or at least a portion of an outer surface of a plant, plant part or plant seed.
  • An effective amount of a modified plant-associated Bacillus strain, or a population, culture, or composition thereof may be applied or coated on or to the plant, plant part or plant seed to provide insecticidal activity against one or more insects within the Lepidopteran order or the Coleopteran order, or a combination thereof.
  • the modified plant-associated Bacillus strain may provide insecticidal activity against one or more of the following: Black cutworm (BCW), Colorado Potato Beetle (CPB), Diamondback moth (DBM), and Western corn rootworm (WCR).
  • a method for providing an insecticidal activity for a plant comprising: applying a modified plant-associated Bacillus strain as provided herein, or a bacterial population or culture of a modified plant-associated Bacillus strain as provided herein, or a composition of any of the foregoing, to a growth medium or soil associated with a plant, plant part or plant seed.
  • An effective amount of the modified plant-associated Bacillus strain, or a population, culture, or composition thereof, is applied to the growth medium or soil to provide insecticidal activity against one or more insects within the Lepidopteran order or the Coleopteran order, or a combination thereof.
  • a method for providing an insecticidal activity for a plant comprising: planting a plant part or seed, wherein the plant part or seed is at least partially coated or associated with a modified plant-associated Bacillus strain as provided herein, or a bacterial population or culture of a modified plant-associated Bacillus strain as provided herein, or a composition of any of the foregoing.
  • the method may further comprise: growing or regenerating a plant from the plant part or seed.
  • FIG. 1 shows a Western Blot analysis, showing transformed B. megaterium (Bm) expressing the insecticidal TIC2463 protein;
  • FIG. 2 shows a Western Blot analysis, showing transformed B. thurigenisis (Bt) expressing the insecticidal TIC2463 protein;
  • FIG. 3 shows a Western Blot analysis, showing transformed B. megaterium (Bm) expressing the insecticidal CrylAc protein;
  • FIG. 4 shows transformed B. thurigenisis (Bt) and B. megaterium (Bm) supernatant and pellet expressing the TIC2463 protein in an artificial diet assay for Western Corn Rootworm (WCR);
  • FIG. 5 shows TIC2463 expression and Western Corn Rootworm (WCR) mortality in an artificial diet assay with either its native (TIC2463) or TIC1201 promoter;
  • FIG. 6 shows Diamondback Moth (DBM) mortality in an artificial diet assay with wild-type and transformed B. thurigenisis (Bt), B. megaterium (Bm), and B. butanolivorans (Bb) with the CrylAc gene cultured for different time periods (26, 40, and 64 hours);
  • BBM Diamondback Moth
  • FIG. 7 shows Diamondback Moth (DBM) mortality in an artificial diet assay with transformants at a variety of concentrations (from 3 to 2430-fold dilutions);
  • FIG. 8 shows Black Cutworm (BCW) mortality in an artificial diet assay with transformants at a variety of concentrations (from 3 to 2430-fold dilutions);
  • FIG. 9 shows an ELISA analysis quantifying the production of CrylAc protein in the B. butanolivorans transformant (Bb-C y 1 Ac) cultured for different time periods (40 and 80 hours) in supernatant and pellet;
  • FIG. 10 shows Diamondback Moth mortality in an artificial diet assay for the B. butanolivorans transformants and conjugants cultured for different time periods (40 and 80 hours) and concentrations;
  • FIG. 11 shows both Bm_N2463 transformant and Bm conjugant (N2463xCrylAc) expressing TIC2463 caused WCR mortality in an artificial diet assay
  • FIG. 12 shows Bm conjugant (N2463xCrylAc) expressing CrylAc caused DBM mortality in the artificial diet assay, whereas Bm_N2463 had no CrylAc gene, did not express CrylAc protein, and did not cause DBM mortality;
  • FIG. 13 shows the Bm_N2463 transformant and Bm conjugant (N2463xCrylAc) reduced Colorado Potato Beetle (CPB) neonate feeding and increased mortality up to 9-fold dilution (0.1 IX) in a potato leaf disc assay;
  • FIG. 14 shows whole tomato plant assays where the Bm_N2463 transformant and Bm conjugant (N2463xCrylAc) increased CPB mortality, reduced total weight of CPB, and decreased defoliation in a bacterial dose dependent manner;
  • FIG. 15 shows the results of a whole tomato plant assay wherein Bm-CrylAc conjugate treatment reduced leaf damage
  • FIG. 16 shows a whole com plant assay wherein Bm-CrylAc treatment reduced leaf damage severity
  • FIG. 17 shows a whole cabbage assay wherein the Bm conjugant controls Diamondback Moth (DBM) in a dose dependent manner
  • FIG. 18 shows a photograph of the whole cabbage assay wherein the Bm conjugant controls Diamondback Moth (DBM) in in a dose dependent manner
  • FIG. 19 shows confocal images of com roots colonized by Bacillus megaterium_TIC2463 cells expressing green fluorescent protein.
  • the term “and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items.
  • the expression “A and/or B” is intended to mean either or both of A and B - i.e., A alone, B alone, or A and B in combination.
  • the expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
  • the present disclosure provides a modified plant-associated bacterium, bacterial cell or bacteria that express(es) one or more insecticidal proteins.
  • any properties, traits, compositions or components of a “bacterium” or “bacterial cell” may be further applied or used to describe “bacteria” in the plural - i.e., more than one bacterium or bacterial cell, and “bacteria” includes a plurality, population, colony or culture of bacteria or bacterial cells.
  • a “plant-associated bacterium” or “plant-associated bacterial cell” is a bacterium or bacterial cell that can reside, proliferate and survive for multiple generations in (or on) one or more plant tissues (e.g., endosphere or phyllosphere of a plant), or in the immediate environment (e.g., rhizosphere) of a plant, although a plant-associated bacterium or plant-associated bacterial cell may reside in an environment not associated with a plant tissue and/or may not need to be associated with a plant tissue to survive and/or proliferate, and “plant-associated bacteria” include a plurality, population, colony or culture of a plant-associated bacterium or plant-associated bacterial cell.
  • the “plant-associated bacterium” or “plant- associated bacterial cell” is a Bacillus bacterium or a Bacillus bacterial strain or cell.
  • a modified plant-associated bacterium (or bacterial cell) or bacteria may comprise a modified plant-associated bacterial strain or isolate (or modified plant-associated strain or isolate).
  • a bacterial strain has been “isolated” if it has been removed and/or purified from the environment.
  • an “isolate” is a particular microbial strain that has been removed and/or purified from the environment.
  • Any description herein of a modified plant-associated bacterium (or bacterial cell) or bacteria shall be construed as also referring to and contemplating a modified plant-associated bacterial strain or isolate (or modified plant-associated strain or isolate), and a plurality, population, colony or culture thereof.
  • a plant-associated bacterium can be modified to comprise a DNA molecule or plasmid from another bacterial cell comprising a coding sequence encoding an insecticidal protein, wherein the coding sequence is operably linked to a bacterial promoter.
  • a “bacterial promoter” is a promoter that is functional in a bacterial strain or cell, such as a plant-associated bacterial cell, to direct, cause and/or permit the expression of an associated coding sequence, such as a coding sequence for an insecticidal protein, in the bacterial strain or cell.
  • a “plant-associated bacterial promoter” is a promoter that is functional in a plant-associated bacterial strain or cell, to direct, cause and/or permit the expression of an associated coding sequence, such as a coding sequence for an insecticidal protein, in the plant-associated bacterial strain or cell.
  • a “Bacillus promoter” or a “plant-associated Bacillus promoter” is a promoter that is functional in a Bacillus bacterial strain or cell, or a plant-associated Bacillus bacterial strain or cell, respectively, to direct, cause and/or permit the expression of an associated coding sequence, such as a coding sequence for an insecticidal protein, in the Bacillus bacterial strain or cell, or the plant-associated Bacillus bacterial strain or cell.
  • a DNA molecule or plasmid may be introduced or transferred into a plant-associated bacterial cell from another bacterial cell through horizontal gene transfer (HGT), such as via transformation, transduction or conjugation, to produce a modified plant-associated bacterial cell comprising the DNA molecule or plasmid.
  • HHT horizontal gene transfer
  • a “horizontal gene transfer technique” refers to a process of introducing or transferring a DNA molecule or plasmid into a plant-associated bacterial cell via horizontal gene transfer enabled through human intervention, such as by culturing a plant-associated bacterial cell in the presence of (i) the DNA molecule or plasmid (in the case of transformation), (ii) another bacterial cell having the DNA molecule or plasmid (in the case of conjugation), and/or (iii) a virus or bacteriophage having the DNA molecule or plasmid (in the case of transduction).
  • a modified plant-associated bacterial cell can be created by intentionally combining a plant- associated bacterial cell with a DNA molecule or plasmid, another bacterial cell having the DNA molecule or plasmid, or a virus or bacteriophage having the DNA molecule or plasmid.
  • the modified plant-associated bacterial cell can then be selected or isolated based on one or more selectable or screenable markers, by any suitable molecular assay or method of detection, and/or by the expression of one or more proteins, such as insecticidal proteins, expressed from the DNA molecule or plasmid.
  • the DNA molecule or plasmid can be described as comprising an expression cassette including a coding sequence encoding an insecticidal protein associated with and operably linked to a bacterial promoter.
  • the DNA molecule or plasmid may further comprise other regulatory and/or expression elements or sequences, such as an enhancer, sequence, termination sequence, and/or a prokaryotic origin or replication.
  • the DNA molecule or plasmid may further comprise a selectable or screenable marker gene to allow for selection or isolation of bacterial cells containing the DNA molecule or plasmid.
  • the plant-associated bacterial cell Prior to introduction or transfer of the DNA molecule or plasmid, or at least the coding sequence and its associated and operably linked promoter, from the other bacterial cell, the plant-associated bacterial cell does not comprise or contain the DNA molecule or plasmid including the coding sequence for an insecticidal protein and its associated promoter.
  • a DNA molecule or plasmid including its expression cassette, coding sequence, promoter, etc., may be described as being heterologous with respect to a plant-associated bacterial cell, or vice versa.
  • heterologous in reference to a DNA molecule or plasmid is defined as being from a different source, bacterial cell or microorganism, and not naturally present in the plant-associated bacterial cell prior to its introduction or transfer into the plant-associated bacterial cell through a horizontal gene transfer technique.
  • a coding sequence of a DNA molecule or plasmid may be described as being heterologous with respect to its associated or operably linked promoter, or vice versa.
  • heterologous in reference to a coding sequence and its associated or operably linked promoter is defined as being from different sources, bacterial cells or microorganisms, and not physically associated or operably linked in nature prior to being combined or brought together via molecular biology techniques.
  • a DNA molecule or plasmid comprising the expression cassette encoding an insecticidal protein may be naturally occurring in the donor bacterium or bacterial cell, but not naturally occurring in the modified plant-associated bacterial cell receiving the DNA molecule or plasmid via a horizontal gene transfer technique.
  • the DNA molecule or plasmid introduced or transferred into the plant-associated bacterial cell using a horizontal gene transfer technique may be a recombinant DNA molecule.
  • the term “recombinant” is defined as a DNA molecule, plasmid or sequence that is man-made and not normally found in nature, and/or is present in a context in which it is not normally found in nature, including a DNA molecule or plasmid comprising a combination of two or more DNA or polynucleotide sequences that would not naturally occur together in the same manner, without human intervention, such as a DNA molecule or plasmid comprising at least two polynucleotide sequences that are operably linked but heterologous with respect to each other.
  • the term “recombinant” can refer to any combination or arrangement of two or more DNA sequences in the same molecule (e.g., a plasmid, construct, vector, chromosome, etc.) where such a combination is man-made and not normally found in nature.
  • the phrase “not normally found in nature” means not found in nature without human introduction.
  • a recombinant DNA molecule, construct, plasmid, etc. may comprise DNA sequence(s) that is/are (i) separated from other DNA sequence(s) that exist in proximity to each other in nature, and/or (ii) adjacent to (or contiguous with) other DNA sequence(s) that are not naturally in proximity with each other.
  • Such a recombinant DNA molecule, construct, plasmid, etc. may also refer to a DNA molecule or sequence that has been genetically engineered and/or constructed outside of a cell.
  • a recombinant DNA molecule may comprise any engineered or man-made plasmid, vector, etc., and may include a linear or circular DNA molecule.
  • Such plasmids, vectors, etc. may contain various maintenance elements including a prokaryotic origin of replication, a selectable marker gene, one or more insecticidal and/or other genes or expression cassettes, etc.
  • the modified plant-associated bacteria are a pure or substantially pure population or culture.
  • a pure population or culture of a modified plant- associated bacteria refers to a population or culture of that modified plant-associated bacteria that is essentially free from contamination of other microorganisms, such that the population or culture has sufficient genetic uniformity in terms of sequence identity, and different subcultures taken therefrom will exhibit substantially identical phenotypes and/or effects on insects.
  • a modified plant-associated bacteria may be “purified”, and the purified modified plant-associated bacteria may be combined with one or more other ingredients to form a composition or formulation.
  • a purified population or culture of the modified plant-associated bacteria may comprise a clonal population or culture of the modified plant-associated bacteria.
  • any suitable method known in the art may be used to isolate and/or purify a modified plant- associated bacteria to form a pure or substantially pure population or culture of the modified plant- associated bacteria.
  • the modified plant-associated bacteria may be mixed with other microorganisms to varying extents or proportions.
  • the modified plant- associated bacteria may be present in a composition or formulation in an amount or concentration that is between 10-30%, 10-40%, 10-50%, 20-40%, 30-50%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 97%, or greater than 99% of the total population (e.g., in terms of colony forming units or cfu’s) of microorganisms present in the composition or formulation.
  • a composition comprises a “substantially pure” population or culture of a modified plant-associated bacteria if the strain or isolate accounts for greater than 95% of the total population of microorganisms present in the composition.
  • Plants host diverse communities and species of microorganisms including bacteria.
  • the relationships between plants and microorganisms including bacteria can be defined in part by the different compartments or environments of a plant in which the communities and species of microorganisms or bacteria reside, which may be divided into different “spheres.”
  • the outer surfaces of the above-ground portion of a plant is referred to as the “episphere” or “phyllosphere” including the leaf surfaces of a plant referred to as the “phylloplane,” and the external zone or region of soil immediately surrounding the roots of a plant referred to as the “rhizosphere,” which includes the m ore immediate rhizoplane surfaces of the roots.
  • microorganisms can also occupy and reside within the internal tissues of a plant referred to collectively as the “endosphere.”
  • a plant-associated microbes or plant-associated bacteria are able to proliferate and survive on one or more surfaces, environments or tissues of a plant over multiple divisions or generations of the microbe or bacteria. Accordingly, it is presently proposed that a plant- associated bacterial cell, such as a bacterial cell of a plant-associated Bacillus species, may be modified to include a DNA molecule or plasmid comprising an insecticidal gene.
  • insecticidal protein can persist in association with a plant for a greater length of time, in comparison to expression from the bacterial (donor) species from which the DNA molecule or plasmid was derived or transferred, due to the insecticidal protein being expressed from a modified plant-associated bacterial cell that is able to persist and survive on a plant for a longer duration of time following its application to the plant.
  • a modified plant-associated bacterial cell or bacteria can be applied to the surface of a seed, or to the above-ground (foliar) tissues or roots of a plant, or to the surrounding soil of a plant.
  • modified plant-associated bacterial cell, or modified plant-associated bacteria Following application of a modified plant-associated bacterial cell, or modified plant-associated bacteria, to a seed, plant or soil, the modified plant- associated bacterial cell, or modified plant-associated bacteria, can migrate to, or proliferate and expand to occupy or reside on or in, different plant surfaces, tissues or environments, perhaps at later stages of plant development, to express an insecticidal protein at those new locations and/or during those later development stages.
  • a plant-associated bacterium or a modified plant-associated bacterium is a Bacillus species, which may be a Bacillus amyloliquefaciens (Ba), Bacillus butanolivorans (Bb), Bacillus megaterium (Bm), Bacillus pumilus (Bp), Bacillus simplex (Bsp), Bacillus velezensis (Bv), and Bacillus subtilis (Bs) species.
  • a modified plant-associated bacterial cell or bacteria is Bacillus butanolivorans (Bb) or Bacillus megaterium (Bm).
  • a modified plant-associated bacterial cell or bacteria is a particular strain or isolate of a Bacillus species modified to include a DNA molecule or plastid comprising an expression cassette encoding an insecticidal protein.
  • Bt strains Bacillus thuringiensis (Bt) have been used as a source for insecticidal proteins since it was discovered that Bt strains show a high toxicity against specific insects.
  • Bt strains are known to produce delta-endotoxins (e.g., Cry proteins) that may be localized within parasporal crystalline inclusion bodies at the onset of sporulation and during the stationary growth phase, and are also known to produce secreted insecticidal protein.
  • delta-endotoxins e.g., Cry proteins
  • delta-endotoxins e.g., Cry proteins
  • Ls Lysinibacillus sphaericus
  • Paenibacillus popilliae Genes encoding insecticidal proteins have also been identified in bacterial species other than Bt, including other Bacillus species and a diversity of other bacterial species, such as Brevibacillus laterosporus, Lysinibacillus sphaericus (“Ls” formerly known as Bacillus sphaericus) and Paenibacillus popilliae.
  • insecticidal proteins from Bt or other Bacillus strains have been identified. Any such insecticidal proteins can be expressed by a modified plant-associated microorganism or bacteria.
  • the insecticidal protein is chosen from an Lepidopteran inhibiting protein, such as, but not limited to, CrylA (see, e.g., U.S. Patent No. 5,880,275), CrylAb, CrylAc, CrylA.105, CrylAe, CrylB (see, e.g., U.S. Patent Application No. 10/525,318), CrylC (see, e.g., U.S. Patent No.
  • CrylD, CrylDa and variants thereof, CrylE, CrylF, and CrylA/F chimeras see, e.g., U.S. Patent Nos. 7,070,982; 6,962,705; and 6,713,063
  • CrylG, CrylH, Cryll, CrylJ, CrylK, CrylL Cryl-type chimeras such as, but not limited to, TIC836, TIC860, TIC867, TIC869, and TIC1100 (see, e.g., International Application Publication No. WO 2016/061391), TIC2160 (see, e.g., International Application Publication No.
  • WO 2016/061392 Cry2A, Cry2Ab (see, e.g., U.S. Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cryl5, Cry43A, Cry43B, Cry51Aal, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AND AXMI-045 (see, e.g., U.S. Patent Publication No.
  • AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 see, e.g., U.S. Patent Application Publication No. 2013/0310543 Al
  • AXMI-115, AXMI-113, AXMI-005 see, e.g., U.S. Patent Application Publication No. 2013/0104259 Al
  • AXMI-134 see, e.g., U.S. Patent Application Publication No. 2013/0167264 Al
  • AXMI-150 see, e.g., U.S. Patent Application Publication No.
  • AXMI-184 see, e.g., U.S. Patent Application Publication No. 2010/0004176 Al
  • AXMI-196 see, e.g., U.S. Patent Application Publication No. 2010/0004176 Al
  • AXMI-204 see, e.g., U.S. Patent Application Publication No. 2010/0004176 Al
  • AXMI-204 see, e.g., AXMI-204
  • AXMI-207 see, e.g., U.S. Patent Application Publication No. 2011/0030096
  • AXMI-218, AXMI-220 see, e.g., U.S. Patent Application Publication No. 2014/0245491
  • AXMI-221z see, e.g., AXMI-222z, AXMI-223z, AXMI- 224z, AXMI-225z
  • AXMI- 238 see, e.g., U.S. Patent Application Publication No. 2014/0033363
  • AXMI-270 see, e.g., U.S. Patent Application Publication No. 2014/0223598
  • AXMI-345 see, e.g., U.S. Patent Application Publication No. 2014/0373195
  • AXMI-335 see, e.g., International Application Publication No. WO2013/134523
  • DIG-3 see, e.g., U.S. Patent Application Publication No. 2013/0219570
  • DIG-5 see, e.g., U.S. Patent Application Publication No.
  • DIG-11 see, e.g., U.S. Patent Application Publication No. 2010/0319093
  • AfIP-lA and derivatives thereof see, e.g., U.S. Patent Application Publication No. 2014/0033361
  • AfIP-lB and derivatives thereof see, e.g., U.S. Patent Application Publication No. 2014/0033361
  • PIP-1APIP-1B see, e.g., U.S. Patent Application Publication No. 2014-0007292
  • PSEEN3174 see, e.g., U.S. Patent Application Publication No. 2014/0007292
  • AECFG-592740 see, e.g., U.S. Patent Application Publication No.
  • 2012/0192310 GS155 and derivatives thereof (see, e.g., U.S. Patent Application Publication Nos. 2012/0192310, 2012/0167259, 2012/0047606, 2011/0154536, 2011/0112013, 2010/0192256, 2010/0077507, 2010/0077508, 2009/0313721, 2010/0269221, and U.S. Patent No. 7,772,465), CF161_0085 and derivatives thereof (see, e.g., International Application Publication No. WO 2014/008054), Lepidopteran toxic proteins and their derivatives (see, e.g., US Patent Application Publications Nos. 2008/0172762, 2011/0055968, and 2012/0117690; and U.S. Patent Nos. 7,510,878 and 7,812, 129). All patent references cited above are incorporated herein by reference in their entirety.
  • the insecticidal protein is chosen from a Coleopteran inhibiting protein, such as, but not limited to, Cry3Bb (see, e.g., U.S. Patent No. 6,501,009), CrylC variants, Cry3A variants, Cry3, Cry3B, Cry34/35, 5307, AXMI134 (see, e.g., U.S. Patent Application Publication No. 2013/0167264) AXMI-184 (see, e.g., U.S. Patent Application Publication No. 2010/0004176), AXMI-205 (see, e.g., U.S. Patent Application Publication No.
  • an insecticidal protein may be CrylAc or TIC2463 from Bacillus thuringiensis, or a combination thereof.
  • an insecticidal protein encoded by a DNA molecule or plasmid introduced into a plant-associated Bacillus bacterium via a horizontal gene transfer technique is a CrylAc protein.
  • an insecticidal protein comprises or consists of a protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or similar to SEQ ID NO: 2.
  • an insecticidal protein comprises or consists of SEQ ID NO: 2.
  • a DNA molecule or plasmid introduced into a plant-associated Bacillus bacterium via a horizontal gene transfer technique comprises a coding sequence encoding a CrylAc protein.
  • the coding sequence of a DNA molecule or plasmid comprises or consists of a DNA sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.
  • the coding sequence of a DNA molecule or plasmid comprises or consists of SEQ ID NO: 1.
  • an insecticidal protein encoded by a DNA molecule or plasmid introduced into a plant-associated Bacillus bacterium via a horizontal gene transfer technique is a TIC2463 protein.
  • an insecticidal protein comprises or consists of a protein sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or similar to SEQ ID NO: 4.
  • an insecticidal protein comprises or consists of SEQ ID NO: 4.
  • a DNA molecule or plasmid introduced into a plant- associated Bacillus bacterium via a horizontal gene transfer technique comprises a coding sequence encoding a TIC2463 protein.
  • the coding sequence of a DNA molecule or plasmid comprises or consists of a DNA sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3.
  • the coding sequence of a DNA molecule or plasmid comprises or consists of SEQ ID NO: 3.
  • a coding sequence of a DNA molecule or plasmid encoding an insecticidal protein is associated and operably linked to a bacterial promoter, a plant-associated bacterial promoter, plant-associated Bacillus promoter, which may be heterologous with respect to the coding sequence.
  • the bacterial promoter is a TIC1201 promoter.
  • the bacterial promoter comprises a DNA sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5.
  • the coding sequence of a DNA molecule or plasmid comprises or consists of SEQ ID NO: 5.
  • the bacterial promoter is a TIC2463 promoter.
  • the bacterial promoter comprises a DNA sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.
  • the coding sequence of a DNA molecule or plasmid comprises or consists of SEQ ID NO: 6.
  • a modified plant-associated bacteria comprises a DNA molecule or plastid encoding an insecticidal protein with activity against insects, which may be within one or more of the Anoplura, Coleoptera, Dermaptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera, Mallophaga, Orthroptera, Siphonaptera, Thysanoptera, and/or Trichoptera taxonomic orders of insects, and any combination thereof.
  • the modified plant-associated bacteria provides insecticidal activity against insects chosen from the Lepidopterans and Coleopterans orders, or a combination thereof.
  • Insects of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the Family Noctuidae, e.g., fall armyworm (Spodoptera frugiperda).
  • insect pests of order Lepidoptera include, e.g., Alabama argillacea (cotton leaf worm), Archips argyrospila (fruit tree leaf roller), Archips rosana (European leafroller) and other Archips species, Chilo suppressalis (Asiatic rice borer, or rice stem borer), Cnaphalocrocis medinalis (rice leaf roller), Crambus caliginosellus (com root webworm), Crambus teterrellus (bluegrass webworm), Diatraea grandiosella (s Wyoming corn borer), Diatraea saccharalis (surgarcane borer), Earias insulana (spiny bollworm), Earias vittella (spotted bollworm), Helicoverpa armigera (American bollworm), Helicoverpa zea (com earworm or cotton bollworm), Heliothis virescens (tobacco budworm), Herpetogramma licarsisalis (sod web
  • Insects of the order Coleoptera include, but are not limited to, Agrotis spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata (Colorado potato beetle, CPB), Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabaeidae, Sitophilus spp., Sitotroga spp., Tenebri
  • Diabrotica virgifera virgifera Western Com Rootworm, WCR
  • Diabrotica barberi Northern Com Rootworm, NCR
  • Diabrotica virgifera zeae MCR
  • Diabrotica balteata Brazilian Corn Rootworm, BZR
  • Diabrotica undecimpunctata howardii Southern Corn Rootworm, SCR
  • a Brazilian Corn Rootworm complex BCR consisting of Diabrotica viridula and Diabrotica speciosa.
  • a modified plant-associated bacteria comprises a DNA molecule or plastid encoding an insecticidal protein with activity against an insect chosen from Black cutworm (BCW), Colorado Potato Beetle (CPB), Diamondback moth (DBM), or Western corn rootworm (WCR), or a combination thereof.
  • composition or formulation comprising a modified plant-associated bacterial cell, or a population, etc., of modified plant- associated bacteria, and an agriculturally acceptable carrier, wherein the modified plant-associated bacterial cell or bacteria express(es) at least one insecticidal protein.
  • compositions of the present disclosure may further comprise an agriculturally acceptable carrier in combination with the modified plant-associated bacteria.
  • the term “agriculturally acceptable” in reference to a carrier, material, ingredient or substance of a composition comprising a modified plant-associated bacteria means that the carrier, material, ingredient or substance, as the case may be, (i) is compatible with other ingredients of the composition at least for the purpose in which the composition will be used, (ii) can be included in the composition to effectively and viably deliver the modified plant-associated bacteria to a plant, plant part, plant seed, or plant growth medium (e.g., soil), (iii) is not normally associated with the plant-associated bacteria in nature (at least in the form in which it will be used), and (iv) is not deleterious to a plant, plant part, or plant seed to which the composition will be associated or applied (at least in the manner and amount in which it will be applied to, or associated with, the plant, plant part, or plant seed).
  • a “carrier” is defined as any substance or material that may be used and/or combined with a modified plant-associated bacteria to improve the delivery or effectiveness of the modified plant-associated bacteria to a plant, plant part, plant seed or soil.
  • An agriculturally acceptable carrier may include a soil-compatible carrier, a seed-compatible carrier, and/or a foliar- compatible carrier.
  • soil-compatible carrier refers to a material that can be added or applied to a soil without causing/having an unduly adverse effect on plant yield, soil structure, soil drainage, or the like.
  • seed-compatible carrier refers to a material that can be added or applied to a seed without causing/having an unduly adverse effect on the seed, seed germination, the plant that grows from the seed, or the like.
  • foliar-compatible carrier refers to a material that can be added or applied to an above ground portion of a plant or plant part without causing/having an unduly adverse effect on plant yield, plant health, or the like. Selection of appropriate carrier materials will depend on the intended application(s) and the microorganism(s) present in the composition.
  • the carrier material(s) may be selected and/or combined to provide a composition or formulation in the form of a liquid, gel, slurry, or solid.
  • compositions may comprise one or more liquid and/or gel carriers, and/or one or more aqueous and/or non-aqueous solvents.
  • non-aqueous may refer to a composition, solvent or substance that comprises no more than a trace amount of water (e.g., no more than 0.5% water by weight).
  • compositions may be in solid or powder form and/or comprise one or more solid carriers.
  • compositions may comprise one or more powders (e.g., wettable powders) and/or granules.
  • powders e.g., wettable powders
  • solid carriers Non-limiting examples of solid carriers that can be useful in compositions of the present disclosure include peat-based powders and granules, freeze-dried powders, spray-dried powders, and combinations thereof.
  • compositions of the present disclosure can be found in Burges, H.D., “Formulation of Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments,” Springer Science & Business Media (2012), the content and disclosure of which are incorporated herein by reference.
  • compositions in some embodiments may be in liquid or gel form and/or comprise one or more liquid and/or gel carriers.
  • Carriers in compositions or formulations may comprise a growth medium suitable for culturing one or more of the microorganisms in the composition.
  • compositions may comprise a Czapek-Dox medium, a glycerol yeast extract, a mannitol yeast extract, a potato dextrose broth, and/or a YEM media.
  • Commercial carriers may be used in accordance with a manufacturer’s recommended amounts or concentrations.
  • compositions may comprise one or more various solvents, such as organic, inorganic, non-aqueous and/or aqueous solvent(s).
  • inorganic solvents include decane, dodecane, hexylether, and nonane.
  • organic solvents include pentadecane, ISOPARTM M, ISOPARTM V, and ISOPARTM L (Exxon Mobil). Additional examples of solvents that may be included in compositions and formulations can be found in Burges, supra; Inoue & Horikoshi, J. Fermentation Bioeng. 71(3):194 (1991), the contents and disclosures of which are incorporated herein by reference.
  • an aqueous solvent such as water
  • a co-solvent such as ethyl lactate, methyl soyate/ethyl lactate cosolvent blends (e.g., STEPOSOL®, available from Stepan), isopropanol, acetone, 1,2-propanediol, n-alkylpyrrolidones (e.g., the AGSOLEX® series, available from ISP), a petroleum based-oil (e.g., AROMATICTM series and SOLVESSOTM series available from Exxon Mobil), isoparaffinic fluids (e.g., ISOPARTM series, available from Exxon Mobil), cycloparaffinic fluids (e.g., NAPPARTM 6, available from Exxon Mobil), mineral spirits (e.g., VARSOLTM series available from Exxon Mobil), and mineral oils (e.g., paraffin oil).
  • ethyl lactate methyl soyate/ethyl
  • compositions may comprise one or more co-solvent(s) in addition to an aqueous solvent or water.
  • co-solvent(s) may include, for example, non-aqueous solvents, such as one or more the foregoing non-aqueous solvents.
  • compositions including formulations may have a desired pH in a range from about 4.5 to about 9.5.
  • compositions may have a pH in a range from about 6 to about 8, or a pH of about 5, 5.5, 6, 6.5, 7, 7.5, 8 or 8.5.
  • a composition may comprise a buffer solution. pH buffers may be selected to provide an aqueous composition having a pH of less than 10, typically from about 5 to about 9, from about 6 to about 8, or about 7. Buffer solutions suitable for a variety of pH ranges are known in the art.
  • compositions may comprise one or more thickeners, rheology modifying agents, or stabilizing agents (“stabilizers”).
  • stabilizers include anionic polysaccharides and cellulose derivatives.
  • a stabilizer may comprise, for example, a clay, a silica, or a colloidal hydrophilic silica.
  • Non-limiting examples of commercially available stabilizers include KELZAN® CC (Kelco), methyl cellulose, carboxymethylcellulose and 2- hydroxyethylcellulose, hydroxymethylcellulose, kaolin, maltodextrin, malt extract, and microcrystalline cellulose.
  • a non-limiting example of a commercially available colloidal hydrophilic silica is AEROSIL® (Evonik).
  • a stabilizer may also include a disaccharide, such as maltose, trehalose, lactose, sucrose, cellobiose, and any combination thereof.
  • a stabilizer component may comprise from about 0.05% to about 10% by weight of a composition.
  • a stabilizer component may comprise from about 0.1% to about 5%, from about 0.1% to about 2%, or from about 0.1% to about 1% by weight of a composition.
  • compositions in some embodiments may comprise one or more oxidation control components, which may include one or more antioxidants (e.g., one or more of: ascorbic acid, ascorbyl palmitate, ascorbyl stearate, calcium ascorbate, carotenoids, lipoic acid, phenolic compounds (e.g., one or more flavonoids, flavones and/or flavonols), potassium ascorbate, sodium ascorbate, one or more thiols (e.g., glutathione, lipoic acid and/or N-acetyl cysteine), tocopherols, one or more tocotrienols, ubiquinone and/or uric acid) and/or one or more oxygen scavengers, such as ascorbic acid and/or sodium hydrogen carbonate.
  • antioxidants e.g., one or more of: ascorbic acid, ascorbyl palmitate, ascorbyl stearate, calcium ascorbate, carotenoids
  • compositions in some embodiments may comprise one or more agriculturally acceptable polymers, such as agar, alginate, carrageenan, cellulose, guar gum, locust bean gum, methylcellulose, pectin, polycaprolactone, polylactide, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, starch and/or xanthan gum.
  • agriculturally acceptable polymers such as agar, alginate, carrageenan, cellulose, guar gum, locust bean gum, methylcellulose, pectin, polycaprolactone, polylactide, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, starch and/or xanthan gum.
  • the one or more polymers is a natural polymer (e.g., agar, starch, alginate, pectin, cellulose, etc.), a synthetic polymer, a biodegradable polymer (e.g., polycaprolactone, polylactide, polyvinyl alcohol, etc.), or a combination thereof.
  • a natural polymer e.g., agar, starch, alginate, pectin, cellulose, etc.
  • a synthetic polymer e.g., a synthetic polymer, a biodegradable polymer (e.g., polycaprolactone, polylactide, polyvinyl alcohol, etc.), or a combination thereof.
  • a biodegradable polymer e.g., polycaprolactone, polylactide, polyvinyl alcohol, etc.
  • compositions in some embodiments may comprise one or more agriculturally acceptable wetting agents or dispersants (“dispersants”), which may include one or more surfactants.
  • Dispersants may be used to maintain a homogeneous or even distribution of particles or cells in a suspension, such as an even or homogeneous distribution of a modified plant- associated bacteria, which may be particularly useful for solid or dried formulations of a microbe and/or liquid formulations or fermentates.
  • a dispersant or wetting agent may also facilitate mixing of a microbe with other ingredients and solvents of a microbial formulation or composition and avoid aggregation or clumping of particles, or their adherence to container walls, etc., during formulation of a microbial composition.
  • Compositions may comprise a primary dispersant in combination with one or more secondary dispersants, and the primary and secondary dispersants may be different types (e.g., non-ionic, cationic, and/or anionic).
  • Wetting agents may be used with compositions applied to soils, particularly hydrophobic soils, to improve the infiltration and/or penetration of water into a soil.
  • the wetting agent or dispersant may be an adjuvant, oil, surfactant, buffer, acidifier, or combination thereof.
  • the wetting agent or dispersant may be a surfactant, such as one or more non-ionic surfactants, one or more cationic surfactants, one or more anionic surfactants, or any combination thereof.
  • anionic surfactants include one or more alkyl carboxylates (e.g., sodium stearate), alcohol ether carboxylates, phenol ether carboxylates, alkyl sulfates (e.g., alkyl lauryl sulfate and/or sodium lauryl sulfate), alkyl ether sulfates, alcohol sulfates, alcohol ether sulfates, alkyl amido ether sulfates, alkyl aryl ether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates, alkyl aryl sulfonates, alkyl sulfonates, alkyl amide sulfonates, aryl sulfonates, alkyl benzene sulfonates, alkyl diphenyloxide sulfonate, alpha-olefin sul
  • Anionic surfactants may be either water soluble anionic surfactants, water insoluble anionic surfactants, or a combination of water soluble anionic surfactants and water insoluble anionic surfactants.
  • Other non-limiting examples of commercially available anionic surfactants include sodium dodecylsulfate (Na-DS, SDS), MORWET® D-425 (a sodium salt of alkyl naphthalene sulfonate condensate, available from Akzo Nobel), MORWET® D-500 (a sodium salt of alkyl naphthalene sulfonate condensate with a block copolymer, available from Akzo Nobel), sodium dodecylbenzene sulfonic acid (Na-DBSA) (Aldrich), diphenyloxide disulfonate, naphthalene formaldehyde condensate, DOWFAXTM (Dow), dihexylsulfosuccinate, and
  • non-ionic surfactants include sorbitan esters, ethoxylated sorbitan esters, alkoxylated alkylphenols, alkoxylated alcohols, block copolymer ethers, and lanolin derivatives.
  • the surfactant comprises an alkylether block copolymer.
  • water insoluble nonionic surfactants include alkyl and aryl: glycerol ethers, glycol ethers, ethanolamides, sulfoanylamides, alcohols, amides, alcohol ethoxylates, glycerol esters, glycol esters, ethoxylates of glycerol ester and glycol esters, sugar- based alkyl polyglycosides, polyoxyethylenated fatty acids, alkanolamine condensates, alkanolamides, tertiary acetylenic glycols, polyoxyethylenated mercaptans, carboxylic acid esters, polyoxyethylenated polyoxyproylene glycols, sorbitan fatty esters, or combinations thereof. Also included are EO/PO block copolymers (EO is ethylene oxide, PO is propylene oxide), EO polymers and copolymers, polyamines, and polyvinylene
  • water soluble non-ionic surfactants include sorbitan fatty acid alcohol ethoxylates and sorbitan fatty acid ester ethoxylates.
  • Further nonlimiting examples of commercially available non-ionic surfactants include SPAN® 20, SPAN® 40, SPAN® 80, SPAN® 65, and SPAN® 85 (Aldrich); TWEEN® 20, TWEEN® 40, TWEEN® 60, TWEEN® 80, and TWEEN® 85 (Aldrich); IGEPAL® CA-210, IGEPAL® CA-520, IGEPAL® CA- 720, IGEPAL® CO-210, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CO-720, IGEPAL® CO- 890, and IGEPAL® DM-970 (available from Aldrich); TRITONTM X-100 (Aldrich); BRIJ® S10, BRIJ® S20, BRIJ® 30, BRIJ® 52, B
  • compositions may comprise at least one or more nonionic surfactants, such as at least one water-insoluble nonionic surfactant, at least one water soluble nonionic surfactant, or combinations thereof.
  • the compositions comprise a combination of nonionic surfactants having hydrocarbon chains of substantially the same length.
  • Non-limiting examples of cationic surfactants include mono alkyl quaternary amine, fatty acid amide surfactants, amidoamine, imidazoline, and polymeric cationic surfactants.
  • Surfactants may also include soaps, organosilicone surfactants, and silicone- based antifoams used as surfactants in silicone-based and mineral-oil based antifoams.
  • compositions may also comprise alkali metal salts of fatty acids (e.g., water soluble alkali metal salts of fatty acids and/or water insoluble alkali metal salts of fatty acids).
  • compositions in some embodiments may comprise at least 5 g/L, at least 10 g/L, at least 15 g/L, at least 20 g/L, at least 25 g/L, at least 30 g/L, at least 35 g/L, at least 40 g/L, at least 45 g/L, or at least 50 g/L of a dispersant(s).
  • the dispersant may be from about 1 to about 100 g/L, from about 5 to about 75 g/L, or from about 20 to about 50 g/L.
  • the amount of dispersants may also be expressed as a percentage by weight of a composition, such as about 0.5% to about 20%, from about 0.5% to about 10%, from about 0.5% to about 5%, from about 0.5% to about 8%, from about 0.5% to about 5%, or from about 1% to about 4% by weight of the composition.
  • compositions in some embodiments may comprise one or more agriculturally acceptable drying agents, such as calcium stearate, one or more clays, graphite, magnesium stearate, magnesium sulfate, powdered milk, one or more silica powders, soy lecithin and/or talc.
  • agriculturally acceptable drying agents such as calcium stearate, one or more clays, graphite, magnesium stearate, magnesium sulfate, powdered milk, one or more silica powders, soy lecithin and/or talc.
  • compositions in some embodiments may comprise one or more anti-freezing agents.
  • an anti-freezing agent may include one or more of ethylene glycol, alcohol, butanediol, pentanediol, mannitol, sorbitol, glycerol (glycerine), propylene glycol and/or urea.
  • the antifreeze agent may be present in a composition at a concentration of at least 5 g/L, at least 10 g/L, at least 15 g/L, at least 20 g/L, at least 30 g/L, at least 40 g/L, at least 50 g/L, at least 60 g/L, at least 70 g/L, or at least 80 g/L, such as from about 1 to about 150 g/L, from about 10 to about 100 g/L, or from about 20 to about 80 g/L.
  • compositions in some embodiments may comprise one or more functionalized dendrimers to enhance the efficacy and/or stability of the composition.
  • functionalized dendrimers include poly(amidoamine) (PAMAM, Generations 0-7), poly(amidoamine-organosilicone) (PAMAMOS), polypropylene imidine) (PPI, Generations 0-5), poly(benzylethers) (Frechet-type), Arobols (Newkome type), poly(phenylacetylenes) and surface engineered dendrimers (e.g., PEGylated dendrimers, glycodendrimers, peptide funtionalized dendrimers, and galabiose-functionalized dendrimers).
  • Dendrimer(s) may comprise at least 0.1% and up to 10% or more, or from about 1% to about 10%, of the composition by weight.
  • compositions in some embodiments may comprise one or more antifoam agents.
  • antifoam agents include organosilicone or silicone-free compounds.
  • Non- limiting examples of commercially available antifoam products include BREAK-THRU® OE441 (Evonik), BREAK-THRU® AF9905 (Evonik), AGNIQUE® DF 6889 (Cognis), AGNIQUE® DFM 111S (Cognis), BYK®-016 (BYK), FG-10 antifoam emulsion (Dow Corning), 1520-US (Dow Coming), 1510-US (Dow Coming), SAG® 1538 (Momentive), and SAG® 1572 (Momentive).
  • compositions in some embodiments may comprise a crystallization inhibitor(s).
  • exemplary crystallization inhibitors include acrylic copolymers, polyethylene glycol, polyethylene glycol hydrogenated castor oil, and any combination thereof.
  • the crystallization inhibitor may be present, for example, at a concentration from about 1% to about 10% by weight of the composition.
  • compositions in some embodiments may comprise one or more viscosity modifying agents.
  • viscosity modifying agents include humic acid salts, fulvic acid salts, humin, and lignin salts, such as the sodium or potassium salt of humic acid.
  • compositions in some embodiments may comprise one or more additional excipients that improve the adhesion of the composition to a substrate or surface, such as a plant seed or other plant material, such as to provide a successful coating of the substrate or surface or otherwise impart improved characteristics to the adhesion or coating.
  • additional excipients that improve the adhesion of the composition to a substrate or surface, such as a plant seed or other plant material, such as to provide a successful coating of the substrate or surface or otherwise impart improved characteristics to the adhesion or coating.
  • Other substances may be added to a composition (e.g., coloring agents) to provide a visual indication of successful coating of the substrate or surface, such as the outer surface of a plant seed or other plant material.
  • compositions and formulations may further comprise one or more pesticidal agents.
  • Pesticidal agents include chemical pesticides and biopesticides or biocontrol agents.
  • Various types of chemical pesticides include acaricides, insecticides, nematicides, fungicides, gastropodicides, herbicides, virucides, bactericides, and combinations thereof.
  • Biopesticides or biocontrol agents may include bacteria, fungi, beneficial nematodes, and viruses that exhibit pesticidal activity.
  • Compositions may comprise other agents for pest control, such as microbial extracts, plant growth activators, and/or plant defense agents.
  • compositions in some embodiments may comprise one or more chemical acaricides, insecticides, and/or nematicides.
  • chemical acaricides, insecticides, and/or nematicides may include one or more carbamates, diamides, macrocyclic lactones, neonicotinoids, organophosphates, phenylpyrazoles, pyrethrins, spinosyns, synthetic pyrethroids, tetronic acids and/or tetramic acids.
  • Non-limiting examples of chemical acaricides, insecticides and nematicides that can be useful in compositions of the present disclosure include abamectin, acrinathrin, aldicarb, aldoxycarb, alpha-cypermethrin, betacyfluthrin, bifenthrin, cyhalothrin, cypermethrin, deltamethrin, csfenvalcrate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, fosthiazate, lambda-cyhalothrin, gamma-cyhalothrin, permethrin, tau-fluvalinate, transfluthrin, zeta-cypermethrin, cyfluthri, bifenthrin, tefluthrin, eflusilanat, fubfenprox, pyrethr
  • Additional non-limiting examples of chemical acaricides, insecticides, and/or nematicides may include one or more of abamectin, aldicarb, aldoxycarb, bifenthrin, carbofuran, chlorantraniliporle, chlothianidin, cyfluthrin, cyhalothrin, cypermethrin, cyantraniliprole, deltamethrin, dinotefuran, emamectin, ethiprole, fenamiphos, fipronil, flubendiamide, fosthiazate, imidacloprid, ivermectin, lambda-cyhalothrin, milbemectin, nitenpyram, oxamyl, permethrin, spinetoram, spinosad, spirodichlofen, spirotetramat, tefluthrin, thiacloprid,
  • acaricides, insecticides and nematicides that may be included or used in compositions may be found in Steffey and Gray, “Managing Insect Pests,” ILLINOIS AGRONOMY HANDBOOK (2008); and Niblack, Nematodes, ILLINOIS AGRONOMY HANDBOOK (2008), the contents and disclosures of which are incorporated herein by reference.
  • Non-limiting examples of commercial insecticides which may be suitable for the compositions disclosed herein include CRUISER® (Syngenta, Wilmington, Del.), GAUCHO® and PONCHO® (Gustafson, Plano, TX).
  • Active ingredients in these and other commercial insecticides may include thiamethoxam, clothianidin, and imidacloprid.
  • Commercial acaricides, insecticides, and/or nematicides may be used in accordance with a manufacturer’s recommended amounts or concentrations.
  • compositions may comprise one or more biopesticidal microorganisms, the presence and/or output of which is toxic to an acarid, insect and/or nematode.
  • compositions may comprise one or more of Bacillus firmus 1-1582, Bacillus mycoides AQ726, NRRL B-21664; Beauveria bassiana ATCC-74040, Beauveria bassiana ATCC-74250, Burkholderia sp. A396 sp.
  • NRRL B-50319 Chromobacterium subtsugae NRRL B-30655, Chromobacterium vaccinii NRRL B-50880, Flavobacterium H492, NRRL B-50584, Metarhizium anisopliae F52 (also known as Metarhizium anisopliae strain 52, Metarhizium anisopliae strain 7, Metarhizium anisopliae strain 43, and/ or Metarhizium anisopliae BIO-1020, TAE-001; deposited as DSM 3884, DSM 3885, ATCC 90448, SD 170 and ARSEF 7711), Paecilomyces fumosoroseus FE991, and combinations thereof.
  • Metarhizium anisopliae F52 also known as Metarhizium anisopliae strain 52, Metarhizium anisopliae strain 7, Metarhizium anisopliae strain 43, and/ or Metarhizium anisopliae BIO-1020, T
  • compositions in some embodiments may comprise one or more chemical fungicides.
  • chemical fungicides may include one or more aromatic hydrocarbons, benzthiadiazole, carboxylic acid amides, morpholines, phenylamides, phosphonates, thiazolidines, thiophene, quinone outside inhibitors and strobilurins, such as azoxystrobin, coumethoxystrobin, coumoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl, metominostrobin, orysastrobin, picoxystrobin, pyraclostrobin, pyrametostrobin, pyraoxystrobin, pyribencarb, trifloxystrobin, 2 [2-(2,5-dimethyl-phenoxymethyl)-phenyl]-3- methoxy- acrylic acid methyl ester, and 2 (2-(3-(2,6-dichlor
  • organometal compounds e.g., fentin salts, such as fentin-acetate, fentin chloride, fentin hydroxide); sulfur-containing heterocyclyl compounds (e.g., dithianon, isoprothiolane), organophosphorus compounds (e.g., edifenphos, fosetyl, iprobenfos, phosphorus acid and its salts, pyrazophos, tolclofos-methyl), organochlorine compounds (e.g., chlorothalonil, dichlofluanid, dichlorophen, flusulfamide, hexachlorobenzene, pencycuron, pentachlorphenole and its salts, phthalide, quintozene, thiophanate-methyl, thiophanates, tolylfluanid, N-(4-chloro- 2-nitro-phenyl)-N-ethyl-4-methyl-benzenesulfonamide) and
  • compositions of the present disclosure comprise acibenzolar-S-methyl, azoxystrobin, benalaxyl, bixafen, boscalid, carbendazim, cyproconazole, dimethomorph, epoxiconazole, fludioxonil, fluopyram, fluoxastrobin, flutianil, flutolanil, fluxapyroxad, fosetyl-Al, ipconazole, isopyrazam, kresoxim-methyl, mefenoxam, metalaxyl, metconazole, myclobutanil, orysastrobin, penflufen, penthiopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, sedaxane, silthiofam, tebuconazole, thiabendazole, thifluzamide, thiophanate, tolclofos-methyl
  • fungicides that may be included in compositions, see, e.g., Bradley, Managing Diseases, ILLINOIS AGRONOMY HANDBOOK (2008), the content and disclosure of which are incorporated herein by reference.
  • Fungicides useful for compositions in some embodiments may exhibit activity against one or more fungal plant pathogens, including but not limited to Phytophthora, Rhiwctonia, Fusarium, Pythium, Phomopsis, Selerotinia or Phakopsora, and combinations thereof.
  • fungal plant pathogens including but not limited to Phytophthora, Rhiwctonia, Fusarium, Pythium, Phomopsis, Selerotinia or Phakopsora, and combinations thereof.
  • Non-limiting examples of commercial fungicides which may be suitable for the compositions include PROTEGETM, RIVALTM or ALLEGIANCETM FL or LS (Gustafson, Plano, Texax), WARDENTM RTA (Agrilance, St.
  • Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides may be used in accordance with a manufacturer’s recommended amounts or concentrations.
  • compositions may comprise one or more biopesticidal microorganisms, the presence and/or output of which is toxic to at least one fungus, bacteria, or both.
  • compositions may comprise one or more of Ampelomyces quisqualis AQ 10® (Intrachem Bio GmbH & Co.
  • catenulata also referred to as Gliocladium catenulatum J1446 (PRESTOP®, Verdera, Finland), Coniothyrium minitans CONTANS® (Prophyta, Germany), Cryphonectria parasitica (CNICM, France), Cryptococcus albidus YIELD PLUS® (Anchor Bio Technologies, South Africa), Fusarium oxysporum BIOFOX® (from S.I.A.P.A., Italy) and FUSACLEAN® (Natural Plant Protection, France), Metschnikowia fructicola SHEMER® (Agrogreen, Israel), Microdochium dimerum ANTIBOT® (Agrauxine, France), Muscodor albus NRRL 30547, Muscodor roseus NRRL 30548, Phlebiopsis gigantea ROTSOP® (Verdera, Finland), Pseudozyma flocculosa SPORODEX® (Plant Products Co.
  • Trichoderma viride TV 1 Agribiotec srl, Italy
  • Trichoderma viride ICC080 Trichoderma viride ICC080
  • Ulocladium oudemansii HRU3 BOTRY ZEN®, Botry Zen Ltd, NZ
  • compositions in some embodiments may comprise one or more chemical gastropodicides.
  • chemical gastropodicides include one or more iron phosphates, metaldehydes, methiocarbs and/or salts.
  • examples of commercial gastropodicides that may be useful in compositions include DEADLINE® M-PsTM, MESUROL PRO®, MESUROL® 75-W, METAREX®, SLUGGO®, and combinations thereof. Additional examples of gastropodicides that can be included in compositions of the present disclosure can be found in Capinera, Handbook of Vegetable Pests (2001), the content and disclosure of which is incorporated herein by reference.
  • Commercial gastropodicides may be used in accordance with a manufacturer’s recommended amounts and concentrations.
  • compositions in some embodiments may comprise one or more chemical herbicides.
  • the herbicides may be a pre-emergent herbicide, a post-emergent herbicide, or a combination thereof.
  • Non-limiting examples of chemical herbicides may comprise one or more acetyl CoA carboxylase (ACCase) inhibitors, acetolactate synthase (ALS) inhibitors, acetanilides, acetohydroxy acid synthase (AHAS) inhibitors, photosystem II inhibitors, photosystem I inhibitors, protoporphyrinogen oxidase (PPO or Protox) inhibitors, carotenoid biosynthesis inhibitors, enolpyruvyl shikimate- 3 -phosphate synthase (EPSPS) inhibitors, glutamine synthetase inhibitors, dihydropteroate synthetase inhibitors, mitosis inhibitors, 4-hydroxyphenyl-pyruvate- dioxygenase (4-HPPD) inhibitors, synthetic
  • Non-limiting examples of chemical herbicides that can be useful in compositions of the present disclosure include 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5- trichlorophenoxyacetic acid (2,4,5-T), ametryn, amicarbazone, aminocyclopyrachlor, acetochlor, acifluorfen, alachlor, atrazine, azafenidin, bentazon, benzofenap, bifenox, bromacil, bromoxynil, butachlor, butafenacil, butroxydim, carfentrazone-ethyl, chlorimuron, chlorotoluro, clethodim, clodinafop, clomazone, cyanazine, cycloxydim, cyhalofop, desmedipham, desmetryn, dicamba, diclofop, dimefuron, diflufenican, diuron, dithiopyr, ethof
  • compositions of the present disclosure comprise acetochlor, clethodim, dicamba, flumioxazin, fomesafen, glyphosate, glufosinate, mesotrione, quizalofop, saflufenacil, sulcotrione, S-3100 and/or 2,4-D, and combinations thereof.
  • herbicides that may be included in compositions in some embodiments may be found in Hager, Weed Management, Illinois Agronomy Handbook (2008); and Loux et al., Weed Control Guide for Ohio, Indiana and Illinois (2015), the contents and disclosures of which are incorporated herein by reference. Commercial herbicides may be used in accordance with a manufacturer’s recommended amounts or concentrations.
  • compositions in some embodiments may comprise one or more virucides.
  • compositions may comprise one or more biopesticidal or herbicidal microorganisms, the presence and/or output of which is toxic to at least one insect, plant (weed), or phytopathogenic virus.
  • biopesticides that may be included or used in compositions may be found in BURGES, supra-, HALL & MENN, “BIOPESTICIDES: USE AND DELIVERY” (Humana Press) (1998); McCoy et al., “Entomogenous fungi,” in CRC HANDBOOK OF NATURAL PESTICIDES. MICROBIAL PESTICIDES, PART A. ENTOMOGENOUS PROTOZOA AND FUNGI (C. M. Inoffo, ed.), Vol.
  • a biocontrol microbe may comprise a bacterium of the genus Actinomycetes, Agrobacterium, Arthrobacter, Alcaligenes, Aureobacterium, Azobacter, Bacillus, Beijerinckia, Brevibacillus, Burkholderia, Chromobacterium, Clostridium, Clavibacter, Comamonas, Corynebacterium, Curtobacterium, Enterobacter, Flavobacterium, Gluconobacter, Hydrogenophaga, Klebsiella, Methylobacterium, Paenibacillus, Pasteuria,, Photorhabdus, Phyllobacterium, Pseudomonas, Rhizobium, Serratia, Sphingobacterium, Stenotrophomonas, Variovorax, and Xenorhabdus, or any combination thereof.
  • a biopesticidal microbe may include one or more of Bacillus amyloliquefaciens, Bacillus cereus, Bacillus firmus , Bacillus lichenformis, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis , Chromobacterium suttsuga, Pasteuria penetrans, Pasteuria usage, and Pseudomona fluorescens.
  • a biopesticidal microbe may comprise a fungus of the genus Alternaria, Ampelomyces, Aspergillus, Aureobasidium, Beauveria, Colletotrichum, Coniothyrium, Gliocladium, Metarhizium, Muscodor, Paecilomyces, Trichoderma, Typhula, Ulocladium, and Verticillium.
  • a fungus is Beauveria bassiana, Coniothyrium minitans, Gliocladium virens, Muscodor albus, Paecilomyces lilacinus, or Trichoderma polysporum.
  • a composition in some embodiments may comprise one or more biocidal agents.
  • a biocidal component may be included or used to prevent fungal and/or bacterial growth in the composition, particularly when the composition is placed in storage.
  • biocidal agents include dichlorophen or benzyl alcohol hemiformal based compounds, benzoisothiazolinones and rhamnolipids.
  • Non-limiting examples of commercially available biocidal agents include ACTICIDE® (Thor), PROXEL® (Arch Chemical), and ZONIXTM (Jeneil).
  • compositions and formulations may further comprise one or more agriculturally beneficial agents, such as biostimulants, nutrients, plant signal molecules, or biologically active agents.
  • compositions may comprise one or more beneficial biostimulants.
  • Biostimulants may enhance metabolic or physiological processes such as respiration, photosynthesis, nucleic acid uptake, ion uptake, nutrient delivery, or a combination thereof.
  • the biostimulants may comprise one or more Azospirillum extracts (e.g., an extract of media comprising A.
  • brasilense INTA Az-39 one or more Bradyrhizobium extracts (e.g., an extract of media comprising B. elkanii SEMIA 501, B. elkanii SEMIA 587, B. elkanii SEMIA 5019, B. japonicum NRRL B-50586 (also deposited as NRRL B-59565), B. japonicum NRRL B-50587 (also deposited as NRRL B-59566), B. japonicum NRRL B-50588 (also deposited as NRRL B-59567), B. japonicum NRRL B-50589 (also deposited as NRRL B-59568), B.
  • a Bradyrhizobium extracts e.g., an extract of media comprising B. elkanii SEMIA 501, B. elkanii SEMIA 587, B. elkanii SEMIA 5019, B. japonicum NRRL B-50586 (
  • japonicum NRRL B-50590 also deposited as NRRL B-59569
  • B. japonicum NRRL B-50591 also deposited as NRRL B-59570
  • B. japonicum NRRL B-50592 also deposited as NRRL B-59571
  • B. japonicum NRRL B-50593 also deposited as NRRL B- 59572
  • B. japonicum NRRL B-50594 also deposited as NRRL B-50493
  • B. japonicum NRRL B-50609 B. japonicum NRRL B-50610
  • japonicum NRRL B-50612 B. japonicum NRRL B-50726, B. japonicum NRRL B- 50727, B. japonicum NRRL B-50728, B. japonicum NRRL B-50729, B. japonicum NRRL B- 50730, B. japonicum SEMIA 566, B. japonicum SEMIA 5079, B. japonicum SEMIA 5080, B. japonicum USDA 6, B. japonicum USDA 110, B. japonicum USDA 122, B. japonicum USDA 123, B. japonicum USDA 127, B. japonicum USDA 129 and/or B.
  • Rhizobium extracts e.g., an extract of media comprising R. leguminosarum SO12A-2
  • Sinorhizobium extracts e.g., an extract of media comprising S. fredii CCBAU114 and/or S. fredii USDA 205
  • Penicillium extracts e.g., an extract of media comprising P. bilaiae ATCC 18309, P. bilaiae ATCC 20851, P. bilaiae ATCC 22348, P. bilaiae NRRL 50162, P. bilaiae NRRL 50169, P.
  • bilaiae NRRL 50787 P. bilaiae NRRL 50788, P. bilaiae RS7B-SD1, P. brevicompactum AgRF18, P. canescens ATCC 10419, P. expansum ATCC 24692, P. expansum YT02, P. fellatanum ATCC 48694, P. gaestrivorus NRRL 50170, P. glabrum DAOM 239074, P. glabrum CBS 229.28, P. janthinellum ATCC 10455, P. lanosocoeruleum ATCC 48919, P. radicum ATCC 201836, P. radicum FRR 4717, P.
  • radicum FRR 4719, P. radicum N93/47267 and/or P. raistrickii ATCC 10490 one or more Pseudomonas extracts (e.g., an extract of media comprising P.jessenii PS06), one or more acaricidal, insecticidal and/or nematicidal extracts (e.g., an extract of media comprising Bacillus firmus 1-1582, Bacillus mycoides AQ726, NRRL B-21664; Beauveria bassiana ATCC-74040, Beauveria bassiana ATCC-74250, Burkholderia sp. A396 sp. nov.
  • Pseudomonas extracts e.g., an extract of media comprising P.jessenii PS06
  • acaricidal, insecticidal and/or nematicidal extracts e.g., an extract of media comprising Bacillus firmus 1-1582, Bac
  • rinojensis NRRL B-50319, Chromobacterium subtsugae NRRL B-30655, Chromobacterium vaccinii NRRL B-50880, Flavobacterium H492, NRRL B-50584, Metarhizium anisopliae F52 (also known as Metarhizium anisopliae strain 52, Metarhizium anisopliae strain 7, Metarhizium anisopliae strain 43 and Metarhizium anisopliae BIO-1020, TAE-001; deposited as DSM 3884, DSM 3885, ATCC 90448, SD 170 and ARSEF 7711) and/or Paecilomyces fumosoroseus FE991), and/or one or more fungicidal extracts (e.g., an extract of media comprising Ampelomyces quisqualis AQ 10® (Intrachem Bio GmbH & Co.
  • fungicidal extracts e.g., an extract of media
  • catenulata also referred to as Gliocladium catenulatum J1446 (PRESTOP®, Verdera, Finland), Coniothyrium minitans CONTANS® (Prophyta, Germany), Cryphonectria parasitica (CNICM, France), Cryptococcus albidus YIELD PLUS® (Anchor Bio Technologies, South Africa), Fusarium oxysporum BIOFOX® (from S.I.A.P.A., Italy) and FUSACLEAN® (Natural Plant Protection, France), Metschnikowia fructicola SHEMER® (Agrogreen, Israel), Microdochium dimerum ANTIBOT® (Agrauxine, France), Muscodor albus NRRL 30547, Muscodor roseus NRRL 30548, Phlebiopsis gigantea ROTSOP® (Verdera, Finland), Pseudozyma flocculosa SPORODEX® (Plant Products Co.
  • compositions in some embodiments may comprise one or more biologically active ingredients.
  • Non- limiting examples of biologically active ingredients include plant growth regulators, plant signal molecules, growth enhancers, microbial stimulating molecules, biomolecules, soil amendments, nutrients, plant nutrient enhancers, etc., such as lipo- chitooligosaccharides (LCO), chitooligosaccharides (CO), chitinous compounds, flavonoids, jasmonic acid or derivatives thereof (e.g., jasmonates), cytokinins, auxins, gibberellins, absiscic acid, ethylene, brassinosteroids, salicylates, macro- and micro-nutrients, linoleic acid or derivatives thereof, linolenic acid or derivatives thereof, karrikins, etc.) and beneficial microorganisms (e.g., Rhiz.ohium spp., Bradyrhizobium spp., Sinorhizobium spp., Azorhizobium spp., Glomus spp.
  • compositions in some embodiments may comprise one or more lipo- chitooligosaccharides (LCOs), chitooligosaccharides (COs), and/or chitinous compounds.
  • LCOs sometimes referred to as symbiotic nodulation (Nod) signals (or Nod factors) or as Myc factors, consist of an oligosaccharide backbone of P-l,4-linked N-acetyl D glucosamine (“GlcNAc”) residues with an N-linked fatty acyl chain condensed at the non-reducing end.
  • LCOs differ in the number of GlcNAc residues in the backbone, in the length and degree of saturation of the fatty acyl chain and in the substitutions of reducing and non-reducing sugar residues. See, e.g., Denarie et al., Ann. Rev. Biochem. 65:503 (1996); Diaz et al., Mol. Plant Microbe Interactions 13:268 (2000); Hungria et al., Soil Biol. Biochem. 29:819 (1997); Hamel et al., Planta 232:787 (2010); and Prome et al., Pure & Appl. Chem. 70( 1) :55 (1998), the contents and disclosures of which are incorporated herein by reference.
  • LCOs may be synthetic or obtained from any suitable source. See, e.g., WO 2005/063784, WO 2007/117500 and WO 2008/071674, the contents and disclosures of which are incorporated herein by reference.
  • a synthetic LCO may have the basic structure of a naturally occurring LCO but contains one or more modifications or substitutions, such as those described in Spaink, Crit. Rev. Plant Sci. 54:257 (2000).
  • LCOs and precursors for the construction of LCOs e.g., COs, which may themselves be useful as a biologically active ingredient
  • LCOs may be included or utilized in compositions in various forms of purity and can be used alone or in the form of a culture of LCO producing bacteria or fungi.
  • OPTIMIZE® commercially available from Monsanto Company (St. Louis, MO) contains a culture of Bradyrhizobium japonicum that produces LCO.
  • Methods to provide substantially pure LCOs include removing the microbial cells from a mixture of LCOs and the microbe, or continuing to isolate and purify the LCO molecules through LCO solvent phase separation followed by HPLC chromatography as described, for example, in U.S. Patent No. 5,549,718.
  • the LCO(s) included in compositions of the present disclosure is/are at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.
  • Compositions and methods described herein may comprise analogues, derivatives, hydrates, isomers, salts and/or solvates of LCOs.
  • compositions of the present disclosure may be incorporated into compositions of the present disclosure in any suitable amount(s)/concentration(s).
  • compositions of the present disclosure comprise about 1 x IO -20 M to about 1 x 10 1 M LCO(s).
  • compositions of the present disclosure can comprise about 1 x IO -20 M, 1 x IO -19 M, 1 x 10 -18 M, 1 x IO -17 M, 1 x 10 16 M, 1 x 10 15 M, 1 x 10 14 M, 1 x 10 13 M, 1 x IO 12 M, 1 x 10 11 M, 1 x IO 10 M, 1 x IO 9 M, 1 x 10 8 M, 1 x IO 7 M, 1 x IO -6 M, 1 x 10 -5 M, 1 x 10 -4 M, 1 x 10 -3 M, 1 x IO -2 M, 1 x 10 1 M of one or more LCOs.
  • the LCO concentration is 1 x 10 14 M to 1 x 10 -5 M, 1 x 10 12 M to 1 x IO -6 M, or 1 x 10" 10 M to 1 x 10’ 7 M.
  • the LCO concentration is 1 x 10 14 M to 1 x 10’ 5 M, 1 x 10 12 M to 1 x IO -6 M, or 1 x IO -10 M to 1 x IO -7 M.
  • the amount/concentration of LCO may be an amount effective to impart a positive trait or benefit to a plant, such as to enhance the growth and/or yield of the plant to which the composition is applied.
  • compositions in some embodiments may comprise any suitable COs, perhaps in combination with one or more LCOs.
  • COs differ from LCOs in that they lack the pendant fatty acid chain that is characteristic of LCOs.
  • COs, sometimes referred to as N acetylchitooligosaccharides, are also composed of GlcNAc residues but have side chain decorations that make them different from chitin molecules [(CSHBNOS) ⁇ CAS No. 1398 61 4] and chitosan molecules [(CsHnNO ⁇ n, CAS No.
  • COs may be obtained from any suitable source.
  • the CO may be derived from an LCO.
  • compositions of the present disclosure comprise one or more COs derived from an LCO obtained (i.e., isolated and/or purified) from a strain of Azorhizobium, Bradyrhizobium (e.g., B. japonicum), Mesorhizobium, Rhizobium (e.g., R. leguminosarum), Sinorhizobium (e.g., S. meliloti), or mycorhizzal fungi (e.g., Glomus intrar adieus).
  • the CO may be synthetic. Methods for the preparation of recombinant COs are known in the art.
  • COs may be included or utilized in compositions in some embodiments in various forms of purity and can be used alone or in the form of a culture of CO producing bacteria or fungi.
  • the CO(s) included in compositions may be at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more pure.
  • compositions and methods of the present disclosure can comprise hydrates, isomers, salts and/or solvates of COs.
  • COs may be incorporated into compositions of the present disclosure in any suitable amount(s)/concentration(s).
  • compositions of the present disclosure may comprise about 1 x 10-20 M to about 1 x 10-1 M COs, such as about 1 x IO’ 20 M, 1 x 10- 19 M, 1 x IO 18 M, 1 x IO 17 M, 1 x IO 16 M, 1 x IO 15 M, 1 x 10" 14 M, 1 x 10 13 M, 1 x IO 12 M, 1 x 10 11 M, 1 x IO 10 M, 1 x IO’ 9 M, 1 x 10’ 8 M, 1 x IO’ 7 M, 1 x IO -6 M, 1 x 10 -5 M, 1 x 10 -4 M, 1 x 10 -3 M, 1 x IO -2 M, or 1 x 10 1 M
  • the CO concentration may be 1 x 10 14 M to 1 x 10’ 5 M, 1 x 10 12 M to 1 x 10’ 6 M, or 1 x 10’ 10 M to 1 x 10’ 7 M.
  • the amount/concentration of CO may be an amount effective to impart or confer a positive trait or benefit to a plant, such as to enhance the soil microbial environment, nutrient uptake, or increase the growth and/or yield of the plant to which the composition is applied.
  • a CO amount/concentration may not be effective to enhance the growth of the plant without beneficial contributions from one or more other ingredients of the composition, such as LCO and/or one or more inoculants, biomolecules, nutrients, or pesticides.
  • compositions in some embodiments may comprise one or more suitable chitinous compounds, such as, for example, chitin (IUPAC: N-[5-[[3-acetylamino-4,5-dihydroxy- 6-(hydroxymethyl)oxan-2yl]methoxymethyl]-2-[[5-acetylamino-4,6 dihydroxy-2-
  • suitable chitinous compounds such as, for example, chitin (IUPAC: N-[5-[[3-acetylamino-4,5-dihydroxy- 6-(hydroxymethyl)oxan-2yl]methoxymethyl]-2-[[5-acetylamino-4,6 dihydroxy-2-
  • Chitins and chitosans which are major components of the cell walls of fungi and the exoskeletons of insects and crustaceans, are composed of GlcNAc residues. Chitins and chitosans may be obtained commercially or prepared from insects, crustacean shells, or fungal cell walls. Methods for the preparation of chitin and chitosan are known in the art. See, e.g., U.S. Patent Nos. 4,536,207 (preparation from crustacean shells) and 5,965,545 (preparation from crab shells and hydrolysis of commercial chitosan); and Pochanavanich et al., Lett. Appl. Microbiol. 35:17 (2002) (preparation from fungal cell walls).
  • Deacetylated chitins and chitosans may be obtained that range from less than 35% to greater than 90% deacetylation and cover a broad spectrum of molecular weights, e.g., low molecular weight chitosan oligomers of less than 15 kD and chitin oligomers of 0.5 to 2 kD; “practical grade” chitosan with a molecular weight of about 15 kD; and high molecular weight chitosan of up to 70 kD.
  • Chitin and chitosan compositions formulated for seed treatment are commercially available. Commercial products include, for example, ELEXA® (Plant Defense Boosters, Inc.) and BEYONDTM (Agrihouse, Inc.)
  • compositions in some embodiments may comprise one or more suitable flavonoids, including, but not limited to, anthocyanidins, anthoxanthins, chaicones, coumarins, flavanones, flavanonols, flavans and isoflavonoids, as well as analogues, derivatives, hydrates, isomers, polymers, salts and solvates thereof.
  • Flavonoids are phenolic compounds having the general structure of two aromatic rings connected by a three-carbon bridge. Classes of flavonoids are known in the art. See, e.g., Jain et al., J. Plant Biochem. & Biotechnol. 11:1 (2002); and Shaw et al., Environ.
  • Flavonoid compounds may be isolated from plants or seeds, e.g., as described in U.S. Patent Nos. 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds may also be produced by genetically engineered organisms, such as yeast. See, e.g., Ralston et al., Plant Physiol. 137:1375 (2005).
  • compositions may comprise one or more flavanones, such as one or more of butin, eriodictyol, hesperetin, hesperidin, homoeriodictyol, isosakuranetin, naringenin, naringin, pinocembrin, poncirin, sakuranetin, sakuranin, and/or sterubin, one or more flavanonols, such as dihydrokaempferol and/or taxifolin, one or more flavans, such as one or more flavan-3-ols (e.g., catechin (C), catechin 3-gallate (Cg), epicatechins (EC), epigallocatechin (EGC) epicatechin 3-gallate (ECg), epigallcatechin 3-gallate (EGCg), epiafzelechin, fisetinidol, gallocatechin (GC), gallcatechin 3-gallate (GCg),
  • flavanones such as one
  • Flavonoids and their derivatives may be included in compositions in any suitable form, including, but not limited to, polymorphic and crystalline forms. Flavonoids may be included in compositions in any suitable amount(s) or concentration(s).
  • the amount/concentration of a flavonoid(s) may be an amount effective to impart a benefit to a plant, which may be indirectly through activity on soil microorganisms or other means, such as to enhance plant nutrition and/or yield.
  • a flavonoid amount/concentration may not be effective to enhance the nutrition or yield of the plant without the beneficial contributions from one or more other ingredients of the composition, such as LCO, CO, and/or one or more pesticides.
  • compositions in some embodiments may comprise one or more suitable nonflavonoid nod-gene inducer(s), including, but not limited to, jasmonic acid ([lR-[la,2P(Z)]]-3- oxo-2-(pentenyl)cyclopentaneacetic acid; JA), linoleic acid ((Z,Z) -9, 12- Octadecadienoic acid) and/or linolenic acid ((Z,Z,Z)-9,12,15-octadecatrienoic acid), and analogues, derivatives, hydrates, isomers, polymers, salts and solvates thereof.
  • suitable nonflavonoid nod-gene inducer(s) including, but not limited to, jasmonic acid ([lR-[la,2P(Z)]]-3- oxo-2-(pentenyl)cyclopentaneacetic acid; JA), linoleic acid ((Z,Z
  • Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecanoid based compounds that occur naturally in some plants (e.g., wheat), fungi (e.g., Botryodiplodia theobromae, Gibbrella fujikuroi), yeast (e.g., Saccharomyces cerevisiae) and bacteria (e.g., Escherichia coli). Linoleic acid and linolenic acid may be produced during the biosynthesis of jasmonic acid.
  • fungi e.g., Botryodiplodia theobromae, Gibbrella fujikuroi
  • yeast e.g., Saccharomyces cerevisiae
  • bacteria e.g., Escherichia coli
  • Jasmonates, linoleic acid and linolenic acid (and their derivatives) are reported to be inducers of nod gene expression or LCO production by rhizobacteria. See, e.g., Mabood et al., PLANT PHYSIOL. BIOCHEM. 44(11):759 (2006); Mabood et al., AGR. J. 98(2):289 (2006); Mabood et al., FIELD CROPS RES.95(2-3):412 (2006); and Mabood & Smith, Linoleic and linolenic acid induce the expression of nod genes in Bradyrhizobium japonicum USDA 3, PLANT BIOL. (2001).
  • esters are compounds in which the carboxyl group of linoleic acid, linolenic acid, or jasmonic acid has been replaced with a COR group, where R is an OR 1 group, in which R 1 is: an alkyl group, such as a Ci Cs unbranched or branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a C2 Cs unbranched or branched alkenyl group; an alkynyl group, such as a C2 Cs unbranched or branched alkynyl group; an aryl group having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon
  • Representative amides are compounds in which the carboxyl group of linoleic acid, linolenic acid, or jasmonic acid has been replaced with a -COR group, where R is an NR 2 R 3 group, in which R 2 and R 3 are each independently: a hydrogen; an alkyl group, such as a Ci Cs unbranched or branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a C2 Cs unbranched or branched alkenyl group; an alkynyl group, such as a C2 Cs unbranched or branched alkynyl group; an aryl group having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroaryl group can be, for example, N, O, P, or S.
  • R is an NR 2 R 3
  • Esters may be prepared by known methods, such as acid catalyzed nucleophilic addition, wherein the carboxylic acid is reacted with an alcohol in the presence of a catalytic amount of a mineral acid.
  • Amides may also be prepared by known methods, such as by reacting the carboxylic acid with the appropriate amine in the presence of a coupling agent, such as dicyclohexyl carbodiimide (DCC), under neutral conditions.
  • Suitable salts of linoleic acid, linolenic acid and jasmonic acid include, for example, base addition salts.
  • the bases that may be used as reagents to prepare metabolically acceptable base salts of these compounds include those derived from cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts may be readily prepared by mixing a solution of linoleic acid, linolenic acid, or jasmonic acid with a solution of the base. The salts may be precipitated from solution and collected by filtration, or may be recovered by other means such as by evaporation of the solvent.
  • alkali metal cations e.g., potassium and sodium
  • alkaline earth metal cations e.g., calcium and magnesium
  • Non-flavonoid nod-gene inducers may be incorporated into compositions in some embodiments in any suitable amount(s)/concentration(s).
  • the amount/concentration of non-flavonoid nod-gene inducers may be an amount effective to impart or confer a positive trait or benefit to a plant, such as to enhance the growth and/or yield of the plant to which the composition is applied.
  • the amount/concentration of non-flavonoid nod-gene inducers may not be effective to enhance the growth and/or yield of the plant without beneficial contributions from one or more other ingredients of the composition, such as a LCO, CO and/or one or more pesticides.
  • compositions in some embodiments may comprise karrakins, including but not limited to 2H-furo[2,3-c]pyran-2-ones, as well as analogues, derivatives, hydrates, isomers, polymers, salts and solvates thereof.
  • biologically acceptable salts of karrakins include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulfonate, benzenesulfonate and p-toluenesulfonic acid.
  • Additional biologically acceptable metal salts may include alkali metal salts, with bases, examples of which include the sodium and potassium salts.
  • Karrakins may be incorporated into compositions in any suitable amount(s) or concentration(s).
  • the amount/concentration of a karrakin may be an amount or concentration effective to impart or confer a positive trait or benefit to a plant, such as to enhance the growth and/or yield of the plant to which the composition is applied.
  • a karrakin amount/concentration may not be effective to enhance the growth and/or yield of the plant without beneficial contributions from one or more other ingredients of the composition, such as a LCO, CO and/or one or more pesticides.
  • compositions in some embodiments may comprise one or more anthocyanidins and/or anthoxanthins, such as one or more of cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, flavones (e.g., apigenin, baicalein, chrysin, 7,8-dihydroxyflavone, diosmin, flavoxate, 6-hydroxyflavone, luteolin, scutellarein, tangeritin and/or wogonin) and/or flavonols (e.g., amurensin, astragalin, azaleatin, azalein, fisetin, furanoflavonols galangin, gossypetin, 3- hydroxyflavone, hyperoside, icariin, isoquercetin, kaempferide, kaempferitrin, kaempferol, is
  • compositions in some embodiments may comprise gluconolactone and/or an analogue, derivative, hydrate, isomer, polymer, salt and/or solvate thereof.
  • Gluconolactone may be incorporated into compositions in any suitable amount(s)/concentration(s).
  • the amount/concentration of a gluconolactone amount/concentration may be an amount effective to impart or confer a positive trait or benefit to a plant, such as to enhance the growth and/or yield of the plant to which the composition is applied.
  • the gluconolactone amount/concentration may not be effective to enhance the growth and/or yield of the plant without beneficial contributions from one or more other ingredients of the composition, such as a LCO, CO and/or one or more pesticides.
  • compositions and formulations may comprise one or more suitable nutrient(s) and/or fertilizer(s), such as organic acids (e.g., acetic acid, citric acid, lactic acid, malic acid, taurine, etc.), macrominerals (e.g., phosphorous, calcium, magnesium, potassium, sodium, iron, etc.), trace minerals (e.g., boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, etc.), vitamins, (e.g., vitamin A, vitamin B complex (i.e., vitamin Bi, vitamin B2, vitamin B3, vitamin B5, vitamin Be, vitamin B7, vitamin Bs, vitamin B9, vitamin B12, choline) vitamin C, vitamin D, vitamin E, vitamin K), and/or carotenoids (a-carotene, P-carotene, cryptoxanthin, lutein,
  • compositions of the present disclosure may comprise macro- and micronutrients of plants or microbes, including phosphorous, boron, chlorine, copper, iron, manganese, molybdenum and/or zinc. In some embodiments, compositions may comprise one or more beneficial micronutrients.
  • Non-limiting examples of micronutrients for use in compositions described herein may include vitamins, (e.g., vitamin A, vitamin B complex (i.e., vitamin Bi, vitamin B2, vitamin B3, vitamin B5, vitamin Be, vitamin B7, vitamin Bs, vitamin B9, vitamin B12, choline) vitamin C, vitamin D, vitamin E, vitamin K, carotenoids (a-carotene, P-carotene, cryptoxanthin, lutein, lycopene, zeaxanthin, etc.), macrominerals (e.g., phosphorous, calcium, magnesium, potassium, sodium, iron, etc.), trace minerals (e.g., boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, etc.), organic acids (e.g., acetic acid, citric acid, lactic acid, malic aclid, taurine, etc.), and combinations thereof.
  • compositions may comprise phosphorous, boron, chlorine, copper, iron, manganese, molybdenum, and/or zinc, and combinations thereof.
  • phosphorous may be derived from a rock phosphate source, such as monoammonium phosphate, diammonium phosphate, monocalcium phosphate, super phosphate, triple super phosphate, and/or ammonium polyphosphate, an organic phosphorous source, or a phosphorous source capable of solubilization by one or more microorganisms (e.g., Penicillium bilaiae).
  • a composition comprising an agricultural plant, plant part, plant seed, plant seedling, or plant growth medium or soil, and a population of modified plant-associated bacteria that expresses at least one insecticidal protein, wherein the population of modified plant-associated bacteria is associated with the agricultural plant, plant part, plant seed, plant seedling, or plant growth medium or soil.
  • a plant or crop plant that may be treated or associated with compositions or formulations provided herein may include a variety of monocotyledonous (monocot) and dicotyledonous (dicot) agricultural plants. Examples may include row crops, such as maize (corn), wheat, rice, barley, other cereal crops, soybean, cotton, canola, sugar beets, alfalfa, and vegetables.
  • row crops such as maize (corn), wheat, rice, barley, other cereal crops, soybean, cotton, canola, sugar beets, alfalfa, and vegetables.
  • Amaranthaceae e.g., chard, spinach, sugar beet, quinoa
  • Asteraceae e.g., artichoke, asters, chamomile, chicory, chrysanthemums, dahlias, daisies, echinacea, goldenrod, guayule, lettuce, marigolds, safflower, sunflowers, zinnias
  • Brassicaceae e.g., arugula, broccoli, bok choy, Brussels sprouts, cabbage, cauliflower, canola, collard greens, daikon, garden cress, horseradish, kale, mustard, radish, rapeseed, rutabaga, turnip, wasabi, watercress, Arabidopsis thaliana
  • Cucurbitaceae e.g., cantaloupe, cucumber, honeydew, melon, pumpkin, squash (e.g., acorn squash, butter
  • a “plant part” refers to any organ or intact tissue of a plant, such as a meristem, shoot organ/structure (e.g., leaf, stem or node), root, flower or floral organ/structure (e.g., bract, sepal, petal, stamen, carpel, anther and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), propagule, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, and the like), or any portion thereof.
  • shoot organ/structure e.g., leaf, stem or node
  • root e.g., flower or floral organ/structure (e.g., bract, sepal, petal, stamen, carpel, anther and ovule)
  • seed e.g., embryo, endosperm, and seed coat
  • fruit e.g., the mature ovary
  • propagule e.g.
  • Plant parts may be viable, nonviable, regenerable, and/or non-regenerable, and plant parts may in some cases be developed, regenerated and/or grown into a plant.
  • a “propagule” may include any plant part that can grow into an entire plant, and may include, for example, cuttings, rhizomes, and tubers, depending on the plant species.
  • Plant parts that may be treated or associated with a microbial composition may further include other cultured plant tissues or propagation materials, such as somatic embryos and callus, which may be regenerated, developed or grown into a plant.
  • a plant, plant part or plant seed may be transgenic or non-transgenic and/or contain one or more genetic changes or mutations.
  • a “plant” refers to a plant at any stage of development including an embryo, seedling, and mature plant whether grown or developed from a seed, regenerated from a cultured tissue, or propagated in any manner.
  • Plants, plant parts or plant seeds that may be treated or associated with compositions provided herein may include commercial products, such as plant seeds, sold by Monsanto Company (St. Louis, Missouri) or others, such as commercial crop seed sold or distributed under the GENUITY®, DROUGHTGARD®, SMARTSTAX®, RIB COMPLETE®, ROUNDUP READY®, VT DOUBLE PRO®, VT TRIPLE PRO®, BOLLGARD® II, ROUNDUP READY® 2 YIELD, YIELDGARD®, ROUNDUP READY 2 XTEND®, INTACTA RR2 PRO®, VISTIVE® GOLD, and/or XTENDFLEXTM trade names.
  • commercial products such as plant seeds, sold by Monsanto Company (St. Louis, Missouri) or others, such as commercial crop seed sold or distributed under the GENUITY®, DROUGHTGARD®, SMARTSTAX®, RIB COMPLETE®, ROUNDUP READY
  • the phrases “associated with”, “in association with”, or “associated therewith” in reference to (i) a composition, formulation or modified plant-associated bacteria described herein and (ii) a plant, plant part, plant seed or growth medium or soil refer to at least a juxtaposition or close proximity of the composition, formulation or modified plant- associated bacteria, and at least a portion of the plant, plant part, plant seed or growth medium or soil.
  • Such a juxtaposition may be achieved by contacting or applying a composition, formulation or modified plant-associated bacteria to the plant, plant part, or plant seed, such as by spraying or coating the plant, plant part, or plant seed with the composition, formulation or modified plant- associated bacteria, as a foliar application applied to one or more above-ground tissues of the plant, and/or by applying the composition, formulation or modified plant-associated bacteria to the soil or growth medium at, near or surrounding the plant, plant part or plant seed.
  • the composition, formulation or modified plant-associated bacteria is applied as a coating to the outer surface of a plant part or plant seed, which may exist as a layer around most or all the plant part or plant seed.
  • composition, formulation or modified plant-associated bacteria may be applied as a foliar spray or as a soil drench or application at or near the base of a crop plant. In some embodiments, the composition, formulation or modified plant-associated bacteria may be applied at or near the site of a plant seed in (or on) the soil or ground before, simultaneously with, or after planting of the plant seed.
  • a plant-associated bacterium may be conjugated, transduced, or transformed with a DNA molecule or plasmid encoding the insecticidal protein using any method or technique known in the art.
  • a plant-associated bacterium (or bacteria) is/are mixed or contacted with a DNA molecule or plasmid comprising the expression cassette encoding an insecticidal protein, and transformation of the plant-associated bacterium (or bacteria) with the DNA molecule or plasmid may be driven, promoted, or increased by a physical or electrical perturbation method, such as electroporation, shaking, sonication, etc.
  • plant-associated bacteria may be transformed as protoplasts (i.e., with their cell walls removed) according to methods known in the art. See, e.g., Biedendieck, R.
  • a (recipient) plant-associated bacterium, bacterial cell or bacteria is/are mixed or contacted with another (donor) bacterium, bacterial cell or bacteria comprising, having or containing a DNA molecule or plasmid comprising an expression cassette encoding an insecticidal protein, and conjugation between a plant- associated bacterium or bacterial cell and the other (donor) bacterium or bacterial cell can occur to allow, permit or cause the DNA molecule or plasmid to be introduced or transferred into the plant-associated bacterium or bacterial cell recipient to produce a modified plant-associated bacterium or bacterial cell.
  • Introduction or transfer of DNA molecules or plasmids into plant- associated bacteria is possible via conjugation using methods known in the art.
  • a DNA molecule or plasmid may be introduced or transferred into a plant-associated bacterium or bacterial cell recipient by a virus or bacteriophage comprising, having or containing a DNA molecule or plasmid comprising an expression cassette encoding an insecticidal protein using any transduction method known in the art.
  • a (recipient) plant-associated bacterium, bacterial cell or bacteria is/are mixed or contacted with a virus or bacteriophage comprising, having or containing a DNA molecule or plasmid comprising an expression cassette encoding an insecticidal protein, and transfer or introduction of the DNA molecule or plasmid into the plant- associated bacterium or bacterial cell can occur via infection of the plant-associated bacterium, bacterial cell or bacteria by the virus or bacteriophage.
  • a modified plant- associated bacterium or bacterial cell that received a DNA molecule or plasmid comprising the expression cassette for an insecticidal protein can be grown and proliferated to produce a plurality, population, culture or colony of modified plant-associated bacteria, which may be subjected to additional selection pressure with a selection agent, such as an antibiotic, if the DNA molecule or plasmid further comprises a selectable marker gene, such as an antibiotic resistance gene.
  • a selection agent such as an antibiotic
  • a selectable marker gene such as an antibiotic resistance gene.
  • a screenable marker or a molecular assay may be used to isolate positive clones or bacteria having the DNA molecule or plasmid.
  • modified plant-associated bacteria can be screened or selected to identify or isolate transformants by any method known in the art.
  • a “transformant” in reference to bacteria includes a bacterium that receives a DNA molecule or plasmid from an outisde source via any transformation, conjugation or transduction technique. Preparations of the modified plant- associated bacteria and/or insecticidal proteins expressed therefrom may be assayed for activity against various insect pests.
  • compositions or formulations comprising a modified plant-associated bacterium (or bacteria) that comprises a DNA molecule or construct expressing at least one insecticidal protein.
  • the composition may further comprise one or more additional ingredients, components, etc., such as an agriculturally acceptable carrier.
  • an agronomic benefit such as insect tolerance or resistance due to the insecticidal protein expressed by the modified plant-associated bacterium or bacteria, or progeny bacteria thereof, is provided to a plant or plant part thereof, which may be grown or developed from a seed, plant part, plantlet or plant that was treated, etc., with the composition, and/or which is another part of the same plant or plantlet that was treated, etc., with the composition.
  • a kit or container comprising a modified plant-associated bacterium or a population of modified plant-associated bacteria that express(es) at least one insecticidal protein, and written instructions for protecting a plant, seed, or seedling from an insect pest infestation, such as instructions for how to treat or administer to a plant, seed, or seedling or to a crop, field or soil an effective amount of a population of modified plant-associated bacteria that express at least one insecticidal protein.
  • Bt thuringiensis
  • Bb and Bm B. megaterium
  • Table 1 provides the results of insect feeding or diet assay experiments using these modified bacterial strains described in reference to Table 1. The results in FIG. 2 (including the fraction of transformants) are pooled for each construct with the different origins of replication.
  • N.T. No Transformants
  • N.A. Not Attempted
  • Insect Diet Bioassay positive # replicates I total # replicates
  • WCR Western Com Rootworm
  • DBM Diamondback Moth
  • BCW Black Cutworm.
  • FIG. 1 shows TIC2463 protein expression in the B. megaterium transformant (Bm/TIC2463), and the protein was primarily secreted into the supernatant.
  • TIC2463 protein was expressed in the B. thurigenisis transformant (B//TIC2463), and expression levels were dependent upon the promoter.
  • the N2463 (pTIC2463::TIC2463) construct i.e., having the TIC2463 gene under the control of, and operably linked to, its native TIC2463 promoter
  • expressed higher levels of the TIC2463 insecticidal protein than a second TIC2463 (pTIC1201::TIC2463) construct (i.e., having the TIC2463 gene under the control of, and operably linked to, a heterologous TIC1201 promoter).
  • the CrylAc construct was transformed into Bm, Bb, and Bt.
  • the transformants were cultured for different time periods (26, 40, and 60 hrs.); cells were spun down to separate supernatant from pellet, and subjected to Western blot, as shown in FIG. 3. Lanes on the left side of pictures were loaded with different amount of purified CrylAc, and used as positive loading controls for quantifying CrylAc expression in supernatant and pellet of transformed Bm, Bb, and Bt. Untransformed wild-type bacteria were used as negative controls (labeled Bm, Bb or Bt). The data indicate that CrylAc is expressed at higher levels in in Bm transformants compared to the Bb and Bt transformants.
  • TIC2463 expressed by Bt and Bm transformants bacterial cultures were separated into supernatant and pellet, and then overlaid on artificial WCR diet, and fed to WCR. One week later WCR mortality was scored. TIC2463- transformed Bt had significantly higher mortality than untransformed Bt control, and TIC2463- transformed Bm showed numerically higher mortality over the untransformed Bm control, as shown in FIG. 4.
  • Both constructs showed activity against Coleopteran pests. However, as further shown in FIG. 5, feeding assays indicated that the pTIC2463::TIC2463 construct caused a higher Colorado Potato Beetle (CPB) mortality than the pTIC1201::TIC2463 construct. Both /?6-Cry I Ac and Bm-CrylAc constructs showed activity against Lepidopteran pests, as shown in FIG. 6. Both the transformants and wild-type bacteria were cultured for different time periods (26, 40, and 64 hrs.), and fed to Diamondback Moth (DBM).
  • DBM Diamondback Moth
  • the B. megaterium transformant with the CrylAc gene (Bm-CrylAc) showed activity in a Lepidoptera feeding assay.
  • the transformant had a 100% mortality rate against Diamondback Moth (DBM) at all tested concentrations (up to 2430-fold titrations) as shown in FIG. 7.
  • DBM Diamondback Moth
  • the Bm-CrylAc expressed CrylAc protein, and caused Black Cutworm mortality, with greater than 40% mortality at 90-fold dilution, as shown in FIG. 8.
  • ELISA analysis confirmed the production of the insecticidal CrylAc protein in the B. butanolivorans transformant (Bb-Cr 1 Ac) as shown in FIG. 9.
  • Feeding assays confirmed the transformant’s pesticidal activity.
  • B. butanolivorans transformants and conjugants cultured for different time periods (40 and 80 hrs.) and concentrations kill Diamondback Moth (DBM) with 100% mortality in an artificial diet assay as shown in FIG. 10.
  • Conjugation was brought about by combining actively growing cultures of a donor strain containing an insecticidal plasmid and a plant-associated acceptor strain. The ratio of donor and recipient cells may be manipulated to maximize transconjugant yield. Bacterial mixes were transferred to a cellophane membrane placed on a nonselective LB agar plate and incubated for three hours at 30°C. Cells on the membrane were suspended and plated onto selective media that favored the growth of the transconjugants.
  • B. thuringiensis strains containing plasmids coding for tetracycline resistance and CrylAc were mated with B. megaterium strains having plasmids coding for chloramphicol resistance and TIC2463. Only cells with containing both plasmids for chloramphicol and tetracycline resistance survive to form colonies on the selection medium.
  • the candidate transconjugants were plated out as single cell colonies and checked by amplification for the presence of both the Tic2463 and CrylAc genes; and confirmed as the B. megaterium strain.
  • the transconjugants contained both the TIC2463 and CrylAc genes, and showed the expected activity against Western corn rootworm (WCR), as well as Black cutworm (BCW) and Diamondback Moth (DBM).
  • Example 3 Artificial Diet Assays for Western Corn Rootworms, Colorado Potato Beetles, Black Cutworms and Diamondback Moths
  • Modified plant-associated microbes were serially diluted in water and 20 pL aliquots were added to 200 pL artificial diet in each well of a 96-well plate. The plates were allowed to stand in a sterile biohood for 24 hours at 10°C after application of test samples to allow the reagents to diffuse into the diet to allow the surface of the diet to dry. One neonate larva was deposited in each well with a fine paintbrush. Plates were sealed with Mylar and ventilated using an insect pin. From 12 to 72 larvae were tested per dose, depending on the design of the assay. The bioassay plates were incubated at 27°C, 60% relative humidity, in complete darkness. Mortality or stunting of the larvae was observed 5-7 after infestation.
  • Bm_N2463 transformant and Bm conjugant expressed TIC2463, and caused WCR mortality in the artificial diet assay as shown in FIG. 11.
  • Bm_N2463 had no CrylAc gene and did not cause Diamondback Moth (DBM) mortality.
  • Bm conjugant (N2463xCrylAc) expressed CrylAc, and caused Diamondback Moth (DBM) mortality as shown in FIG. 12.
  • Example 4 Potato Leaf Disc Assay for Colorado Potato Beetles
  • Modified plant-associated microbes were serially diluted in a SIL WET® L-77 solution (0.05% in water).
  • Leaf discs (90 mm diameter), were punched from fresh potato plant leaves, treated with 10 pL aliquots of the diluted cultures, allowed to dry for 20 minutes, and placed put onto 2% agar in 48-well plates.
  • One Colorado Potato Beetle (CPB) larvae was placed onto each leaf disc. The dish was covered and transferred to a holding room at 25°C. Mortality and leaf consumption was determined 3 days after infestation.
  • both the Bm_N2463 transformant and Bm conjugant reduced Colorado Potato Beetle (CPB) neonate feeding and increased mortality up to 9-fold dilution (0.1 IX) as shown in FIG. 13.
  • Tomato plants (2-weeks old) were treated with cultures of the modified plant- associated microbes. Each plant was treated with 500 pL of culture, allowed to dry, and caged for 24 hours. Each plant was infested with 10 neonates of Colorado Potato Beetle (CPB). Mortality and leaf consumption was scored one week following infestation.
  • CPB Colorado Potato Beetle
  • Corn plants (2-weeks old) were treated with cultures of the modified plant- associated microbes in a 0.2% SILWET® solution. A 9-mL culture sample was centrifuged at 4700 rpm for 10 minutes at 25° C, the supernatant removed, and diluted with SILWET® L-77 (6 pL) and water to yield 3 mL of 0.2% SILWET® L-77. Each plant was treated with 60 pL of the SILWET® culture, including both sides of leaf and stem. After 24 hours, each plant was infested with 10 neonates of BCW. Mortality and leaf consumption was scored 11 days following infestation.
  • Cabbage plants (2-weeks old) were treated with cultures of the modified plant- associated microbes in a 0.25% SILWET solution. A culture sample was centrifuged at 220 rpm for 10 minutes at 28°C, the supernatant removed, and diluted with SILWET® L-77 and water to yield 4 mL of 0.25% SILWET® L-77. Each plant was treated with 1 mL of the SILWET®culture, infested with 20 neonates of Diamondback Moth (DBM), and incubated in a cage with block design. Photographs of the assay were taken 7, 9, and 12 days’ post-infestation. Mortality and leaf consumption was scored 12 days following infestation.
  • DBM Diamondback Moth
  • Root tissue sections ( ⁇ 40 pm) from aseptically gathered root tips of com were treated with Live/Dead bacterial viability kit components SYTOTM 9 (S9) and propidium iodide (PI) followed by confocal laser scanning microscopy (CLSM).
  • SYTOTM 9 S9
  • PI propidium iodide
  • FIG. 19 shows the GFP expressing cells (highlighted with an arrow). This study provides microscopic evidence of an endophytic plant-associated bacterial cells expressing a GFP transgene when present in a plant tissue.

Abstract

La divulgation concerne des bactéries Bacillus modifiées associées à une plante qui expriment des protéines insecticides, ainsi que des procédés de protection d'une plante contre toute infestation par des insectes nuisibles. L'invention concerne également des compositions comprenant une population de bactéries Bacillus modifiées associées à une plante et des procédés de fabrication d'une bactérie Bacillus associée à une plante par l'intermédiaire d'une technique de transfert horizontal de gènes.
PCT/US2021/062372 2020-12-08 2021-12-08 Bactéries modifiées associées à une plante et leurs procédés d'utilisation WO2022125639A1 (fr)

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