WO2021030400A1 - Combinaisons pesticides de yersinia et de protéases - Google Patents

Combinaisons pesticides de yersinia et de protéases Download PDF

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Publication number
WO2021030400A1
WO2021030400A1 PCT/US2020/045868 US2020045868W WO2021030400A1 WO 2021030400 A1 WO2021030400 A1 WO 2021030400A1 US 2020045868 W US2020045868 W US 2020045868W WO 2021030400 A1 WO2021030400 A1 WO 2021030400A1
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optionally
yersinia
entomophaga
plant
protease
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PCT/US2020/045868
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English (en)
Inventor
Calum RUSSELL
Jarrod LELAND
Mark Robert Holmes HURST
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Novozymes Bioag A/S
Agresearch Limited
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Publication of WO2021030400A1 publication Critical patent/WO2021030400A1/fr

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    • 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

Definitions

  • the present disclosure contains references to biological materials deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Agricultural Research Service Culture Collection, 1815 North University Street, Peoria, Illinois 61604, U.S.A.
  • Yersinia entomophaga is a Gram-negative bacterium that exhibits pesticidal activity against a wide range of targets, including, but not limited to, coleopteran, dipteran, hymenopteran, lepidopteran orthopteran and thysanopteran insects. See, e.g., Hurst et al., INTL. J. SYS. EVOL. MICROBIOL. 61:844 (2011); WO 2007/142543; WO 2008/041863. Previous studies have shown that Yersinia entomophaga may be used synergistically with myriad chemical pesticides. See. e.g., WO 2018/175677).
  • the present disclosure provides novel and inventive uses and methods for Yersinia entomophaga (and Yersinia nurmii), as well as compositions useful for practicing such methods.
  • a first aspect of the present disclosure is use of Yersinia entomophaga, a cell-free Yersinia entomophaga culture filtrate, or an isolated Yersinia entomophaga toxin for improving the insecticidal efficacy of a protease against a pest.
  • a second aspect of the present disclosure is use of Yersinia nurmii, a cell-free Yersinia nurmii culture fdtrate, or an isolated Yersinia nurmii toxin for improving the insecticidal efficacy of a protease against a pest.
  • a third aspect of the present disclosure is use of a protease for improving the insecticidal efficacy of a Yersinia entomophaga, a cell-free Yersinia entomophaga culture filtrate, or an isolated Yersinia entomophaga toxin against a pest.
  • a fourth aspect of the present disclosure is use of a protease for improving the insecticidal efficacy of a Yersinia nurmii, a cell-free Yersinia nurmii culture filtrate, or an isolated Yersinia nurmii toxin against a pest.
  • a fifth aspect of the present disclosure is a method comprising applying one or more Yersinia entomophaga, one or more cell-free Yersinia entomophaga culture filtrates and/or one or more isolated Yersinia entomophaga toxins, as well as one or more proteases to a plant or plant part.
  • a sixth aspect of the present disclosure is a method comprising applying one or more Yersinia nurmii, one or more cell-free Yersinia nurmii culture filtrates and/or one or more isolated Yersinia nurmii toxins, as well as one or more proteases to a plant or plant part.
  • a seventh aspect of the present disclosure is a method comprising applying one or more proteases to a plant or plant part that expresses one or more Yersinia entomophaga toxins or components thereof (e.g., Chil, Chi2, YenAl, YenA2, YenB, YenCl and/or YenC2).
  • An eighth aspect of the present disclosure is a method comprising applying one or more proteases to a plant or plant part that expresses one or more Yersinia nurmii toxins or components thereof.
  • a ninth aspect of the present disclosure is synthetic consortium comprising, consisting essentially of or consisting of one or more strains of Yersinia entomophaga and/or one or more strains of Yersinia nurmii and/or one or more proteases.
  • a tenth aspect of the present disclosure is composition comprising, consisting essentially of, or consisting of one or more strains of Yersinia entomophaga (and/or one or more toxins or other components derived therefrom) and/or one or more strains of Yersinia nurmii (and/or one or more toxins or other components derived therefrom) and/or one or more proteases.
  • a twelth aspect of the present disclosure is a transgenic plant comprising genetic material derived from Yersinia entomophaga and/or Yersinia nurmii and/or a protease.
  • a thirteenth aspect of the present disclosure is a transgenic plant that expresses one or more toxins of/from Yersinia entomophaga and/or Yersinia nurmii.
  • acaricide and “acaricidal” refer to an agent or combination of agents the application of which is toxic to an acarid (i.e., kills an acarid, inhibits the growth of an acarid and/or inhibits the reproduction of an acarid).
  • additive when used in reference to a combination of two or more independent effects (e.g., a first effect produced by application of a Yersinia entomophaga and a second effect produced by application of a protease), means the cumulative effect of the two or more independant effects is (about) the same as the theoretical sum of the independent effects in combination.
  • the term "agriculturally acceptable carrier” refers to a substance or composition that can be used to deliver an agriculturally beneficial agent to a plant, plant part or plant growth medium (e.g., soil) without causing/having an unduly adverse effect on plant growth and/or yield.
  • foliar- compatible carrier refers to a material that can be foliarly applied to a plant or plant part without causing/having an unduly adverse effect on the plant, plant part, plant growth, plant health, or the like.
  • seed-compatible carrier refers to a material that can be applied to a seed without causing/having an unduly adverse effect on the seed, the plant that grows from the seed, seed germination, or the like.
  • soil-compatible carrier refers to a material that can be added to a soil without causing/having an unduly adverse effect on plant growth, soil structure, soil drainage, or the like.
  • the term "agriculturally beneficial agent” refers to any agent (e.g., chemical or biological agent) or combination of agents the application of which causes or provides a beneficial and/or useful effect in agriculture including, but not limited to, agriculturally beneficial microorganisms, biostimulants, nutrients, pesticides (e.g., acaricides, fungicides, herbicides, insecticides, and nematicides) and plant signal molecules.
  • agent e.g., chemical or biological agent
  • agents the application of which causes or provides a beneficial and/or useful effect in agriculture including, but not limited to, agriculturally beneficial microorganisms, biostimulants, nutrients, pesticides (e.g., acaricides, fungicides, herbicides, insecticides, and nematicides) and plant signal molecules.
  • the term "agriculturally beneficial microorganism” refers to a microorganism having at least one agriculturally beneficial property (e.g., the ability to fix nitrogen, the ability to solubilize phosphate and/or the ability to produce an agriculturally beneficial agent, such as a plant signal molecule).
  • antagonistic when used in reference to a combination of two or more independent effects (e.g., a first effect produced by application of a Yersinia entomophaga and a second effect produced by application of a protease), means the cumulative effect of the two or more independant effects is decreased as compared to the theoretical sum of the independent effects in combination.
  • the terms "associated with,” in association with” and “associated therewith,” when used in reference to a relationship between a microbial strain or inoculant composition of the present disclosure and a plant or plant part, refer to at least a juxtaposition or close proximity of the microbial strain or inoculant composition and the plant or plant part. Such a juxtaposition or close proximity may be achieved by contacting or applying the microbial strain or inoculant composition directly to the plant or plant part and/or by applying the microbial strain or inoculant composition to the plant growth medium (e.g., soil) in which the plant or plant part will be grown (or is currently being grown).
  • the plant growth medium e.g., soil
  • the microbial strain or inoculant composition is applied as a coating to the outer surface of the plant or plant part. According to some embodiments, the microbial strain or inoculant composition is applied to soil at, near or surrounding the site in which the plant or plant part will be grown (or is currently being grown).
  • aqueous refers to a composition that contains more than a trace amount of water (i.e., more than 0.5% water by weight, based upon the total weight of the composition).
  • biologically pure culture refers to a microbial culture that is free or essentially free of biological contamination and that has genetic uniformity such that different subcultures taken therefrom will exhibit identical or substantially identical genotyopes and phenotypes.
  • the biologically pure culture is 100% pure (i.e., all subcultures taken therefrom exhibit identical genotypes and phenotypes).
  • the biologically pure culture is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% pure (i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% of the subcultures taken therefrom exhibit identical genotypes and phenotypes).
  • biostimulant refers to an agent or combination of agents the application of which enhances one or more metabolic and/or physiological processes of a plant or plant part (e.g., carbohydrate biosynthesis, ion uptake, nucleic acid uptake, nutrient delivery, photosynthesis and/or respiration).
  • BRADY' is to be interpreted as a shorthand substitute for the phrase " Bradyrhizobium spp.
  • colony forming unit and “cfu” refer to a microbial cell/spore capable of propagating on or in a suitable growth medium or substrate (e.g., a soil) when conditions (e.g., temperature, moisture, nutrient availability, pH, etc.) are favorable for germination and/or microbial growth.
  • a suitable growth medium or substrate e.g., a soil
  • conditions e.g., temperature, moisture, nutrient availability, pH, etc.
  • diazotroph refers to an organism capable of converting atmospheric nitrogen (N 2 ) into a form that may be utilized by a plant or plant part (e.g., ammonia (N3 ⁇ 4), ammonium (NH4+), etc.).
  • the term "dispersant” refers to an agent or combination of agents the application of which reduces the cohesiveness of like particles, the surface tension of a liquid, the interfacial tension between two liquids and/or the interfacial tension between or a liquid and a solid.
  • the terms "effective amount,” “effective concentration” and “effective amount/concentration” refer to an amount or concentration that is sufficient to cause a desired effect (e.g. , toxicity to a pest and/or enhanced crop yield).
  • the absolute value of the amount/concentration that is sufficient to cause the desired effect may be affected by factors such as the type and magnitude of effect desired, the type, size and volume of material to which the composition will be applied, the type(s) of microorganisms in the composition, the number of microorganisms in the composition, the stability of the microorganism(s) in the composition and the storage conditions (e.g., temperature, relative humidity, duration). Those skilled in the art will understand how to select an effective amount/concentration using routine dose-response experiments.
  • an effective amount of a substance when used alone may be different than an effective amount of the same substance when used as part of a combination.
  • an effective amount of a combination of Yersinia entomophaga and protease is an amount of the two agents that is capable of producing an unexpected insecticidal result, as compared to the insecticidal effects of Yersinia entomophaga and the insecticidal effects of protease alone.
  • an effective amount of an insecticidal agent may be determined through use of the agent alone.
  • the amount of the protease used is generally considered an effective amount of the protease for the purpose in which it was used.
  • an effect e.g., insect killing
  • the amount of the agent e.g., protease
  • the amount of the agent may produce a measurable effect when used with another agent (e.g., Yersinia).
  • the effect of the combination may be different than the effect of the protease alone and the Yersinia alone.
  • an effective amount may be defined in terms of the specific use (e.g., insect used against, active ingredients used in combination, etc.). Effective amounts may be determined empirically and this is within the scope of one of skill in the art.
  • the term "enhanced dispersion” refers to an improvement in one or more characteristics of microbial dispersion as compared to one or more controls (e.g., a control composition that is identical to an inoculant composition of the present disclosure except that it lacks one or more of the components found in the inoculant composition of the present disclosure).
  • exemplary microbial dispersion characteristics include, but are not limited to, the percentage of microbes that exist as single cells/spores when the inoculant composition is diluted in water.
  • An inoculant composition that improves one or more microbial dispersion characteristics of the microorganism(s) contained therein as compared to a control composition (e.g., a control composition that is identical to the inoculant composition except that it lacks one or more of the components found in the inoculant composition) provides enhanced dispersion and can be referred to as a "readily dispersable inoculant composition.”
  • the terms “enhanced growth” and “enhanced plant growth” refer to an improvement in one or more characteristics of plant growth and/or development as compared to one or more control plants (e.g., a plant germinated from an untreated seed or an untreated plant).
  • Exemplary plant growth/development characteristics include, but are not limited to, biomass, carbohydrate biosynthesis, chlorophyll content, cold tolerance, drought tolerance, height, leaf canopy, leaf length, leaf mass, leaf number, leaf surface area, leaf volume, lodging resistance, nutrient uptake and/or accumulation (e.g., ammonium, boron, calcium, copper, iron, magnesium, manganese, nitrate, nitrogen, phosphate, phosphorous, potassium, sodium, sulfur and/or zinc uptake/accumulation), rate(s) of photosynthesis, root area, root diameter, root length, root mass, root nodulation (e.g., nodule mass, nodule number, nodule volume), root number, root surface area, root volume, salt tolerance, seed germination, seedling emergence, shoot diameter, shoot length, shoot mass, shoot number, shoot surface area, shoot volume, spread, stand, stomatal conductance and survival rate.
  • nutrient uptake and/or accumulation e.g., ammonium, boron, calcium, copper
  • references to enhanced plant growth are to be interpreted as meaning that microbial strains, inoculant compositions and methods of the present disclosure enhance plant growth by enhancing nutrient availability, improving soil characteristics, etc. and are not to be interpreted as suggesting that microbial strains, inoculant compositions and methods of the present disclosure act as plant growth regulators.
  • the terms “enhanced stability” and “enhanced microbial stability” refer to an improvement in one or more characteristics of microbial stability as compared to one or more controls (e.g., a control composition that is identical to an inoculant composition of the present disclosure except that it lacks one or more of the components found in the inoculant composition of the present disclosure).
  • exemplary microbial stability characteristics include, but are not limited to, the ability to germinate and/or propagate after being coated on a seed and/or stored for a defined period of time and the ability to cause a desired effect (e.g., enhanced plant yield and/or increased pesticidal activity) after being coated on a seed and/or stored for a defined period of time.
  • An inoculant composition that improves one or more microbial stability characteristics of the microorganism(s) contained therein as compared to a control composition e.g., a control composition that is identical to the inoculant composition except that it lacks one or more of the components found in the inoculant composition
  • the terms “enhanced survival” and “enhanced microbial survival” refer to an improvement in the survival rate of one or more microorganisms in an inoculant composition as compared to one or more microorganisms in a control composition (e.g., a control composition that is identical to an inoculant composition of the present disclosure except that it lacks one or more of the components found in the inoculant composition of the present disclosure).
  • An inoculant composition that improves the survival rate of one or more of the microorganisms contained therein as compared to a control composition provides enhanced survival and can be referred to as a stable inoculant composition.
  • the terms “enhanced yield” and “enhanced plant yield” refer to an improvement in one or more characteristics of plant yield as compared to one or more control plants (e.g., a control plant germinated from an untreated seed).
  • Exemplary plant yield characteristics include, but are not limited to, biomass; bushels per acre; grain weight per plot (GWTPP); nutritional content; percentage of plants in a given area (e.g., plot) that fail to produce grain; yield at standard moisture percentage (YSMP), such as grain yield at standard moisture percentage (GYSMP); yield per plot (YPP), such as grain weight per plot (GWTPP); and yield reduction (YRED).
  • references to enhanced plant yield are to be interpreted as meaning that microbial strains, inoculant compositions and methods of the present disclosure enhance plant yield by enhancing nutrient availability, improving soil characteristics, etc. and are not to be interpreted as suggesting that microbial strains, inoculant compositions and methods of the present disclosure act as plant growth regulators.
  • foliage refers to those portions of a plant that normally grow above the ground, including, but not limited to, leaves, stalks, stems, flowers, fruiting bodies and fruits.
  • foliar application and “foliarly applied” refer to the application of one or more active ingredients to the foliage of a plant (e.g., to the leaves of the plant).
  • Application may be effected by any suitable means, including, but not limited to, spraying the plant with a composition comprising the active ingredient(s).
  • the active ingredient(s) is/are applied to the leaves, stems and/or stalk of the plant and not to the flowers, fruiting bodies or fruits of the plant.
  • fungicide and “fungicidal” refer to an agent or combination of agents the application of which is toxic to a fungus (i.e., kills a fungus, inhibits the growth of a fungus and/or inhibits the reproduction of a fungus).
  • gastropodicide refers to an agent or combination of agents the application of which is toxic to a gastropod (e.g., snails, slugs) (kills a gastropod, inhibits the growth of a gastropod and/or inhibits the reproduction of a gastropod).
  • molluscicide is generally interchangeable with gastropodicide.
  • the term "fulvic acid” encompasses pure fulvic acids and fulvic acid salts (fulvates).
  • fulvic acids include ammonium fulvate, boron fulvate, potassium fulvate, sodium fulvate, etc.
  • the fulvic acid comprises, consists essentially of or consists MDL Number MFCD09838488 (CAS Number 479-66-3).
  • the terms "herbicide” and “herbicidal” refer to an agent or combination of agents the application of which is toxic to a weed (i.e., kills a weed, inhibits the growth of a weed and/or inhibits the reproduction of a weed).
  • the term "humic acid” encompasses pure humic acids and humic acid salts (humates).
  • humic acids include ammonium humate, boron humate, potassium humate, sodium humate, etc.
  • the humic acid comprises, consists essentially of or consists of one or more of MDL Number MFCD00147177 (CAS Number 1415-93-6), MDL Number MFCD00135560 (CAS Number 68131-04-4), MDL Number MFCS22495372 (CAS Number 68514-28-3), CAS Number 93924-35-7 and CAS Number 308067-45-0.
  • inoculant composition and “inoculum” refer to a composition comprising microbial cells and/or spores, said cells/spores being capable of propagating/germinating on or in a suitable growth medium or substrate (e.g., a soil) when conditions (e.g., temperature, moisture, nutrient availability, pH, etc.) are favorable for germination and/or microbial growth.
  • a suitable growth medium or substrate e.g., a soil
  • conditions e.g., temperature, moisture, nutrient availability, pH, etc.
  • insecticide and “insecticidal” refer to an agent or combination of agents the application of which is toxic to an insect (i.e., kills an insect, inhibits the growth of an insect and/or inhibits the reproduction of an insect).
  • isolated microbial strain refers to a microbe that has been removed from the environment in which it is normally found.
  • the term "isomer” includes all stereoisomers of the compounds and/or molecules to which it refers, including enantiomers and diastereomers, as well as all conformers, roatmers and tautomers, unless otherwise indicated.
  • Compounds and/or molecules disclosed herein include all enantiomers in either substantially pure levorotatory or dextrorotatory form, or in a racemic mixture, or in any ratio of enantiomers. Where embodiments disclose a (D) -enantiomer, that embodiment also includes the (L) -enantiomer; where embodiments disclose a (L)-enantiomer, that embodiment also includes the (D)-enantiomer.
  • embodiments disclose a (+)- enantiomer, that embodiment also includes the (-)-enantiomer; where embodiments disclose a (-)-enantiomer, that embodiment also includes the (+)-enantiomer.
  • embodiments disclose a (S)-enantiomer, that embodiment also includes the (R)-enantiomer; where embodiments disclose a (R)-enantiomer, that embodiment also includes the (S)-enantiomer.
  • Embodiments are intended to include any diastereomers of the compounds and/or molecules referred to herein in diastereomerically pure form and in the form of mixtures in all ratios. Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the chemical structure or chemical name is intended to embrace all possible stereoisomers, conformers, retainers and tautomers of compounds and/or molecules depicted.
  • miticide and “miticidal” refer to an agent or combination of agents the application of which is toxic to an mite (i.e., kills a mite, inhibits the growth of a mite and/or inhibits the reproduction of a mite).
  • modified microbial strain refers to a microbial strain that is modified from a strain isolated from nature. Modified microbial strains may be produced by any suitable method(s), including, but not limited to, chemical or other form of induced mutation to a polynucleotide within any genome within the strain; the insertion or deletion of one or more nucleotides within any genome within the strain, or combinations thereof; an inversion of at least one segment of DNA within any genome within the strain; a rearrangement of any genome within the strain; generalized or specific transduction of homozygous or heterozygous polynucleotide segments into any genome within the strain; introduction of one or more phage into any genome of the strain; transformation of any strain resulting in the introduction into the strain of stably replicating autonomous extrachromosomal DNA; any change to any genome or to the total DNA composition within the strain isolated from nature as a result of conjugation with any different microbial strain; and any combination of the foregoing.
  • modified microbial strains includes a strain with (a) one of more heterologous nucleotide sequences, (b) one or more non-naturally occurring copies of a nucleotide sequence isolated from nature (i.e., additional copies of a gene that naturally occurs in the microbial strain from which the modified microbial strain was derived), (c) a lack of one or more nucleotide sequences that would otherwise be present in the natural reference strain by for example deleting nucleotide sequence, and (d) added extrachromosomal DNA.
  • modified microbial strains comprise a combination of two or more nucleotide sequences (e.g., two or more naturally occurring genes that do not naturally occur in the same microbial strain) or comprise a nucleotide sequence isolated from nature at a locus that is different from the natural locus.
  • nematicide and “nematicidal” refer to an agent or combination of agents the application of which is toxic to a nematode (i.e., kills a nematode, inhibits the growth of a nematode and/or inhibits the reproduction of a nematode).
  • nitrogen fixing organism refers to an organism capable of converting atmospheric nitrogen (N 2 ) into a form that may be utilized by a plant or plant part (e.g., ammonia (NH 3 ), ammonium (NHA), etc.).
  • N 2 atmospheric nitrogen
  • NHA ammonium
  • non-aqueous refers to a composition that comprises no more than a trace amount of water (i.e., no more than 0.5% water by weight, based upon the total weight of the composition).
  • the term "nutrient” refers to a compound or element useful for nourishing a plant (e.g., vitamins, macrominerals, micronutrients, trace minerals, organic acids, etc. that are necessary for plant growth and/or development).
  • Penicillium bilaiae is intended to include all iterations of the species name, such as “ Penicillium bilaji " and " Penicillium bilaii.”
  • percent identity refers to the relatedness of two or more nucleotide or amino acid sequences, which may be calculated by (i) comparing two optimally aligned sequences over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity.
  • the percent identity is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present invention, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the "percent identity" for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%.
  • the term “pest” includes any organism or virus that negatively affects a plant, including, but not limited to, organisms and viruses that spread disease, damage host plants and/or compete for soil nutrients.
  • the term “pest” encompasses organisms and viruses that are known to associate with plants and to cause a detrimental effect on the plant's health and/or vigor.
  • Plant pests include, but are not limited to, arachnids (e.g., mites, ticks, spiders, etc.), bacteria, fungi, gastropods (e.g., slugs, snails, etc.), invasive plants (e.g., weeds), insects (e.g., white flies, thrips, weevils, etc.), nematodes (e.g., root-knot nematode, soybean cyst nematode, etc.), rodents and viruses (e.g., tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV), cauliflower mosaic virus (CaMV), etc.).
  • arachnids e.g., mites, ticks, spiders, etc.
  • bacteria fungi
  • gastropods e.g., slugs, snails, etc.
  • invasive plants e.g., weeds
  • insects e.g., white
  • pesticide and “pesticidal” refer to agents or combinations of agents the application of which is toxic to a pest (i.e., kills a pest, inhibits the growth of a pest and/or inhibits the reproduction of a pest).
  • pesticides include acaricides, fungicides, herbicides, insecticides, and nematicides, etc.
  • phosphate-solubilizing microorganism refers to a microorganism capable of converting insoluble phosphate into a soluble form of phosphate.
  • plant includes all plant populations, including, but not limited to, agricultural, horticultural and silvicultural plants.
  • the term “plant” encompasses plants obtained by conventional plant breeding and optimization methods (e.g., marker-assisted selection) and plants obtained by genetic engineering, including cultivars protectable and not protectable by plant breeders' rights.
  • plant cell refers to a cell of an intact plant, a cell taken from a plant, or a cell derived from a cell taken from a plant.
  • plant cell includes cells within seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen and microspores.
  • plant growth regulator refers to an agent or combination of agents the application of which accelerates or retards the growth/maturation rate of a plant through direct physiological action on the plant or which otherwise alters the behavior of a plant through direct physiological action on the plant.
  • Plant growth regulator shall not be interpreted to include any agent or combination of agents excluded from the definition of "plant regulator” that is set forth section 2(v) of the Federal Insecticide, Fungicide, and Rodenticide Act (7 U.S.C. ⁇ 136(v)).
  • plant growth regulator does not encompass microorganisms applied to a plant, plant part or plant growth medium for the purpose of enhancing the availability and/or uptake of nutrients, nutrients necessary to normal plant growth, soil amendments applied for the purpose of improving soil characteristics favorable for plant growth or vitamin hormone products as defined by 40 C.F.R. ⁇ 152.6(f).
  • plant part refers to any part of a plant, including cells and tissues derived from plants.
  • plant part may refer to any of plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, plant cells and seeds.
  • plant parts include, but are not limited to, anthers, embryos, flowers, fruits, fruiting bodies, leaves, ovules, pollen, rhizomes, roots, seeds, shoots, stems and tubers, as well as scions, rootstocks, protoplasts, calli and the like.
  • plant propagation material refers to a plant part from which a whole plant can be generated.
  • plant propagation materials include, but are not limited to, cuttings (e.g., leaves, stems), rhizomes, seeds, tubers and cells/tissues that can be cultured into a whole plant.
  • peptidase As used herein, the terms “peptidase,” “protease” and “proteinase” refer to an agent or combination of agents capable of catalyzing proteolysis.
  • spore and "microbial spore” refer to a microorganism in its dormant, protected state.
  • stabilizing compound refers to an agent or combination of agents the application of which enhances the survival and/or stability of a microorganism in an inoculant composition.
  • stable refers to an inoculant composition in which microorganisms exhibit enhanced stability and/or enhanced survival.
  • an inoculant composition may be labeled “stable” if it improves the survival rate and/or at least one microbial stability characteristic of at least one microorganism contained therein.
  • the term "survival rate” refers to the percentage of microbial cell/spore that are viable (i.e., capable of propagating on or in a suitable growth medium or substrate (e.g., a soil) when conditions (e.g., temperature, moisture, nutrient availability, pH, etc.) are favorable for germination and/or microbial growth) at a given period of time.
  • Yersinia strains of the present disclosure encompasses Yersinia entomophaga MH96, which has previously been described (see, e.g., Hurst, M.R.H. et al., TOXINS 8: 143 (2016); GenBank Accession No. DQ400782), and which has previously been deposited as in the Leibniz Institute DSMZ -German Collection of Microorganisms and Cell Cultures as DSM 22339, in the American Type Culture Collection as ATCC BAA-1678, and in the U.S. Department of Agriculture's Agricultural Research Service Culture Collection as NRRL B-67598, progeny of Y.
  • entomophaga MH96 modified microbial strains derived from Y. entomophaga MH96, modified microbial strains derived from progeny of Y. entomophaga MH96, Yersinia entomophaga 024G3R, which has a whole genome sequence that is 99.74% identical to that of MH96 and which has previously been deposited as NRRL B-67599, progeny of Y. entomophaga 024G3R, modified microbial strains derived from Y. entomophaga 024G3R, modified microbial strains derived from progeny of Y. entomophaga 024G3R,
  • Yersinia entomophaga 024KEK which has a whole genome sequence that is 99.69% identical to that of MH96 and which has previously been deposited as NRRL B-67600, progeny of Y. entomophaga 024KEK, modified microbial strains derived from Y. entomophaga 024KEK, modified microbial strains derived from progeny of Y. entomophaga 024KEK, Yersinia entomophaga 0333A4, which has a whole genome sequence that is 99.7% identical to that of MH96 and which has previously been deposited as NRRL B-67601, progeny of Y.
  • entomophaga 0333A4 modified microbial strains derived from Y. entomophaga 0333A4, modified microbial strains derived from progeny of Y. entomophaga 0333A4, Yersinia entomophaga 023ZMJ, which has a whole genome sequence that is 99.59% identical to that of MH96, progeny of Y. entomophaga 023ZMJ, modified microbial strains derived from Y. entomophaga 023ZMJ, modified microbial strains derived from progeny of Y. entomophaga 023ZMJ, Yersinia entomophaga 0348UX, progeny of Y.
  • entomophaga 0348UX modified microbial strains derived from Y. entomophaga 0348UX, modified microbial strains derived from progeny of Y. entomophaga 0348UX, Yersinia entomophaga 033ZDX, progeny of Y. entomophaga 033ZDX, modified microbial strains derived from Y. entomophaga 033ZDX, and modified microbial strains derived from progeny of Y. entomophaga 033ZDX.
  • Progeny may be produced using any suitable method(s), including, but not limited to, protoplast fusion, traditional breeding programs and combinations thereof.
  • Modified microbial strains may be produced using suitable method(s), including, but not limited to, chemically-induced mutation of a polynucleotide within any genome within one of the aformentioend strains; the insertion or deletion of one or more nucleotides within any genome within one of the aformentioend strains, or combinations thereof; an inversion of at least one segment of DNA within any genome within one of the aformentioend strains; a rearrangement of any genome within one of the aformentioend strains; generalized or specific transduction of homozygous or heterozygous polynucleotide segments into any genome within one of the aformentioend strains; introduction of one or more phage into any genome of one of the aformentioend strains; transformation of one of the aformentioend strains resulting in the introduction into one of the aformentioend strains of stably replicating autonomous extrachromosomal DNA; any change to
  • the present disclosure extends to close relatives of Yersinia entomophaga strains of the present disclosure, including, but not limited to, closely related progeny of Y. entomophaga MH96, (e.g., progeny having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05,
  • modified microbial strains derived from Y. entomophaga MH96 e.g., modified microbial strains derived from Y. entomophaga MH96and having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75,
  • modified microbial strains derived from progeny of Y. entomophaga MH96 e.g., modified microbial strains derived from one or more progeny of Y. entomophaga MH96 and having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25, 96.3, 96.35, 96.4, 96.45, 96.5, 96.55, 96.6, 96.65, 96.7, 96.75, 96.8, 96.85, 96.9, 96.95, 97,
  • modified microbial strains derived from Y. entomophaga 024G3R e.g., modified microbial strains derived from Y. entomophaga 024G3R and having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25, 96.3, 96.35, 96.4, 96.45, 96.5, 96.55, 96.6, 96.65, 96.7, 96.75, 96.8, 96.85, 96.9, 96.95, 97, 97.5, 97.55, 97.6, 97.65, 97.7, 97.75, 97.8, 97.85, 97.9, 97.95, 98, 98.
  • entomophaga 024G3R and/or a whole genome sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96,
  • modified microbial strains derived from progeny of Y. entomophaga MH96 e.g., modified microbial strains derived from one or more progeny of Y. entomophaga 024G3R and having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75,
  • entomophaga 024KEK and having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25, 96.3, 96.35, 96.4, 96.45, 96.5, 96.55, 96.6, 96.65, 96.7, 96.75, 96.8, 96.85, 96.9, 96.95, 97, 97.5, 97.55, 97.6, 97.65,
  • entomophaga 024KEK and/or a whole genome sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25, 96.3, 96.35, 96.4, 96.45, 96.5, 96.55, 96.6, 96.65, 96.7, 96.75, 96.8, 96.85, 96.9, 96.95, 97, 97.5, 97.55,
  • Y. entomophaga 024KEK 99.95% identical to the whole genome sequence of Y. entomophaga 024KEK
  • other closely related strains e.g., Yersinia strains having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8,
  • entomophaga 0333A4 and/or a whole genome sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25, 96.3, 96.35, 96.4, 96.45, 96.5, 96.55, 96.6, 96.65, 96.7, 96.75, 96.8, 96.85, 96.9, 96.95, 97, 97.5, 97.55, 97.6, 97.65, 97.7,
  • modified microbial strains derived from Y. entomophaga 0333A4 e.g., modified microbial strains derived from Y. entomophaga 0333A4 and having a 16S sequence that is about/at least
  • entomophaga 0333A4 and having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25, 96.3, 96.35, 96.4, 96.45, 96.5, 96.55, 96.6, 96.65, 96.7, 96.75, 96.8, 96.85, 96.9,
  • entomophaga 0333A4 e.g., Yersinia strains having a 16S sequence that is about/at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9,
  • Y. entomophaga 0333A4 99.95% identical to the whole genome sequence of Y. entomophaga 0333A4), which may themselves exhibit insecticidal or other pesticidal activities and be useful for protecting and/or enhancing the growth and/or yield of various plants, including, but not limited to, cereals and pseudocereals, such as barley, buckwheat, com, millet, oats, quinoa, rice, rye, sorghum and wheat, and legumes, such as alfalfa, beans, carob, clover, guar, lentils, mesquite, peas, peanuts, soybeans, tamarind, tragacanth and vetch.
  • cereals and pseudocereals such as barley, buckwheat, com, millet, oats, quinoa, rice, rye, sorghum and wheat
  • legumes such as alfalfa, beans, carob, clover, guar,
  • the present disclosure extends to cultures comprising, consisting essentially of or consisting of one or more Yersinia. In some embodiments, at least 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25, 96.3, 96.35, 96.4, 96.45, 96.5, 96.55, 96.6, 96.65, 96.7, 96.75, 96.8, 96.85,
  • the culture is a biologically pure culture of the Yersinia.
  • the present disclosure encompasses isolated microbial strains, biologically pure cultures, inoculant compostions, non-naturally occurring compositions, plants, plant parts, processed products, crops, kits, methods and uses, such as those set forth herein, in which one or more closely related progeny of Y. entomophaga MH96, Y. entomophaga 024G3R, Y. entomophaga 024KEK, Y. entomophaga 0333A4, Y. entomophaga 023ZMJ, Y. entomophaga 0348UX and/or Y.
  • entomophaga 033ZDX one or more closely related modified microbial strains derived from Y. entomophaga MH96, Y. entomophaga 024G3R, Y. entomophaga 024KEK, Y. entomophaga 0333A4, Y. entomophaga 023ZMJ, Y. entomophaga 0348UX and/or Y. entomophaga 033ZDX, one or more closely related modified microbial strains derived from progeny of Y. entomophaga MH96, Y. entomophaga 024G3R, Y. entomophaga 024KEK, Y.
  • entomophaga 0333A4 Y entomophaga 023ZMJ, Y. entomophaga 0348UX and/or Y. entomophaga 033ZDX, and/or one or more other close relatives of Y. entomophaga MH96, Y. entomophaga 024G3R, Y. entomophaga 024KEK, Y. entomophaga 0333A4, Y. entomophaga 023ZMJ, Y. entomophaga 0348UX and/or Y. entomophaga 033ZDX is/are substituted for Y. entomophaga MH96, Y. entomophaga 024G3R, Y. entomophaga 024KEK and/or Y. entomophaga 0333A4.
  • the present disclosure describes uses, methods and compositions wherein one or more Yersinia entomophaga (and/or one or more toxins or other components derived therefrom) and/or one or more Yersinia nurmii (and/or one or more toxins or other components derived therefrom) are combined with one or more proteases.
  • the desired effect is achieved using a first composition comprising one or more Yersinia entomophaga (and/or one or more toxins or other components derived therefrom) and/or one or more Yersinia nurmii (and/or one or more toxins or other components derived therefrom) and a second composition comprising one or more proteases.
  • the desired effect is achieved using a single composition comprising one or more Yersinia entomophaga (and/or one or more toxins or other components derived therefrom) and/or one or more Yersinia nurmii (and/or one or more toxins or other components derived therefrom) as well as one or more proteases.
  • the desired effect is achieved using a transgenic microorganism (e.g., a Yersinia entomophaga strain that has been modified to express one or more proteases).
  • the desired effect is achieved using a transgenic plant that expresses both Yersinia components and protease components (e.g., a plant that has been modified to express one or more Yersinia entomophaga and/or Yersinia nurmii toxins as well as one or more proteases).
  • the desired effect is achieved using a transgenic plant that expresses one or more Yersinia components and is treated with one or more protease components (e.g. a plant that has been modified to express one or more Yersinia entomophaga toxins and is treated with one or more proteases).
  • the desired effect is achieved using a transgenic plant that expresses one or more protease components and is treated with one or more Yersinia components (e.g. a plant that has been modified to (over)express one or more proteases and is treated with one or more Yersinia entomophaga (and/or one or more toxins or other components derived therefrom)).
  • Yersinia components e.g. a plant that has been modified to (over)express one or more proteases and is treated with one or more Yersinia entomophaga (and/or one or more toxins or other components derived therefrom).
  • the genes/proteins expressed in such transgenic microorganisms/plants may be variants of naturally occurring genes/proteins.
  • Uses, methods and compositions of the present disclosure may comprise/utilize any suitable Yersinia entomophaga and/or Yersinia nurmii, including, but not limited to, those set forth herein as " Yersinia strains of the present disclosure” and "close relatives of Yersinia entomophaga strains of the present disclosure.”
  • Yersinia entomophaga is a Gram-negative, pesticidal bacterium with activity against a wide range of insects. See, e.g., WO 2007/142543; WO 2008/041863; Hurst et al., INT. J. SYST. EVOL. MICROBIOL. 61:844-849 (2011).
  • Yersinia entomophaga express a toxin complex (TC) called Yen-TC.
  • TC toxin complex
  • Yen-TC is reportedly composed of seven subunits — YenAl and YenA2 and the chitinases Chil and Chi2 reportedly form a pentameric cage, into which YenB, and one of YenCl or YenC2, bind to form the active Yen-TC.
  • YenAl and YenA2 the chitinases Chil and Chi2 reportedly form a pentameric cage, into which YenB, and one of YenCl or YenC2, bind to form the active Yen-TC.
  • J. MOL. BIOL. 415:359- 371 (2012) Genes encoding other putative toxins have been identified in Yersinia entomophaga. See, e.g., Hurst et al., TOXINS 8: 143 (2016).
  • Toxins and other components may be found in and isolated from the supernatants of media in which Yersinia entomophaga and/or Yersinia nurmii has been cultured.
  • toxins and other Yersinia components are present in (and applied as part of) a cell-free Yersinia culture filtrate.
  • toxins and other Yersinia components are present (and applied as) isolate molecules that are substantially purified from the cells and culture media from which they are derived.
  • MH96 also called MH96 T
  • MH-1 or SpK The type strain of Yersinia entomophaga
  • Y. entomophaga MH96 has been deposited in the Leibniz Institute DSMZ -German Collection of Microorganisms and Cell Cultures as DSM 22339, in the American Type Culture Collection as ATCC BAA-1678, and in the U.S. Department of Agriculture's Agricultural Research Service Culture Collection as NRRL B-67598.
  • the genome sequence of MH96 was published by Hurst, M.R.H. et al., TOXINS 8: 143 (2016).
  • the 16S rRNA sequence of MH96 is designated as GenBank Accession No. DQ400782 and is set forth herein as SEQ ID NO: 1.
  • Yersinia entomophaga are related to Yersinia nurmii.
  • the combinations of Yersinia and other substances, described herein, may include both Yersinia entomophaga and Yersinia nurmii.
  • the use of the word Yersinia alone generally is meant to include both Yersinia entomophaga and Yersinia nurmii.
  • Yersinia entomphaga and Yersinia nurmii are taxonomically distant from other Yersinia, including Yersinia that are pathogenic for humans. Yersinia entomphaga and Yersinia nurmii form a distinct clade away from other Yersinia species (Reuter, S. et al., PNAS 111:6768-6773 (2014)). Bioinformatic analysis of the Yersinia entomphaga genome failed to identify orthologs of know Yersinia pestis or Yersinia pseudotuberculosis virulence determinants, for example (Hurst, M.R.H. et al., TOXINS 8: 143 (2016)).
  • Yersinia may be cultured using any suitable method(s), including, but not limited to, liquid-state fermentation and solid-state fermentation.
  • Yersinia entomophaga may be grown on/in Luria (LB) agar/medium.
  • the compositions described herein may contain Yersinia organisms and/or may contain a toxin from the organisms (cell free filtrate).
  • the toxin may be purified or partially purified away from other, non-toxin components. Purification or partial purification of the toxin and/or subunits may use standard protein purification methodologies that are known in the art.
  • Yersinia may be harvested during any suitable growth phase. In some embodiments, Yersinia is allowed to reach the stationary growth phase and is harvested as vegetative cells.
  • Yersinia may be harvested and/or concentrated using any suitable method(s), including, but not limited to, centrifugation (e.g., density gradient centrifugation, disc stack centrifugation, tubular bowl centrifugation), coagulation, decanting, felt bed collection, filtration (e.g., drum filtration, sieving, ultrafiltration), flocculation, impaction and trapping (e.g., cyclone spore trapping, liquid impingement).
  • centrifugation e.g., density gradient centrifugation, disc stack centrifugation, tubular bowl centrifugation
  • coagulation coagulation
  • decanting felt bed collection
  • filtration e.g., drum filtration, sieving, ultrafiltration
  • flocculation e.g., cyclone spore trapping, liquid impingement
  • Uses, methods and compositions of the present disclosure may comprise/utilize any suitable toxin (or other component) of/from Yersinia entomophaga and/or Yersinia nurmii, including, but not limited to, Yen-TC and components thereof (e.g., Chil, Chi2, YenAl, YenA2, YenB, YenCl, YenC2).
  • Yersinia and Yersinia toxins may be incorporated into compositions in any suitable amount/concentration.
  • the absolute value of the amount/concentration that is/are sufficient to cause the desired effect(s) may be affected by factors such as the type, size and volume of material to which the composition will be applied and storage conditions (e.g., temperature, relative humidity, duration). Those skilled in the art will understand how to select an effective amount/concentration using routine dose-response experiments.
  • compositions of the present disclosure comprise Yersinia in an amount ranging from about 1 x 10 1 to about 1 x 10 15 , optionally about 1 x 10 3 to about 1 x 10 7 , colony-forming units (CFU) per gram and/or milliliter of composition.
  • CFU colony-forming units
  • compositions of the present disclosure may comprise about/at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 or more CFU of Yersinia per gram and/or milliliter of composition.
  • compositions of the present disclosure comprise about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 ,
  • Yersinia and/or Yersinia toxins comprise about 0.00000001 to about 95% (by weight) of the composition. In some embodiments, Yersinia and/or Yersinia toxins comprise about/at least 1 x 10 x 10- 2 , 1 x 10 1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25 4.5, 4.75, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45
  • Yersinia and/or Yersinia toxins comprise about 1 to about 25%, about 5 to about 20%, about 5 to about 15%, about 5 to about 10% or about 8 to about 12% (by weight) of the composition.
  • compositions of the present disclosure comprise Yersinia and/or Yersinia toxins in an effective amount/concentration for enhancing plant growth/yield and/or for insecticidal or other pesticidal activity when the composition is introduced into a plant growth medium (e.g., a soil).
  • a plant growth medium e.g., a soil
  • compositions of the present disclosure comprise Yersinia and/or Yersinia toxins in an effective amount/concentration for enhancing plant growth/yield and/or for insecticidal or other pesticidal activity when the composition is applied to a plant or plant part.
  • Uses, methods and compositions of the present disclosure may comprise/utilize any suitable protease(s), including, but not limited to, asparigine peptide lyases, aspartic proteases, catalytic RNA molecules, cysteine proteases, glutamic proteases, metalloproteases (e.g., proteases in MEROPS families M4, M5, M7 and M8), serine proteases (e.g., proteases in MEROPS families SI, S8 and S53), threonine proteases, and combinations thereof.
  • suitable protease(s) including, but not limited to, asparigine peptide lyases, aspartic proteases, catalytic RNA molecules, cysteine proteases, glutamic proteases, metalloproteases (e.g., proteases in MEROPS families M4, M5, M7 and M8), serine proteases (e.g., proteases in MEROPS families SI,
  • uses, methods and compositions of the present disclosure comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct proteases.
  • the protease(s) is/are toxic to multiple orders, genuses and/or species of insects. In some embodiments, the protease(s) exhibit(s) toxicity to one or more insects belonging to the order(s) Coleoptera, Diptera, Hymenoptera, Lepidoptera, Orthoptera and/or Thysanoptera.
  • the protease(s) exhibit(s) toxicity to only certain types of cells (e.g., cells that are part of certain tissues) within an insect or insect larva.
  • the protease(s) exhibit(s) toxicity to all cells within an insect or insect larva.
  • the protease(s) itself/themselves exhibit(s) insecticidal or other pesticidal activity.
  • the protease(s) exhibit(s) insecticidal or other pesticidal activity only when used in conjunction with one or more additional agents (e.g., one or more Yersinia entomophaga and/or Yersinia nurmii toxins).
  • additional agents e.g., one or more Yersinia entomophaga and/or Yersinia nurmii toxins.
  • the protease(s) may be produced using any suitable method(s) known in the art, including, but not limited to, production in and isolation from bacteria/fungi (e.g., Alicyclohaeiilus ; Arthrobacier; Aspergillus, such as A. oryzae Bacillus, such as B. alkalophilus , B. amyloliquefaciens , B. clausii, B. gibsonii, B. lentus ( B . lentus DSM 5483), B. licheniformis, B. mojavensis, B. pumilus and B. subtilis; Cellumonas ; Dichomitus, such as D.
  • bacteria/fungi e.g., Alicyclohaeiilus ; Arthrobacier; Aspergillus, such as A. oryzae Bacillus, such as B. alkalophilus , B. amyloliquefaciens , B. clausii, B
  • the protease(s) is/are recombinant.
  • the protease(s) may be purified, partially purified of unpurified.
  • the protease(s) may or may not be concentrated.
  • Non-limiting examples of proteases that may be used in methods and compositions of the present disclosure include those described in International Patent Publications WO 1992/019729, WO 1992/175177, WO 1993/018140, WO 1996/034946, WO 1998/20115, WO 1998/20116, WO 1999/011768, WO 2001/016285, WO 2001/044452, WO 2002/016547, WO 2002/026024, WO 2003/006602, WO 2004/03186, WO 2004/041979, WO 2004/067737, WO 2007/006305, WO 2011/036263, WO 2011/036264, WO 2011/140051, WO 2016/000671, WO 2016/001449, WO 2019/157061, WO 2019/236687 and WO 2019/236717.
  • Non-limiting examples of commercially available proteases that may be used in methods and compositions of the present disclosure include those sold under the trade names Alcalase®, DuralaseTM, DurazymTM, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Neutrase®, Everlase®, Esperase®, Maxatase®, Maxacal®, Maxapem®, Purafast®, Purafect Ox®, Purafect OxP®, Puramax®, FN2®, FN3®, FN4®, Excellase®, Excellenz® P1000, Excellenz® P1250, Eraser®, Peltec®
  • the protease comprises, consists essentially of or consists of an amino acid sequence that is at least 60, 65, 70, 75, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5 94, 94.5, 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25,
  • the protease comprises, consists essentially of or consists of an amino acid sequence that is at least 60, 65, 70, 75, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5 94, 94.5, 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25,
  • the protease comprises, consists essentially of or consists of an amino acid sequence that is at least 60, 65, 70, 75, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5 94, 94.5, 95, 95.5, 95.55, 95.6, 95.65, 95.7, 95.75, 95.8, 95.85, 95.9, 95.95, 96, 96.05, 96.1, 96.15, 96.2, 96.25,
  • the protease is a substilase or subtilase variant (e.g., subtilisin A, subtilisin B, subtilisin 147, subtilisin 168, subtilisin 309 and variants thereof).
  • the protease comprises, consists essentially of or consists of an amino acid sequence that is at least 60, 65, 70, 75, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5,
  • the protease is a variant of a naturally occurring protease (e.g., a naturally occurring bacterial/fungal protease).
  • the protease is a subtilase variant comprising a substitution at one or more of the following positions (wherein each position corresponds to the corresponding position of the polypeptide set forth herein as SEQ ID NO: 11): 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268 and 269.
  • the protease comprises one or more of the following substitutions: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E,
  • Such variants preferably comprise, consist essentially of or consist of an amino acid sequence that is at least 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5,
  • proteases useful for methods and compositions of the present disclosure may be present as enzymatically/pesticidally active fragments of larger proteases.
  • uses, methods and compositions of the present disclosure may comprise/utilize proteolytic fragments derived from one or more of the polypeptides set forth herein as SEQ ID NOs: 2-3, 5 and 10-26.
  • Proteases may be incorporated into compositions in any suitable amount/concentration.
  • the absolute value of the amount/concentration that is/are sufficient to cause the desired effect(s) may be affected by factors such as the type, size and volume of material to which the composition will be applied and storage conditions (e.g., temperature, relative humidity, duration). Those skilled in the art will understand how to select an effective amount/concentration using routine dose-response experiments.
  • compositions of the present disclosure comprise one or more proteases in an amount ranging from about 1 x 10 1 to about 1 x 10 15 enzyme units (at optimum conditions) per gram and/or milliliter of composition.
  • compositions of the present disclosure may comprise about/at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 or 1 x 10 15 enzyme units (at optimum conditions) per gram and/or milliliter of composition.
  • compositions of the present disclosure comprise about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , or 1 x 10 12 enzyme units (at optimum conditions) per gram and/or milliliter of composition.
  • compositions of the present disclosure comprise one or more proteases in an amount ranging from about 0.001 to about 100 milligrams per gram and/or milliliter of composition.
  • compositions of the present disclosure may comprise about/at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80
  • compositions of the present disclosure comprise about/at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 milligrams of protease per gram and/or milliliter of composition.
  • one or more proteases comprise about 0.00000001 to about 95% (by weight) of the composition. In some embodiments, one or more proteases comprise about/at least 1 x 10 15 , 1 x 10 14 , 1 x 10 13 , 1 x 10 12 , 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 , 1 x 10 6 , 1 x 10 5 , 1 x 10 4 , 1 x 10 3 , 1 x 10 2 , 1 x 10 1 ,
  • compositions of the present disclosure comprise one or more proteases in an effective amount/concentration for insecticidal or other pesticidal activity and/or for enhancing plant growth/yield when the composition is introduced into a plant growth medium (e.g., a soil).
  • a plant growth medium e.g., a soil
  • compositions of the present disclosure comprise one or more proteases in an effective amount/concentration for insecticidal or other pesticidal activity and/or for enhancing plant growth/yield when the composition is applied to a plant or plant part.
  • Combinations of Yersinia and proteases provide advantages as insecticides, as compared to Yersinia alone and protease alone.
  • the insecticidal activity e.g., the magnitude of insecticidal activity
  • the insecticidal activity of the combination is unexpected as compared to insecticidal activity of Yersinia entomophaga alone and protease alone.
  • combinations of Yersinia and protease may restore insecticidal activity and/or slow/prevent development of this resistance among one or more types of insects.
  • combinations of Yersinia and protease may provide a different insect host range as compared to the combined insect host range of the Yersinia alone and the protease alone.
  • the amount of one of the agents may be reduced as compared to the amount of the agent used to produce the same effect alone.
  • the effects of the combinations, insecticidal or otherwise may be determined using assays known in the art. Generally, these assays, including the assays used in the studies described in the Examples of this application, robustly reflect efficacy/activity/re suits of the compositions in the field.
  • the effects of the combinations of Yersinia and protease may be unexpected, additive or antagonistic on insect control and/or plant growth and/or yield (e.g., enhanced plant growth and/or enhanced plant yield). Although unexpected or even additive effects of the combinations are thought to be most advantageous, In some embodiments, there may be advantages to antagonistic effects of the combinations.
  • compositions of the present disclosure may contain one or more additional components.
  • the disclosed compositions may contain one or more pesticides and/or one or more other substances.
  • Pesticidal agents may include chemical pesticides and biopesticides or biocontrol agents.
  • Various types of chemical pesticides and biopesticides include acaricides, insecticides, nematicides, fungicides, gastropodicides, herbicides, virucides, bactericides, miticides 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. Biostimulants and/or plant signal molecules may be part of the compositions in some examples.
  • one or more of the other pesticides or other substances may be specifically excluded from the disclosed compositions.
  • the number of different Yersinia organisms or strains and/or proteases in the disclosed compositions is not limited.
  • the number of different pesticides and/or other substances within a disclosed combination is not limited.
  • a composition may contain one Yersinia, one protease, and one pesticide or other substance.
  • a combination may contain multiple Yersinia strains, multiple proteases, and multiple pesticides, insecticides and/or other substances.
  • Individual components of a combination may be combined as part of a manufacturing or formulation process or may be combined immediately prior to use. In some embodiments, individual components of a combination may not be combined until they are applied to a plant (e.g., individual components are applied separately to plants, but simultaneously or at about the same time).
  • the Yersinia (organisms and/or toxins) and protease are applied to plants at the same time. In some embodiments, the Yersinia is applied to the plant before the protease is applied to the plant.
  • the protease is applied to the plant before the Yersinia is applied to the plant.
  • the Yersinia organism(s) and/or toxin(s) is/are applied minutes, hours, days, weeks, or months before the protease(s).
  • the protease(s) is/are applied minutes, hours, days, weeks, or months before the Yersinia organism(s) and/or toxin(s).
  • the time between application of the the Yersinia organism(s) and/or toxin(s) and applications of the protease(s) is about 5, 15 or 30 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6, 7 or 8 weeks, or 1, 2, 3, 4, 5, 6, 8, 10 or 12 months.
  • the individual components are present in at least an effective amount within the composition.
  • the compositions containing these individual components may produce unexpected effects as compared to the effects of individual components of the combination. These effects may be one or more of pesticidal, insecticidal, enhanced plant growth, enhanced plant yield, and the like. In some embodiments, the compositions containing these individual components produce additive effects as compared to the effects of individual components of the combination. The additive effects may be one or more of pesticidal, insecticidal, enhanced plant growth, enhanced plant yield, and the like. In some embodiments, the compositions containing these individual components may produce antagonistic effects as compared to the effects of individual components of the combination.
  • the disclosed pesticides/other substances generally are grouped (e.g., Group 1, fungicides; Group 2, gastropodicides; Group 3, herbicides; Group 4, insecticides and/or nematicides; Group 5, acaracides and/or miticides; Group 6, biostimulants; Group 7, plant signal molecules; Group 8, other microbes) for the purposes of this disclosure.
  • Each group generally contains pesticides/other substances that generally produce similar activities, within the contexts applicable herein (e.g., individual fungicides generally are active against fungi).
  • Fungicides may be selected to provide effective control against a broad spectrum of phytopathogenic fungi (and fungus-like organisms), including, but not limited to, soil-borne fungi from the classes Ascomycetes, Basidiomycetes, Chytridiomycetes, Deuteromycetes (syn. Fungi imperfecti), Peronosporomycetes (syn. Oomycetes), Plasmodiophoromycetes and Zygomycetes.
  • the compositions comprise a fungicide (or combination of fungicides) that is toxic to one or more strains of Albugo (e.g., A.
  • Candida Alternaria (c.g. , A. alternatd), Aspergillus (e.g., A. candidus,A. clavatus,A.flavus,A.fumigatus,A. parasiticus,A. restrictus, A. sojae,A. solani), Blumeria (e.g., B. graminis), Botrytis (e.g., B. cinerea), Cladosporum (e.g., C. cladosporioides), Colletotrichum (e.g., C. acutatum, C. boninense, C. capsid, C. caudatum, C. coccodes, C. crassipes, C. dematium, C.
  • Aspergillus e.g., A. candidus,A. clavatus,A.flavus,A.fumigatus,A. parasiticus,A. restrictus, A. sojae,A. solani
  • Mycosphaerella e.g ,M. graminicola
  • Nematospora Penicillium (e.g., F*. rugulosum, P. verrucosum), Phakopsora (e.g., P. pachyrhizi), Phomopsis, Phytiphtoria (e.g., P. infestans), Puccinia (e.g., P. graminis, P. striiformis, P. tritici, P. triticina), Pucivinia (e.g., P. graministice), Pythium, Pytophthora, Rhizoctonia (e.g., R.
  • compositions of the present disclosure comprise one or more chemical fungicides.
  • chemical fungicides include 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-acry lie acid methyl ester and 2-(2-(3-(2,6-dichlorophenyl)-l -methyl - allybdeneaminooxymethyl)-phenyl)-2-methoxyimino-N-methyl-acetamide; carboxamides, such as carboxanil
  • 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-
  • compositions of the present disclosure comprise azoxystrobin, pyraclostrobin, fluoxastrobin, trifloxystrobin, ipconazole, prothioconazole, sedaxane, fludioxonil, metalaxyl, mefenoxam, thiabendazole, fluxapyroxad and/or fluopyram.
  • compositions of the present disclosure comprise one or more aromatic hydrocarbons, benzimidazoles, benzthiadiazole, carboxamides, carboxylic acid amides, morpholines, phenylamides, phosphonates, quinone outside inhibitors (e.g. strobilurins), thiazolidines, thiophanates, thiophene carboxamides and/or triazoles.
  • one or more of these fungicides may be specifically excluded from the compositions and methods disclosed herein.
  • Gastropodicides Group 21
  • the substances described in this section are part of Group 2.
  • one or more of these gastropodicides may be specifically excluded from the compositions and methods disclosed herein.
  • the composition comprises an herbicide (or combination of herbicides) that is toxic to one or more strains of Echinochloa (e.g., E. brevipedicellata, E. callopus, E. chacoensis, E. colona, E. crus-galli, E. crus-pavonis, E. elliptica, E. esculenta, E. frumentacea, E.
  • Echinochloa e.g., E. brevipedicellata, E. callopus, E. chacoensis, E. colona, E. crus-galli, E. crus-pavonis, E. elliptica, E. esculenta, E. frumentacea, E.
  • glabrescens E. haploclada, E. helodes, E. holciformis, E. inundata, E. jaliscana, E. Jubata, E. kimberleyensis, E. lacunaria, E. macrandra, E. muricata, E. obtusiflora, E. oplismenoides, E. orzyoides, E. paludigena, E. picta, E. pithopus, E. polystachya, E. praestans, E. pyramidalis, E. rotundiflora, E. stagnina, E. telmatophila, E. turneriana, E. ugandensis, E.
  • Fallopia e.g., F. baldschuanica, F. japonica, F. sachalinensis
  • Stellaria e.g., S. media
  • Taraxacum e.g., T. albidum, T. aphrogenes, T. brevicorniculatum, T. californicum, T. centrasiatum, T. ceratophorum, T. erythrospermum, T. farinosum, T. holmboei, T. japonicum, T. kok-saghyz, T. laevigatum T. officinale, T. platycarpum).
  • compositions of the present disclosure may be found in Hager, Weed Management, in ILLINOIS AGRONOMY HANDBOOK (2008) and Loux ET AL., WEED CONTROL GUIDE FOR OHIO, INDIANA AND ILLINOIS (2015).
  • compositions of the present disclosure comprise one or more chemical herbicides.
  • chemical herbicides 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, diuron, dithiopyr, fe
  • compositions of the present disclosure comprise acetochlor, clethodim, dicamba, flumioxazin, fomesafen, glyphosate, glufosinate, mesotrione, quizalofop, saflufenacil, sulcotrione, S-3100 and/or 2,4-D.
  • compositions of the present disclosure comprise glyphosate, glufosinate, dicamba, 2,4-D, acetochlor, metolachlor, pyroxasulfone, flumioxazin, fomesafen, lactofen, metribuzin, mesotrione, and/or ethyl 2-((3-(2-chloro-4-fluoro-5-(3-methyl-2,6- dioxo-4-(trifluoromethyl)-2,3-dihydropyrimidin-l(6H)-yl)phenoxy)pyridin-2-yl)oxy)acetate.
  • compositions of the present disclosure comprise one or more acetyl CoA carboxylase (ACCase) inhibitors, acetolactate synthase (ALS) inhibitors, acetohydroxy acid synthase (AHAS) inhibitors, photosystem II inhibitors, photosystem I inhibitors, protoporphyrinogen oxidase (PPO or Protox) inhibitors, carotenoid biosynthesis inhibitors, enolpyruvyl shikimate-3 -phosphate (EPSP) synthase inhibitor, glutamine synthetase inhibitor, dihydropteroate synthetase inhibitor, mitosis inhibitors, 4-hydroxyphenyl-pyruvate-dioxygenase (4- HPPD) inhibitors, synthetic auxins, auxin herbicide salts, auxin transport inhibitors, nucleic acid inhibitors and/or one or more salts, esters, racemic mixtures and/or resolved isomers thereof.
  • ACCase acetyl CoA carboxylase
  • one or more of these herbicides may be specifically excluded from the compositions and methods disclosed herein.
  • Insecticides and/or Nematicides (Group 4)
  • Insecticides may be selected so as to provide effective control against a broad spectrum of insects, including, but not limited to, insects from the orders Coleoptera, Dermaptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera, Orthoptera and Thysanoptera.
  • compositions of the present disclosure may comprise one or more insecticides toxic to insects from the families Acrididae, Aleytodidae, Anobiidae, Anthomyiidae, Aphididae, Bostrichidae, Bruchidae, Cecidomyiidae, Cerambycidae, Cercopidae, Chrysomelidae, Cicadellidae, Coccinelbdae, Cryllotalpidae, Cucujidae, Curculionidae, Dermestidae, Elateridae, Gelechiidae, Lygaeidae, Meloidae, Membracidae, Miridae, Noctuidae, Pentatomidae, Pyralidae, Scarabaeidae, Silvanidae, Spingidae, Tenebrionidae and/or Thripidae.
  • the composition comprises an insecticide (or combination of insecticides) that is toxic to one or more species of Acalymma, Acanthaoscelides (e.g., A. obtectus, ), Anasa (e.g., A. tristis), Anastrepha (e.g., A. ludens), Anoplophora (e.g., A. glabripennis), Anthonomus (e.g., A. eugenii), Acyrthosiphon (e.g., A. pisum), Bactrocera (e.g. , B. dosalis), Bemisia (e.g., B. argentifolii, B.
  • Acanthaoscelides e.g., A. obtectus,
  • Anasa e.g., A. tristis
  • Anastrepha e.g., A. ludens
  • Anoplophora e.g., A
  • Brevicoryne e.g., B. brassicae
  • Bruchidius e.g., B. atrolineatus
  • Bruchus e.g., B. atomarius, B. dentipes, B. lends, B. pisorum and/or B. rufipes
  • Callosobruchus e.g., C. chinensis, C. maculatus, C. rhodesianus, C. subinnotatus , C. theobromae
  • Caryedon e.g., C. serratus
  • Ceratitis e.g., C.
  • Epiphyas e.g., E. postvidana
  • Eumolpinae Galerucinae
  • Helicoverpa e.g., H. zea
  • Heteroligus e.g., H. meles
  • Iobesia e.g., I. botrana
  • Lamprosomatinae Lasioderma (e.g., L. serricorne)
  • Leptinotarsa e.g., E decemlineata
  • Leptoglossus Liriomyza (e.g., L. trifolii)
  • Manducca Melittia (e.g ,M. cucurbitae), Myzus (e.g.,
  • Nezara e.g., N. viridula
  • Orzaephilus e.g., O. merator, O. surinamensis
  • Ostrinia e.g., O. nubilalis
  • Phthorimaea e.g., P. operculella
  • Pieris e.g., P. rapae
  • Plodia e.g., P. interpunctella
  • Plutella e.g., P. xylostella
  • Popillia e.g., P. japonica
  • Prostephanus e.g., P. truncates
  • Psila Rhizopertha (e.g., R.
  • Rhopalosiphum e.g., R. maidis
  • Sagrinae e.g., Solenopsis
  • Spilopyrinae e.g., Sitophilus (e.g., S. granaries, S. oryzae and/or S. zeamais)
  • Sitotroga e.g., S. cerealella
  • Spodoptera e.g., S. frugiperda
  • Stegobium e.g., S. paniceum
  • Synetinae e.g., Tenebrio (e.g., T. malens and/or T. molitor), Thrips (e.g., T. tabaci), Trialeurodes (e.g., T., T.
  • Tribolium e.g., T. castaneum and/or T. confusum
  • Trichoplusia e.g., T. ni
  • Trogoderma e.g., T. granarium
  • Trogossitidae e.g., T. mauritanicus
  • Additional species of insects that may be targeted by compositions of the present disclosure may be found in CAPINERA, HANDBOOK OF VEGETABLE PESTS (2001) and Steffey and Gray , Managing Insect Pests , in ILLINOIS AGRONOMY HANDBOOK (2008).
  • Nematicides may be selected so as to provide effective control against a broad spectrum of nematodes, including, but not limited to, phytoparasitic nematodes from the classes Chromadorea and Enoplea.
  • the composition comprises a nematicide (or combination of nematicides) that is toxic to one or more strains of Anguina, Aphelenchoides, Belonolaimus , Bursaphelenchus , Ditylenchus, Globodera, Helicotylenchus , Heterodera, Hirschmanniella, Meloidogyne, Naccobus, Pratylenchus, Radopholus, Rotylenshulus , Trichodorus, Tylenchulus and/or Xiphinema. Additional species that may be targeted by compositions of the present disclosure may be found in CAPINERA, HANDBOOK OF VEGETABLE PESTS (2001) and Niblack, Nematodes , in ILLINOIS AGRONOMY H
  • compositions of the present disclosure comprise one or more chemical insecticides and/or nematicides.
  • chemical insecticides and nematicides include acrinathrin, alpha- cypermethrin, betacyfluthrin , cyhalothrin , cypermethrin, deltamethrin , csfenvalcrate , etofenprox , fenpropathrin , fenvalerate, flucythrinate, fosthiazate, lambda-cyhalothrin, gamma-cyhalothrin, permethrin, tau- fluvalinate, transfluthrin, zeta-cypermethrin, cyfluthri, bifenthrin, tefluthrin, eflusilanat, fubfenprox, pyrethrin, resmethrin
  • compositions of the present disclosure comprise 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, thiamethoxam and/or thiodicarb.
  • compositions of the present disclosure comprise one or more carbamates, diamides, macrocyclic lactones, neonicotinoids, organophosphates, phenylpyrazoles, pyrethrins, spinosyns, synthetic pyrethroids, tetronic acids and/or tetramic acids.
  • compositions of the present disclosure comprise an insecticide selected from the group consisting of clothianidin, thiamethoxam, imidacloprid, cyantraniliprole, chlorantraniliprole, fluopyram and tioxazafen.
  • insecticides include methomyl, examples of which are Lannate® and Acinate 24 L, which has activity at least against armyworms; oxamyl, one example of which is Vydate®, which has activity at least against armyworms; carbaryl, one of which is Sevin®, which has activity at least against codling moths; acephate, one of which is Orthene®, which has activity at least against armyworms; Lambda-cyhalothrin, examples of which are Mustang Max®, Baythroid®, and Karate, which have activity at least against armyworms; esfenvalerate, one of which is Asana®; fenpropathrin, one of which is Danitol®, which has activity at least against armyworms; spinosad, examples of which are Entrust®, PESTANAL® and Monterey Garden Insect Spray, which has activity at least against armyworms and/or codling moths; spinetoram, one of which is Radi
  • one or more of these insecticides/nematicides may be specifically excluded from the compositions and methods disclosed herein.
  • acaracides/mitides may include carvacrol, sanguinarine, azobenzene, benzoximate, benzyl benzoate, bromopropylate, chlorbenside, chlorfenethol, chlorfenson, chlorfensulphide, chlorobenzilate, chloropropylate, cyflumetofen, DDT, dicofol, diphenyl sulfone, dofenapyn, fenson, fentrifanil, fluorbenside, genit, hexachlorophene, phenproxide, proclonol, tetradifon, tetrasul, benomyl, carbanolate, carbaryl, carbofiiran, methiocarb, me
  • one or more of these acaracides/miticides may be specifically excluded from the compositions and methods disclosed herein.
  • compositions of the present disclosure may comprise any suitable biostimulant(s), including, but not limited to, seaweed extracts (e.g., Ascophyllum nodosum extracts, such as alginate, Ecklonia maxima extracts, etc.), myo-inositol, glycine and combinations thereof.
  • seaweed extracts e.g., Ascophyllum nodosum extracts, such as alginate, Ecklonia maxima extracts, etc.
  • myo-inositol glycine and combinations thereof.
  • compositions of the present disclosure comprise one or more biostimulants in an amount/concentration of about 0.0001 to about 5% or more (by weight) of the composition.
  • the biostimulant(s) e.g., glycine and/or seaweed extract
  • the biostimulant(s) comprise(s) about about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.02, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 to about 1, 1.1, 1.2
  • compositions of the present disclosure may comprise about 0.0005, 0.00075, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,
  • biostimulants e.g., glycine and/or seaweed extract.
  • biostimulants may be specifically excluded from the compositions and methods disclosed herein.
  • Plant Signal Molecules (Group 71
  • compositions of the present disclosure may comprise any suitable plant signal molecule(s), including, but not limited to, lipo- chitooligosaccharides (LCOs), chitooligosaccharides (COs), chitinous compounds, flavonoids, non-flavonoid node-gene inducers, jasmonic acid or derivatives thereof, linoleic acid or derivatives thereof, linolenic acid or derivatives thereof and karrikins.
  • LCOs lipo- chitooligosaccharides
  • COs chitooligosaccharides
  • chitinous compounds flavonoids, non-flavonoid node-gene inducers, jasmonic acid or derivatives thereof, linoleic acid or derivatives thereof, linolenic acid or derivatives thereof and karrikins.
  • Compositions of the present disclosure may comprise any suitable LCO(s).
  • LCOs sometimes referred to as symbiotic nodulation (Nod) signals or Nod factors, consist of an oligosaccharide backbone of -l,4-linked N- acetyl-D-glucosamine ("GIcNAc”) residues with an N-linked fatty acyl chain condensed at the non-reducing end.
  • LCOs differ in the number of GIcNAc 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, el al, ANN. REV. BIOCHEM. 65:503 (1996); Hamel, etal, PLANTA 232:787 (2010); Prome, etal, PURE &APPL. CHEM. 70(1):55 (1998).
  • compositions of the present disclosure may comprise any suitable CO(s).
  • COs sometimes referred to as N-acetylchitooligosaccharides, are also composed of GIcNAc residues but have side chain decorations that make them different from chitin molecules [(Cs-Hi3N05) n , CAS No.1398-61-4] and chitosan molecules [(CsHnNO n , CAS No. 9012-76-4] See, e.g., D'Haeze et al., GLYCOBIOL. 12(6):79R (2002); Demont-Caulet et al., PLANT PHYSIOL.
  • COs differ from LCOs in that they lack the pendant fatty acid chain that is characteristic of LCOs.
  • compositions of the present disclosure may comprise any suitable chitinous compound(s), including, but not limited to, chitin (IUPAC: N-[5-[[3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2yl]methoxymethyl]- 2-[[5-acetylamino-4,6-dihydroxy-2-(hydroxymethyl)oxan-3-yI]methoxymethyl]-4-hydroxy-6- (hydroxymethyl)oxan-3-ys]ethanamide), chitosan(IUPAC: 5-amino-6-[5-amino-6-[5-amino-4,6-dihydroxy- 2(hydroxymethyl)oxan-3-yl]oxy-4-hydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-2(hydroxymethyl)oxane-3,4-diol) and isomers, salts and solvates thereof.
  • chitin IUPAC: N-[5-[[3-acety
  • compositions of the present disclosure may comprise any suitable flavonoid(s), including, but not limited to, anthocyanidins, anthoxanthins, chalcones, coumarins, flavanones, flavanonols, flavans and isoflavonoids, as well as analogues, derivatives, hydrates, isomers, polymers, salts and solvates thereof.
  • suitable flavonoid(s) including, but not limited to, anthocyanidins, anthoxanthins, chalcones, 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 include are known in the art. See, e.g., Jain et al., J. PLANT BIOCHEM. & BlOTECHNOL. 11: 1 (2002); Shaw et al., ENVIRON. MICROBIOL. 11: 1867 (2006). Flavonoid compounds are commercially available, e.g., from Novozymes BioAg, Saskatoon, Canada; Natland International Corp., Research Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Wobum MA. Flavonoid compounds may be isolated from plants or seeds, e.g., as described in U.S.
  • Flavonoid compounds may also be produced by genetically engineered organisms, such as yeast, as described in Ralston et al., PLANT PHYSIOL. 137: 1375 (2005).
  • compositions of the present disclosure comprise one or more anthocyanidins.
  • the composition comprises cyanidin, delphinidin, malvidin, pelargonidin, peonidin and/or petunidin.
  • compositions of the present disclosure comprise one or more anthoxanthins.
  • the composition comprises one or more 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, isorhamnetin, morin, myricetin, myricitrin, natsudaidain, pachypodol,
  • flavones e
  • compositions of the present disclosure comprise one or more flavanones.
  • the composition comprises butin, eriodictyol, hesperetin, hesperidin, homoeriodictyol, isosakuranetin, naringenin, naringin, pinocembrin, poncirin, sakuranetin, sakuranin and/or sterubin.
  • compositions of the present disclosure comprise one or more flavanonols. According to some embodiments, the composition comprises dihydrokaempferol and/or taxifolin.
  • compositions of the present disclosure comprise one or more flavans.
  • the composition comprises 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), guibourtinidol, mesquitol, robinetinidol, theaflavin-3-gallate, theaflavin-3'-gallate, theflavin-3,3'-digallate, thearubigin), flavan-4-ols (e.g., apiforol and/or luteoforol) and/or flavan-3,4-diols (e.g., leucocyanidin, leucodel
  • compositions of the present disclosure comprise one or more isoflavonoids.
  • the composition comprises one or more isoflavones (e.g, biochanin A, daidzein, formononetin, genistein and/or glycitein), isoflavanes (e.g., equol, ionchocarpane and/or laxifloorane), isoflavandiols, isoflavenes (e.g., glabrene, haginin D and/or 2-methoxyjudaicin), coumestans (e.g., coumestrol, plicadin and/or wedelolactone), pterocarpans and/or roetonoids.
  • isoflavones e.g, biochanin A, daidzein, formononetin, genistein and/or glycitein
  • isoflavanes e.g., equol, ionchocarpane and
  • compositions of the present disclosure may comprise any suitable flavonoid derivative, including, but not limited to, neoflavonoids (e.g, calophyllolide, coutareagenin, dalbergichromene, dalbergin, nivetin) and pterocarpans (e.g., bitucarpin A, bitucarpin B, erybraedin A, erybraedin B, erythrabyssin II, erthyrabissin-1, erycristagallin, glycinol, glyceollidins, glyceollins, glycyrrhizol, maackiain, medicarpin, morisianine, orientanol, phaseolin, pisatin, striatine, trifolirhizin).
  • neoflavonoids e.g, calophyllolide, coutareagenin, dalbergichromene, dalbergin
  • Flavonoids and derivatives thereof may be incorporated into compositions of the present disclosure in any suitable form, including, but not limited to, polymorphic and crystalline forms.
  • compositions of the present disclosure may comprise any suitable non-flavonoid nod-gene inducer(s), including, but not limited to, jasmonic acid (
  • jasmonic acid
  • JA linoleic acid
  • 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 in the course of 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 ah, AGR. J. 98(2):289 (2006); Mabood, et al., FIELD CROPS RES.95(2-3):412 (2006); Mabood & Smith, Linoleic and linolenic acid induce the expression of nod genes in Bradyrhizobium japonicum USD A 3, PLANT BIOL. (2001).
  • Non-limiting examples of derivatives of jasmonic acid, linoleic acid, linolenic acid include esters, amides, glycosides and salts.
  • Representative 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 C1-C8 unbranched or branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a C2-C8 unbranched or branched alkenyl group; an alkynyl group, such as a C2-C8 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,
  • 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 independently: hydrogen; an alkyl group, such as a C1-C8 unbranched or branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a C2-C8 unbranched or branched alkenyl group; an alkynyl group, such as a C2-C8 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 group
  • 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 e.g., 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 together a solution of linoleic acid, linolenic acid, or jasmonic acid with a solution of the base. The salts may be precipitated from solution and be collected by fdtration 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
  • compositions of the present disclosure may comprise any suitable karrakin(s), 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.
  • suitable karrakin(s) 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.
  • one or more of these plant signal molecules may be specifically excluded from the compositions and methods disclosed herein.
  • Yersinia entomophaga and/or Yersinia nurmii are the only microbes in the compositions of the present disclosure.
  • compositions of the present disclosure comprise one or more additional microorganisms.
  • Any suitable microorganism(s) may be added, including, but not limited to, agriculturally beneficial microorganisms such as diazotrophs, phosphate-solubilizing microorganisms, mycorrhizal fungi and biopesticides. Selection of additional microbes (if any) will depend on the intended application(s).
  • Non-limiting examples of bacteria that may be included in compositions of the present disclosure include Azospirillum brasilense INTA Az-39, Bacillus amyloliquefaciens D747, Bacillus amyloliquefaciens NRRL B 50349, Bacillus amyloliquefaciens TJ1000, Bacillus amyloliquefaciens FZB24, Bacillus amyloliquefaciens FZB42, Bacillus amyloliquefaciens IN937a, Bacillus amyloliquefaciens IT-45, Bacillus amyloliquefaciens TJ1000, Bacillus amyloliquefaciens MBI600, Bacillus amyloliquefaciens BS27 (deposited as NRRF B-5015), Bacillus amyloliquefaciens BS2084 (deposited as NRRF B-50013), Bacillus amyloliquefaciens 15AP4 (deposited
  • Non-limiting examples of fungi include Gliocladium virens ATCC 52045, Gliocladium virens GL-21, Glomus intraradices RTI-801, Metarhizium anisopliae F52, PENI, Trichoderma asperellum SKT-1, Trichoderma asperellum ICC 012, Trichoderma atroviride LC52, Trichoderma atroviride CNCM 1-1237, Trichoderma fertile JM41R, Trichoderma gamsii ICC 080, Trichoderma hamatum ATCC 52198, Trichoderma harzianum ATCC 52445, Trichoderma harzianum KRL- AG2, Trichoderma harzianum T-22, Trichoderma harzianum TH-35, Trichoderma harzianum T-39, Trichoderma harzianum ICC012, Trichoderma reesi ATCC 28217, Trichoderma viren
  • ITS internal transcribed spacer
  • COl cytochrome c oxidase
  • mycorrhizal fungi that may be included in compositions of the present disclosure include mycorrhizal strains such as Gigaspora margarita, Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus intraradices , Glomus monosporum, Glomus mosseae, Laccaria bicolor, Laccaria laccata, Paraglomus brazilianum, Pisolithus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosuli, Scleroderma cepa and Scleroderma citrinum and combinations thereof.
  • mycorrhizal strains such as Gigaspora margarita, Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus intraradices , Glo
  • Additional microorganisms may be incorporated into compositions of the present disclosure in any suitable amount(s)/concentration(s).
  • the absolute value of the amount/concentration that is/are sufficient to cause the desired effect(s) may be affected by factors such as the type, size and volume of material to which the composition will be applied, the microorganisms in the composition, the number of microorganisms in the composition, the stability of the microorganisms in the composition and storage conditions (e.g., temperature, relative humidity, duration).
  • factors e.g., temperature, relative humidity, duration.
  • one or more additional microorganisms is/are present in an effective amount/concentration for fixing atmospheric nitrogen, solubilizing phosphate, controlling one or more phytopathogenic pests, enhancing stress tolerance and/or enhancing plant growth/yield when the composition is introduced into a plant growth medium (e.g., a soil).
  • a plant growth medium e.g., a soil
  • one or more additional microorganisms is/are present in an effective amount/concentration for fixing atmospheric nitrogen, solubilizing phosphate, controlling one or more phytopathogenic pests, enhancing stress tolerance and/or enhancing plant growth/yield when the composition is applied to a plant or plant part.
  • one or more additional microorganisms is/are present in an amount ranging from about 1 x 10 1 to about 1 x 10 12 colony-forming units (CFU) per gram and/or milliliter of composition.
  • the composition comprises about 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 or more CFU of one or more additional microorganisms per gram and/or milliliter of composition (e.g., about 1 x 10 4 to about 1 xo 10 9 CFU/g of Bacillus amyloliquefaciens TJ1000 (also known as 1BE, isolate ATCC BAA-390), Bradyrhizobium, Metarhizium anisopliae F52, PENI, Tri
  • compositions of the present disclosure comprise at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 CFU of one or more additional microorganisms per gram and/or milliliter of composition.
  • spores from one or more additional microorganisms comprise about 0.1 to about 90% (by weight) of the composition. According to some embodiments, the composition comprises about 0.1, 0.2,
  • microbial spores from one or more additional microorganisms (e.g., about 10% Bacillus amyloliquefaciens TJ1000, Metarhizium anisopliae F52, Penicillium bilaiae ATCC 20851,
  • the amount/concentration of microbial spores from one or more additional microorganisms is about 1 to about 25%, about 5 to about 20%, about 5 to about 15%, about 5 to about 10% or about 8 to about 12% (by weight) of the composition.
  • compositions of the present disclosure may comprise vegetative cells and/or dormant spores. According to some embodiments, at least 1, 5, 10, 15, 20, 25,
  • compositions of the present disclosure as vegetative cells. According to some embodiments, at least 1,
  • compositions of the present disclosure as spores.
  • compositions of the present disclosure may comprise any suitable microbial extract(s), including, but not limited to, bacterial extracts, fungal extracts and combinations thereof.
  • compositions of the present disclosure comprise one or more extracts of media comprising one or more diazotrophs, phosphate- solubilizing microorganisms and/or biopesticides.
  • compositions of the present disclosure comprise an extract of media comprising one or more of the microbial strains.
  • one or more of these other microbes may be specifically excluded from the compositions and methods disclosed herein.
  • compositions and methods disclosed herein may be active against any type of insect, including insects that are members of the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Orthoptera and Thysanoptera.
  • Yersinia entomophaga and protease may not be effective against one or more insects in one or more of these orders.
  • combinations of Yersinia entomophaga and protease may be effective against insects that include Aedes mosquitos, cotton leafhoppers, Anopheline mosquitos, melon and cotton aphids, tobacco whiteflys, rice stem borers, bed bugs, cockroaches, house mosquitos, codling moths, Asian citrus psyllids, sugarcane borers, green-belly stink bugs, stink bugs, western flower thrips, tsetse flies, cotton bollworms, com earworms, tobacco budworms, Colorado potato beetles, eggplant fruit borers, American serpentine leafminers, European grapevine moths, African cowpea thrips, pollen beatles, houseflies, green peach aphids, currant-lettuce aphids, brown planthoppers, European com borers, European red mites, diamondback moths, cabbage stem flea beetles, birdcherry-
  • Yersinia entomophaga and protease may not be effective against one or more insects that include Aedes mosquitos, cotton leafhoppers, Anopheline mosquitos, melon and cotton aphids, tobacco whiteflys, rice stem borers, bed bugs, cockroaches, house mosquitos, codling moths, Asian citrus psyllids, sugarcane borers, green-belly stink bugs, stink bugs, western flower thrips, tsetse flies, cotton bollworms, com earworms, tobacco budworms, Colorado potato beetles, eggplant fruit borers, American serpentine leafminers, European grapevine moths, African cowpea thrips, pollen beatles, houseflies, green peach aphids, currant-lettuce aphids, brown planthoppers, European com borers, European red mites, diamondback moths, cabbage stem flea beetles, birdcher
  • Yersinia entomophaga and protease may be effective against insects that include chewing pests, examples of which include: 1) Lepidoptera - codling moths, sugarcane borers, cotton bollworms, com earworms, tobacco budworms, eggplant fruit borers, European grapevine moths, European com borers, diamondback moths, beet armyworms, fall armyworms, cotton leafworms, cabbage loopers, black cutworms, Agrotis spp., black cutworm, cutworm, Helicoverpa spp., tomato fruitworm, Heliothis spp., swift moth, strawberry root worm, Egyptian cotton leafworm, armyworms, velvetbean caterpillar, southwestern com borer, soybean looper, southern armyworm and green cloverworms; 2) Coleoptera - rice stem borers, Colorado potato beetles, pollen beetles, cabbage stem flea beetles, red flour beetles,
  • Yersinia entomophaga and protease may be effective against insects that include rasping pests, examples of which include: 1) Thysanoptera - western flower thrips, African cowpea thrips, avocado thrips, onion thrips, privet thrips, eastern flower thrips and chili thrips.
  • Yersinia entomophaga and protease may be effective against insects that include sucking pests, examples of which include: 1) Hemiptera - cotton leafhoppers, melon and cotton aphids, tobacco whiteflys, bed bugs, asian citrus psyllids, green-belly stink bugs, stink bugs, green peach aphids, currant-lettuce aphids, brown planthoppers, birdcherry-oat aphids, English grain aphids, white-backed planthoppers, glasshouse whiteflies, kissing bugs, root aphids, grape phylloxera, brown marmorated stink bug, lygus, bagruda bug, three cornered alfalfa hopper, chinch bugs, potato psyllid, cabbage aphid, green leafhopper and potato leafhopper; 2) Acari: European red mites, twospotted spider mites, rust mite, livestock ticks, blacklegged tick
  • Yersinia entomophaga and protease may be effective against insects that include root aphids, grape phylloxera, brown marmorated stink bugs, lygus, bagrada bugs, three cornered alfalfa hoppers, green cloverworms, chinch bugs and blister beetles.
  • any of the above-listed insects may be specifically excluded from the range of insects against which the compositions and methods disclosed herein exhibit insecticidal or other pesticidal activity.
  • Yersinia entomophaga and proteases may be formulated into any suitable type of composition, including, but not limited to, compositions for foliar applications, seed coatings and soil applications.
  • the Yersinia entomophaga and protease may be formulated separately, and then combined before application, or the formulated compositions may be separately applied, to a plant for example.
  • Separate compositions for prolonging viability/activity of the organisms and enzymes may take into account the differences between the organisms/enzymes and/or different forms of the organisms/enzymes that may be used.
  • compositions of the present disclosure may comprise any suitable carrier(s), including, but not limited to, foliar-compatible carriers, seed-compatible carriers and soil-compatible carriers. Selection of appropriate carrier materials will depend on the intended application(s) and the microorganism(s) present in the composition.
  • the carrier material(s) will be selected to provide a composition in the form of a liquid, gel, slurry, or solid.
  • the carrier will consist essentially of or consist of one or more stabilizing compounds.
  • the composition comprises one or more solid carriers.
  • the composition comprises one or more powders (e.g., wettable powders) and/or granules.
  • solid carriers include clays (e.g., attapulgite clays, montmorillonite clay, etc.), peat-based powders and granules, freeze-dried powders, spray-dried powders, spray-freeze-dried powders and combinations thereof.
  • the formulated composition comprises one or more liquid and/or gel carriers.
  • the composition comprises one or more non-aqueous solvents.
  • the composition comprises one or more aqueous solvents (e.g., water).
  • an aqueous solvent such as water
  • a co-solvent such as ethyl lactate, methyl soyate/ethyl lactate co-solvent blends (e.g., STEPOSOLTM, Stepan), isopropanol, acetone, 1,2- propanediol, n-alkylpyrrolidones (e.g., AGSOLEXTM wetting agents; Ashland, Inc., Covington, KY ), petroleum based-oils (e.g., AROMATICTM and SOLVESSOTM fluids; ExxonMobil Chemical Company, Spring, TX), isoparrafmic hyydrocarbons (e.g., ISOPARTM fluids; ExxonMobil Chemical Company, Spring, TX), cycloparaffinic hydrocarbons (e.g., NAPPARTM 6; ExxonMobil Chemical Company, Spring, TX), mineral spirits (e.g.,
  • the composition comprises one or more inorganic solvents, such as decane, dodecane, hexylether and nonane.
  • the composition comprises one or more organic solvents, such as acetone, dichloromethane, ethanol, hexane, methanol, propan-2-ol and trichloroethylene.
  • Non limiting examples of liquid/gel carriers include oils (e.g., mineral oil, olive oil, peanut oil, soybean oil, sunflower oil), polyethylene glycols (e.g., PEG 200, PEG 300, PEG 400, etc.), propylene glycols (e.g., PPG-9, PPG-10, PPG-17, PPG-20, PPG-26, etc.), ethoxylated alcohols (e.g., TOMADOL® (Air Products and Chemicals, Inc., Allentown, PA), TERGITOLTM 15-S surfactants such as TERGITOLTM15-S-9 (The Dow Chemical Company, Midland, MI), etc.), isoparrafmic hyydrocarbons (e.g., ISOPARTM, ISOPARTM L, ISOPARTM M, ISOPARTM V; ExxonMobil Chemical Company, Spring, TX), pentadecane, polysorbates (e.g. polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, etc
  • compositions of the present disclosure may comprise any suitable stabilizing compound(s), including, but not limited to, maltodextrins, monosaccharides, disaccharides, oligosaccharides, sugar alcohols, humic acids, fulvic acids, malt extracts, peat extracts, betaines, prolines, sarcosines, peptones, skim milks, oxidation control components, hygroscopic polymers and UV protectants.
  • suitable stabilizing compound(s) including, but not limited to, maltodextrins, monosaccharides, disaccharides, oligosaccharides, sugar alcohols, humic acids, fulvic acids, malt extracts, peat extracts, betaines, prolines, sarcosines, peptones, skim milks, oxidation control components, hygroscopic polymers and UV protectants.
  • the composition comprises one or more maltodextrins (e.g., one or more maltodextrins having a dextrose equivalent value (DEV) of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25).
  • the composition comprises one or more maltodextrins having a DEV of about 5 to about 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 or 20, about 10 to about 11, 12, 14, 15, 16, 17, 18, 19 or 20, or about 15 to about 16, 17, 18, 19 or 20.
  • the composition comprises a combination of maltodextrins having a DEV of about 5 to about 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 or 20, about 10 to about 11, 12, 14, 15, 16, 17, 18, 19 or 20, or about 15 to about 16, 17, 18, 19 or 20.
  • the composition comprises one or more monosaccharides (e.g., allose, altrose, arabinose, fructose, galactose, glucose, gulose, iodose, lyxose, mannose, ribose, talose, threose and/or xylose).
  • the composition comprises glucose.
  • the composition does not comprise glucose.
  • the composition comprises one or more disaccharides (e.g., cellobiose, chitobiose, gentiobiose, gentiobiulose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose (e.g., maltose monohydrate, anhydrous maltose), maltulose, mannobiose, melibiose, melibiulose, nigerose, palatinose, rutinose, rutinulose, sophorose, sucrose, trehalose, turanose and/or xylobiose).
  • the composition comprises maltose.
  • the composition does not comprise maltose.
  • the composition comprises trehalose.
  • the composition does not comprise trehalose.
  • the composition comprises one or more oligosaccharides (e.g., fructo- oligosaccharides, galacto-oligosaccharides, mannon-oligosaccharides and/or raffmose).
  • oligosaccharides e.g., fructo- oligosaccharides, galacto-oligosaccharides, mannon-oligosaccharides and/or raffmose.
  • the composition comprises one or more sugar alcohols (e.g., arabitol, erythritol, fucitol, galactitol, glycerol, iditol, inositol, isomalt, lactitol, maltitol, maltotetraitol, maltotriitol, mannitol, polyglycitol, ribitol, sorbitol, threitol, volemitol and/or xylitol).
  • sugar alcohols e.g., arabitol, erythritol, fucitol, galactitol, glycerol, iditol, inositol, isomalt, lactitol, maltitol, maltotetraitol, maltotriitol, mannitol, polyglycitol, ribitol, sorbito
  • the composition comprises one or more humic acids (e.g., one or more leonardite humic acids, lignite humic acids, peat humic acids and water-extracted humic acids).
  • the composition comprises ammonium humate, boron humate, potassium humate and/or sodium humate.
  • one or more of ammonium humate, boron humate, potassium humate and sodium humate is/are excluded from the composition.
  • Nonlimiting examples of humic acids that may be useful in embodiments of the present disclosure include MDL Number MFCD00147177 (CAS Number 1415-93-6), MDL Number MFCD00135560 (CAS Number 68131-04-4), MDL Number MFCS22495372 (CAS Number 68514-28-3), CAS Number 93924-35-7, and CAS Number 308067-45-0.
  • the composition comprises one or more fulvic acids (e.g., one or more leonardite fulvic acids, lignite fulvic acids, peat fulvic acids and/or water-extracted fulvic acids).
  • the composition comprises ammonium fulvate, boron fulvate, potassium fulvate and/or sodium fulvate.
  • one or more of ammonium fulvate, boron fulvate, potassium fulvate and sodium fulvate is/are excluded from compositions of the present disclosure.
  • fulvic acids that may be useful in embodiments of the present disclosure include MDL Number MFCD09838488 (CAS Number 479-66-3).
  • the composition comprises one or more betaines (e.g., trimethylglycine).
  • the composition comprises one or more peptones (e.g., bacterial peptones, meat peptones, milk peptones, vegetable peptones and yeast peptones).
  • peptones e.g., bacterial peptones, meat peptones, milk peptones, vegetable peptones and yeast peptones.
  • the composition comprises one or more oxidation control components (e.g., one or more antioxidants and/or oxygen scavengers).
  • the composition comprises one or more oxygen scavengers, such as ascorbic acid, ascorbate salts, catechol and/or sodium hydrogen carbonate.
  • the composition comprises one or more antioxidants, such as ascorbic acid, ascorbyl palmitate, ascorbyl stearate, calcium ascorbate, carotenoids, lipoic acid, phenolic compounds (e.g., flavonoids, flavones, flavonols), potassium ascorbate, sodium ascorbate, thiols (e.g., glutathione, lipoic acid, N- acetyl cysteine), tocopherols, tocotrienols, ubiquinone and/or uric acid.
  • antioxidants such as ascorbic acid, ascorbyl palmitate, ascorbyl stearate, calcium ascorbate, carotenoids, lipoic acid, phenolic compounds (e.g., flavonoids, flavones, flavonols), potassium ascorbate, sodium ascorbate, thiols (e.g., glutathione, lipoic acid, N- acetyl cysteine
  • Non-limiting examples of antioxidants include those that are soluble in the cell membrane (e.g., alpha tocopherol (vitamin E), ascorbyl palmitate) and those that are soluble in water (e.g., ascorbic acid and isomers or ascorbic acid, sodium or potassium salts of ascorbic acid or isomers or ascorbic acid, glutathione, sodium or potassium salts of glutathione).
  • vitamin E vitamin E
  • ascorbyl palmitate those that are soluble in water
  • water e.g., ascorbic acid and isomers or ascorbic acid, sodium or potassium salts of ascorbic acid or isomers or ascorbic acid, glutathione, sodium or potassium salts of glutathione.
  • use of a membrane -soluble antioxidant necessitates the addition of one or more surfactants to adequately disperse the antioxidant within the composition.
  • the composition is/comprises ascorbic acid and/or glutathione.
  • the composition comprises one or more hygroscopic polymers (e.g., hygroscopic agars, albumins, alginates, carrageenans, celluloses, gums (e.g., cellulose gum, guar gum, gum arabic, gum combretum, xantham gum), methyl celluloses, nylons, pectins, polyacrylic acids, polycaprolactones, polycarbonates, polyethylene glycols (PEG), polyethylenimines (PEI), polylactides, polymethylacrylates (PMA), polyurethanes, polyvinyl alcohols (PVA), polyvinylpyrrolidones (PVP), propylene glycols, sodium carboxymethyl celluloses and/or starches).
  • hygroscopic polymers e.g., hygroscopic agars, albumins, alginates, carrageenans, celluloses, gums (e.g., cellulose gum, gu
  • Non-limiting examples of polymers include AGRIMERTM polymers (e.g., 30, AL-10 LC, AL-22, AT/ATF, VA 3E, VA 31, VA 5E, VA 51, VA 6, VA 6E, VA 7E, VA 71, VEMA AN- 216, VEMA AN-990, VEMA AN-1200, VEMA AN-1980, VEMA H-815MS; Ashland Specialty Ingredients, Wilmington, DE), EASYSPERSETM polymers (Ashland Specialty Ingredients, Wilmington, DE); DISCOTM AG polymers (e.g., L-250, L-280, L-285, L-286, L-320, L-323, L-517, L-519, L-520, L800; Incotec Inc., Salinas,
  • the composition comprises one or more UV protectants (e.g., one or more aromatic amino acids (e.g., tryptophan, tyrosine), carotenoids, cinnamates, lignosulfonates (e.g., calcium lignosulfonate, sodium lignosulfonate), melanins, mycosporines, polyphenols and/or salicylates).
  • UV protectants e.g., one or more aromatic amino acids (e.g., tryptophan, tyrosine), carotenoids, cinnamates, lignosulfonates (e.g., calcium lignosulfonate, sodium lignosulfonate), melanins, mycosporines, polyphenols and/or salicylates).
  • UV protectants include Borregaard LignoTechTM lignosulfonates (e.g., Borresperse 3A, Borresperse CA, Borresperse NA, Marasperse AG, Norlig A, Norlig 1 ID, Ufoxane 3A, Ultrazine NA, Vanisperse CB; Borregaard Lignotech, Sarpsborg, Norway) and combinations thereof. Additional examples of UV protectants may be found in Burges, composition of Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments (Springer Science & Business Media) (2012).
  • Compositions of the present disclosure may comprise any suitable nutrient(s), including, but not limited to, 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 B ( .
  • 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,
  • composition of the present disclosure comprises phosphorous, boron, chlorine, copper, iron, manganese, molybdenum and/or zinc.
  • compositions of the present disclosure may comprise any suitable pest attractant(s) and/or feeding stimulant(s), including, but not limited to, brevicomin, ceralure, codlelure, cue-lure, disparlure, dominicalure, eugenol, frontalin, gossyplure, grandlure, hexalure, ipsdienol, ipsenol, japonilure, latitlure, lineatin, litlure, looplure, medlure, megatomic acid, methyl eugenol, moguchun, a-multistriatin, muscalure, orfalure, oryctalure, ostramone, rescalure, siglure, sulcatol, trimedlure and/or trunc -call.
  • suitable pest attractant(s) and/or feeding stimulant(s) including, but not limited to, brevicomin, ceralure, codlelure, cue-
  • compositions of the present disclosure may comprise gluconolactone and/or one or more analogues, derivatives, hydrates, isomers, polymers, salts and/or solvates thereof.
  • compositions of the present disclosure may comprise any suitable excipient(s), including, but not limited to, dispersants, drying agents, anti-freezing agents, seed flowability agents, safeners, anti-settling agents, pH buffers and adhesives.
  • compositions of the present disclosure may comprise any suitable agriculturally acceptable dispersant(s), including, but not limited to, surfactants and wetting agents. Selection of appropriate dispersants will depend on the intended application(s) and the microorganism(s) present in the composition. In general, the dispersant(s) will have low toxicity for the microorganism(s) in the composition and for the plant part(s) to which the composition is to be applied. In some embodiments, the dispersant(s) will be selected to wet and/or emulsify one or more soils.
  • Non-limiting examples of dispersants include AtloxTM (e.g., 4916, 4991; Croda International PLC, Edison, NJ), Atlox METASPERSETM (Croda International PLC, Edison, NJ), BIO-SOFT® (e.g., N series, such as Nl-3, Nl-7, Nl-5, Nl-9, N23-3, N2.3-6.5, N25-3, N25-7, N25-9, N91-2.5, N91-6, N91-8; Stepan Company, Northfield, IL), MAKON® nonionic surfactants (e.g., DA-4, DA-6 and DA-9; Stepan Company, Northfield, IL), MORWET® powders (Akzo Nobel Surface Chemistry LLC, Chicago, IL), MULTIWETTM surfactants (e.g., MO-85P-PW- (AP); Croda International PLC, Edison, NJ), SILWET® L-77 (Helena Chemical Company, Collierville, TN), SPANTM
  • Soil Facts Using wetting Agents (Nonionic Surfactants) on Soil (North Carolina Cooperative Extension Service Publication AG-439-25) (1993); Burges, composition of Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments (Springer Science & Business Media) (2012); McCarty, Wetting Agents (Clemson University Cooperative Extension Service Publication) (2001).
  • compositions of the present disclosure comprise one or more anionic surfactants.
  • the composition comprises one or more water-soluble anionic surfactants and/or one or more water-insoluble anionic surfactants, optionally one or more anionic surfactants selected from the group consisting of alkyl carboxylates (e.g., sodium stearate), alkyl sulfates (e.g., alkyl lauryl sulfate, sodium lauryl sulfate), alkyl ether sulfates, alkyl amido ether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates, alkyl aryl sulfonates, alkyl sulfonates, alkyl amide sulfonates, alkyl aryl sulfonates, alkyl benzene sulfonates, alkyl diphenyloxide sulfon
  • compositions of the present disclosure comprise one or more cationic surfactants.
  • the composition comprises one or more pH-dependent amines and/or one or more quaternary ammonium cations, optionally one or more cationic surfactants selected from the group consisting of alkyltrimethylammonium salts (e.g., cetyl trimethylammonium bromide, cetyl trimethylammonium chloride), cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, 5-Bromo-5-nitro-l,3- dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide and/or octenidine dihydrochloride.
  • alkyltrimethylammonium salts e.g., cetyl trimethylammonium bromide, cetyl trimethylammonium chloride
  • compositions of the present disclosure comprise one or more nonionic surfactants.
  • the composition comprises one or more water-soluble nonionic surfactants and/or one or more water-insoluble nonionic surfactants, optionally one or more nonionic surfactants selected from the group consisting of alcohol ethoxylates (e.g., TERGITOLTM 15-S surfactants, such as TERGITOLTM15- S-9 (The Dow Chemical Company, Midland, MI)), alkanolamides, alkanolamine condensates, carboxylic acid esters, cetostearyl alcohol, cetyl alcohol, cocamide DEA, dodecyldimethylamine oxides, ethanolamides, ethoxylates of glycerol ester and glycol esters, ethylene oxide polymers, ethylene oxide-propylene oxide copolymers, glucoside alkyl ethers, glycerol alkyl ethers, glycerol esters
  • compositions of the present disclosure comprise at least one nonionic surfactant.
  • the composition comprises at least one water insoluble nonionic surfactant and at least one water soluble nonionic surfactant.
  • compositions of the present disclosure comprise a combination of nonionic surfactants having hydrocarbon chains of substantially the same length.
  • compositions of the present disclosure comprise one or more zwitterionic surfactants.
  • the composition comprises one or more betaines and/or one or more sultaines, optionally one or more zwitterionic surfactants selected from the group consisting of 3-[(3- Cholamidopropyl)dimethylammonio]-l-propanesulfonate, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and/or one or more sphingomyelins.
  • compositions of the present disclosure comprise one or more soaps and/or organosilicone surfactants.
  • the composition comprises one or more alkali metal salts of fatty acids.
  • compositions of the present disclosure comprise one or more wetting agents.
  • the composition comprises one or more naphthalene sulfonates, optionally one or more alkyl naphthalene sulfonates (e.g., sodium alkyl naphthalene sulfonate), one or more isopropyl naphthalene sulfonates (e.g., sodium isopropyl naphthalene sulfonate) and/or one or more butyl naphthalene sulfonates (e.g., sodium n-butyl naphthalene sulfonate).
  • alkyl naphthalene sulfonates e.g., sodium alkyl naphthalene sulfonate
  • isopropyl naphthalene sulfonates e.g., sodium isopropyl naphthalene sulfonate
  • compositions of the present disclosure may comprise any suitable drying agent(s), including, but not limited to, drying powders.
  • drying agents include AEROSIL® hydrophobic fumed silica powders (Evonik Corporation, Parsippany, NJ), BENTOLITE® powders (BYK-Chemie GmbH, Wesel, Germany), INCOTEC® powders (INCOTEC Inc., Salinas, CA), SIPERNAT® silica powders (Evonik Corporation, Parsippany, NJ) and combinations thereof. Additional examples of drying agents may be found in Burges, composition of Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments (Springer Science & Business Media) (2012).
  • compositions of the present disclosure comprise calcium stearate, clay (e.g., attapulgite clay, montmorillonite clay), graphite, magnesium stearate, magnesium sulfate, powdered milk, silica (e.g., fumed silica, hydrophobically-coated silica, precipitated silica), soy lecithin and/or talc.
  • clay e.g., attapulgite clay, montmorillonite clay
  • graphite e.g., graphite
  • magnesium stearate e.g., magnesium stearate
  • magnesium sulfate e.g., magnesium stearate
  • powdered milk e.g., silica (e.g., fumed silica, hydrophobically-coated silica, precipitated silica), soy lecithin and/or talc.
  • silica e.g., fumed silica, hydrophobically-co
  • compositions of the present disclosure may comprise any suitable anti-freezing agent(s), including, but not limited to, ethylene glycol, glycerin, propylene glycol and urea.
  • Compositions of the present disclosure may comprise any seed flowability agent to improve the lubricity of the treated seeds.
  • the flowability agent may comprise one or more liquid lubricants, solid lubricants, liquid emulsions, or suspensions of solid lubricants.
  • Non-limiting examples of flowability agents include, for example, lubricants such as fats and oils, natural and synthetic waxes, graphite, talc, fluoropolymers (e.g., polytetrafluoroethylene), and solid lubricants such as molybdenum disulfide and tungsten disulfide.
  • the flowability agent comprises a wax material.
  • Non-limiting examples of wax materials that can be incorporated into the liquid seed treatment composition include plant and animal -derived waxes such as camauba wax, candelilla wax, ouricury wax, beeswax, spermaceti, and petroleum derived waxes, such as paraffin wax.
  • the flowability agent comprises camauba wax.
  • the flowability agent comprises an oil.
  • the flowability agent may comprise soybean oil.
  • Non-limiting examples of commercially available wax materials suitable for use as flowability agents include AQUAKLEAN 418 supplied by Micro Powders, Inc. (an anionic aqueous emulsion comprising extra light camauba wax at 35% solids content).
  • compositions of the present disclosure may comprise any suitable safener(s), including, but not limited to, napthabc anhydride.
  • compositions of the present disclosure may comprise any suitable pH buffer(s), including, but not limited to, potassium phosphate monobasic and potassium phosphate dibasic.
  • the composition comprises one or more pH buffers selected to provide a composition having a pH of less than 10, typically from about 4.5 to about 9.5, from about 6 to about 8, or about 7.
  • compositions of the present disclosure may comprise any suitable anti-settling agent(s), including, but not limited to, polyvinyl acetate, polyvinyl alcohols with different degrees of hydrolysis, polyvinylpyrrolidones, polyacrylates, acrylate-, polyol- or polyester-based paint system binders which are soluble or dispersible in water, moreover copolymers of two or more monomers such as acrylic acid, methacrylic acid, itaconic acid, maleic acid, fumaric acid, maleic anhydride, vinylpyrrolidone, ethylenically unsaturated monomers such as ethylene, butadiene, isoprene, chloroprene, styrene, divinylbenzene, ot-methylstyrene or p-methylstyrene, further vinyl halides such as vinyl chloride and vinylidene chloride, additionally vinyl esters such as vinyl acetate, vinyl propionate or vinyl stearate, moreover vinyl methyl
  • compositions of the present disclosure may comprise any suitable adhesive(s), including, but not limited to, adhesive compositions comprising, consisting essentially of or consisting of one or more disaccharides (e.g. maltose), gums (e.g., cellulose gum, guar gum, gum arabic, gum combretum, xantham gum), maltodextrins (e.g., one or more maltodextrins (each and/or collectively) having a DEV of about 10 to about 20), monosaccharides, oils (e.g., mineral oil, olive oil, peanut oil, soybean oil and/or sunflower oil) and/or oligosaccharides.
  • adhesive compositions comprising, consisting essentially of or consisting of one or more disaccharides (e.g. maltose), gums (e.g., cellulose gum, guar gum, gum arabic, gum combretum, xantham gum), maltodextrins (e.g., one
  • compositions of the present disclosure may comprise any suitable effect pigment(s).
  • Effect pigments which are sometimes also referred to in the art as “pearl pigments,” are a class of materials that provide reflectivity, shine, and/or a pearlescent effect when applied as a coating.
  • the effect pigment is in the form of a powder comprising a substrate material and a metal oxide coating.
  • the effect pigment may comprise a substrate material including but not limited to talc, silicate materials (e.g., mica), clay minerals, calcium carbonate, kaolin, phlogopite, alumina, and similar substances.
  • the substrate material comprises a hydrophilic material.
  • the substrate material may be coated with a semi-transparent layer of a metal oxide, including but not limited to titanium dioxide, iron oxide, chromium oxide, or zirconium oxide.
  • the effect pigment comprises metal powder or metal flakes.
  • the metal powder or metal flakes may comprise a metal including, but not limited to aluminum, copper, silver, or bronze.
  • the effect pigment comprises a silicate based substrate.
  • particulate silicates that can be incorporated into the dry powder coating include mica coated with titanium dioxide (e.g., SUNMICA FINE WHITE 2800102, which is commercially available from Sun Chemical Corp.).
  • the dry powder has a mean particle size of from about 1 to about 25 microns.
  • compositions of the present disclosure may comprise any suitable growth medium suitable for culturing one or more of the microorganisms in the composition.
  • suitable growth medium suitable for culturing one or more of the microorganisms in the composition.
  • compositions of the present disclosure comprise Czapek-Dox medium, glycerol yeast extract, mannitol yeast extract, potato dextrose broth and/or YEM media.
  • Carriers, stabilizing compounds, biostimulants, microbial extracts, nutrients, pest attractants and/or feeding stimulants, pesticides, plant signal molecules, dispersants, drying agents, safeners, flowability agents, anti-settling agents, buffers, adhesives, etc. may be incorporated into compositions of the present disclosure in any suitable amount(s)/concentration(s).
  • the absolute value of the amount/concentration that is/are sufficient to cause the desired effect(s) may be affected by factors such as the type, size and volume of material to which the compositon will be applied, the type(s) of microorganisms in the composition, the number of microorganisms in the composition, the stability of the microorganisms in the composition and storage conditions (e.g., temperature, relative humidity, duration).
  • factors such as the type, size and volume of material to which the compositon will be applied, the type(s) of microorganisms in the composition, the number of microorganisms in the composition, the stability of the microorganisms in the composition and storage conditions (e.g., temperature, relative humidity, duration).
  • compositions of the present disclosure comprise one or more carriers in an amount/concentration of about 1 to about 99% or more (by weight, based upon the total weight of the composition).
  • compositions of the present disclosure may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% (by weight) of one or more non-aqueous carriers.
  • compositions of the present disclosure comprise one or more stabilizing compounds in an amount/concentration of about 0.0001 to about 95% or more (by weight, based upon the total of the composition).
  • compositions of the present disclosure may comprise about 0.0001 to about 0.001, about 0.001 to about 1%, about 0.25 to about 5%, about 1 to about 10%, about 5 to about 25%, about 10% to about 30%, about 20% to about 40%, about 25% to about 50%, about 30 to about 60%, about 50 to about 75%, or about 75 to about 95% (by weight), optionally about 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,
  • maltodextrins monosaccharides, disaccharides, sugar alcohols, humic acids, betaines, prolines, sarcosines, peptones, oxidation control components, hygroscopic polymers and/or UV protectants.
  • compositions of the present disclosure comprise one or more stabilizing compounds at a concentration of about 1 x 10 20 M to about 1 x 10 1 M.
  • compositions of the present disclosure may comprise about 1 x 10 15 M to about 1 x 10 10 M, about 1 x 10 14 M to about 1 x 10 8 M, about 1 x 10 14 M to about 1 x 10 6 M, about 1 x 10 12 M to about 1 x 10 8 M, about 1 x 10 12 M to about 1 x 10 6 M, about 1 x 10 10 M to about 1 x 10 6 M, or about 1 x 10 8 M to about 1 x 10 2 M, optionally about 1 x 10 20 M, 1 x 10 19 M, 1 x Kb 18 M, 1 x Kb 17 M, 1 x Kb 16 M, 1 x 10 15 M, 1 x 10 14 M, 1 x 10 13 M, 1 x 10 12 M, 1 x 10 11 M, 1 x 10 10 M, 1 x Kb 9 M, 1
  • compositions of the present disclosure comprise one or more monosaccharides in an amount/concentration of about 0.005 to about 50% (by weight) of the composition.
  • compositions of the present disclosure may comprise about/at least/less than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 15, 20, 25% (by weight) of one or more monosaccharides (e.g., arabinose, fructose and/or glucose).
  • monosaccharides e.g., arabinose, fructose and/or glucose
  • one or more monosaccharides is/are present in a concentration ranging from about 1 x 10 20 M to about 1 x 10 1 M.
  • one or more monosaccharides may be included at a concentration of about/at least/less thanl x lO 20 M, 1 x 10 19 M, 1 x 10 18 M, 1 x 10 17 M, 1 x 10 16 M, 1 x 10 15 M, 1 x 10 14 M, 1 x 10 13 M,
  • compositions of the present disclosure comprise one or more disaccharides in an amount/concentration of about 0.005 to about 50% (by weight) of the composition.
  • compositions of the present disclosure may comprise about/at least/less than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
  • one or more disaccharides e.g., maltose, sucrose and/or trehalose.
  • one or more disaccharides is/are present in a concentration ranging from about 1 x 10 20 M to about 1 x 10 1 M.
  • one or more disaccharides may be included at a concentration of about/at least/less than 1 x 10- 20 M 1 x 10- 19 M 1 x 10 ⁇ TVl 1 x 10 TVl 1 x 10 ⁇ TVl 1 x 10 ⁇ TVl 1 x 10 ⁇ TVl 1 x 10 TVl 1 x 10 ⁇ 2 TVl 1 x 10 11 M, 1 x 10 10 M.
  • compositions of the present disclosure comprise one or more maltodextrins in an amount/concentration of about 0.001 to about 95% or more (by weight) of the composition.
  • the maltodextrin(s) comprise(s) about 0.001 to about 1%, about 0.25 to about 5%, about 1 to about 10%, about 5 to about 25%, about 10% to about 30%, about 20% to about 40%, about 25% to about 50%, about 50 to about 75%, or about 75 to about 95% (by weight) of the composition.
  • compositions of the present disclosure may comprise about/at least/less than 0.01, 0.02, 0.03, 0.04, 0.05.
  • one or more maltodextrins e.g., one or more maltodextrins (each and/or collectively) having a DEV value of about 15 to about 20).
  • compositions of the present disclosure comprise one or more sugar alcohols in an amount/concentration of about 0.001 to about 95% or more (by weight) of the composition.
  • the sugar alcohol(s) e.g., arabitol, mannitol, sorbitol and/or xylitol
  • the sugar alcohol(s) comprise(s) about 0.001 to about 1%, about 0.25 to about 5%, about 1 to about 10%, about 5 to about 25%, about 10% to about 30%, about 20% to about 40%, about 25% to about 50%, about 50 to about 75%, or about 75 to about 95% (by weight) of the composition.
  • compositions of the present disclosure may comprise about/at least/less than 0.01,
  • sugar alcohols e.g., arabitol, mannitol, sorbitol and/or xylitol.
  • compositions of the present disclosure comprise one or more humic acids in an amount/concentration of about 0.001 to about 95% or more (by weight) of the composition.
  • the humic acid(s) e.g., potassium humate
  • the humic acid(s) comprise(s) about 0.001 to about 1%, about 0.25 to about 5%, about 1 to about 10%, about 5 to about 25%, about 10% to about 30%, about 20% to about 40%, about 25% to about 50%, about 50 to about 75%, or about 75 to about 95% (by weight) of the composition.
  • compositions of the present disclosure may comprise about/at least/less than 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15,
  • humic acids e.g., potassium humate and/or sodium humate.
  • compositions of the present disclosure comprise one or more UV protectants in an amount/concentration of about 0.0001 to about 5% or more (by weight) of the composition.
  • the UV protectant(s) e.g., calcium lignosulfate and/or sodium lignosulfate
  • compositions of the present disclosure may comprise about/at least/less than 0.0005, 0.001, 0.002,
  • UV protectants e.g., calcium lignosulfate and/or sodium lignosulfate.
  • compositions of the present disclosure comprise one or more oxidation control components in an amount/concentration of about 0.0001 to about 5% or more (by weight) of the composition.
  • compositions of the present disclosure may comprise about/at least/less than 0.0005, 0.001, 0.002,
  • the amount/concentration of oxidation control components is about 0.005 to about 2% (by weight) of the composition.
  • the oxidation control component(s) is/are present in a concentration ranging from about 1 x 10 20 M to about 1 x 10 1 M.
  • compositions of the present disclosure comprise one or more commercial antioxidants used in accordance with the manufacturer's recommended amounts/concentrations.
  • compositions of the present disclosure comprise one or more commercial oxygen scavengers used in accordance with the manufacturer's recommended amounts/concentrations .
  • compositions of the present disclosure comprise one or more stabilizing compounds in an amount/concentration sufficient to ensure Yersinia remain viable and/or proteases remain stable and active therein.
  • compositions of the present disclosure comprise one or more stabilizing compounds in an amount/concentration sufficient to ensure the deliquescence relative humidity (DRH) of the composition is less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 at the temperature(s) at which the composition is to be stored (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and/or 40°C).
  • DRRH deliquescence relative humidity
  • Stablizing compounds may be incorporated into compositions of the present disclosure in any suitable ratio(s).
  • compositions of the present disclosure comprise one or more maltodextrins and one or more monosaccharides, disaccharides, sugar alcohols and/or humic acids in a maltodextrin: (monosaccharide, disaccharide, sugar alcohol and/or humic acid) ratio of about 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5.
  • maltodextrin: (monosaccharide, disaccharide, sugar alcohol and/or humic acid) ratio of about 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90
  • compositions of the present disclosure may comprise one or more maltodextrins (e.g., one or more maltodextrins (each and/or collectively) having a DEV of about 15 to about 20) and one or more sugar alcohols (e.g., sorbitol and/or xylitol) and/or humic acids (e.g., potassium humate) in a maltodextrin: (sugar alcohol/humic acid) ratio of about 5:95, about 15:85, about 25:75 or about 50:50.
  • maltodextrins e.g., one or more maltodextrins (each and/or collectively) having a DEV of about 15 to about 20
  • sugar alcohols e.g., sorbitol and/or xylitol
  • humic acids e.g., potassium humate
  • compositions of the present disclosure comprise one or more microbial and/or viral extracts in an amount/concentration of about 0.0001 to about 5% or more (by weight) of the composition.
  • the microbial extract(s) comprise(s) about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.02, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 to about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
  • compositions of the present disclosure may comprise about 0.0005, 0.00075, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1,
  • compositions of the present disclosure comprise one or more nutrients in an amount/concentration of about 0.0001 to about 5% or more (by weight) of the composition.
  • the nutrient(s) e.g., phosphorous, boron, chlorine, copper, iron, manganese, molybdenum and/or zinc
  • the nutrient(s) comprise(s) about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.02, 0.3, 0.4, 0.5, 0.6,
  • compositions of the present disclosure may comprise about 0.0005, 0.00075, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25,
  • nutrients e.g., phosphorous, boron, chlorine, copper, iron, manganese, molybdenum and/or zinc.
  • compositions of the present disclosure comprise one or more pest attractant(s) and/or feeding stimulant(s) in an amount/concentration of about 0.0001 to about 5% or more (by weight) of the composition.
  • the pest attractant(s) and/or feeding stimulant(s) comprise(s) about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025,
  • compositions of the present disclosure may comprise about 0.0005, 0.00075, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55,
  • compositions of the present disclosure comprise one or more dispersants in an amount/concentration of about 0.001 to about 25% or more (by weight) of the composition.
  • the dispersant(s) comprise(s) 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.02, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,
  • dispersants e.g., one or more surfactants and/or wetting agents.
  • compositions of the present disclosure comprise one or more drying agents in an amount/concentration of about 0.001 to about 95% or more (by weight) of the composition.
  • the drying agent(s) comprise(s) about ) 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.02, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
  • compositions of the present disclosure may comprise about 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1,
  • compositions of the present disclosure comprise about 0.5 to about 10 grams of drying powder per liter of composition.
  • compositions of the present disclosure may comprise about 0.5, 1, 1.25,
  • compositions of the present disclosure comprise one or more buffers in an amount/concentration of about 0.0001 to about 5% or more (by weight) of the composition.
  • the buffer(s) comprise(s) about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.02, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 to about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,
  • compositions of the present disclosure may comprise about 0.0005, 0.00075, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4., 4.5, 4.6, 4.7, 4.8, 4.9, 5% or more (by weight) of one or more buffers (
  • compositions of the present disclosure comprise one or more commercial carriers, antioxidants, oxygen scavengers, hygroscopic polymers, UV protectants, biostimulants, microbial extracts, nutrients, pest attractants and/or feeding stimulants, pesticides, plant signal molecules, disperants, drying agents, anti-freezing agents, buffers and/or adhesives used in accordance with the manufacturer's recommended amounts/concentrations .
  • compositions of the present disclosure may be formulated as any suitable type of composition, including, but not limited to, foliar compositions, seed coatings and soil composition.
  • compositions of the present disclosure are formulated as amorphous solids.
  • compositions of the present disclosure are formulated as amorphous liquids.
  • compositions of the present disclosure are formulated as wettable powders.
  • compositions of the present disclosure are formulated as liquid compositions that are subsequently dried to produce a powder or granule.
  • liquid compositions of the present disclosure are drum dried, evaporation dried, fluidized bed dried, freeze dried, spray dried, spray- freeze dried, tray dried and/or vacuum dried to produce powders/granules.
  • Such powders/granules may be further processed using any suitable method(s), including, but not limited to, flocculation, granulation and milling, to achieve a desired particle size or physical format.
  • the precise method(s) and parameters of processing dried powders/granules that are appropriate in a given situation may be affected by factors such as the desired particle size(s), the type, size and volume of material to which the composition will be applied, the type(s) of microorganisms in the composition, the number of microorganisms in the composition, the stability of the microorganisms in the composition and the storage conditions (e.g., temperature, relative humidity, duration). Those skilled in the art will understand how to select appropriate methods and parameters using routine experiments.
  • compositions of the present disclosure are formulated as granules/particles comprising a core and one or more coatings (outer layers) surrounding the core.
  • compositions of the present disclosure are formulated as granules/particles comprising a core that comprises, consists essentially of or consists of an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
  • compositions of the present disclosure are formulated as granules/particles comprising a core having a diameter of about 20 to about 2000 pm, optionally about 50 to about 1500 pm, preferably about 100 to about 1500 pm, more preferably about 250 to about 1200 pm.
  • the core of a granule/particle may include binders (e.g., polymers, waxes, fats), fillers, fiber materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants, fragrances, etc.
  • the core comprises a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
  • the core of a granule/particle may be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation: a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material.
  • granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation: a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in
  • Very small particles can be produced this way; b) Layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle.
  • Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found; c) Absorbed core particles, wherein rather than coating the enzyme as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core; d) Extrusion or pelletized products, wherein an enzyme-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening.
  • Prilled products wherein an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomiser, into a cooling chamber where the droplets quickly solidify.
  • the product obtained is one wherein the enzyme is uniformly distributed throughout an inert material instead of being concentrated on its surface;
  • Mixer granulation products wherein a liquid is added to a dry powder composition of, e.g., conventional granulating components, the enzyme being introduced either via the liquid or the powder or both.
  • the liquid and the powder are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme.
  • various high-shear mixers can be used as granulators, granulates consisting of enzyme as enzyme, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to give the so-called T-granulate. Reinforced particles, being more robust, release less enzymatic dust; g) Size reduction, wherein the cores are produced by milling or crashing of larger particles, pellets, tablets, briquettes etc. containing the enzyme.
  • the wanted core particle fraction is obtained by sieving the milled or crashed product. Over and undersized particles can be recycled; h) Fluid bed granulation, involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them and form a granule; or i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or detergent industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C.
  • the cores comprising the enzyme contain a low amount of water before coating. If water sensitive enzymes are coated before excessive water is removed, it will be trapped within the core and it may affect the activity of the enzyme negatively. After drying, the cores may contain 0.1-10 % w/w water.
  • the core of a granule/particle may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule/particle.
  • the optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy -propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
  • the coating of a granule/particle may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, 1% or 5%.
  • the amount may be at most 100%, 70%, 50%, 40% or 30%.
  • the coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm.
  • the thickness of the coating is below 100 pm.
  • the thickness of the coating is below 60 pm.
  • the total thickness of the coating is below 40 pm.
  • the coating may encapsulate the core unit by forming a substantially continuous layer.
  • a substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit it is encapsulating/enclosing has few or none uncoated areas.
  • the layer or coating should be homogeneous in thickness.
  • the coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
  • a salt coating may comprise at least 60% by weight w/w of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight w/w.
  • the salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles is less than 50 pm, such as less than 10 pm or less than 5 pm.
  • the salt coating may comprise a single salt or a mixture of two or more salts.
  • the salt may be water soluble, in particular having a solubility at least 0.1 grams in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
  • the salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate.
  • Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium.
  • Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate.
  • alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
  • the salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate).
  • the salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization
  • a hydrated salt i.e. a crystalline salt hydrate with bound water(s) of crystallization
  • Specific examples include anhydrous sodium sulfate (Na 2 S0 4 ), anhydrous magnesium sulfate (MgS0 4 ), magnesium sulfate heptahydrate (MgS0 4 -7H 2 0), zinc sulfate heptahydrate (ZnS0 4 -7H 2 0), sodium phosphate dibasic heptahydrate (Na 2 HP0 4 -7H 2 0), magnesium nitrate hexahydrate (Mg(N0 3 ) 2 (6H 2 0)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
  • the salt is applied as a solution of the salt, e.g., using a fluid bed.
  • Non-dusting granulates may be produced and may be coated by methods known in the art.
  • waxy coating materials are polyethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG polyethylene oxide
  • ethoxylated nonylphenols having from 16 to 50 ethylene oxide units
  • ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units
  • fatty alcohols fatty acids
  • mono- and di- and triglycerides of fatty acids are given in GB 1483591.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods, generally prior to forming granules.
  • a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods, generally prior to forming granules.
  • Protected enzymes may be prepared.
  • the present disclosure provides a granule/particle comprising (a) a core that comprises one or more Yersinia entomophaga (and/or one or more toxins or other components derived therefrom) and/or one or more Yersinia nurmii (and/or one or more toxins or other components derived therefrom) and/or one or more proteases, and, optionally, (b) a coating consisting of one or more layer(s) surrounding the core.
  • compositions of the present disclosure are frozen for cryopreservation.
  • liquid compositions of the present disclosure are flash-frozen and stored in a cryopreservation storage unit/facility.
  • the precise method(s) and parameters of freezing and preserving compositions of the present disclosure that are appropriate in a given situation may be affected by factors such as the type(s) of microorganisms in the composition, the number of microorganisms in the composition, the stability of the microorganisms in the composition and the storage conditions (e.g., temperature, relative humidity, duration). Those skilled in the art will understand how to select appropriate methods and parameters using routine experiments.
  • compositions of the present disclosure may be formulated as aqueous or non-aqueous compositions.
  • compositions of the present disclosure comprise no water.
  • compositions of the present disclosure comprise a trace amount of water.
  • compositions of the present disclosure comprise less than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25,
  • compositions of the present disclosure are formulated to have a pH of about 4.5 to about 9.5. In some embodiments, compositions of the present disclosure have a pH of about 6 to about 7.5. In some embodiments, compositions of the present disclosure have a pH of about 5, 5.5, 6, 6.5, 7, 7.5, 8 or 8.5.
  • compositions of the present disclosure may contain a variety of carriers, stabilizers, nutrients, pesticides, plant signal molecules, dispersants, etc. It is to be understood that the components to be included in the composition and the order in which components are incorporated into the composition may be chosen or designed to maintain or enhance the dispersion, stability and/or survival of Yersinia bacteria during storage, distribution, and/or application of the composition.
  • compositions of the present disclosure are non-naturally occurring compositions.
  • the composition comprises one or more non-naturally occurring components.
  • the composition comprises a non-naturally occurring combination of naturally occurring components.
  • Yersinia and proteases may be applied to any plant type, including, but not limited to, row crops and vegetables.
  • the compositions of the present disclosure are formulated for the treatment of one or more plants selected from the families 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, butternut squash, summer squash), watermelon, zucchini
  • Fabaceae e.g., alfalf
  • compositions of the present disclosure are formulated for the treatment of one or more plants with which the Yersinia and/or protease is/are not naturally associated (e.g., one or more plants that does not naturally exist in the geographical location(s) from which the Yersinia and/or protease was/were isolated).
  • compositions of the present disclosure are formulated for the treatment of one or more acaricide-, fungicide-, gastropodicide-, herbicide-, insecticide-, nematicide-, rodenticide- and/or virucide-resistant plants (e.g., one or more plants resistant to acetolactate synthase inhibitors (e.g., imidazolinone, pryimidinyoxy(thio)benzoates, sulfonylaminocarbonyltriazolinone, sulfonylurea, triazolopyrimidines), bialaphos, glufosinate, glyphosate, hydroxyphenylpyruvatedioxygenase inhibitors and/or phosphinothricin).
  • acetolactate synthase inhibitors e.g., imidazolinone, pryimidinyoxy(thio)benzoates, sulfonylaminocarbonyltriazolinone, s
  • Non-limiting examples of plants that may be treated with compositions of the present disclosure include plants sold by Monsanto Company (St. Louis, MO) under the BOLLGARD II®, DROUGHTGARD®, GENUITY®, RIB COMPLETE®, ROUNDUP READY®, ROUNDUP READY 2 YIELD®, ROUNDUP READY 2 EXTENDTM, SMARTSTAX®, VT DOUBLE PRO®, VT TRIPLE PRO®, YIELDGARD®, YIELDGARD VT ROOTWORM/RR2®, YIELDGARD VT TRIPLE® and/or XTENDFLEXTM tradenames.
  • compositions of the present disclosure may be applied to any part/portion of a plant.
  • the compositions are applied to plant propagation materials (e.g., cuttings, rhizomes, seeds and tubers).
  • the compositions are applied to the roots of a plant.
  • the compositions are applied to the foliage of a plant.
  • the compositions are applied to both the roots and the foliage of a plant.
  • the compositions are applied to plant propagation materials and to the plants that grow from said plant propagation materials.
  • compositions of the present disclosure may be applied to any plant growth medium, including, but not limited to, soil.
  • compositions of the present disclosure may be applied to plants, plant parts and/or plant growth media in any suitable manner, including, but not limited to, on-seed application, in-furrow application and foliar application.
  • compositions of the present disclosure may be applied using any suitable method(s), including, but not limited to, coating, dripping, dusting, encapsulating, immersing, spraying and soaking.
  • Batch systems in which predetermined batch sizes of material and composition are delivered into a mixer, may be employed.
  • Continuous treatment systems which are calibrated to apply composition at a predefined rate in proportion to a continuous flow of material, may also be employed.
  • the compositions are applied directly to plant propagation material (e.g., seeds).
  • plant propagation materials are soaked in a composition comprising the compositions for at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, 24, 36, 48 hours.
  • plant propagation materials are coated with the compositions.
  • Plant propagation materials may be coated with one or more additional layers (e.g., one or more protective layers that serve to enhance the stability and/or survival of Yersinia and/or stability/activity of proteases and/or one or more sequestration layers comprising substances that may reduce the stability and/or survival of Yersinia and/or stability/activity of proteaes if included in the same layer as the Ye rs in ia/p ro tease ( s )) .
  • the coating comprises, consists essentially of, or consists of a composition of the present disclosure and a drying powder.
  • the compositions are applied directly to a plant growth medium (e.g., a soil). According to some embodiments, the compositions are applied in the vicinity of a plant propagation material (e.g., a seed). According to some embodiments, the compositions are applied to the root zone of a plant.
  • a plant growth medium e.g., a soil
  • a plant propagation material e.g., a seed
  • the compositions are applied to the root zone of a plant.
  • the compositions are applied using a drip irrigation system. In some embodiments, the compositions are applied directly to plants. According to some embodiments, the compositions are sprayed and/or sprinkled on the plant(s) to be treated.
  • foliar application e.g., application to leaves
  • Individual components of the compositions e.g., Yersinia and protease
  • Combinations of some components of the compositions may be separately applied by foliar means. All components of the compositions may be applied by foliar means.
  • one of the agents may be applied to one part of a plant (e.g., seed coating) and the other agent may be applied to another part of the plant (e.g., foliar application to leaves).
  • the two agents may be applied at the same time, or may be applied at different times during the life cycle of the plant (e.g., seeds first and leaves subsequently).
  • the compositions are freeze- spray- or spray-freeze-dried and then applied to plants/plant parts.
  • a composition comprising the compositions and one or more stabilizing components (e.g., one or more maltodextrins having a DEV of about 15 to about 20) is freeze- spray- or spray-freeze-dried, mixed with a drying powder (e.g., a drying powder comprising calcium stearate, attapulgite clay, montmorillonite clay, graphite, magnesium stearate, silica (e.g., fumed silica, hydrophobically- coated silica and/or precipitated silica) and/or talc), then coated on seed that was been pre-treated with one or more adhesives (e.g., an adhesive composition comprising one or more maltodextrins, one or more mono-, di- or oligosaccharides, one or more peptones, etc.), one or more pesticides
  • a drying powder e.g
  • compositions of the present disclosure may be applied to plants, plant parts and/or plant growth media in any suitable amount(s)/concentration(s).
  • compositions of the present disclosure comprise one or more proteases in an amount ranging from about 0.001 to about 100 milligrams per gram and/or milliliter of composition.
  • compositions of the present disclosure may comprise about/at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80
  • compositions of the present disclosure comprise about/at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 milligrams of protease per gram and/or milliliter of composition.
  • one or more proteases comprise about 0.00000001 to about 95% (by weight) of the composition. In some embodiments, one or more proteases comprise about/at least 1 x 10 15 , 1 x 10 14 , 1 x 10 13 , 1 x 10 12 , 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 , 1 x 10 6 , 1 x 10 5 , 1 x 10 4 , 1 x 10 3 , 1 x 10 2 , 1 x 10 1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70,
  • the composition is applied at a rate that is equivalen to about 1 x 10 1 to about 1 x 10 20 CFU of Yersinia per kilogram of plant propagation material.
  • compositions of the present disclosure are applied in an amount sufficient to ensure the plant propagation materials are coated with about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 ,
  • compositions of the present disclosure is/are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 CFU of Yersinia is applied to each seed.
  • compositions of the present disclosure are applied in an amount sufficient to ensure each plant is treated with about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 ,
  • compositions of the present disclosure are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 CFU of Yersinia are applied to each plant.
  • compositions of the present disclosure are applied in an amount sufficient to ensure each acre of treated crops is treated with about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 CFU of
  • compositions of the present disclosure are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 CFU of Yersinia are applied to each acre of treated crops.
  • the composition is applied at a rate that is equivalen to about 1 x 10 1 to about 1 x 10 20 CFU of Yersinia per acre of plant growth media.
  • compositions of the present disclosure are applied in an amount sufficient to ensure each acre of plant growth media is treated with about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 CFU of Yersinia.
  • compositions of the present disclosure are are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 CFU of Yersinia are applied to each acre of plant growth media.
  • the composition is applied at a rate that is equivalen to about 1 x 10 1 to about 1 x 10 15 enzyme units (at optimum conditions) of each protease per kilogram of plant propagation material.
  • compositions of the present disclosure are applied in an amount sufficient to ensure the plant propagation materials are coated with about/at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 or 1 x 10 15 enzyme units (at optimum conditions) of each protease per kilogram of plant propagation material.
  • one or more compositions of the present disclosure is/are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , or 1 x 10 12 enzyme units (at optimum conditions) of each protease is applied to each seed.
  • the composition is applied at a rate that is equivalen to about 1 x 10 1 to about 1 x 10 15 enzyme units (at optimum conditions) of each protease per plant.
  • compositions of the present disclosure are applied in an amount sufficient to ensure each plant is treated with about/at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 or 1 x 10 15 enzyme units (at optimum conditions) of each protease.
  • compositions of the present disclosure are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , or 1 x 10 12 enzyme units (at optimum conditions) of each protease is applied to each plant.
  • the composition is applied at a rate that is equivalen to about 1 x 10 1 to about 1 x 10 15 enzyme units (at optimum conditions) of each protease per acre of treated crops.
  • compositions of the present disclosure are applied in an amount sufficient to ensure each acre of treated crops is treated with about/at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 or 1 x 10 15 enzyme units (at optimum conditions) of each protease.
  • compositions of the present disclosure are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , or 1 x 10 12 enzyme units (at optimum conditions) of each protease is applied to each acre of treated crops.
  • the composition is applied at a rate that is equivalen to about 1 x 10 1 to about 1 x 10 15 enzyme units (at optimum conditions) of each protease per acre of plant growth media.
  • compositions of the present disclosure are applied in an amount sufficient to ensure each acre of plant growth media is treated with about/at least 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , l x 10 4 , 1 x 10 5 , l x 10 6 , l x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 or 1 x 10 15 enzyme units (at optimum conditions) of each protease.
  • compositions of the present disclosure are are applied in an amount sufficient to ensure that an average of about/at least 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , or 1 x 10 12 enzyme units (at optimum conditions) of each protease is applied to each acre of plant growth media.
  • compositions of the present disclosure are applied in an amount sufficient to ensure the plant propagation materials are coated with about/at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,
  • compositions of the present disclosure is/are applied in an amount sufficient to ensure that an average of about/at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 milligrams of protease is applied to each seed.
  • compositions of the present disclosure are applied at a rate that is equivalen to about 0.001 to about 100 milligrams of protease per plant. According to some embodiments, compositions of the present disclosure are applied in an amount sufficient to ensure each plant is treated with about/at least 0.001, 0.002, 0.003, 0.004,
  • compositions of the present disclosure are applied in an amount sufficient to ensure that an average of about/at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 milligrams of protease are applied to each plant.
  • compositions of the present disclosure are applied at a rate that is equivalen to about 0.001 to about 100 milligrams of protease per acre of treated crops. According to some embodiments, compositions of the present disclosure are applied in an amount sufficient to ensure each acre of treated crops is treated with about/at least
  • compositions of the present disclosure are applied in an amount sufficient to ensure that an average of about/at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 milligrams of protease are applied to each acre of treated crops.
  • compositions of the present disclosure are applied in an amount sufficient to ensure each acre of plant growth media is treated with about/at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40
  • compositions of the present disclosure are are applied in an amount sufficient to ensure that an average of about/at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 milligrams of protease are applied to each acre of plant growth media.
  • compositions of the present disclosure are applied at a rate of about 0.05 to about 100 milliliters and/or grams of composition per kilogram of plant propagation material. According to some embodiments, one or more compositions of the present disclosure is/are applied in an amount sufficient to ensure the plant propagation materials are coated with about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5,
  • compositions of the present disclosure is/are applied in an amount sufficient to ensure that an average of about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2,
  • compositions of the present disclosure are applied at a rate of about 0.5 to about 100 milliliters and/or grams of composition per plant. According to some embodiments, one or more compositions of the present disclosure is/are applied in an amount sufficient to ensure each plant is treated with about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475 ; 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25,
  • compositions of the present disclosure is/are applied in an amount sufficient to ensure that an average of about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25,
  • composition 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75 or 5 milliliters and/or grams of composition is applied to each plant.
  • compositions of the present disclosure are applied at a rate of about 0.5 to about 100 milliliters and/or grams of composition per acre of treated crops.
  • one or more compositions of the present disclosure is/are applied in an amount sufficient to ensure each acre of treated crops is treated with about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3,
  • compositions of the present disclosure is/are applied in an amount sufficient to ensure that an average of about/at least 0.05, 0.1,
  • compositions of the present disclosure are applied at a rate of about 0.5 to about 100 milliliters and/or grams of composition per acre of plant growth media.
  • one or more compositions of the present disclosure is/are applied in an amount sufficient to ensure each acre of plant growth media is treated with about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 milliliters and/or grams of composition.
  • one or more compositions of the present disclosure is
  • composition 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75 or 5 milliliters and/or grams of composition is applied to each acre of plant growth media.
  • compositions of the present disclosure may be applied to plants, plant parts and/or plant growth media at any time, including, but not limited to, prior to planting, at the time of planting, after planting, prior to germination, at the time of germination, after germination, prior to seedling emergence, at the time of seedling emergence, after seedling emergence, prior to the vegetative stage, during the vegetative stage, after the vegetative stage, prior to the reproductive stage, during the reproductive stage, after the reproductive stage, prior to flowering, at the time of flowering, after flowering, prior to fruiting, at the time of fruiting, after fruiting, prior to ripening, at the time of ripening, and after ripening.
  • compositions are applied to plant propagation materials (e.g., seeds) about/at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104 weeks priorto planting.
  • plant propagation materials e.g., seeds
  • compositions are applied to plant propagation materials (e.g., seeds) at the time of planting.
  • compositions are applied to plant propagation materials (e.g., seeds) after planting but before germination.
  • compositions are applied to plants following emergence.
  • the present disclosure extends to plants and plant parts (e.g., coated plant propagation materials) that have been treated with the compositions, to plants that grow from plant parts (e.g., coated plant propagation materials) that have been treated with the compositions, to plant parts harvested from plants that have been treated with the compositions, to plant parts harvested from plants that grow from plant parts (e.g., coated plant propagation materials) that have been treated with the compositions, to processed products derived from plants that have been treated with the compositions, to processed products derived from plants that grow from plant parts (e.g., coated plant propagation materials) that have been treated with the compositions, to crops comprising a plurality of plants that have been treated with the compositions, and to crops comprising a plurality of plants that grow from plant parts (e.g., coated plant propagation materials) that have been treated with the compositions.
  • plants and plant parts e.g., coated plant propagation materials
  • the present disclosure provides coated plant propagation materials comprising, consisting essentially of, or consisting of a plant propagation material and a coating that covers at least a portion of the outer surface of the plant propagation material, said coating comprising, consisting essentially of, or consisting the compositions of the present disclosure.
  • the coating comprises two, three, four, five or more layers. According to some embodiments, the coating comprises an inner layer that contains Yersinia! protease and one or more outer layers free or substantially free of microorganisms and/or protease. In some embodiments, the coating comprises an inner layer that is a composition of the present disclosure and an outer layer that is equivalent to a composition of the present disclosure except that it does not contain Yersinia! protease.
  • some seeds may be coated with Yersinia and not with protease.
  • Other seeds may be coated with protease and not with Yersinia.
  • the Yersinia- coated seeds may be mixed with the protease-coated seeds. The seed mixture may be planted.
  • the coating comprises, consists essentially of, or consists of a composition of the present disclosure and a drying powder. Drying powders may be applied in any suitable amount(s)/concentration(s). The absolute value of the amount/concentration that is/are sufficient to cause the desired effect(s) may be affected by factors such as the type, size and volume of material to which the composition will be applied, the type(s) of microorganisms in the composition, the number of microorganisms in the composition, the stability of the microorganisms in the composition and storage conditions (e.g., temperature, relative humidity, duration). Those skilled in the art will understand how to select an effective amount/concentration using routine dose-response experiments.
  • the drying powder is applied in an amount ranging from about 0.5 to about 10 grams of drying powder per kilogram of plant propagation material.
  • drying powder e.g., drying powder comprising magnesium stearate, magnesium sulfate, powdered milk, silica, soy lecithin and/or talc
  • drying powder comprising magnesium stearate, magnesium sulfate, powdered milk, silica, soy lecithin and/or talc
  • a drying powder comprising calcium stearate, attapulgite clay, montmorillonite clay, graphite, magnesium stearate, silica (e.g., fumed silica, hydrophobically-coated silica and/or precipitated silica) and/or talc is applied to seeds coated with a composition of the present disclosure at a rate of about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 grams per kilogram of seed.
  • silica e.g., fumed silica, hydrophobically-coated silica and/or precipitated silica
  • the coating completely covers the outer surface of the plant propagation material.
  • the average thickness of the coating is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4, 4.5, 5 pm or more. In some embodiments, the average thickness of the coating is about 1.5 to about 3.0 pm.
  • kits comprising, consisting essentially of, or consisting of one or more plants and/or plant parts (e.g., coated plant propagation materials) that have been treated with the compositions of the present disclosure and a container housing the treated plant(s) and/or plant part(s).
  • the kit further comprises one or more oxygen scavengers, such as activated carbon, ascorbic acid, iron powder, mixtures of ferrous carbonate and metal halide catalysts, sodium chloride and/or sodium hydrogen carbonate.
  • the container may comprise any suitable material(s), including, but not limited to, materials that reduce the amount of light, moisture and/or oxygen that contact the coated plant propagation material when the container is sealed.
  • the container comprises, consists essentially of, or consists of a material having light permeability of less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75%.
  • the container comprises, consists essentially of, or consists of a material having an oxygen transmission rate of less than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 cm 3 /m 2 day (as measured in accordance with ASTM D3985).
  • the container reduces the amount of ambient light that reaches said coated plant propagation material by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% when sealed.
  • the container reduces the amount of ambient moisture that reaches said plant propagation material by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% when sealed.
  • the container reduces the amount of ambient oxygen that reaches said plant propagation material by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% when sealed.
  • kits of the present disclosure comprise 1, 2, 3, 4, 5 or more additional containers.
  • the additional containers may comprise any suitable component(s) or composition(s), including, but not limited to, agriculturally beneficial microorganisms, biostimulants, drying agents, nutrients, oxidation control components and pesticides. Examples of agriculturally beneficial microorganisms, biostimulants, drying agents, nutrients, oxidation control components and pesticides that may be included in the additional containers are described above.
  • the present disclosure extends to animal feed compositions comprising, consisting essentially of or consisting of a food component and a microbial component, said microbial component comprising, consisting essentially of, or consisting of the compositions of the present disclosure.
  • Animal feed compositions of the present disclosure may comprise any suitable food component, including, but not limited to, fodder (e.g., grains, hay, legumes, silage and/or straw) and forage (e.g., grass).
  • fodder e.g., grains, hay, legumes, silage and/or straw
  • forage e.g., grass
  • Animal feed compositions of the present disclosure may be fed to any suitable animal, including, but not limited to, farm animals, zoo animals, laboratory animals and/or companion animals.
  • the animal feed composition is formulated to meet the dietary needs of birds (e.g., chickens, ducks, quails and/or turkeys), bovids (e.g., antelopes, bison, cattle, gazelles, goats, impala, oxen, sheep and/or wildebeests), canines, cervids (e.g., caribou, deer, elk and/or moose), equines (e.g., donkeys, horses and/or zebras), felines, fish, pigs, rabbits, rodents (e.g., guinea pigs, hamsters, mice and/or rats) and the like.
  • birds e.g., chickens, ducks, quails and/or turkeys
  • bovids e.g.,
  • the present disclosure extends to methods and uses for the compositions of the present disclosure.
  • methods and uses of the present disclosure comprise, consist essentially of or consist of applying the compositions disclosed herein to a plant or plant part (e.g., plant propagation material).
  • a plant or plant part e.g., plant propagation material
  • the compositions of the present disclosure may be applied to any type of plant, to any part/portion of a plant, in any suitable manner, in any suitable amount(s)/concentration(s) and at any suitable time(s).
  • methods and uses of the present disclosure comprise, consist essentially of or consist of applying the compositions to a monocotyledonous plant or plant part (e.g., a cereal or pseudocereal plant or plant part, optionally, barley, buckwheat, com, millet, oats, quinoa, rice, rye, sorghum or wheat).
  • a monocotyledonous plant or plant part e.g., a cereal or pseudocereal plant or plant part, optionally, barley, buckwheat, com, millet, oats, quinoa, rice, rye, sorghum or wheat.
  • methods and uses of the present disclosure comprise, consist essentially of or consist of applying the disclosed compositions to a plant growth medium.
  • the compositions of the present disclosure may be applied to any plant growth medium, in any suitable manner, in any suitable amount(s)/concentration(s) and at any suitable time(s).
  • methods and uses of the present disclosure comprise, consist essentially of or consist of introducing a plant or plant part (e.g., plant propagation material) that has been treated with the disclosed compositions into a plant growth medium (e.g., a soil). Such methods may further comprise introducing one or more nutrients (e.g., nitrogen and/or phosphorous) into the plant growth medium. Any suitable nutrient(s) may be added to the growth medium, including, but not limited to, rock phosphate, monoammonium phosphate, diammonium phosphate, monocalcium phosphate, super phosphate, triple super phosphate, ammonium polyphosphate, fertilizers comprising one or more phosphorus sources, and combinations thereof.
  • methods and uses of the present disclosure comprise, consist essentially of or consist of growing a plant from a plant propagation material that has been treated with the compositions of the present disclosure.
  • the compositions may be used to kill pests (e.g., insects), retard their grown, or prevent pests from infecting, infesting, killing/destroying or retarding growth of a plant.
  • the compositions may enhance plant growth.
  • the compositions disclosed herein are combinations of one or more Yersinia! protease and one or more substances, like a pesticide or an insecticide.
  • one or more of the effects of the compositions, on plants or insects for example are less than additive (e.g., antagonistic) as compared to the effects of the individual components of the composition, or groups of individual of the components that are less than all of the components of the composition.
  • one or more of the effects of the compositions, on plants or insects for example are additive as compared to the effects of the individual components of the composition, or groups of individual of the components that are less than all of the components of the composition.
  • one or more of the effects of the compositions, on plants or insects for example give unexpected results as compared to the effects of the individual components of the composition, or groups of individual of the components that are less than all of the components of the composition.
  • unexpected results of the compositions as compared to individual components or groups of individual components of a composition may be described by a performance index.
  • the performance index may be the effect of the combination divided by the sum of effects of the individual components of the composition.
  • compositions may be used to enhance growth and/or yield of plants.
  • application of the compositions enhances 1, 2, 3, 4, 5 or more growth characteristics and/or 1, 2, 3, 4, 5 or more yield characteristics by about/at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 150, 175, 200, 225, 250% or more as compared to one or more controls (e.g., untreated control plants and/or plants treated with an alternative microbial strain).
  • application of the compositions enhances plant yield by about/at least 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
  • compositions may likewise be used to enhance plant growth and/or yield.
  • application of an composition of the present disclosure enhances 1, 2, 3, 4, 5 or more plant growth characteristics and/or 1, 2, 3, 4, 5 or more plant yield characteristics by about/at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 150, 175, 200, 225, 250% or more as compared to a control composition (e.g., a control composition that is identical to the composition of the present disclosure except that it lacks the disclosed compositions).
  • application of a composition of the present disclosure enhances plant yield by about/at least 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
  • methods and uses of the present disclosure comprise, consist essentially of or consist of applying the disclosed compositions to seeds, to the plant growth medium in which said seeds are being or will be grown, and/or to the plant(s) that grow(s) from said seeds.
  • compositions are applied to seeds in an amount/concentration effective to enhance 1, 2, 3, 4, 5 or more plant growth characteristics (e.g., biomass) and/or 1, 2, 3, 4, 5 or more plant yield characteristics (e.g., bushels per acre) of the plant that grows from said seed by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 150, 175, 200, 225, 250% or more as compared to one or more control plants (e.g., plants grown from untreated seeds and/or plants grown from seeds treated with a control).
  • plant growth characteristics e.g., biomass
  • plant yield characteristics e.g., bushels per acre
  • the disclosed compositions are applied to seeds in an amount effective to enhance yield by about/at least 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or
  • compositions are introduced into a plant growth medium (e.g., soil) in an amount/concentration effective to enhance 1, 2, 3, 4, 5 or more plant growth characteristics (e.g., biomass) and/or 1, 2, 3, 4, 5 or more plant yield characteristics (e.g., bushels per acre) of plants grown therein by at least about 5,
  • a plant growth medium e.g., soil
  • 1, 2, 3, 4, 5 or more plant growth characteristics e.g., biomass
  • plant yield characteristics e.g., bushels per acre
  • the compositions are introduced into the plant growth medium in an amount effective to enhance plant yield by about/at least 0.25, 0.5 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 bushels per acre.
  • Y. entomophaga NRRL B-67598, Y. entomophaga NRRL B-67599, Y. entomophaga NRRL B-67600 and Y. entomophaga NRRL B-67601 were deposited on March 15, 2018, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Agricultural Research Service Culture Collection, 1815 North University Street, Peoria, Illinois 61604, U.S.A.
  • Y. entomophaga NRRL B-67598, Y. entomophaga NRRL B-67599, Y. entomophaga NRRL B-67600 and Y. entomophaga NRRL B-67601 were deposited under conditions that assure access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. ⁇ 1.14 and 35 U.S.C. ⁇ 122. Each deposit represents a pure culture of the deposited strain. Each deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are fried. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
  • Yersinia entomophaga NRRL B-67598 was cultured in Cell-free Luria-Bertani (LB) medium at 30°C. The culture was filtered to remove Yersinia entomophaga NRRL B-67598, and the filtrate was analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis (SDA-PAGE). A prominent band at approximately 81 kD was excised from the gel and subjected to LC-MS/MSN sequencing. The resultant amino acid sequence was cross-referenced with the genome sequence of MH96 (Hurst, M.R.H.
  • SEQ ID NO: 4 The nucleotide sequence set forth herein as SEQ ID NO: 4 was identified. The longest open reading frame within the identified sequence encodes the amino acid sequence set forth herein as SEQ ID NO: 5.
  • the polypeptide sequence of SEQ ID NO: 5 is similar to that of zinc metallopeptidases belonging to the Peptidase M66 family (Pfam 10462).
  • a targeted mutation of the gene encoding the putative protease was made and recombined into the genome of Yersinia entomophaga NRRL B-67598.
  • Primers set forth herein as SEQ ID NO: 6 and SEQ ID NO: 7 were used to PCR amplify the relevant region from the Yersinia genome.
  • the resultant 2643 bp amplicon was cloned into the vector pGEM®-T Easy to generate pGEMBandl.
  • pGEMBandl was transformed into the Escherichia coli methylation-minus strain, GM2929.
  • the spectinomycin resistance gene from the plasmid was amplified by PCR.
  • the spectinomycin-resistance amplicon, flanked by cleavage sites for the Agel restriction endonuclease, was digested with Agel and spectinomycin-containing fragment was ligated into the analogous site of pGEMBandl (this released a 579 bp fragment within the open reading frame encoding the putative protease).
  • the resulting plasmid was called pGEMBlAGSP.
  • pGEMBlAGSP was then digested with BamHI restriction endonuclease and cloned into the analogous site of the suicide vector, pJP5608, to form pJP5608::Bandl.
  • the mutation was then recombined into the Yersinia entomophaga NRRL B-67598 genome using homologous recombination and the recombinant was validated. Disruption of the gene was confirmed by analyzing cultured medium in which the mutant strain was grown, as described above, and determining that the 81 kD band was absent from the medium.
  • PM were obtained after cutting the head and hind regions of larvae, measuring 25-30 mm in size, with a sterile scalpel. Using dissecting tweezers, the PM was gently pulled from the remainder. The PM was cut into 5 cross sections and individually placed into 300 m ⁇ drops of phosphate buffered saline (PBS) containing 0.02% sodium azide (antibacterial) and nystatin (1 m ⁇ of 1000 unit stock as antifungal) to wash the tissue. The PM sections were then transferred to wells of 96-well microplates containing 45 m ⁇ of PBS, sodium azide and nystatin.
  • PBS phosphate buffered saline
  • Cabbage leaf disks were dipped in phosphate buffer or phosphate buffer containing Yersinia entomophaga NRRL B-67598 (1 x 10 5 cfii/ml), a Bacillus licheniformis serine protease (SEQ ID NO: 2; 0.205 mg/ml), or a Nocardiopsis prasina serine protease (SEQ ID NO: 3; 0.35 mg/ml). After the cabbage disks had dried for 1 hr, a single 3 rd instar cabbage looper larva was added to each individual cabbage disk. A total of 20 insects were evaluated for each treatment. The results are shown in Table 1 below.

Abstract

L'invention concerne des combinaisons pesticides, y compris des combinaisons de Yersinia entomophaga (et/ou de toxine(s) de celle-ci) et/ou de Yersinia nurmii (et/ou de toxine(s) de celle-ci) avec une protéase. De telles combinaisons peuvent fournir des effets pesticides inattendus et peuvent être utiles pour traiter des insectes et d'autres organismes nuisibles et pour améliorer la croissance et/ou le rendement de plantes.
PCT/US2020/045868 2019-08-13 2020-08-12 Combinaisons pesticides de yersinia et de protéases WO2021030400A1 (fr)

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