FIELD OF THE INVENTION

[0001] Provided herein are compositions and methods to manage the development of pest resistance to a pesticidal agent or product, such as a pesticidal product produced by a plant comprising a transgene encoding for the pesticidal product. For example, provided herein are seed blends comprising pest-resistant seeds and refuge seeds, and methods of deploying a crop of the seeds.

BACKGROUND

[0002] Government regulatory agencies often require that genetically modified, pest- resistant crops be planted alongside a smaller proportion of crop plants that do not express the same pest-resistant trait. These plants, which are referred to as a "refuge" crop, support a population of pests (e.g., insect or nematode pests) that are susceptible to the pest-resistant trait. When properly managed, refuge crops are effective in delaying the evolution of resistant pest progeny and improving the durability of pest-resistant crop species.

[0003] There is a continuing need in the art to develop improved insect resistance management (IRM) practices, and in particular to develop methods that improve the effectiveness of refuge crops in delaying the evolution of trait-resistant pest populations.

SUMMARY

[0004] Provided herein is a seed blend comprising refuge crop seeds and pest-resistant crop seeds in a uniform mixture, wherein the pest-resistant crop seeds comprise a gene that confers resistance against a rootworm or nematode pest, and the refuge seeds lack the gene; and wherein the refuge crop seeds are treated with a treatment composition comprising a first component that improves or increases the root biomass, root growth or root health of plants grown from the refuge seeds.

[0005] Also provided is a seed blend comprising refuge crop seeds and pest-resistant crop seeds in a uniform mixture, wherein the pest-resistant crop seeds comprise a gene that confers resistance against one or more targeted insect or nematode pests, and the refuge crop seeds lack the gene; and wherein the refuge crop seeds are treated with a treatment composition comprising a first component that improves or increases the root biomass, root growth or root health of plants grown from the refuge crop seeds. [0006] Also provided is a method for deploying a refuge crop in a field of pest-resistant crop plants, the method comprising planting a seed blend as described herein in a field.

[0007] Also provided is a population of plants produced by growing a seed blend as described herein.

[0008] Also provided is a method of managing pest resistance in a plot of pest-resistant crop plants, the method comprising (a) planting pest-resistant crop seeds in a plot, the pest- resistant crop seeds comprising a gene that confers resistance against a nematode or rootworm pest; and (b) planting refuge crop seeds in the same plot or an adjacent plot, the refuge crop seeds lacking the gene; wherein the refuge crop seeds are treated with a treatment composition comprising a first component that improves or increases the root biomass, root growth or root health of crop plants grown from the refuge crop seeds.

[0009] Also provided is a method of managing pest resistance in a plot of pest-resistant crop plants, the method comprising (a) planting pest-resistant crop seeds in a plot, the pest- resistant crop seeds comprising a gene that confers resistance against crop rootworm; and (b) planting refuge crop seeds in the same plot or an adjacent plot, the refuge crop seeds lacking the gene; and (c) treating the soil in the locus surrounding the planted refuge crop seeds with a treatment composition comprising a first component that improves or increases the root biomass, root growth or root health of crop plants grown from the refuge crop seeds.

[0010] Also provided is a method of managing pest resistance in a plot of pest-resistant crops, the method comprising (a) planting pest-resistant crop seeds in a plot, the pest-resistant crop seeds comprising a gene that confers resistance against one or more targeted insect or nematode pests; and (b) planting refuge crop seeds in the same plot or an adjacent plot, the refuge crop seeds lacking the gene; wherein the refuge crop seeds are treated with a treatment composition comprising a first component that improves the root growth or root health of plants grown from the refuge crop seeds.

[0011] Also provided is a method of managing pest resistance in a plot of pest-resistant crops, the method comprising (a) planting pest-resistant crop seeds in a plot, the pest-resistant crop seeds comprising a gene that confers resistance against one or more targeted insect or nematode pests; (b) planting refuge crop seeds in the same plot or an adjacent plot, the refuge crop seeds lacking the gene; and (c) treating the soil in the locus surrounding the planted refuge crop seeds with a treatment composition comprising a first component that improves the root growth or root health of plants grown from the refuge crop seeds. [0012] Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figs. 1 and 2 provide diagrams related to aspects of the com rootworm adult emergence trial described in Example 1.

[0014] Corresponding reference characters indicate corresponding parts throughout the drawings.

DETAILED DESCRIPTION

[0015] Provided herein are improved insect resistance management practices, such as improved methods for deploying a crop refuge.

[0016] It has been observed that in some refuge areas with high pest pressure, the refuge plants may provide insufficient feeding material to sustain a desired number of trait-susceptible pests to adulthood. For example, in areas where the available refuge feeding material is substantially or completely consumed by high numbers of insect pests, young or larval pests may be killed by starvation or, particularly in the case of an integrated refuge, may migrate to neighboring, non-refuge plants where they are predominantly killed as a result of the expressed pesticidal trait. If a refuge is unable to sustain a sufficient number of trait-susceptible pests to adulthood, it is less effective at managing the spread of resistance to the pesticidal trait.

[0017] The effectiveness of a refuge may be improved by treating the refuge seeds, or the soil in the locus surrounding the planted refuge crop seeds, with a treatment composition comprising a first component. As described in further detail below, the first component acts to increase the amount of pest feeding material provided by the refuge plant, and enhances the plant's ability to provide an effective refuge.

[0018] In a preferred embodiment, plants grown from seeds treated with the first component may exhibit increased below-ground or root biomass, larger roots, greater root mass, or otherwise improved root production. For refuge seeds treated with the first component, the increased feeding material provided by the refuge plants will allow for additional young or larval pests to survive on the plants, thus increasing the effectiveness of the refuge relative to otherwise similar plants grown from seeds that were not treated with the first component.

[0019] The first component may be applied to the refuge crop seeds prior to planting.

For example, provided herein are seed blends comprising refuge crop seeds and pest-resistant crop seeds in a uniform mixture, wherein the refuge crop seeds are treated with a first component. In some embodiments, the pest-resistant crop seeds are not treated with the first component. Alternatively, in some embodiments, both the pest-resistant crop seeds and the refuge seeds are treated with the first component.

[0020] Alternatively, the first component may be applied to the refuge crop seeds after planting. For example, the soil in the locus surrounding the planted refuge crop seeds may be treated with the first component. In some embodiments, the soil surrounding the pest-resistant crop seeds is not treated with the first component. Alternatively, in some embodiments, the soil surrounding both the pest-resistant crop seeds and the refuge seeds is treated with the first component. According to some embodiments, the first component may be applied as a foliar application to the refuge crop plant.

Seed Blends and Compositions

[0021] In certain embodiments, the pest-resistant seed and the refuge seed are provided as a seed mixture or seed blend. The seed blend may be in the form of a refuge-in-a-bag (RIB) composition or package. Seeds in such a mixture may be planted to produce a refuge-integrated field.

[0022] The seed blends described herein comprise at least a first genetically modified pest-resistant seed, which contains at least a first gene that provides a pesticidal trait against a target pest, and at least one type of refuge plant seed. The refuge plant seed can be uniform in nature, in that it is composed of a single type of seed from a single variety of plant, or can be non-uniform in nature and consist of two or more varieties of plant. In one embodiment, the refuge seed is similar in variety (or agronomic characteristics) to the first transgenic crop seed.

[0023] For example, the pest-resistant seed may express a trait that is active against target insect and/or nematode pests.

[0024] The pest-resistant seed can incorporate, in addition to the first gene that provides a pesticidal trait, one or more additional genes that confer additional traits. For example, the pest-resistant seed can incorporate one or more additional genes that confer, for example, tolerance to a particular herbicide or combination of herbicides, increased disease resistance, enhanced tolerance to insects, drought, stress and/or enhanced yield. The pest-resistant seed may comprise a breeding trait, including for example, in one embodiment a disease tolerant breeding trait. In some instances, the pest-resistant seed seed includes at least one transgenic and breeding trait. [0025] The refuge seed can be genetically modified or non-genetically modified. For example, the refuge seed may be a transgenic seed from which a transgenic plant can grow and incorporates a transgenic event that confers, for example, tolerance to a particular herbicide or combination of herbicides, increased disease resistance, enhanced tolerance to insects, drought, stress and/or enhanced yield. The refuge seed may comprise a breeding, genome editing or mutant trait, including for example, in one embodiment, a disease tolerant breeding trait. In some instances, the refuge seed includes at least one transgenic trait and at least one breeding, genome editing or mutant trait.

[0026] A refuge seed that is a genetically modified seed can contain any gene so long as it is not the gene that provides the pest-resistant trait in the pest-resistant crop seed. For example, the refuge seed may comprise a genetic trait that provides insecticidal activity against a different pest. Alternatively, the refuge seed may comprise a genetic trait that provides insecticidal activity against the same pest, but by a different mode of action than the genetic trait in the pest- resistant crop seed.

[0027] The refuge seed may comprise an insecticidal gene, an herbicide tolerance gene, a fungicide tolerance gene, a fragment of an insect gene, or a combination thereof.

[0028] In some embodiments, the refuge seed is of a variety that is rated to reach reproductive maturity at a different respective time in the field than that of the pest-resistant crop seed. Methods of deploying a differential maturing refuge are described in US 2014/0366786 Al, the disclosure of which is herein incorporated by reference.

[0029] Refuge seeds may be grown into plants that act as a refuge for pests that either feed directly on a particular crop species, or other pests, the presence of which within the local proximity of a particular crop species, results in the damage, decrease in viability, infertility, or decrease in yield of a crop produced from such crop species.

[0030] In various embodiments, the contribution of refuge crop seed to the seed mixture or seed blend can be measured by percentage count of refuge seed to total count of the seed mixture. Seed mixtures in accordance with the present invention for deployment in a field may comprise from about 1% to about 50% refuge crop seed, from about 1% to about 10% refuge crop seed, or from about 5% to about 10% refuge crop seed. Refuge seed contribution can therefore be about 50%, 45%, 40%, 35%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%,

22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14.5%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%.

6%, 5%, 4%, 3%, 2%, or 1% (by total number of seeds) of the seed mixture. That is, the pest- resistant seed might comprise about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85.5%, 90%, 95% or up to about 99% of the seed mixture. The respective ratio of pest-resistant seed to refuge seed in seed mixtures of the present invention may be about 50:50 to about 99: 1, about 70:30 to about 99: 1, about 80:20 to about 97:3, or about 90: 10 to about 95:5, and any integer or fraction ratio in between, for example, about 50:50, 55:45, 60:40, 65:34, 70:30, 75:25, 80:20, 85: 15, 85.5: 14.5, 90: 10, 95:5, 97:3, or 99: 1.

[0031] Also provided herein is a field or population of plants grown from a seed blend as described herein.

Seeds and Plant Species

[0032] The compositions and methods described herein can be used in connection with any species of plant and/or the seeds thereof. The compositions and methods are typically used in connection with seeds that are agronomically important.

[0033] The transgenic, pest-resistant seed and the refuge seed may each independently be selected from any suitable seed type, including, but not limited to, row crops and vegetables. In some embodiments, one or more plants are selected from 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., acom squash, butternut squash, summer squash), watermelon, zucchini), Fabaceae (e.g., alfalfa, beans, carob, clover, guar, lentils, mesquite, peas, peanuts, soybeans, tamarind, tragacanth, vetch), Malvaceae (e.g., cacao, cotton, durian, hibiscus, kenaf, kola, okra), Poaceae (e.g., bamboo, barley, com or maize, fonio, lawn grass (e.g., Bahia grass, Bermudagrass, bluegrass, Buffalograss, Centipede grass, Fescue, or Zoysia), millet, oats, ornamental grasses, rice, rye, sorghum, sugar cane, triticale, wheat), Polygonaceae (e.g., buckwheat), Rosaceae (e.g., almonds, apples, apricots, blackberry, blueberry, cherries, peaches, plums, quinces, raspberries, roses, strawberries), Solanaceae (e.g., bell peppers, chili peppers, eggplant, petunia, potato, tobacco, tomato) and Vitaceae (e.g., grape).

[0034] Non-limiting examples of seeds that may be utilized in the compositions and methods described herein 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 EXTEND™,

SMARTSTAX®, SMARTSTAX PRO™, VT DOUBLE PRO®, VT TRIPLE PRO®,

YIELDGARD®, YIELDGARD VT ROOTWORM/RR2®, YIELDGARD VT TRIPLE® and/or XTENDFLEX™ tradenames.

[0035] In a preferred embodiment, the transgenic, pest-resistant seed and the refuge seed are each com or maize seeds.

[0036] In some embodiments, the transgenic, pest-resistant seed comprises an event selected from the group consisting of MIR 604, DAS-59122-7, 5307, MON 88017, MON 87411, MON 853, and MON 863. In other embodiments, the the transgenic, pest-resistant seed comprises a transgene encoding for a pesticidal product selected from the group consisting of Cry34Abl/Cry35Abl, Cry3A, mCry3A, eCry3TAb, Cry3Bbl, DvSnf7 dsRNA, and DvSnf7 RNA.

[0037] For example, in one embodiment, the pest-resistant crop seeds are com seeds comprising a Cry3Bb.11231 protein having activity against one or more targeted insect pests.

[0038] The pest-resistant crop seeds may comprise a transgene that confers resistance against one or more targeted pests. For example, the pest-resistant crop seeds may comprise a transgene that confers resistance against one or more targeted below-ground pests. In some embodiments, the pest-resistant crop seeds comprise a transgene that confers resistance against one or more insects from the orders Coleoptera or Lepidoptera. In a preferred embodiment, the pest-resistant crop seeds comprise a transgene that confers resistance against com rootworm ( Diabrotica spp ).

First Components

[0039] The first component acts to increase the amount of pest feeding material provided by the refuge plant, and enhances the plant's ability to provide an effective refuge. First components can include, without limitation, microbes, chemical compounds, and molecules. The first component may enhance the growth of the plant through any mechanism, for example by providing nutritional enhancements, growth regulator activity, or a combination thereof.

[0040] The first component may comprise, without limitation, one or more microbes, chemical compounds, or molecules, including but not limited to plant inoculants, nutrient enhancing products, soil amendments, plant biostimulants, signaling molecules, plant regulators, or plant growth regulators. For example, the first component may comprise a plant growth regluator, plant a nutrient enhancing product, a plant innoculant, or a combination thereof. [0041] The first component should be selected to increase the growth of the portion(s) of the plant that provide(s) sustenance for those pests targeted by the pesticidal trait of the genetically modified pest-resistant seed. For example, in a preferred embodiment, refuge plants treated with the first component may exhibit larger roots, greater root mass, or otherwise improved root health or production.

[0042] Non-limiting examples of the first component include lipo-chitooligosaccharides (LCOs), chitooligosaccharides (COs), chitinous compounds, NOD factors, MYC factors, Strigalactones, flavonoids, jasmonic acid or derivatives thereof, linoleic acid or derivatives thereof, linolenic acid or derivatives thereof, and karrikins.

[0043] The first component may comprise any suitable LCO(s).

[0044] LCOs, sometimes referred to as symbiotic nodulation (Nod) signals or Nod factors, consist of an oligosaccharide backbone of b-1_4-1 i nked /V-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, et al, ANN. REV. BIOCHEM. 65:503 (1996); Hamel, et al, PLANTA 232:787 (2010); Prome, et al, PURE & APPL. CHEM. 70(l):55 (1998).

[0045] In some embodiments, the first component comprises one or more LCOs represented by formula I:

in which G is a hexosamine which can be substituted, for example, by an acetyl group on the nitrogen, a sulfate group, an acetyl group and/or an ether group on an oxygen; Ri, R ₂ , R ₃ , Rs, R ₆ and R ₇ , which may be identical or different, represent H, CH ₃ CO—, C _x H _y CO— where x is an integer between 0 and 17 and y is an integer between 1 and 35, or any other acyl group such as, for example, a carbamoyl; R ₄ represents a saturated or mono-, di- or tri-unsaturated aliphatic chain containing at least 12 carbon atoms; and n is an integer between 1 and 4.

[0046] In some embodiments, the first component comprises one or more LCOs represented by formula II:

in which R represents H or CLL CO- and n is equal to 2 or 3. See, e.g., U.S. Patent No.

5,549,718. A number of Bradyrhizobium japonicum-dQn\Qd LCOs have also been described, including BjNod-V (Cis _:i ), BjNod-V (Ac, Ci8 _:i ), BjNod-V (Ci6 _:i ) and BjNod-V (Ac, Ci6 _: o) (with "V" indicating the presence of five N-acetylglucos amines, "Ac" an acetylation, the number following the "C" indicating the number of carbons in the fatty acid side chain and the number following the indicating the number of double bonds). See, e.g., U.S. Patent Nos. 5,175,149 and 5,321,011. Additional LCOs obtained from bacterial strains include NodRM, NodRM-l, NodRM-3. When acetylated (the R=CLb CO—), they become AcNodRM-l and AcNodRM-3, respectively (U.S. Patent No. 5,545,718).

[0047] In some embodiments, the first component comprises one or more LCOs represented by formula III:

in which n = 1 or 2; Ri represents C16, Cl6:0, Cl6: l, Cl6:2, Cl8:0, Cl8:lA9Z or Cl8: lAl lZ; and R.2 represents hydrogen or SO3H.

[0048] LCOs included in compositions and methods of the present disclosure may be obtained from any suitable source.

[0049] In some embodiments, the LCO is obtained (i.e., isolated and/or purified) from a bacterial strain. For example, in some embodiments, the first component comprises one or more LCOs obtained from a strain of Azorhizobium, Bradyrhizobium (e.g., B. japonicum),

Mesorhizobium, Rhizobium (e.g.. R. leguminosarum), or Sinorhizobium (e.g., S. meliloti).

[0050] In some embodiments, the LCO is obtained (i.e., isolated and/or purified) from a mycorrhizal fungus. For example, in some embodiments, the first component comprises one or more LCOs obtained from a strain of Glomerocycota (e.g., Glomus intraradicus). See, e.g., WO 2010/049751 (in which the LCOs are referred to as "Myc factors").

[0051] In some embodiments, the LCO is synthetic. For example, in some embodiments, the first component comprises one or more of the synthetic LCOs described in

WO 2005/063784, WO 2007/117500 and/or WO 2008/071674. In some embodiments, the synthetic LCO contains one or more modifications or substitutions, such as those described in Spaink, CRIT. REV. PLANT SCI. 54:257 (2000) and D'Haeze, supra. LCOs and precursors for the construction of LCOs may be synthesized by genetically engineered organisms. See, e.g.,

Samain et al, CARBOHYDRATE RES. 302:35 (1997); Cottaz, et al, METH. ENG. 7(4):311 (2005); and Samain, et al., J. BIOTECHNOL. 72:33 (1999) (e.g., Fig. 1 therein, which shows structures of COs that can be made recombinantly in E. coli harboring different combinations of genes nodBCHL).

[0052] Further examples of LCOs (and derivatives thereof) that may be useful in compositions and methods of the present disclosure are provided below as formula IV :

in which Ri represents Cl4:0, 3OH-Cl4:0, iso-Cl5:0, Cl6:0, 3-OH-Cl6:0, iso-Cl5:0, Cl6: l, Cl6:2, Cl6:3, iso-Cl7:0, iso-Cl7: l, Cl8:0, 3OH-Cl8:0, Cl8:0/3-OH, Cl8: l, OH-Cl8: l, Cl8:2, Cl8:3, Cl8:4, Cl9: l carbamoyl, C20:0, C20: l, 3-OH-C20: l, C20: l/3-OH, C20:2, C20:3, C22: l and Cl8-26(co-l)-OH (which according to D'Haeze, et al., Glycobiology 72:79R-l05R (2002), includes C18, C20, C22, C24 and C26 hydroxylated species and Cl6:lA9, Cl6:2 (D2,9) and Cl6:3 (D2,4,9)); R ₂ represents hydrogen or methyl; R ₃ represents hydrogen, acetyl or carbamoyl; R ₄ represents hydrogen, acetyl or carbamoyl; R ₅ represents hydrogen, acetyl or carbamoyl; R ₆ represents hydrogen, arabinosyl, fucosyl, acetyl, SO3H, sulfate ester, 3-0-S-2-0- MeFuc, 2-ri-MeFuc and 4-9-AcFuc; R7 represents hydrogen, mannosyl or glycerol; Rx represents hydrogen, methyl, or -CH2OH; R9 represents hydrogen, arabinosyl, or fucosyl; Rio represents hydrogen, acetyl or fucosyl; and n represents 0, 1, 2 or 3. Naturally occurring LCOs embraced by this structure are described in D'Haeze, et al, supra.

[0053] Further examples of LCOs (and derivatives thereof) that may be useful in compositions and methods of the present disclosure are provided below as structures V-XXXIII:

(XXIII)

(XXXII)

(XXXIII).

[0054] It is to be understood that compositions and methods of the present disclosure may comprise analogues, derivatives, hydrates, isomers, salts and/or solvates of LCOs.

[0055] Thus, in some embodiments, the first component comprises one, two, three, four, five, six, seven, eight, nine, ten, or more LCOs represented by one or more of formulas I-IV and/or structures V-XXXIII and/or one, two, three, four, five, six, seven, eight, nine, ten, or more analogues, derivatives, hydrates, isomers, salts and/or solvates of LCOs represented by one or more of formulas I-IV and/or structures V-XXXIII.

[0056] LCOs may be incorporated into treatment compositions described herein in any suitable amount(s)/ concentration(s).

[0057] In some embodiments, the treatment composition comprises from about 1 x 10 ²⁰ M to about 1 x 10 ¹ M LCO. For example, the treatment compositions described herein may comprise about 1 x 10 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 Kb ¹³ M, 1 x Kb ¹² M, 1 x 10 ¹¹ M, 1 x 10 ¹⁰ M, 1 x 10 ⁹ M, 1 x 10 ⁸ M, 1 x 10 ⁷ M, 1 x 10- ⁶ M, 1 x 10- ⁵ M, 1 x 10 ⁴ M, 1 x 10 ³ M, 1 x 10 ² M, 1 x 10 ¹ M of one or more LCOs. In some embodiments, the LCO concentration is 1 x 10 ¹⁴ M to 1 x 10 ⁵ M, 1 x 10 ¹² M to 1 x 10 ⁶ M, or 1 x 10 ¹⁰ M to 1 x 10 ⁷ M. In some embodiments, the LCO concentration is 1 x 10 ¹⁴ M to 1 x Kb ⁵ M, 1 x Kb ¹² M to 1 x 10 ⁶ M, or 1 x lO ¹⁰ M to 1 x lO ⁷ M.

[0058] The first component may comprise any suitable CO(s).

[0059] 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 [(C ₈ 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. l2(6):79R (2002); Demont-Caulet et al, PLANT PHYSIOL. l20(l): 83 (1999); Hanel et al., PLANTA 232:787 (2010); Muller et al, PLANT PHYSIOL.124:733 (2000); Robina et al, TETRAHEDRON 58:521-530 (2002); Rouge et al, Docking of Chitin Oligomers and Nod Factors on Lectin Domains of the LysM-RLK Receptors in the Medicago-Rhizobium Symbiosis, in THE MOLECULAR IMMUNOLOGY OF COMPLEX

CARBOHYDRATES-3 (Springer Science, 2011); Van der Holst et al, CURR. OPIN. STRUC. BIOL. 11 :608 (2001); and Wan et al, PLANT CELL 21 : 1053 (2009); PCT/F 100/00803 (2000). COs differ from LCOs in that they lack the pendant fatty acid chain that is characteristic of LCOs.

[0060] In some embodiments, inoculant compositions of the present disclosure comprise one or more COs represented by formula XXXIV:

in which Ri represents hydrogen or methyl; FC represents hydrogen or methyl; R represents hydrogen, acetyl or carbamoyl; R ₄ represents hydrogen, acetyl or carbamoyl; Rs represents hydrogen, acetyl or carbamoyl; Rr, represents hydrogen, arabinosyl, fucosyl, acetyl, sulfate ester, 3-ri-S-2-ri-MeFuc, 2-ri-MeFuc and 4-ri-AcFuc; R ₇ represents hydrogen, mannosyl or glycerol; Rs represents hydrogen, methyl, or -CH2OH; R9 represents hydrogen, arabinosyl, or fucosyl; Rio represents hydrogen, acetyl or fucosyl; and n represents 0, 1, 2 or 3.

[0061] COs included in compositions and methods of the present disclosure may be obtained from any suitable source.

[0062] In some embodiments, the CO is derived from an LCO. For example, in some embodiments, the first component comprises one or more COs derived from an LCO obtained (i.e., isolated and/or purified) from a strain of Azorhizobium, Bradyrhizobium (e.g., B.

japonicum), Mesorhizobium, Rhizobium (e.g , R. leguminosarum), Sinorhizobium (e.g., S.

meliloti), or mycorhizzal fungus (e.g., Glomus intraradicus). In some embodiments, the CO is derived from an LCO represented by one or more of formulas I-IV and/or structures V-XXXIII. Thus, in some embodiments, the first component described herein may comprise one or more COs represented by one or more of formulas I-IV and/or structures V-XXXIII except that the pendant fatty acid is replaced with a hydrogen or methyl group.

[0063] In some embodiments, the CO is synthetic. Methods for the preparation of recombinant COs are known in the art. See, e.g., Cottaz et al, METH. ENG. 7(4):3l 1 (2005); Samain et al, CARBOHYDRATE RES. 302:35 (1997.); and Samain et al, J. BIOTECHNOL. 72:33 (1999).

[0064] Examples of COs (and derivatives thereof) that may be useful in compositions and methods of the present disclosure are provided below as formula XXXV:

in which n = 1 or 2; Ri represents hydrogen or methyl; and R2 represents hydrogen or SO3H. .

[0065] Further examples of COs (and derivatives thereof) that may be useful in compositions and methods of the present disclosure are provided below as structures XXXVI-

XXXIX:

(XXXVII)

(XXXIX)

[0066] COs (and derivatives thereof) may be utilized in various forms of purity and may be used alone or in the form of a culture of CO-producing bacteria or fungi. In some embodiments, the CO(s) included in the treatment composition described herein is/are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more pure.

[0067] It is to be understood that compositions and methods of the present disclosure may comprise hydrates, isomers, salts and/or solvates of COs.

[0068] Thus, in some embodiments, the first component comprises one, two, three, four, five, six, seven, eight, nine, ten, or more COs represented by one or more of formulas XXXIV- XXXV and/or structures XXXVI-XXXIX and/or one, two, three, four, five, six, seven, eight, nine, ten, or more analogues, derivatives, hydrates, isomers, salts and/or solvates of COs represented by one or more of formulas XXXIV-XXXV and/or structures XXXVI-XXXIX.

[0069] COs may be incorporated into the treatment compositions described herein in any suitable amount(s)/concentration(s). In some embodiments, the treatment composition described herein comprises about 1 x 10 ²⁰ M to about 1 x 10 ¹ M CO. For example, the treatment composition described herein may comprise about 1 x 10 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, 1 x 10 ¹² M, 1 x 10 ¹¹ M, 1 x 10 ¹⁰ M, 1 x 10- ⁹ M, 1 x 10- ⁸ M, 1 x 10- ⁷ M, 1 x 10- ⁶ M, 1 x 10 ⁵ M, 1 x 10 ⁴ M, 1 x 10 ³ M, 1 x 10 ² M, 1 x 10 ¹ M of one or more COs. In some embodiments, the CO concentration is 1 x 10 ¹⁴ M to 1 x 10 ⁵ M, 1 x 10 ¹² M to 1 x 10 ⁶ M, or 1 x 10 ¹⁰ M to 1 x 10 ⁷ M. In some embodiments, the CO concentration is 1 x 10 ¹⁴ M to 1 x 10 ⁵ M, 1 x 10 ¹² M to 1 x 10 ⁶ M, or 1 x 10 ¹⁰ M to 1 x 10 ⁷ M.

[0070] The treatment composition described herein 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.

[0071] 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. & Biotechnol. 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, Woburn MA. Flavonoid compounds may be isolated from plants or seeds, e.g., as described in U.S. Patents 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds may also be produced by genetically engineered organisms, such as yeast, as described in Ralston et al, Plant Physiol. 137: 1375 (2005).

[0072] In some embodiments, the treatment composition described herein comprises one or more anthocyanidins. For example, in some embodiments, the treatment composition described herein comprises cyanidin, delphinidin, malvidin, pelargonidin, peonidin and/or petunidin.

[0073] In some embodiments, the treatment composition described herein comprises one or more anthoxanthins. For example, in some embodiments, the treatment composition described herein 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, pyranoflavonols quercetin, quericitin, rhamnazin, rhamnetin, robinin, rutin, spiraeoside, troxerutin and/or zanthorhamnin).

[0074] In some embodiments, the treatment composition described herein comprises one or more flavanones. For example, in some embodiments, the treatment composition described herein comprises butin, eriodictyol, hesperetin, hesperidin, homoeriodictyol, isosakuranetin, naringenin, naringin, pinocembrin, poncirin, sakuranetin, sakuranin and/or sterubin.

[0075] In some embodiments, the treatment composition described herein comprises one or more flavanonols. For example, in some embodiments, the treatment composition described herein comprises dihydrokaempferol and/or taxifolin.

[0076] In some embodiments, the treatment composition described herein comprises one or more flavans. For example, in some embodiments, the treatment composition described herein comprises one or more flavan-3-ols (e.g., catechin (C), catechin 3-gallate (Cg), epicatechins (EC), epigallocatechin (EGC) epicatechin 3-gallate (ECg), epigall catechin 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, leucodelphinidin, leucofisetinidin, leucomalvidin, luecopelargonidin, leucopeonidin, leucorobinetinidin, melacacidin and/or teracacidin) and/or dimers, trimers, oligomers and/or polymers thereof (e.g., one or more proanthocyanidins).

[0077] In some embodiments, the treatment composition comprises one or more isoflavonoids. For example, in some embodiments, the treatment 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.

[0078] The treatment composition described herein may comprise any suitable flavonoid derivative, including, but not limited to, neoflavonoids (e.g, calophyllobde, coutareagenin, dalbergichromene, dalbergin, nivetin) and pterocarpans (e.g., bitucarpin A, bitucarpin B, erybraedin A, erybraedin B, erythrabyssin II, erthyrabissin-l, erycristagallin, glycinol, glyceolbdins, glyceolbns, glycyrrhizol, maackiain, medicarpin, morisianine, orientanol, phaseobn, pisatin, striatine, trifolirhizin).

[0079] Flavonoids and derivatives thereof may be incorporated into inoculant compositons of the present disclosure in any suitable form, including, but not limited to, polymorphic and crystalline forms.

[0080] Flavonoids may be incorporated into the treatment composition described herein in any suitable amount(s)/concentration(s). The treatment composition described herein may comprise any suitable non-flavonoid node-gene inducer(s), including, but not limited to, jasmonic acid (| 1 R-| 1 a.2b(Z)| |-3-oxo-2-(pentenyl)cyclopentaneacetic acid; JA), linoleic acid ((Z,Z)-9,l2-Octadecadienoic acid) and linolenic acid ((Z,Z,Z)-9,l2,l5-octadecatrienoic acid), as well as analogues, derivatives, hydrates, isomers, polymers, salts and solvates thereof.

[0081] 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. 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(l l):759 (2006); Mabood et al, Agr. J. 98(2):289 (2006); Mabood, et al., Field Crops Res.95(2-3):4l2 (2006); Mabood & Smith, Linoleic and linolenic acid induce the expression of nod genes in Bradyrhizobium japonicum USDA 3, Plant Biol. (2001).

[0082] Useful derivatives of jasmonic acid, linoleic acid, linolenic acid that may be useful in compositions of the present disclosure 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 Ci-C ₈ 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.

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 Ci-Cs 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. 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 filtration or may be recovered by other means such as by evaporation of the solvent.

[0083] Non-flavonoid node-gene inducers may be incorporated into the treatment composition described herein in any suitable amount(s)/concentration(s).

[0084] The treatment composition described herein may comprise 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. In some embodiments, the inoculant composition comprises one or more karrakins represented by formula XL:

[0085] in which Z is O, S or NR ₅ ; Ri, FL. R3 and R4 are each independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, CORi,. COOR=, halogen, NR ₆ R7, or NO2; and R5, Rr, and R7 are each independently H, alkyl or alkenyl, or a biologically acceptable salt thereof.

[0086] Examples of biologically acceptable salts of karrakins may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulphonate, benzenesulphonate and p-toluenesulphonic acid. Additional biologically acceptable metal salts may include alkali metal salts, with bases, examples of which include the sodium and potassium salts. Examples of compounds embraced by formula XXXX and which may be suitable for use in the present disclosure include 3-methyl-2H-furo[2,3-c]pyran-2-one (where R _I =CH3, R2, R3, R ₄ =H), 2H-furo[2,3-c]pyran-2-one (where Ri, R2, R3, R4=H), 7-methyl-2H-furo[2,3-c]pyran-2- one (where Ri, R2, R4=H, R3=CH3), 5-methyl-2H-furo[2,3-c]pyran-2-one (where Ri, R2, R3=H, R4=CH3), 3,7-dimethyl-2H-furo[2,3-c]pyran-2-one (where Ri, R3=CH3, R2, R ₄ =H), 3,5- dimethyl-2H-furo[2,3-c]pyran-2-one (where Ri, R ₄ =CH3, R2, R3=H), 3,5,7-trimethyl-2H- furo[2,3-c]pyran-2-one (where Ri, R3, R ₄ =CH3, R2=H), 5-methoxymethyl-3-methyl-2H- furo[2,3-c]pyran-2-one (where R _I =CH3, R2, R3=H, R ₄ =CH ₂ 0CH ₃ ), 4-bromo-3,7-dimethyl-2H- furo[2,3-c]pyran-2-one (where Ri, R3=CH3, R2=Br, R ₄ =H), 3-methylfuro[2,3-c]pyridin-2(3H)- one (where Z=NH, R _I =CH3, R2, R3, R4=H) and 3,6-dimethylfuro[2,3-c]pyridin-2(6H)-one (where Z=N-CH ₃ , R _I =CH ₃ , R ₂ , R3, R ₄ =H). See, e.g., U.S. Patent No. 7,576,213; Halford, Smoke Signals, in Chem. Eng. News (April 12, 2010) (reporting that karrikins or butenolides contained in smoke act as growth stimulants and spur seed germination after a forest fire and can invigorate seeds such as com, tomatoes, lettuce and onions that had been stored).

[0087] Karrakins may be incorporated into the treatment composition described herein in any suitable amount(s)/concentration(s).

[0088] The treatment composition described herein may comprise gluconolactone and/or one or more analogues, derivatives, hydrates, isomers, polymers, salts and/or solvates thereof.

[0089] Gluconolactone may be incorporated into the treatment composition described herein in any suitable amount(s)/concentration(s).

[0090] The first component may comprise a plant growth regulator or plant growth substance. Non-limiting examples of plant growth regulators/substances include cytokinins, auxins, gibberellins, abscisic acid, ethylene, brassinosteroids, salicylates, and jasmonates.

[0091] The first component may comprise one or more microorganisms.

[0092] The first component may comprise, for example, one or more species or strains from the following genera of microorganisms: Rhizobium spp., Bradyrhizobium spp.,

Sinorhizobium spp., Azorhizobium spp., Glomus spp., Gigaspora spp., Hymenoscyphous spp., Oidiodendron spp., Laccaria spp., Pisolithus spp., Rhizopogon spp., Scleroderma spp., Rhizoctonia spp., Acinetobacter spp., Arthrobacter sp Arthrobotrys spp., Aspergillus spp.. Azospirillum spp., Bacillus spp., Burkholder ia spp., Candida spp., Chryseomonas spp., Enterobacter spp., Eupenicillium spp., Exiguobacterium spp., Klebsiella spp., Kluyvera spp., Microbacterium spp., Mucor spp., Paecilomyces spp., Paenibacillus spp., Penicillium spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Streptomyces spp.,

Streptosporangium spp., Swaminathania spp., Thiobacillus spp., Torulospora spp., Vibrio spp., Xanthobacter spp., Xanthomonas spp., or a combinations thereof. According to some embodiments, the first component may comprise a Bradyrhizobium japonicum, a

Bradyrhizobium elkanii, aRhizobium leguminosctrum, aRhizobium meliloti, a Sinorhizobium me li loti, a Penicillium bilcticte, a Trichodermct virens, or a Bacillus amyloliquefaciens species or strain, or a combination thereof.

[0093] For example, the first component may comprise a microorganism that solubilizes phosphorous, phosphate, or phytate, or which otherwise enhances the availability of

phosphorous. In a preferred embodiment, the first component comprises Penicillium bilaiae.

[0094] The first component may comprise one or more mycorrhizal fungi. The mycorrhizal fungi may be an arbuscular mycorrhizal fungus (AMF) or an

ectomycorrhizal fungus. According to some embodiments, the first component may comprise a species or strain within one or more of the following families, Glomeraceae,

Claroideoglomeraceae, Gigasporaceae, Acaulosporaceae, Paraglomeraceae, or

Ambisporaceae, or within the genus, Glomus. According to some embodiments, the first component may comprise a Glomus intrar adieus species or strain.

[0095] The first component may comprise one or more nitrogen reducing, nitrogen fixing, or otherwise nitrogen availability-enhancing microorganisms. For example, the first component may comprise one or more nitrogen-fixing bacteria. For example, the first component may comprise one or more bacterial species or strain from one or more of the following orders, families, and/or genera: a Rhizobiales sp. including a Bradyrhizobiaceae sp., such as a Bosea sp. or a Bradyrhizobium sp., a Brucellaceae sp., a Hyphomicrobiaceae sp., Methylobacteriaceae sp., a Phyllobacteriaceae sp., such as a Aminobacter sp., Mesorhizobium sp., or a Phyllobaterium sp., a Rhizobiaceae sp., such as aRhizobium species, Burkholderiaceae or Burkholderia sp., or a Xanthobacter aceae sp., or any combination thereof. According to some embodiments, the first component may comprise one or more Rhizobium or

Bradyrhizobium species.

[0096] The first component may comprise a one or more bacteria and/or fungi or one or more compounds or molecules that is/are beneficial to the growth or health of a plant or plant roots, which is/are a part of a commercial product, such as, for example, Ratchet®, Cell-Tech®, Nitragin®, QuickRoots®, JumpStart®, Optimize®, TagTeam®, CUE®, REVV®, Dyna-Start®, First Up®, Acceleron® B-300, Acceleron® B-360, Acceleron® BioRise™, or Acceleron® BioRise2™, or a combination thereof. Additional Components

[0097] Generally, the treatment composition described herein can also comprise an additional component. The additional component can be an additional active ingredient or non active ingredient, including for example any adjuvants, excipients, nutrients, micronutrients, or other desirable components useful in seed treatment formulations.

[0098] The additional component may comprise one or more agrochemicals. For example, the treatment composition described herein may comprise any suitable pesticide(s), including, but not limited to, fungicides, herbicides, insecticides, and nematicides. In some embodiments, the treatment composition described herein comprises one or more biopesticides (e.g., one or more biofungicides, bioinsecticides and/or bionematicides).

[0099] Generally, the refuge seed should not be treated with an insecticide that uses the same mechanism of action as the trait expressed by the pest-resistant seed.

[00100] The treatment composition described herein may comprise any suitable insecticide(s), including, but not limited to, biological insecticides and chemical insecticides. 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. For example, the treatment composition described herein may comprise one or more insecticides toxic to insects from the families Acrididae, Aleytodidae, Anobiidae, Anthomyiidae, Aphididae, Bostrichidae, Bruchidae, Cecidomyiidae, Cerambycidae, Cercopidae, Chrysomelidae,

Cicadellidae, Coccinellidae, Cryllotalpidae, Cucujidae, Curculionidae, Dermestidae, Elateridae, Gelechiidae, Lygaeidae, Meloidae, Membracidae, Miridae, Noctuidae, Pentatomidae, Pyralidae, Scarabaeidae, Silvanidae, Spingidae, Tenebrionidae and/or Thripidae.

[00101] In some embodiments, the treatment composition described herein 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. tabaci), Brevicoryne (e.g., B. brassicae ), Bruchidius (e.g., B. atrolineatus), Bruchus (e.g., B. atomarius, B. dentipes, B. lends, B. pisorum and/or B. ruflpes), Callosobruchus (e.g., C. chinensis, C. maculatus, C.

rhodesianus, C. subinnotatus , C. theobromae), Caryedon (e.g., C. serratus ), Cassadinae, Ceratitis (e.g., C. capitata), Chrysomelinae , Circulifer (e.g., C. tenellus), Criocerinae,

Cryptocephalinae, Cryptolestes (e.g., C. ferrugineus, C. pusillis, C. pussilloides), Cylas (e.g., C. formicarius), Delia (e.g., D. antiqua), Diabrotica, Diaphania (e.g., D. nitidalis), Diaphorina (e.g., D. citri), Donaciinae, Ephestia (e.g, E. cautella, E. elutella, E., keuhniella), Epilachna (e.g., E. varivestris ), Epiphyas (e.g., E. postvittana), 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., L. decemlineata), Leptoglossus, Liriomyza (e.g.. /.. trifolii), Manducca, Melittia (e.g., M cucurbitae), Myzus (e.g., M. persicae), 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. dominica), Rhopalosiphum (e.g., R. maidis ), Sagrinae, Solenopsis (e.g., S. Invicta), Spilopyrinae, 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, Tenebrio (e.g., T. malens and/or T. molitor), Thrips (e.g., T. tabaci), Trialeurodes (e.g., T.

vapor ariorum), Tribolium (e.g., T. castaneum and/or T. confusum ), Trichoplusia (e.g., T. ni), Trogoderma (e.g., T. granarium ) and Trogossitidae (e.g., T. mauritanicus).

[00102] 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).

[00103] The treatment composition described herein may comprise any suitable nematicide(s) including, but not limited to, biological nematicides and chemical nematicides. 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.

[00104] In some embodiments, the treatment composition described herein 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.

[00105] Additional examples of nematodes that may be targeted by the treatment composition described herein may be found in Capinera, Handbook of Vegetable Pests (2001) and Niblack, Nematodes, in Illinois Agronomy Handbook (2008). [00106] As discussed above, the treatment composition described herein may comprise one or more biological insecticides and/or nematicides (i.e., one or more microorganisms the presence and/or output of which is toxic to an acarid, insect and/or nematode).

[00107] In some embodiments, the treatment composition described herein may comprise one or more chemical insecticides and/or nematicides. For example, in some embodiments, the treatment composition described herein comprises one or more carbamates, diamides, macrocyclic lactones, neonicotinoids, organophosphates, phenylpyrazoles, pyrethrins, spinosyns, synthetic pyrethroids, tetronic acids and/or tetramic acids.

[00108] Non-limiting examples of chemical insecticides and nematicides that may be useful in the treatment composition described herein include acrinathrin, alpha-cypermethrin, betacyfluthrin, cyhalothrin, cypermethrin, deltamethrin, esfenvalerate , etofenprox,

fenpropathrin, fenvalerate, flucythrinate, fosthiazate, lambda-cyhalothrin, gamma-cyhalothrin, permethrin, tau-fluvalinate, transfluthrin, zeta-cypermethrin, cyfluthrin, bifenthrin, tefluthrin, eflusilanat, fubfenprox, pyrethrin, resmethrin, imidacloprid, acetamiprid, thiamethoxam, nitenpyram, thiacloprid, dinotefuran, clothianidin, imidaclothiz, chlorfluazuron, diflubenzuron, lufenuron, teflubenzuron, triflumuron, novaluron, flufenoxuron, hexaflumuron, bistrifluoron, noviflumuron, buprofezin, cyromazine, methoxyfenozide, tebufenozide, halofenozide, chromafenozide, endosulfan, fipronil, ethiprole, pyrafluprole, pyriprole, flubendiamide, chlorantraniliprole (e.g., Rynaxypyr ), cyantraniliprole, emamectin, emamectin benzoate, abamectin, ivermectin, milbemectin, lepimectin, tebufenpyrad, fenpyroximate, pyridaben, fenazaquin, pyrimidifen, tolfenpyrad, dicofol, cyenopyrafen, cyflumetofen, acequinocyl, fluacrypyrin, bifenazate, diafenthiuron, etoxazole, clofentezine, spinosad, triarathen, tetradifon, propargite, hexythiazox, bromopropylate, chinomethionat, amitraz, pyrifluquinazon, pymetrozine, flonicamid, pyriproxyfen, diofenolan, chlorfenapyr, metaflumizone, indoxacarb, chlorpyrifos, spirodiclofen, spiromesifen, spirotetramat, pyridalyl, spinctoram, acephate, triazophos, profenofos, oxamyl, spinetoram, fenamiphos, fenamipclothiahos, 4-{[(6-chloropyrid- 3-yl)methyl](2,2-difluoroethyl)amino}furan-2(5H)-one, cadusaphos, carbaryl, carbofuran, ethoprophos, thiodicarb, aldicarb, aldoxycarb, metamidophos, methiocarb, sulfoxaflor, and tioxazafen and combinations thereof. In some embodiments, the treatment composition described herein comprises abamectin, aldicarb, aldoxycarb, bifenthrin, carbofuran,

chlorantraniliprole, clothianidin, cyfluthrin, cyhalothrin, cypermethrin, cyantraniliprole, deltamethrin, dinotefuran, emamectin, ethiprole, fenamiphos, fipronil, flubendiamide, fosthiazate, imidacloprid, ivermectin, lambda-cyhalothrin, milbemectin, nitenpyram, oxamyl, permethrin, spinetoram, spinosad, spirodiclofen, spirotetramat, tefluthrin, thiacloprid, thiamethoxam and/or thiodicarb. In some embodiments, the treatment composition described herein comprises an insecticide selected from the group consisting of clothianidin,

thiamethoxam, imidacloprid, cyantraniliprole, chlorantraniliprole, fluopyram and tioxazafen.

[00109] Additional examples of insecticides and nematicides that may be included in the treatment composition described herein may be found in Steffey and Gray, Managing Insect Pests, in Illinois Agronomy Handbook (2008) and Niblack, Nematodes, in Illinois Agronomy Handbook (2008).

[00110] In some embodiments, the treatment composition described herein comprises one or more commercial insecticides and nematicides used in accordance with the manufacturer's recommended amounts/concentrations.

[00111] The treatment composition described herein may comprise any suitable fungicide(s), including, but not limited to, biological fungicides and chemical fungicides.

Fungicides may be selected so as 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.

[00112] In some embodiments, the treatment composition described herein comprises a fungicide (or combination of fungicides) that is toxic to one or more strains of Albugo (e.g., A. Candida), Alternaria (e.g. _, A. alt ernata), Aspergillus (e.g., A. Candidas, A. clavatus , A. flavus , A. fumigatus , A. parasiticus , A. restrictus, A. sojae, A. solani ), Plumeria (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. destructivum, C. fragariae, C. gloeosporioides, C. graminicola, C. kehawee, C. lindemuthianum, C. musae, C. orbiculare, C. spinaceae, C. sublineolum, C. trifolii, C. truncatum), Fusarium (e.g., F. graminearum, F. moniliforme, F. oxysporum, F. roseum, F. tricinctum),

Helminthosporium, Magnaporthe (e.g., M grisea, M. oryzae), Melamspora (e.g., M lini), Mycosphaerella (e.g., M. graminicola), Nematospora, Penicillium (e.g., P. 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. solani), Scopulariopsis, Selerotinia, Thielaviopsis and/or Ustilago (e.g . JJ. maydis). [00113] Additional examples of fungi that may be targeted by the treatment composition described herein may be found in Bradley, Managing Diseases, in Illinois Agronomy Handbook (2008).

[00114] As discussed above, the treatment composition described herein may comprise one or more biological fungicides (i.e., one or more microorganisms the presence and/or output of which is toxic to a fungus).

[00115] In some embodiments, the treatment composition described herein comprises one or more chemical fungicides. For example, in some embodiments, the treatment composition described herein comprises 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.

[00116] Non-limiting examples of chemical fungicides that may be useful in the treatment composition described herein 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-acrybc acid methyl ester and 2-(2-(3-(2,6-dichlorophenyl)-l-methyl-allybdeneaminooxymethy l)- phenyl)-2-methoxyimino-N-methyl-acetamide; carboxamides, such as carboxanibdes (e.g., benalaxyl, benalaxyl-M, benodanil, bixafen, boscabd, carboxin, fenfuram, fenhexamid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isopyrazam, isotianil, kiralaxyl, mepronil, metalaxyl, metalaxyl-M (mefenoxam), ofurace, oxadixyl, oxycarboxin, penflufen, penthiopyrad, sedaxane, tecloftalam, thifluzamide, tiadinil, 2-amino-4-methyl-thiazole-5-carboxanibde, N-(4'- trifluoromethylthiobiphenyl-2-yl)-3-difluoromethyl-l-methyl- lH-pyra- zole-4-carboxamide, N- (2-(l,3,3-trimethylbutyl)-phenyl)-l,3-dimethyl-5-fluoro-lH-p yrazole-4-carboxamide), carboxylic morphobdes (e.g., dimethomorph, flumorph, pyrimorph), benzoic acid amides (e.g., flumetover, fluopicobde, fluopyram, zoxamide), carpropamid, dicyclomet, mandiproamid, oxytetracycbn, silthiofam and N-(6-methoxy-pyridin-3-yl) cyclopropanecarboxybc acid amide; azoles, such as triazoles (e.g., azaconazole, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, diniconazole-M, epoxiconazole, fenbuconazole, fluquinconazole, flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, oxpoconazole, paclobutrazole, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triticonazole, uniconazole) and imidazoles (e.g., cyazofamid, imazalil, pefurazoate, prochloraz, triflumizol); heterocyclic compounds, such as pyridines (e.g., fluazinam, pyrifenox (cf.Dlb), 3-[5-(4-chloro-phenyl)-2,3-dimethyl- isoxazolidin-3-yl] -pyridine, 3-[5-(4-methyl-phenyl)-2,3-dimethyl-isoxazolidin-3-yl]-pyrid ine), pyrimidines (e.g., bupirimate, cyprodinil, diflumetorim, fenarimol, ferimzone, mepanipyrim, nitrapyrin, nuarimol, pyrimethanil), piperazines (e.g., triforine), pyrroles (e.g., fenpiclonil, fludioxonil), morpholines (e.g., aldimorph, dodemorph, dodemorph-acetate, fenpropimorph, tridemorph), piperidines (e.g., fenpropidin); dicarboximides (e.g., fluoroimid, iprodione, procymidone, vinclozolin), non-aromatic 5-membered heterocycles (e.g., famoxadone, fenamidone, flutianil, octhilinone, probenazole, 5-amino-2-isopropyl-3-oxo-4-ortho-tolyl-2,3- dihydro-pyrazole-l-carbothioic acid S-allyl ester), acibenzolar-S-methyl, ametoctradin, amisulbrom, anilazin, blasticidin-S, captafol, captan, chinomethionat, dazomet, debacarb, diclomezine, difenzoquat, difenzoquat-methylsulfate, fenoxanil, folpet, oxobnic acid, piperabn, proquinazid, pyroquilon, quinoxyfen, triazoxide, tricyclazole, 2-butoxy-6-iodo-3- propylchromen-4-one, 5-chloro-l-(4,6-dimethoxy-pyrimidin-2-yl)-2-methyl-lH-benzoi midazole and 5-chloro-7-(4-methylpiperidin-l-yl)-6-(2,4,6-trifluorophenyl )-[l,2,4]triazolo-[l,5- ajpyrimidine; benzimidazoles, such as carbendazim; and other active substances, such as guanidines (e.g., guanidine, dodine, dodine free base, guazatine, guazatine-acetate,

iminoctadine), iminoctadine-triacetate and iminoctadine-tris(albesilate); antibiotics (e.g., kasugamycin, kasugamycin hydrochloride-hydrate, streptomycin, polyoxine and validamycin A), nitrophenyl derivates (e.g., binapacryl, dicloran, dinobuton, dinocap, nitrothal-isopropyl, tecnazen); organometal compounds (e.g., fentin salts, such as fentin-acetate, fentin chloride, fentin hydroxide); sulfur-containing heterocyclyl compounds (e.g., dithianon, isoprothiolane), organophosphorus compounds (e.g., edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, phosphorus acid and its salts, pyrazophos, tolclofos-methyl), organochlorine compounds (e.g., chlorothalonil, dichlofluanid, dichlorophen, flusulfamide, hexachlorobenzene, pencycuron, pentachlorphenole and its salts, phthalide, quintozene, thiophanate-methyl, thiophanate, tolylfluanid, N-(4-chloro-2-nitro-phenyl)-N-ethyl-4-methyl-benzenesulfonam ide) and inorganic active substances (e.g., Bordeaux mixture, copper acetate, copper hydroxide, copper oxychloride, basic copper sulfate, sulfur) and combinations thereof. In some embodiments, the treatment composition described herein comprises acibenzolar-S-methyl, azoxystrobin, benalaxyl, bixafen, boscabd, carbendazim, cyproconazole, dimethomorph, epoxiconazole, fludioxonil, fluopyram, fluoxastrobin, flutianil, flutolanil, fluxapyroxad, fosetyl-Al, ipconazole, isopyrazam, kresoxim-methyl, mefenoxam, metalaxyl, metconazole, myclobutanil, orysastrobin, penflufen, penthiopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, sedaxane, silthiofam, tebuconazole, thiabendazole, thifluzamide, thiophanate, tolclofos-methyl, trifloxystrobin and triticonazole. In some embodiments, the treatment composition described herein comprises azoxystrobin, pyraclostrobin, fluoxastrobin, trifloxystrobin, ipconazole, prothioconazole, sedaxane, fludioxonil, metalaxyl, mefenoxam, thiabendazole, fluxapyroxad and/or fluopyram.

[00117] Additional examples of fungicides that may be included in the treatment composition described herein may be found in Bradley, Managing Diseases, in Illinois

Agronomy Handbook (2008).

[00118] In some embodiments, the treatment composition described herein comprises one or more commercial fungicides used in accordance with the manufacturer's recommended amounts/ concentrations .

[00119] The treatment composition described herein may comprise any suitable herbicide(s), including, but not limited to, biological herbicides and chemical herbicides.

Herbicides may be selected so as to provide effective control against a broad spectrum of plants, including, but not limited to, plants from the families Asteraceae, Caryophyllaceae, Poaceae and Polygonaceae.

[00120] In some embodiments, the treatment composition described herein 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. 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. walteri), Fallopia (e.g.. // baldschuanica, F. japonica, F.

sachalinensis), Stellaria (e.g., S. media) and/or 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).

[00121] Additional species of plants that may be targeted by the treatment composition described herein 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). [00122] As discussed above, the treatment composition described herein may comprise one or more biological herbicides (i.e., one or more microorganisms the presence and/or output of which is toxic to a plant).

[00123] In some embodiments, the treatment composition described herein comprises one or more chemical herbicides. For example, in some embodiments, the treatment composition described herein comprises 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. Non-limiting examples of chemical herbicides that may be useful in the treatment composition described herein 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, fenoxaprop, fluazifop, fluazifop-P, fluometuron, flufenpyr-ethyl, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluthiacet- methyl, fomesafe, fomesafen, glyphosate, glufosinate, haloxyfop, hexazinone, imazamox, imazaquin, imazethapyr, ioxynil, isoproturon, isoxaflutole, lactofen, linuron, mecoprop, mecoprop-P, mesotrione, metamitron, metazochlor, methibenzuron , metolachlor (and S-metolachlor ), metoxuron, metribuzin, monolinuron, oxadiargyl, oxadiazon, oxyfluorfen, phenmedipham, pretilachlor, profoxydim, prometon, prometry, propachlor, propanil , propaquizafop, propisochlor, pyraflufen-ethyl, pyrazon, pyrazolynate, pyrazoxyfen, pyridate, quizalofop, quizalofop-P (e.g., quizalofop-ethyl, quizalofop-P-ethyl, clodinafop- propargyl, cyhalofop-butyl, diclofop- methyl, fenoxaprop-P-ethyl, fluazifop-P-butyl, haloxyfop- methyl, haloxyfop-R-methyl), saflufenacil, sethoxydim, siduron, simazine, simetryn, sulcotrione, sulfentrazone, tebuthiuron, tembotrione, tepraloxydim, terbacil, terbumeton, terbuthylazine, thaxtomin (e.g., the thaxtomins described in US Patent No.: 7,989,393), thenylchlor, tralkoxydim, triclopyr, trietazine, tropramezone, salts and esters thereof; racemic mixtures and resolved isomers thereof and combinations thereof. In some embodiments, the treatment composition described herein comprises acetochlor, clethodim, dicamba, flumioxazin, fomesafen, glyphosate, glufosinate, mesotrione, quizalofop, saflufenacil, sulcotrione, ethyl 2- ((3-(2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-(trifluoromet hyl)-2,3-dihydropyrimidin-l(6H)- yl)phenoxy)pyridin-2-yl)oxy)acetate and/or 2,4-D. In some embodiments, the treatment composition described herein comprises 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-(trifluorom ethyl)-2,3-dihydropyrimidin- l(6H)-yl)phenoxy)pyridin-2-yl)oxy)acetate.

[00124] Additional examples of herbicides that may be included in the treatment composition described herein 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).

[00125] In some embodiments, the first and second seed treatment compositions described herein comprise one or more commercial herbicides used in accordance with the manufacturer's recommended amounts/concentrations.

Application to Seeds

[00126] A treatment composition comprising a first component as described herein may be applied to a refuge seed, thereby forming a treated refuge seed, which may be combined with a treated or non-treated non-refuge seed. According to some embodiments, the first component may also be applied to a non-refuge seed, thereby forming a treated non-refuge seed, which may be combined with a treated refuge seed.

[00127] The treatment composition may be applied to the seed prior to sowing the seed, so that the sowing operation is simplified. In this manner, seeds can be treated, for example, at a central location and then distributed for planting. This may permit a person who plants the seeds to avoid the complexity and effort associated with handling and applying the treatment compositions, and to merely plant the treated seeds in a manner that is conventional for regular untreated seeds.

[00128] The treatment composition can be applied to seeds by any standard seed treatment methodology, including but not limited to mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, immersion, and solid matrix priming. Seed coating methods and apparatus for their application are disclosed in, for example, U.S. Pat. Nos.

5,918,413, 5,891,246, 5,554,445, 5,389,399, 5,107,787, 5,080,925, 4,759,945 and 4,465,017, among others. Any conventional active or inert material can be used for contacting seeds with the treatment composition, such as conventional film-coating materials including but not limited to water-based film coating materials.

[00129] For example, in one embodiment, a treatment composition can be introduced onto or into a seed by use of solid matrix priming. For example, a quantity of the treatment composition can be mixed with a solid matrix material and then the seed can be placed into contact with the solid matrix material for a period to allow the treatment composition to be introduced to the seed. The seed can then optionally be separated from the solid matrix material and stored or used, or the mixture of solid matrix material plus seed can be stored or planted directly. Non-limiting examples of solid matrix materials which are useful include

polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the treatment composition for a time and releasing the first component of the treatment composition into or onto the seed. It is useful to make sure that the first component and the solid matrix material are compatible with each other. For example, the solid matrix material should be chosen so that it can release the first component at a reasonable rate, for example over a period of minutes, hours, days, or weeks.

[00130] Imbibition is another method of treating seed with the treatment composition. For example, a plant seed can be directly immersed for a period of time in the treatment

composition. During the period that the seed is immersed, the seed takes up, or imbibes, a portion of the treatment composition. Optionally, the mixture of plant seed and the treatment composition can be agitated, for example by shaking, rolling, tumbling, or other means. After imbibition, the seed can be separated from the treatment composition and optionally dried, for example by patting or air drying.

[00131] The treatment composition may be applied to the seeds using conventional coating techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. Other methods, such as spouted beds may also be useful. The seeds may be pre-sized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for sizing. Such procedures are generally known in the art.

[00132] If the treatment composition is applied to the seed in the form of a coating, the seeds can be coated using a variety of methods known in the art. For example, the coating process can comprise spraying the treatment composition onto the seed while agitating the seed in an appropriate piece of equipment such as a tumbler or a pan granulator. [00133] In one embodiment, when coating seed on a large scale (for example a commercial scale), the seed coating may be applied using a continuous process. For example, seed may be introduced into the treatment equipment (such as a tumbler, a mixer, or a pan granulator) either by weight or by flow rate. The amount of treatment composition that is introduced into the treatment equipment can vary depending on the seed weight to be coated, surface area of the seed, the concentration of the first component and/or other active ingredients in the treatment composition, the desired concentration on the finished seed, and the like. The treatment composition can be applied to the seed by a variety of means, for example by a spray nozzle or revolving disc. The amount of liquid may be determined by the assay of the formulation and the required rate of active ingredient necessary for efficacy. As the seed falls into the treatment equipment the seed can be treated (for example by misting or spraying with the treatment composition) and passed through the treater under continual movement/tumbling where it can be coated evenly and dried before storage or use.

[00134] In another embodiment, the seed coating may be applied using a batch process. For example, a known weight of seeds can be introduced into the treatment equipment (such as a tumbler, a mixer, or a pan granulator). A known volume of treatment composition can be introduced into the treatment equipment at a rate that allows the treatment composition to be applied evenly over the seeds. During the application, the seed can be mixed, for example by spinning or tumbling. The seed can optionally be dried or partially dried during the tumbling operation. After complete coating, the treated sample can be removed to an area for further drying or additional processing, use, or storage.

[00135] In an alternative embodiment, the seed coating may be applied using a semi-batch process that incorporates features from each of the batch process and continuous process embodiments set forth above.

[00136] In still another embodiment, seeds can be coated in laboratory size commercial treatment equipment such as a tumbler, a mixer, or a pan granulator by introducing a known weight of seeds in the treater, adding the desired amount of treatment composition, tumbling or spinning the seed and placing it on a tray to thoroughly dry.

[00137] In another embodiment, seeds can also be coated by placing the known amount of seed into a narrow neck bottle or receptacle with a lid. While tumbling, the desired amount of treatment composition can be added to the receptacle. The seed is tumbled until it is coated with the treatment composition. After coating, the seed can optionally be dried, for example on a tray. [00138] In some embodiments, the treated seeds may also be enveloped with a film overcoating to protect the coating. Such overcoatings are known in the art and may be applied using conventional fluidized bed and drum film coating techniques. The overcoatings may be applied to seeds that have been treated with any of the seed treatment techniques described above, including but not limited to solid matrix priming, imbibition, coating, and spraying, or by any other seed treatment technique known in the art.

Methods of Application to Soil

[00139] Alternatively, a treatment composition comprising the first component may be applied to the soil in the locus surrounding the refuge seeds (i.e., the root zone of the plants grown from the refuge crop plants). According to some embodiments, a treatment composition comprising the first component may also be applied to the soil in the locus surrounding the non- refuge seeds.

[00140] The application may be performed using any method or apparatus known in the art, including but not limited to hand sprayer, mechanical sprinkler, or irrigation, including drip irrigation.

[00141] For example, in one embodiment, the treatment composition comprising the first component is applied to the soil using a drip irrigation technique. The composition may be applied through existing drip irrigation systems. This procedure is particularly preferred for use in connection with cotton, strawberries, tomatoes, potatoes, vegetables, and ornamental plants.

[00142] In another embodiment, the treatment composition is applied to plants and/or soil using a drench application. Preferably, a sufficient quantity of the treatment composition is applied such that it drains through the soil to the planted seed, or to the root area of the plant.

The drench application technique is particularly preferred for use in connection with crop plants, turf grasses, and animals.

[00143] In some embodiments, the treatment composition is applied to soil after planting. In other embodiments, however, the treatment composition may be applied to soil during planting. In other embodiments, however, the treatment composition may be applied to soil before planting. When the treatment composition is applied directly to the soil, it may be applied using any method known in the art. For example, it may be tilled into the soil or applied in furrow. Methods of Managing Pest Resistance

[00144] The seed blends, compositions, and methods described herein can be utilized in connection with an improved strategy for insect resistance management. For example, the seed blends, compositions, and methods described herein can be utilized to manage pest resistance in a plot of pest-resistant crops.

[00145] The effectivness of a refuge used to manage pest resistance in a plot of pest- resistant crops may be improved by planting refuge seeds that have been treated with a first component as described herein. For example, the method may comprise (a) planting pest- resistant crop seeds comprising a transgene that confers resistance against one or more targeted insect or nematode pests; (b) planting refuge crop seeds that lack the transgene and are treated with a first component that improves the root growth or root health of plants grown from the refuge crop seeds. In some embodiments, the pest-resistant crop seeds are not treated with the first component. Alternatively, in other embodiments, both the refuge seeds and the pest- resistant crop seeds are treated with the first component.

[00146] The pest-resistant crop seeds and the refuge crop seeds may be planted in the same plot using a seed mixture or blend containing pest-resistant crop seeds and the refuge crop seeds, sometimes referred to a refuge-in-a-bag (RIB) composition or package. Alternatively, the pest-resistant crop seeds and the refuge crop seeds may be planted in adjacent plots in a separate section of the field adjacent the plot in which the pest-resistant crop seed are planted, sometimes referred to as a structured refuge (e.g., block or strip plantings).

[00147] The effectivness of a refuge may be improved by planting a seed blend comprising refuge crop seeds and pest-resistant crop seeds in a uniform mixture, as described herein.

[00148] The effectivness of a refuge may be improved by treating the soil in the locus surrounding the refuge seeds (i.e., the root zone of the plants grown from the refuge crop plants) with a treatment commposition comprising a first component as described herein. For example, the method may comprise (a) planting pest-resistant crop seeds comprising a transgene that confers resistance against one or more targeted insect or nematode pests; (b) planting refuge crop seeds that lack the transgene; and (c) treating the soil in the locus surrounding the planted refuge crop seeds with a first component that improves the root growth or root health of plants grown from the refuge crop seeds. In some embodiments, the soil in the locus surrounding the planted pest-resistant crop seeds is not treated with the first component. Alternatively, in other embodiments, both the refuge seeds and the pest-resistant crop seeds are treated with the first component.

Definitions

[00149] As used herein, the term“genetically modified” broadly encompasses any seed or plant whose genome has been modified using genetic engineering. Genetically modified seeds and plants include, for example, transgenic, cisgenic, and subgenic seeds and plants.

[00150] As used herein, the term“plot” refers to a contiguous area in which a population of crop plants is grown. A first plot is considered“adjacent” to a second plot if the distance between the plots is no greater than 1 mile, for example no greater than 0.5 miles, no greater than 0.25 miles, no greater than 400 yards, no greater than 300 yards, no greater than 200 yards, or no greater than 100 yards.

[00151] As used herein, the term“refuge-integrated” field or“integrated refuge” refers to a field containing a population of genetically modified pest-resistant plants and a population of refuge plants. Populations of pest-resistant plants and refuge plants can be planted from a seed blend, such as a“refuge-in-a-bag” (RIB) or“improved RIB” (iRIB) seed blend. Such a field may include a first population pest-resistant crop that harbors a gene with pesticidal activity against a pest or a number of pests of the crop species, and a second population refuge crop that is not pesticidal against the pest or the number of pests of the crop species or is insecticidal against the same pest or number of pests, but through a different mode of action. As used herein, the first population pest-resistant crop is grown from a“first seed type”,“toxin seed”,“pesticidal seed,” or“plant-integrated protectant seed” or“PIP seed”, and the second population refuge crop is grown from a“second seed type” or“refuge seed”.

[00152] As used herein, the term“refuge” includes an isolated plant or plant population; including the plant parts, such as the seed, developing seed, or seed assemblage of the isolated refuge plant or refuge plant population. The refuge crop (plant population, isolated plant, or plant parts) functions as a dietary refuge or refugia for pest(s) under control by nearby plants or plant parts comprising the pest-resistant trait.

[00153] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. EXAMPLES

[00154] The following non-limiting examples are provided to further illustrate the present invention.

[00155] Example 1

[00156] An experimental trial is conducted to evaluate the effectiveness of seed treatments as applied to refuge com seeds. The refuge com seeds do not comprise a transgene that is active against com rootworm (“CRW”).

[00157] Refuge com seeds treated with two different biological seed treatments (LCO and CO) are compared against refuge com seeds alone, and against refuge com seeds treated with one biological seed treatment formulation only, to determine if the addition of a biological seed treatment impacts com root volume and/or CRW adult emergence.

Table 1: Seed Treatments

Table 2: Corn Treatments for Evaluation and Infestation Rate

Table 3: Planting Details

Table 4: Artificial Infestation with Corn Rootworm

[00158] A final stand count is taken prior to infestation for each emergence plot.

[00159] Prior to flowering (anthesis) or upon initial detection of emerging adult beetles, whichever comes first, plants are cut off above the third or fourth green healthy leaf to a height of 40 to 60 cm from the soil surface. Leaving intact leaves allows the roots to remain alive longer for late developing rootworm larvae. The cut plant tops are placed within the trial area and are disposed of using approved methods for a regulated trial.

[00160] Tents are erected prior to beetle emergence. Tents remain closed during the trial, and the and edges are sealed with soil at their base to ensure insects do not escape. Additionally, the interior and outer perimeters of the tents are kept free of weeds. Each tent is individually labeled with a unique identifier (e.g. tent number linked to a specific treatment).

[00161] Beetles are collected from individual tents starting at first indication of beetle emergence. Collections occur at least weekly, targeting no more than 5-7 days between collections. Collections are continued until no insects emerge for at least 10 days or until deemed appropriate. For each collection date, beetles are placed in labeled collection vials. Collections from different dates or plots are not mixed.

[00162] Prior to beetle collection, the collection vial is labeled and/or bar coded with an identifier. The beetles are collected and placed in vials. Vails containing the beetles are placed in a cooler on wet ice (or similar) or dry ice during collection and between each tent collection. To ensure beetle integrity, vials are placed in a -80°C freezer as soon as possible after beetle collection, and are held in the freezer until the beetles are evaluated. Following collection, the beetles are evaluated and identified by species and sex.

[00163] The roots of the com plants are also evaluated. A series of three successive infested plants are collected from within the tent. As a control, a series of three successive non- infestive plants will be collected from a dedicated row located outside of the tent. The roots are dug, washed, and the total root volume is assessed. [00164] When introducing elements of the present invention or the embodiments(s) thereof, the articles "a", "an", "the" and "the" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to mean that there may be additional elements other than the listed elements.

[00165] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[00166] As various changes could be made in the above compositions and processes without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying draw ing! s | shall be interpreted as illustrative and not in a limiting sense.