High yield minicell and protein producing bacterial strains

Abstract

The present disclosure relates to mixtures of insecticidal minicells containing the insecticidal protein toxins Pir and Cry, and methods relating to their production and use. The present disclosure also relates to mixtures of insecticidal minicells containing the insecticidal protein toxins Pir and Bt-derived active ingredients, and methods relating to their production and use.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 299,321, filed January 13, 2022, and U.S. Provisional Patent Application No. 63 / 322,978, filed March 23, 2022, the contents of which are all incorporated by reference in their entireties as if fully set forth herein.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (165852002940seqlist.xml; size: 305,619 bytes; and creation date: January 10, 2023) are incorporated herein by reference in their entirety.

[0003] This disclosure relates generally to genetically engineered bacterial cells for efficient minicell and protein production. This disclosure also relates to mixtures of insecticidal minicells containing the insecticidal protein toxins Pir and Cry, and methods for making and using the same. This disclosure also relates to mixtures of insecticidal minicells containing the insecticidal protein toxin Pir with Bt-derived active ingredients, and methods for making and using the same. [Background technology]

[0004] There is a need for delivery vectors that can target cells and deliver biological agents. Furthermore, there is a need for methods for delivering the vectors to cells and thereby regulating biological systems, including animal, plant, and insect cells, tissues, and organisms. Delivery vectors that can target cells and deliver biological agents are known, for example, from International Publication No. 2020123569. The applicant desires compositions containing vectors with improved delivery efficacy, including, for example, the delivery of biological agents. Summary of the Invention

[0005] The present disclosure provides compositions and methods for producing minicell particles from parent bacterial cells (e.g., E. coli strains) with various genetic mutations that increase the number of minicell particles produced. Additionally, various expression cassettes for the minicell-producing parent cells are provided that result in high levels of protein expression.

[0006] Some embodiments relate to methods for producing a composition comprising a plurality of minicells substantially free of viable parent bacterial cells, the method comprising: (a) exposing a population of parent bacterial cells exhibiting a reduced level or activity of at least one cell division topology-specific factor to conditions that allow for the formation of minicells, wherein the parent bacterial cells are defective in a gene involved in DNA repair; and (b) separating the minicells from the parent bacterial cells, thereby producing a composition comprising a plurality of minicells substantially free of viable parent bacterial cells. In some embodiments, the cell division topology-specific factor is selected from the group consisting of minC, minD, minE, and ftsZ. In some embodiments, the gene involved in DNA repair is selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD, and DNA polymerase III. In some embodiments, the gene involved in DNA repair is RecA. In some embodiments, the parent bacterial cells comprise one or more of T3, T7, K11, or SP6 polymerase, or a nucleic acid encoding a T3, T7, K11, or SP6 polymerase. In some embodiments, the parent bacterial cells comprise T7 polymerase, or a nucleic acid encoding a T7 polymerase. In some embodiments, the bacterial cells are E. coli DH10BΔminCDE. In some embodiments, the composition comprises at least 10 11 In some embodiments, the composition is an agricultural composition.

[0007] In some aspects, methods are provided for producing a composition comprising a plurality of protein-loaded minicells substantially free of viable parent bacterial cells, the method comprising: (a) exposing a population of parent bacterial cells exhibiting a reduced level or activity of at least one cell division topology specificity factor to conditions that allow for expression of the protein, wherein the parent bacterial cells are deficient in a gene involved in DNA repair; (b) exposing the parent bacterial cells to conditions that allow for the formation of minicells; and (c) separating the minicells from the parent bacterial cells, thereby producing a composition comprising a plurality of protein-loaded minicells that is substantially free of viable parent bacterial cells. In some embodiments, the parent bacterial cells comprise an expression vector. In some embodiments, the expression vector encodes one or more insecticidal proteins. In some embodiments, the gene encoding the protein is expressed under the control of T3, T7, K11, or SP6. In some embodiments, the composition comprises 10 9 Contains at least 100 ng of protein per minicell.

[0008] Some embodiments relate to a composition comprising (a) a plurality of minicells derived from a plurality of parent bacterial cells that exhibit a reduced level or activity of at least one cell division topology-specificity factor, and (b) the plurality of minicells comprises at least 100 ng of protein per 10 minicells. In some embodiments, the cell division topology-specificity factor is selected from the group consisting of minC, minD, minE, and ftsZ. In some embodiments, the parent bacterial cell is further deficient in a gene involved in DNA repair. In some embodiments, the gene involved in DNA repair is selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD, and DNA polymerase III. In some embodiments, the gene involved in DNA repair is RecA. In some embodiments, the parent bacterial cell comprises one or more of T3, T7, K11, or SP6 polymerase, or a nucleic acid encoding a T3, T7, K11, or SP6 polymerase. In some embodiments, the parent bacterial cell comprises T7 polymerase or a nucleic acid encoding T7 polymerase. In some embodiments, the bacterial cell is E. coli DH10BΔminCDE. In some embodiments, the parent bacterial cell comprises an expression vector. In some embodiments, the expression vector encodes one or more insecticidal proteins (e.g., Pir, Cry, BT, etc.). In some embodiments, the gene encoding the protein is expressed under the control of T3, T7, K11, or SP6. In some embodiments, the composition is an agricultural composition. In some embodiments, the agricultural composition comprises one or more heterologous insecticides.

[0009] In some aspects, the disclosure includes a plurality of parent bacterial cells, wherein the parent bacterial cells (a) have a reduced level or activity of at least one cell division topology specificity factor; (b) are deficient in a gene involved in DNA repair; and the parent bacterial cells are at least 10 per liter of parent bacterial cell culture. 11In some embodiments, the gene involved in DNA repair is selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD, and DNA polymerase III. In some embodiments, the gene involved in DNA repair is RecA. In some embodiments, the bacterial cell is E. coli DH10BΔminCDE.

[0010] Some embodiments relate to a delivery vector and a composition containing a mixture of active ingredients for controlling pests. Methods for reducing overall damage, reducing insect populations, and reducing yield losses caused by insects are also disclosed. In many embodiments, the compositions and methods disclosed herein exhibit surprisingly good insecticidal activity. Thus, in some embodiments, the present disclosure enables the production of minicells containing effective concentrations of the insecticidal toxin Pir (e.g., PirAB) or minicells containing effective concentrations of the insecticidal toxin Cry (e.g., Cry1Ac). In many embodiments, compositions of Pir-containing minicells and Cry-containing minicells provide a synergistic effect for killing agricultural pests.

[0011] In other embodiments, the present disclosure allows for the production of minicells containing effective concentrations of the insecticidal toxin Pir (e.g., PirAB). The present disclosure further allows for the manufacture of compositions containing these minicells and a Bt-derived active ingredient (e.g., Thuricide®). In many embodiments, compositions of Pir-containing minicells and a Bt-derived active ingredient provide a synergistic effect for killing agricultural pests.

[0012] Some embodiments relate to a composition comprising a liquid carrier phase and a plurality of minicells dispersed in the carrier phase, the plurality of minicells comprising a mixture of first minicells comprising a first effective amount of a first exogenous protein toxin comprising Pir and second minicells comprising a second effective amount of a second exogenous protein toxin comprising Cry, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In further embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on controlling the at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling the at least one pest when the first effective amount or the second effective amount is administered to the pest. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the first particle concentration of the first minicells is about 1 x 10 2 ~Approx. 8×10 14 and the second particle concentration in the second minicell is in the range of about 1×10 2 ~Approx. 8×10 14 In some embodiments of this aspect that may be combined with any of the preceding embodiments, administering the composition to the pest includes an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the pest with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds on, or infests; or applying the composition to the plant. In further embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the composition is formulated as at least one of a ready-to-use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, and a drench application.

[0013] Some embodiments relate to a composition comprising: a liquid carrier phase; a plurality of minicells dispersed in the carrier phase (wherein the plurality of minicells comprises a first effective amount of an exogenous protein toxin comprising Pir); and a second effective amount of a Bt-derived active ingredient ("AI"), wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In further embodiments of this aspect that can be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on control of the at least one pest when either the first effective amount or the second effective amount is administered to the pest. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the particle concentration of the minicells is about 1×10 2 ~Approx. 8×10 14In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least one of Bacillus thuringiensis, a fermentation solid thereof, a spore thereof, and an insecticidal toxin thereof. In still further embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, a fermentation solid thereof, a spore thereof, and an insecticidal toxin thereof. In some embodiments of this aspect that may be combined with any of the preceding embodiments, administering the composition to the pest includes an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the pest with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds, or infests; or applying the composition to a plant. In further embodiments of this aspect, the composition is applied to plants as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the composition is formulated as at least one of a Ready To Use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, a drench application, a dust, an emulsifiable concentrate, a flowable concentrate, an intermediate formulation, a granule formulation, an impregnated material formulation, a pellet / tablet formulation, a soluble concentrate formulation, an industrial chemical formulation, a water-dispersible granule formulation, a water-soluble package formulation, or a wettable powder formulation.

[0014] In some embodiments that may be combined with any of the preceding embodiments, the first exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group consisting of PirA selected from the group consisting of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, or SEQ ID NO:203; and SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:203. In an additional embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 201, or SEQ ID NO: 204, or a functional component of any of these. In an additional embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 203 and a PirB comprising SEQ ID NO: 204, or the PirAB comprises a PirA comprising SEQ ID NO: 197 and a PirB comprising SEQ ID NO: 178.

[0015] In some embodiments that may be combined with any of the preceding embodiments, the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, or Cry1K, or a functional component thereof. In further embodiments of this aspect, Cry1A comprises a Cry1A selected from the group of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152, or SEQ ID NO:205; Cry1B comprises a Cry1B selected from the group of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, or SEQ ID NO:155; Cry1C comprises a Cry1C selected from the group of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, or SEQ ID NO:156; and Cry1D comprises a Cry1D selected from the group of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175, or SEQ ID NO:180. In an additional embodiment of this aspect, the second exogenous protein toxin comprises a Cry1D selected from the group of SEQ ID NO: 176; Cry1E comprises a Cry1E selected from the group of SEQ ID NO: 128 or SEQ ID NO: 129; Cry1F comprises a Cry1F selected from the group of SEQ ID NO: 130 or SEQ ID NO: 131; Cry1G comprises a Cry1G selected from the group of SEQ ID NO: 132 or SEQ ID NO: 133; Cry1H comprises a Cry1H selected from the group of SEQ ID NO: 134 or SEQ ID NO: 135; Cry1I comprises a Cry1I selected from the group of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139; Cry1J comprises a Cry1J selected from the group of SEQ ID NO: 140 or SEQ ID NO: 141; or Cry1K comprises SEQ ID NO: 142, or any functional component thereof. In an additional embodiment of this aspect, the second exogenous protein toxin comprises a Cry1A comprising SEQ ID NO: 205.

[0016] In some embodiments of the second aspect that may be combined with any of the preceding embodiments, the exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; and SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. 3, SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58, or a functional component of any of these. In additional embodiments of this aspect, PirAB comprises PirA comprising SEQ ID NO: 4 and PirB comprising SEQ ID NO: 5, or PirAB comprises PirA comprising SEQ ID NO: 6 and PirB comprising SEQ ID NO: 7. In still further embodiments of this aspect, PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,and linked to PirB by a linker sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI is selected from the group consisting of Thuricide®, Thuricide® HPC-ORTU, Thuricide® HPC, Thuricide® 48LV Forestry, Thuricide® 76B, Thuricide® 76LV, Bonide Bacillus Thuringiensis (BT) moth larva (caterpillar) control, DiPel®, Security BT powder biological insecticide, Fertilome DiPel powder biological insecticide, Widestrike®, botanical pesticide BT subsp. tenebrionis Colorado potato beetle protein, SA-50 brand DiPel® dust, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 dust for vegetables, Green Light® DiPel® dust, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granule, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits®,BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L Worm Killer, TERRO® Larvicide, VectoMax® FG Biological Larvicide Granules, Chemsico Insect Granules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Microparticles, VectoBac® FG+ Biological Larvicide Microparticles, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE!® G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar® Bti CRG, VBC-60092 (Bactimos Briquette), Summit BtiBRIQUETS, Dunks, Sympatico Biological Insecticide, XenTari® AS Biological Insecticide Aqueous Suspension, B402 Biological Larvicide, VectoBac® 12AS Biological Larvicide Aqueous Suspension, VectoBac® SC Biological Larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F Flowable Biological Insecticide Concentrate, Foray 76B, Safer BT Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. Israelensis Slurry, Bacillus thuringiensis subsp. christaqui subsp. Kurstaki) slurry, Trident® biological insecticide, VBC-60219 biological insecticide slurry,Javelin, Bacillus thuringiensis subspecies Aizawai subsp. aizawai) Slurry, FlorBac® Slurry Biological Insecticide, DiPel® ES, DiPel® ES-NT Biological Insecticide Emulsifier, DiPel® (Worm Killer) Wettable Powder Biological Insecticide, Condor, Leap® ES Bacterial Disease Control Biological Insecticide Emulsifier, Biobit® HPWPII Biological Insecticide, VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wettable Powder, BioBit® HP Biological Insecticide Wettable Powder, DiPel® WDG Biological Insecticide, VBC -60236 Biological Insecticide Dry Flowable, Foray2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, ForayWG, ThyllombeetleGONE!® Biological Insecticide, JavelinWP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, JavelinWG, SAN420IWG, JavelinWG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW,The biological control formulation may be selected from the group consisting of Bonide Bacillus thuringiensis 'O', Chemsico HPC-O Concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide® Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, or BTI Technical Powder BIOInsecticide.

[0017] In some embodiments that may be combined with any of the preceding embodiments, the first minicell comprises a first vector comprising a coding sequence for a first exogenous protein toxin. In further embodiments of this aspect, the first vector comprises a CloDF origin of replication or a pMB1 origin of replication. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a first vector, the coding sequence for the first exogenous protein toxin is operably linked to a first promoter. In other embodiments of this aspect, the first promoter is an inducible promoter or a constitutive promoter. In some embodiments of this aspect, the inducible promoter comprises Ptac, Ptet, or PT7. In some embodiments of this aspect, the constitutive promoter comprises J23119 (SEQ ID NO: 200). In some embodiments, the promoter is selected from T3, T7, K11, or SP6. In an additional embodiment of this aspect that may be combined with any of the preceding embodiments having a first vector, the first exogenous protein toxin comprises PirAB, which PirAB comprises PirA comprising SEQ ID NO: 203 and PirB comprising SEQ ID NO: 204, wherein the coding sequence for PirA comprises SEQ ID NO: 102, and the coding sequence for PirB comprises SEQ ID NO: 103. In a further embodiment of this aspect that may be combined with any of the preceding embodiments having a first vector and an inducible promoter, expression of the first exogenous protein toxin is induced with aTc or IPTG.

[0018] In further embodiments of the second aspect that may be combined with any of the preceding embodiments, the plurality of minicells comprises a vector comprising a coding sequence for an exogenous protein toxin. In further embodiments of this aspect, the vector comprises a pMB1 origin of replication. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a vector, the coding sequence for the exogenous protein toxin is operably linked to a promoter. In other embodiments of this aspect, the promoter is an inducible promoter or a constitutive promoter. In some embodiments of this aspect, the inducible promoter comprises Ptet. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a vector, the exogenous protein toxin comprises PirAB, wherein PirAB comprises PirA comprising SEQ ID NO:4 and PirB comprising SEQ ID NO:5, wherein the coding sequence for PirA comprises SEQ ID NO:2, and the coding sequence for PirB comprises SEQ ID NO:3. In further embodiments of this aspect that may be combined with any of the preceding embodiments having a vector and an inducible promoter, expression of the exogenous protein toxin is induced with aTc.

[0019] In further embodiments that may be combined with any of the preceding embodiments, the second minicell comprises a second vector comprising a coding sequence for a second exogenous protein toxin. In further embodiments of this aspect, the second vector comprises a CloDF origin of replication or a pMB1 origin of replication. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a second vector, the coding sequence for the second exogenous protein toxin is operably linked to a second promoter. In other embodiments of this aspect, the second promoter is an inducible promoter or a constitutive promoter. In some embodiments of this aspect, the inducible promoter comprises Ptac, Ptet, or PT7. In some embodiments of this aspect, the constitutive promoter comprises J23119 (SEQ ID NO: 200). In some embodiments, the promoter is selected from T3, T7, K11, or SP6. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a second vector, the second exogenous protein toxin comprises Cry1A, wherein the Cry1A comprises SEQ ID NO: 205, and the coding sequence for Cry1A comprises SEQ ID NO: 104. In further embodiments of this aspect that may be combined with any of the preceding embodiments having a second vector and an inducible promoter, expression of the second exogenous protein toxin is induced with aTc or IPTG.

[0020] In additional embodiments of all of the foregoing aspects that can be combined with any of the foregoing embodiments involving pest control, the control includes at least one of a reduction in pest numbers, a reduction in pest weight, a reduction in physical damage caused by pests, and an increase in pest mortality. In further embodiments of this aspect, the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction, and the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction. wherein the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction, and the increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase. In some embodiments of this aspect that may be combined with any of the preceding embodiments, control includes observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0021] In all further embodiments of the foregoing aspect that may be combined with any of the foregoing embodiments, the composition further comprises an agrochemical surfactant, which improves at least one of sprayability, spreadability, and pourability properties. In additional embodiments of this aspect that may be combined with any of the foregoing embodiments, the liquid carrier phase is aqueous or oil-based. In some embodiments of this aspect that may be combined with any of the foregoing embodiments involving pest control, the at least one pest is selected from the group consisting of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species.

[0022] A further aspect of the present disclosure relates to a composition comprising a liquid carrier phase and a plurality of minicells dispersed in the carrier phase, the plurality of minicells comprising minicells comprising a first effective amount of a first exogenous protein toxin comprising Pir and a second effective amount of a second exogenous protein toxin comprising Cry1, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In some embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the combined first effective amount and second effective amount have a synergistic effect on control of at least one pest that exceeds the effect of the first effective amount alone and the second effective amount alone on control of the at least one pest when the composition is administered to the pest.

[0023] A further aspect of the present disclosure relates to a composition comprising a liquid carrier phase and a plurality of minicells dispersed in the carrier phase, the plurality of minicells comprising minicells including a first effective amount of an exogenous protein toxin including a Pir and a second effective amount of a Bt-derived AI, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, when the composition is administered to a pest, the combined first effective amount and second effective amount have a synergistic effect on control of at least one pest that exceeds the effect of the first effective amount alone and the second effective amount alone on control of the at least one pest when the composition is administered to the pest.

[0024] A further aspect of the present disclosure includes a method for controlling pests, comprising administering to the pests the composition of any one of the preceding embodiments. In some embodiments of this aspect, administering the composition to the pests includes an administration method selected from the group consisting of delivering the composition to the pests for ingestion; contacting the pests with the composition; delivering the composition to at least one habitat where the pests grow, thrive, reproduce, feed, or infest; or applying the composition to the plant. In other embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment. In further embodiments of this aspect that may be combined with any of the preceding embodiments, the composition is formulated as at least one of a ready-to-use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, a drench treatment, a dust, an emulsifiable concentrate, a flowable concentrate, an intermediate formulation, a granule formulation, an impregnated material formulation, a pellet / tablet formulation, a soluble concentrate formulation, an industrial chemical formulation, a water-dispersible granule formulation, a water-soluble packaged formulation, or a wettable powder formulation. In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the control comprises at least one of a reduction in pest numbers, a reduction in pest weight, a reduction in physical damage caused by the pests, and an increase in pest mortality.In further embodiments of this aspect, the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; and the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction. wherein the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction, and the increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase. In some embodiments of this aspect that may be combined with any of the preceding embodiments, control includes observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the at least one pest is selected from the group of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species.

[0025] Yet another aspect of the present disclosure includes a wettable powder comprising a plurality of dry minicells comprising a mixture of a first effective amount of first minicells comprising a first exogenous protein toxin comprising a Pir and a second effective amount of second minicells comprising a second exogenous protein toxin comprising a Cry, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50, and the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to an insect. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In other embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of the at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. In further embodiments of this aspect that may be combined with any of the preceding embodiments, the first particle concentration of the first minicells is about 1 x 10 2 ~Approx. 8×10 14 and the second particle concentration in the second minicell is in the range of about 1×10 2 ~Approx. 8×10 14In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the hydratable powder further comprises a pesticidally acceptable solid carrier component comprising at least one of a clay component, a kaolin component, a talc component, a chalk component, a calcite component, a quartz component, a pumice component, a diatomaceous earth component, a vermiculite component, a silicate component, a silicon dioxide component, a silica powder component, an aluminum component, an ammonium sulfate component, an ammonium phosphate component, a calcium carbonate component, a urea component, a sugar component, a starch component, a sawdust component, a ground coconut shell component, a ground corn cob component, and a ground tobacco stalk component. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the aqueous carrier comprises water. In further embodiments of this aspect that may be combined with any of the preceding embodiments, administering the composition to the pest includes an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the insect with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds, or infests; and applying the composition to a plant. In some embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, or a post-emergence treatment.

[0026] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the first exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, or SEQ ID NO:203; and SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO: In another embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 201, or SEQ ID NO: 204, or a functional component of any of these. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 203 and a PirB comprising SEQ ID NO: 204, or the PirAB comprises a PirA comprising SEQ ID NO: 197 and a PirB comprising SEQ ID NO: 178.

[0027] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, or Cry1K, or a functional component thereof. In further embodiments of this aspect, Cry1A comprises a Cry1A selected from the group of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152, or SEQ ID NO:205; Cry1B comprises a Cry1B selected from the group of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, or SEQ ID NO:155; Cry1C comprises a Cry1C selected from the group of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, or SEQ ID NO:156; and Cry1D comprises a Cry1D selected from the group of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175, or SEQ ID NO:180. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1D selected from the group of SEQ ID NO: 176; Cry1E comprises a Cry1E selected from the group of SEQ ID NO: 128 or SEQ ID NO: 129; Cry1F comprises a Cry1F selected from the group of SEQ ID NO: 130 or SEQ ID NO: 131; Cry1G comprises a Cry1G selected from the group of SEQ ID NO: 132 or SEQ ID NO: 133; Cry1H comprises a Cry1H selected from the group of SEQ ID NO: 134 or SEQ ID NO: 135; Cry1I comprises a Cry1I selected from the group of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139; Cry1J comprises a Cry1J selected from the group of SEQ ID NO: 140 or SEQ ID NO: 141; or Cry1K comprises SEQ ID NO: 142, or any functional component thereof. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1A comprising SEQ ID NO: 205.

[0028] Yet another aspect of the present disclosure includes a wettable powder comprising a plurality of dry minicells comprising a mixture of a first effective amount of minicells comprising an exogenous protein toxin including Pir and a second effective amount of an AI derived from Bt, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50, and the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to an insect. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In other embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of the at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. In further embodiments of this aspect that may be combined with any of the preceding embodiments, the particle concentration of minicells is about 1 x 10 2 ~Approx. 8×10 1In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least one of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxin. In yet further embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxin. In additional embodiments of this aspect that can be combined with any of the preceding embodiments, the wettable powder further comprises a pesticidally acceptable solid carrier component comprising at least one of a clay component, a kaolin component, a talc component, a chalk component, a calcite component, a quartz component, a pumice component, a diatomaceous earth component, a vermiculite component, a silicate component, a silicon dioxide component, a silica powder component, an aluminum component, an ammonium sulfate component, an ammonium phosphate component, a calcium carbonate component, a urea component, a sugar component, a starch component, a sawdust component, a ground coconut shell component, a ground corn cob component, and a ground tobacco stalk component. In some embodiments of this aspect that can be combined with any of the preceding embodiments, the aqueous carrier comprises water. In further embodiments of this aspect that can be combined with any of the preceding embodiments, administering the composition to the pest comprises an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the insect with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds, or infests; and applying the composition to a plant. In some embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, or a post-emergence treatment.

[0029] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; and SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO:4 and a PirB comprising SEQ ID NO:5, or a PirAB comprising a PirA comprising SEQ ID NO:6 and a PirB comprising SEQ ID NO:7. In another embodiment of this aspect, PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,and linked to PirB by a linker sequence selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI is selected from the group consisting of Thuricide®, Thuricide® HPC-ORTU, Thuricide® HPC, Thuricide® 48LV Forestry, Thuricide® 76B, Thuricide® 76LV, Bonide Bacillus Thuringiensis (BT) moth larva (caterpillar) control, DiPel®, Security BT powder biological insecticide, Fertilome DiPel powder biological insecticide, Widestrike®, botanical pesticide BT subsp. tenebrionis Colorado potato beetle protein, SA-50 brand DiPel® powder, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 powder for vegetables, Green Light® DiPel® powder, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granule, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits®,BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L Worm Killer, TERRO® Larvicide, VectoMax® FG Biological Larvicide Granules, Chemsico Insect Granules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Microparticles, VectoBac® FG+ Biological Larvicide Microparticles, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE!® G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar® Bti CRG, VBC-60092 (Bactimos Briquette, SummitB.tiBRIQUETS, Dunks, Sympatico Biological Insecticide, XenTari® AS Biological Insecticide Aqueous Suspension, B402 Biological Larvicide, VectoBac® 12AS Biological Larvicide Aqueous Suspension, VectoBac® SC Biological Larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F Flowable Biological Insecticide Concentrate, Foray 76B, Safer BT Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. Israelensis Slurry, Bacillus thuringiensis subsp. christaqui subsp. Kurstaki) slurry, Trident® biological insecticide, VBC-60219 biological insecticide slurry,Javelin, Bacillus thuringiensis subspecies Aizawai subsp. aizawai) Slurry, FlorBac® Slurry Biological Insecticide, DiPel® ES, DiPel® ES-NT Biological Insecticide Emulsifier, DiPel® (Worm Killer) Wettable Powder Biological Insecticide, Condor, Leap® ES Bacterial Disease Control Biological Insecticide Emulsifier, Biobit® HPWPII Biological Insecticide, VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wettable Powder, BioBit® HP Biological Insecticide Wettable Powder, DiPel® WDG Biological Insecticide, VBC -60236 Biological Insecticide Dry Flowable, Foray2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, ForayWG, ThyllombeetleGONE!® Biological Insecticide, JavelinWP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, JavelinWG, SAN420IWG, JavelinWG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW,The biological control formulation may be selected from the group consisting of Bonide Bacillus thuringiensis 'O', Chemsico HPC-O Concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide® Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, or BTI Technical Powder BIOInsecticide.

[0030] In additional embodiments of either of the two preceding aspects that can be combined with any of the preceding embodiments, the control includes at least one of a reduction in pest numbers, a reduction in pest weight, a reduction in physical damage caused by the pests, and an increase in pest mortality. In further embodiments of this aspect, the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction, and the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction. wherein the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction, and the increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase. In some embodiments of this aspect that may be combined with any of the preceding embodiments, control includes observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the at least one pest is selected from the group of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species.

[0031] Yet another aspect of the present disclosure includes a method of making a wettable powder comprising the steps of: a) providing a first effective amount of first dry minicells comprising a first exogenous protein toxin comprising Pir, b) a second effective amount of second dry minicells comprising a second exogenous protein toxin comprising Cry, and c) mixing the first effective amount and the second effective amount in a ratio ranging from about 50:1 to 1:50, wherein the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to the pest. In some embodiments of this aspect, the first effective amount and the second effective amount are mixed in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1.

[0032] Yet another aspect of the present disclosure includes a method of making a wettable powder, comprising the steps of: a) providing a first effective amount of first dried minicells comprising an exogenous protein toxin comprising Pir; b) a second effective amount of a dried Bt-derived AI; and c) mixing the first effective amount and the second effective amount in a ratio ranging from about 50:1 to 1:50, wherein the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to the pest. In some embodiments of this aspect, the first effective amount and the second effective amount are mixed in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1.

[0033] A further aspect of the present disclosure is a plantable composition comprising a seed and a coating overlying the seed, the coating comprising a plurality of minicells comprising a mixture of a first effective amount of first minicells comprising a first exogenous protein toxin comprising a Pir and a second effective amount of second minicells comprising a second exogenous protein toxin comprising a Cry, the first effective amount and the second effective amount being present in a ratio ranging from about 50:1 to 1:50, and the mixture comprising a composition sufficient to provide insecticidal activity against at least one pest that feeds on the seed or the seedlings produced therefrom. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In other embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on controlling the at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling the at least one pest when the first effective amount or the second effective amount is administered to the pest. In further embodiments of this aspect that may be combined with any of the preceding embodiments, the first particle concentration of the first minicells is about 1 x 10 2 ~Approx. 8×10 14 and the second particle concentration in the second minicell is in the range of about 1×10 2 ~Approx. 8×10 14 The range is.

[0034] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the first exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, or SEQ ID NO:203; and SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO: In another embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 201, or SEQ ID NO: 204, or a functional component of any of these. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 203 and a PirB comprising SEQ ID NO: 204, or the PirAB comprises a PirA comprising SEQ ID NO: 197 and a PirB comprising SEQ ID NO: 178.

[0035] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, or Cry1K, or a functional component thereof. In further embodiments of this aspect, Cry1A comprises a Cry1A selected from the group of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152, or SEQ ID NO:205; Cry1B comprises a Cry1B selected from the group of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, or SEQ ID NO:155; Cry1C comprises a Cry1C selected from the group of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, or SEQ ID NO:156; Cry1D comprises a Cry1D selected from the group of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175, or SEQ ID NO:180. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1D selected from the group of SEQ ID NO: 176; Cry1E comprises a Cry1E selected from the group of SEQ ID NO: 128 or SEQ ID NO: 129; Cry1F comprises a Cry1F selected from the group of SEQ ID NO: 130 or SEQ ID NO: 131; Cry1G comprises a Cry1G selected from the group of SEQ ID NO: 132 or SEQ ID NO: 133; Cry1H comprises a Cry1H selected from the group of SEQ ID NO: 134 or SEQ ID NO: 135; Cry1I comprises a Cry1I selected from the group of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139; Cry1J comprises a Cry1J selected from the group of SEQ ID NO: 140 or SEQ ID NO: 141; or Cry1K comprises SEQ ID NO: 142, or any functional component thereof. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1A comprising SEQ ID NO: 205.

[0036] A further aspect of the present disclosure is a plantable composition comprising a seed and a coating overlying the seed, the coating comprising a plurality of minicells comprising a mixture of a first effective amount of minicells comprising an exogenous protein toxin including Pir and a second effective amount of a Bt-derived AI, the first effective amount and the second effective amount being present in a ratio ranging from about 50:1 to 1:50, and the mixture comprising a composition sufficient to provide insecticidal activity against at least one pest that feeds on the seed or the resulting seedlings. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In other embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on controlling the at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling the at least one pest when the first effective amount or the second effective amount is administered to the pest. In further embodiments of this aspect that may be combined with any of the preceding embodiments, the particle concentration of minicells is about 1 x 10 2 ~Approx. 8×10 14 In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least one of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxin. In yet further embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxin.

[0037] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; and SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO:4 and a PirB comprising SEQ ID NO:5, or a PirAB comprising a PirA comprising SEQ ID NO:6 and a PirB comprising SEQ ID NO:7. In still further embodiments of this aspect, PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,and linked to PirB by a linker sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI is selected from the group consisting of Thuricide®, Thuricide® HPC-ORTU, Thuricide® HPC, Thuricide® 48LV Forestry, Thuricide® 76B, Thuricide® 76LV, Bonide Bacillus Thuringiensis (BT) moth larva (caterpillar) control, DiPel®, Security BT powder biological insecticide, Fertilome DiPel powder biological insecticide, Widestrike®, botanical pesticide BT subsp. tenebrionis Colorado potato beetle protein, SA-50 brand DiPel® dust, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 dust for vegetables, Green Light® DiPel® dust, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granule, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits®,BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L Worm Killer, TERRO® Larvicide, VectoMax® FG Biological Larvicide Granules, Chemsico Insect Granules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Microparticles, VectoBac® FG+ Biological Larvicide Microparticles, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE!® G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar® Bti CRG, VBC-60092 (Bactimos Briquette, SummitB.tiBRIQUETS, Dunks, Sympatico Biological Insecticide, XenTari® AS Biological Insecticide Aqueous Suspension, B402 Biological Larvicide, VectoBac® 12AS Biological Larvicide Aqueous Suspension, VectoBac® SC Biological Larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F Flowable Biological Insecticide Concentrate, Foray 76B, Safer BT Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. Israelensis Slurry, Bacillus thuringiensis subsp. christaqui subsp. Kurstaki) slurry, Trident® biological insecticide, VBC-60219 biological insecticide slurry,Javelin, Bacillus thuringiensis subspecies Aizawai subsp. aizawai) Slurry, FlorBac® Slurry Biological Insecticide, DiPel® ES, DiPel® ES-NT Biological Insecticide Emulsifier, DiPel® (Worm Killer) Wettable Powder Biological Insecticide, Condor, Leap® ES Bacterial Disease Control Biological Insecticide Emulsifier, Biobit® HPWPII Biological Insecticide, VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wettable Powder, BioBit® HP Biological Insecticide Wettable Powder, DiPel® WDG Biological Insecticide, VBC -60236 Biological Insecticide Dry Flowable, Foray2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, ForayWG, ThyllombeetleGONE!® Biological Insecticide, JavelinWP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, JavelinWG, SAN420IWG, JavelinWG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW,The biological control formulation may be selected from the group consisting of Bonide Bacillus thuringiensis 'O', Chemsico HPC-O Concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide® Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, or BTI Technical Powder BIOInsecticide.

[0038] In some embodiments of any of the preceding two aspects that can be combined with any of the preceding embodiments, the at least one pest is selected from the group of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species. In further embodiments of this aspect that may be combined with any of the preceding embodiments, the seeds are selected from the group consisting of alfalfa, almonds, apples, apricots, artichokes, asparagus, avocados, bananas, barley, beans, blueberries, cranberries, Brazil nuts, cacao, calamansi, canola, Polish canola, broccoli, kale, cabbage, turnips, carnations, carrots, cashews, cassava, celery, cherries, chestnuts, chickpeas, chicory, chili peppers, chrysanthemums, citrons, coconuts, coffee, cotton, cowpeas, broad beans, cucumbers, redcurrants, gooseberries, dates, duckweed, eggplant, elderberries, eucalyptus, flax, geraniums, ginger, ginseng, grapefruit, grapes, guavas, hazelnuts, hemp, cannabis, hops, horseradish, irises, jackfruit, and kiwi. Fruits, kumquats, lemons, lentils, lettuce, limes, litchis, macadamias, corn, mandarins, mangoes, mangosteens, melons, millet, oats, palm, okra, olives, onions, oranges, papayas, parsley, passion fruit, pecans, peaches, nectarines, pears, peas, peanuts, peonies, persimmons, petunias, pineapples, pistachios, plantains, plums, poinsettias , pomelo, poplar, potato, pumpkin, squash, quince, raspberry, rhubarb, rice, rose, rubber, rye, safflower, satsuma mandarin, sesame, sorghum, bitter orange, soursop, soybean, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tamarind, tangerine, tea, tobacco, tomatillo, tomato, tulip, walnut, watermelon, wheat, or yam.

[0039] These and other aspects of the invention are described in more detail in the description of the invention below. [Brief explanation of the drawings]

[0040] [Figure 1A-1B] 1A-1B show the size distribution of minicell particles and protein production by E. coli strain DH10BΔminCDE (FIG. 1A) and E. coli strain DH10B(DE3)ΔminCDE (FIG. 1B). [Figure 2] Figure 2 shows the results of an artificial diet bioassay demonstrating the efficacy against DBM of minicells containing PirAB protein produced by strain AD16 ("AD2") and strain AD17 ("AD3") (DH10B chassis), tested at the indicated PirAB concentrations. [Figure 3] Figure 3 shows the results of an artificial diet bioassay demonstrating the efficacy against DBM of minicells containing Cry1Ac protein produced by strain AD28 ("AD14"; DH10B chassis), tested at the indicated Cry1Ac concentrations. [Figure 4] Figure 4 shows the results of an artificial diet bioassay demonstrating the efficacy against DBM of minicells containing PirAB protein produced by the AD19 strain ("AD5"; (DH10B(DE3) chassis)), tested at the indicated PirAB concentrations. [Figures 5A-5C] Figures 5A-5C show greenhouse (GH) effects on DBM in cabbage with the toxins PirAB (AD18 = "AD4"; AD16 = "AD2") and Cry expressed in the optimized DH10BΔminCDE chassis. Figure 5A shows a comparison of the efficacy of minicells containing PirAB between chassis strains; Figure 5B shows the dose response of minicells containing PirAB in GH; Figure 5C shows the dose response of minicells containing Cry1Ac in GH. [Figure 6] FIG. 6 shows a translational field study of the efficacy of minicell PirAB and minicell Cry1Ac against DBM in cabbage. DETAILED DESCRIPTION OF THE INVENTION

[0041] The following description sets forth example methods, parameters, etc. However, it should be understood that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0042] Some embodiments relate to minicell-producing bacterial chassis strains that maximize minicell production yield and protein production yield. Maximizing the number of minicell particles and expression of the active ingredient provides an effective concentration of the active ingredient. This disclosure describes the creation of bacterial chassis that produce high yields of minicell particle production and protein expression, respectively. Chassis capabilities are demonstrated with several insecticidal toxins, such as PirAB, Cry1Ac, and Vip3Aa19.

[0043] Minicell-producing parent cells Minicells loaded with heterologous functional agents (e.g., insecticidal proteins) can be derived from bacterial parent cells, as described herein. In some aspects, compositions are provided comprising a plurality of minicells derived from parent bacteria having reduced levels, activity, or expression of a cell division topology-specific factor. In some aspects, compositions are provided comprising a plurality of minicells, where the minicells do not comprise a cell division topology-specific factor and the composition is substantially free of live bacterial cells. In some aspects, compositions comprising a plurality of minicells are provided, where the composition is substantially free of live bacterial cells and is produced by a process comprising: (a) producing, providing, or obtaining a plurality of parent bacteria having reduced levels or activity of a cell division topology-specific factor; (b) exposing the parent bacteria to conditions that allow for the formation of minicells, thereby producing highly active minicells; and (c) separating the minicells from the parent bacteria, thereby producing a composition that is substantially free of live bacterial cells. In some embodiments, the cell division topology-specific factor is a minE polypeptide. In some embodiments, the parent bacterium is Escherichia coli (E. coli) and the minE polypeptide is E. coli minE. In other embodiments, the parent bacterium is Salmonella typhimurium and the minE polypeptide is S. typhimurium minE. Examples of species that have minE polypeptides are described in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005, which is incorporated herein in its entirety. In some embodiments, the cell division topology specificity factor is a DivIVA polypeptide. Examples of species that have DivIVA polypeptides are described in Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005, which is incorporated herein in its entirety. In some embodiments, the parent bacterium is Bacillus subtilis and the cell division topology specificity factor is B. subtilis DivIVA.

[0044] In some embodiments, a minicell-producing parent bacterium having a reduced level or activity of a cell division topology specificity factor also has a reduced level of one or more Z-ring inhibitory proteins. In some embodiments, the Z-ring inhibitory protein is a minC polypeptide. In some embodiments, the Z-ring inhibitory protein is a minD polypeptide. In some embodiments, the Z-ring inhibitory protein is an E. coli minD polypeptide. In some embodiments, the ADAS or parent bacterium has a reduced level, activity, or expression of at least two Z-ring inhibitory proteins. In some embodiments, a minicell-producing parent bacterium has reduced expression of a minC polypeptide and a minD polypeptide. In some embodiments, a minicell-producing parent bacterium has reduced expression of a minC polypeptide, a minD polypeptide, and a minE polypeptide, e.g., a deletion of the minCDE operon (ΔminCDE). In some embodiments, a minicell-producing parent bacterium has a reduced level of the RNA polymerase sigma F factor (sigF). In some embodiments, sigF is involved in sporulation.

[0045] The reduction in the level, activity, or expression of the cell division topology specificity factor or Z ring inhibitory protein in the parent bacterial cell can be achieved using any suitable method. For example, in some embodiments, the reduction in level or activity is caused by a loss-of-function mutation, e.g., a gene deletion. In some embodiments, the loss-of-function mutation is an inducible loss-of-function mutation, and the loss-of-function mutation is induced by exposing the parent cell to an inducing condition, e.g., the inducible loss-of-function mutation is a temperature-sensitive mutation, and the inducing condition is a temperature condition.

[0046] In some embodiments, the parent cell has a deletion of the minCDE operon (ΔminCDE) or a homologous operon.

[0047] Increased minicell production due to deletion or mutation of genes involved in DNA repair In some embodiments, the parent bacterial cell has a deletion or mutation in a gene implicated in DNA repair. In some embodiments, the gene involved in DNA repair is selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD, and DNA polymerase III. In some embodiments, the gene involved in DNA repair is RecA, and the parent cell is E. coli DH10B. In some embodiments, minicell production is increased in bacterial cells with a deletion or mutation in a gene involved in DNA repair. In some embodiments, the parent bacterial cell is a Gram-positive bacterial cell. In some embodiments, the parent bacterial cell is Bacillus subtilis or a Bacillus species. In some embodiments, the parent bacterial cell is a Lactobacillus species. In some embodiments, the parent bacterial cell is a Gram-negative bacterial cell. In some embodiments, the parent bacterial cell is selected from the group consisting of Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, and the like. um), Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella,Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Sal Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio (V) ibrio), Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus,It is derived from bacteria of the genus Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium, or Thermoanaerobacter.

[0048] Mini-cell containing cargo In some embodiments, minicells produced by the parent bacteria described herein include cargo contained within the minicell. The cargo can be any moiety located within the minicell (e.g., encapsulated by the minicell) or attached to the surface of the minicell. In some embodiments, the cargo comprises a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immunomodulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP), or a combination thereof. In some aspects, the cargo is delivered by a secretion system (e.g., a T3SS). In other aspects, the cargo is not delivered by a T3SS.

[0049] In some embodiments, the cargo is a polypeptide. Polypeptides useful for agricultural applications include, for example, bacteriocins, ricin, antimicrobial peptides, nodule C-rich peptides, and bacteriostatic peptides. In some embodiments, the polypeptides can be used to alter the levels, activity, or metabolism of target microorganisms to increase the fitness of insects such as honeybees and silkworms. Examples of agriculturally useful polypeptides that can be provided as minicell cargo include peptide toxins, such as those naturally produced by insect pathogenic bacteria (e.g., Bacillus thuringiensis, Photorhabdus luminescens, Serratia entomophila, or Xenorhabdus nematophila).

[0013] Embodiments of agriculturally useful polypeptides that can be provided as minicell cargo include polypeptides (including small peptides such as cyclodipeptides or diketopiperazines) for controlling agriculturally important pests or pathogens, e.g., antibacterial or antifungal polypeptides for controlling plant diseases, or insecticidal polypeptides (e.g., insecticidal and / or nematicidal polypeptides) for controlling harmful invertebrates such as insects or nematodes.

[0014] Embodiments of agriculturally useful polypeptides that can be provided as minicell cargo include antibodies, nanobodies, and fragments thereof, e.g., fragments of antibodies or nanobodies that retain at least some (e.g., at least 10%) of the specific binding activity of the intact antibody or nanobody.

[0015] Embodiments of agriculturally useful polypeptides that can be provided as minicell cargo include transcription factors, e.g., plant transcription factors (see, e.g., the "AtTFDB" database (publicly available at agris-knowledgebase[dot]org / AtTFDB / ), which lists transcription factor families identified in the model plant Arabidopsis thaliana.Embodiments of agriculturally useful polypeptides that can be provided as minicell cargo include nucleases, e.g., exonucleases or endonucleases (e.g., Cas nucleases such as Cas9 or Cas12a). Embodiments of agriculturally useful polypeptides that can be provided as minicell cargo further include cell-penetrating peptides, enzymes (e.g., amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones (e.g., yeast mating pheromones, invertebrate reproductive pheromones, and larval signaling pheromones (see, e.g., Altstein (2004) Peptides, 25:1373-1376).

[0050] In some embodiments, the cargo is a nucleic acid. In some embodiments, the nucleic acid is DNA, RNA, or a plasmid. In some embodiments, the nucleic acid (e.g., DNA, RNA (e.g., mRNA, ASO, circular RNA, siRNA, shRNA, tRNA, dsRNA, or a combination thereof), or a plasmid) encodes a protein. In some embodiments, the protein is transcribed and / or translated in the minicell. In some embodiments, the nucleic acid inhibits translation of a protein or polypeptide, e.g., an siRNA or an antisense oligonucleotide (ASO).

[0051] In some embodiments, the cargo is a peptide. In some embodiments, the peptide is expressed from a vector comprising a peptide coding sequence operably linked to a promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the peptide is expressed from a vector comprising a peptide coding sequence operably linked to a Ptac, Ptet, or PT7 promoter. In some embodiments, the peptide is expressed from a vector comprising a peptide coding sequence operably linked to a T7, Ptac, or Ptet promoter. In some embodiments, the peptide is expressed from a vector comprising a peptide coding sequence operably linked to a T7, Ptac, or Ptet promoter. In some embodiments, the peptide is a cathelicidin (e.g., LL-37 (PDB:4EYC), fauricidin-1 (PDB:2AMN), CAP18 (PDB:1LYP), porcine protoglin (PDB:1KWI), SMAP29 (PDB:1FRY)), cecropin (e.g., papiliosin (PDB:2LA2), cecropin A, cecropin B (PDB:2IGR), CECD (PDB:2M MM), cecropin P1), defensin-beta (e.g., HBD-1 (PDB:1E4S), HBD-2 (PDB:1E4Q), AvBD7 (PDB:5LCS), MBD-7 (PDB:1E4T), MBD-8 (PDB:1E4R)), mammalian defensins (e.g., HNP1 (2PM5), HD5 (PDB:1ZMP), Crp4 (2GWP)), frog AMPs (e.g., gegrin- 4 (PDB:2G9L), palustrin-Ca (PDB:7P4X), ranturin-2CSA (PDB:2K10), esculentin, gaegulin, brevnin, rugosin, ranturin, alpha / beta enterocins, ant antimicrobial peptides, ponericin, aphid megourin, apidaecin, apolipoprotein A-II, attacin, aurein-like, bactericidal permeability-increasing protein (BPI) 1, bactericidal permeability-increasing protein (BPI) 2, bacteriocin class II with double glycine-type leader peptide, bacteriocin class IIc cyclic gassericin A-like, bacteriocin class IId cyclic uberolysin-like, bacteriocin, heterocyclic anthracin, sonorensin, wasp antimicrobial peptides, abaecin, bombinin, caerin 1 protein,CAP18 C-terminal, Class II bacteriocin, clavaninin, choreoptericin, cupienin, culotide, cystatin, cystatin-like domain, defensin-actinodefensin, defensin-alpha, defensin-beta136, defensin-beta2, defensin-beta50, defensin-big, defensin-fungal copsin, defensin-plant thionin, defensin-scorpion-like, defensin-like domain BmBKTx1-like, deltalysin, dermaseptin, delmicidin, DUF325 4, formaesin, frog AMP class 22, frog AMP class 24, frog AMP propeptide, frog AMP: brevinin-1, esculentin-2, temporin, ranaturin-2, gallidermin, gibberellin-regulated protein, gloverin, GPR15L-like AMP, granulin, halocidin, lactocin 705, lactococcin 972, lactococcin G-beta, lactococcin G-beta, enterocin 1071B family bacteriocins, latercin, laterosporin defensin-like peptide, LCI, Bacillus cereus (Bacillus cereus group AMPs, lipid transfer protein, liver-expressed antimicrobial peptide 2, macin, mastoparan, mastoparan 2, melegulin, signin family, melittin, MiAMP1, microcin-V, colicin V, moricin, myotoxin, crotamine, myticin preprotein, nematode AMP25, ocerulatin, pardaxin, pelovaterin, penaidin, pyroslin, plant AMP21, plant thionin, Platypus intermediate defensin-like peptide, pleurocidin, ponericin L, potassium channel toxin, schujin ( Pseudin), lanacycline, saposin type A domain, saposin type B domain, saposin-like type B, region 1, saposin-like type B, region 2, scorpion antimicrobial peptide, ShK domain-like, spider antimicrobial peptide, oxyopinin, stomoxin, stierin, sublancin, subtilosin A, tachystatin A, tachystatin B, type A lantibiotic, uperin, vicilin N-terminal region / MiAMP2, whey acid protein (WAP type) 4-disulfide core, peptide domain, antifungal peptide (e.g., PAFP-S (PDB: 1DKC),ALO3 (1Q3J)), antifungal proteins (e.g., PAF (PDB:2KCN), NFAP (PDB:5OQS), PAFB (PDB:7BAD), AFP (PDB:AFP1)), defensin antifungal-like (e.g., Egk (PDB:7C2P), DEF4 (PDB:2LR3), VvK1 (PDB:7C31), OsAFP1 (6LCQ), PsDef1 (5NCE)), protease inhibitors, seed storage proteins, lipid transfer proteins (e.g., Lentivirus, lipid transfer protein (5LQV), rice nsLTP2 (1L6H), pea lipid transfer protein (2N81), maize amylase inhibitor (1BEA), thermicins (e.g., Pseudacanthotermes thermicin (PDB:1MM0), Nasutitermes thermicin, Tumulitermes thermicin), Berwin family, basal layer antifungal peptides (BAP), beta / gamma crystallin, chitin recognition protein, defensin-plant gamma thionin, diapausin, DUF1962, hepcidin, hevein-like AMP, late nodulin protein, metschnikowin, thaumatin family, agatoxin, protoxin, ctenitoxin, spider neurotoxins (e.g., omega-agatoxin-IVA (PDB:2NDB), omega-agatoxin-IVB (PDB:1AGG), delta-partoxin IT1 (P DB:1V90)), cecropins (e.g., papiliocin (PDB:2LA2), cecropin A, cecropin B (PDB:2IGR), CECD (PDB:2MMM), cecropin P1), defensin-like antifungals (e.g., Egk (PDB:7C2P), DEF4 (PDB:2LR3), VvK1 (PDB:7C31), OsAFP1 (6LCQ), PsDef1 (5NCE)), fungal fruiting body lectins (e.g., Sclerotium rolfsii (Sclerotium rolfsii lectin (PDB:2OFC)), common edible mushroom lectin (PDB:1Y2T), Boletus edulis lectin (PDB:3QDS)), snake nicotinic acetylcholine receptor toxins (e.g., linguin (PDB:4ZQY), MT7: (PDB:3FEV), alpha-bungarotoxin (PDB:1KFH),Atratoxin-b (PDB:1ONJ), bulb-type mannose-specific lectin, conotoxin, cyclotide, D-mannose-binding lectin, Huwentoxin II family, I2-superfamily conotoxin, insecticidal crystal toxin, P42, Janus-atracotoxin, Magi 5 toxic peptide family, Magi peptide toxic family, MIT-like atracotoxin family, omega-atracotoxin, PhTx neurotoxin family, ponericin, Ptu family, putidacin L1 lectin-like bacteriocin, scorpion toxin-like, short spider neurotoxin, spider insecticidal peptide, spider calcium channel neurotoxin, Cry1, Cry2, Cry3, Cry4, Cyt, Cry16A, Cry17A, Crycbm17. 1, Crycbm17.2, ISP1A, ISP2A, Mcf, MrxA, PirA, PirB, PirA, PirB, SepA, SepB, SepC, Sip, Tc complex A, TccomplexB, Tc complex C, Tc cC, txp40, Vip1, Vip2, Vip3, Vip3Aa, Vip3Aa14, XaxA, XaxB, XptA1, XptB1, XptC1, YenA1, YenA2, YenB, YenC1 or YenC2. ,

[0052] In some embodiments, the cargo is an agent capable of modulating the microbiome of a target organism (e.g., the microbiome of a human, animal, plant, or insect), such as a polysaccharide, an amino acid, an antimicrobial agent (e.g., an anti-infective or antibacterial peptide, protein, and / or natural product), a short-chain fatty acid, or a combination thereof. In some examples, the agent capable of modulating the host microbiome is a probiotic agent.

[0053] In some embodiments, the cargo is an enzyme. The enzyme can be an enzyme that performs a catalytic activity in a target cell or organism (e.g., human, animal, plant, or insect). In some embodiments, the catalytic activity is extracellular matrix (ECM) digestion (e.g., the enzyme is hyaluronidase and the catalytic activity is ECM digestion) or toxin removal. In some embodiments, the enzyme is an enzyme replacement therapy, e.g., phenylalanine hydroxylase. In some embodiments, the enzyme is UDP-glucuronosyltransferase. In some embodiments, the enzyme has hepatic enzyme activity (e.g., porphobilinogen deaminase (PBGD), e.g., human PBGD (hPBGD)). In some embodiments, the enzyme is a protease, oxidoreductase, or a combination thereof. In some embodiments, the enzyme alters a substrate to generate a target product. In some embodiments, the substrate is present in the minicell and the target product is produced in the minicell. In other embodiments, the substrate is present in the target cell or the environment to which the minicell is delivered.

[0054] In certain embodiments, the cargo is modified to improve stability compared to an unmodified version of the cargo. The "stability" of a cargo is the unitless ratio of the half-life of the unmodified cargo to the half-life of the modified cargo when measured under the same environmental conditions. In some embodiments, the environment, e.g., simulated body fluid, RNAse-free water, cytoplasm, extracellular space, or "minicelloplasm" (i.e., the contents of the interior volume of the minicell, e.g., after lysis), is experimentally controlled. In some applications, it is an agricultural environment, e.g., variable soil, river water, or seawater. In other embodiments, the environment is a real environment or a simulated environment, i.e., an animal's intestine, animal skin, animal reproductive tract, animal respiratory tract, animal bloodstream, or animal extracellular space. In certain embodiments, the minicell does not substantially degrade the cargo.

[0055] In certain embodiments, the cargo comprises a protein. In certain embodiments, the protein has a stability in the cytoplasm or other environment of greater than about 1.01, 1.1, 10, 100, 1000, 10,000, 100,000, 100,000, or 10,000,000. The protein can be any protein, including growth factors; enzymes; hormones; immunomodulatory proteins; antimicrobial proteins, such as antibacterial, antifungal, or insecticidal proteins; targeting agents, such as antibodies or nanobodies; and the like. In some embodiments, the protein is a hormone, e.g., a paracrine, endocrine, or autocrine protein.

[0056] In some embodiments, the cargo comprises a plant hormone such as abscisic acid, auxin, cytokinin, ethylene, gibberellin, or a combination thereof.

[0057] In some embodiments, the cargo is an anti-inflammatory agent, such as a cytokine (e.g., a heterologously expressed anti-inflammatory cytokine or mutein thereof (e.g., IL-10, TGF-beta, IL-22, IL-2) or an antibody (e.g., an antibody or antibody fragment that targets tumor necrosis factor (TNF)) (e.g., an anti-TNF antibody); an antibody or antibody fragment that targets IL-12 (e.g., an anti-IL-12 antibody); or an antibody or antibody fragment that targets IL-23 (e.g., an anti-IL-23 antibody).

[0058] In certain embodiments, the cargo is an immunomodulatory agent. Immunomodulatory agents include, for example, immunostimulatory agents; checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4); chemotherapeutic agents; immunosuppressants; antigens; superantigens; and small molecules (e.g., cyclosporin A, cyclic dinucleotides (CDNs), or STING agonists (e.g., MK-1454)). In some embodiments, the immunomodulatory agent is a moiety that induces tolerance in a subject, such as an allergen, an autoantigen (e.g., a disease-associated autoantigen), or a microorganism-specific antigen. In some embodiments, the immunomodulatory agent is a vaccine, such as an antigen derived from a pathogen (e.g., a virus (e.g., a viral envelope protein) or a bacterium). In some embodiments, the antigen is a cancer neoantigen. In some embodiments, the pathogen is a coronavirus, such as SARS-CoV-2. In some embodiments, the cargo is an adjuvant, such as an immunomodulatory molecule, or a molecule that alters the compartmentalization, presentation, or profile of one or more costimulatory molecules associated with a vaccine antigen. In some examples, the adjuvant is an activator of an immune pathway upstream of the desired immune response (e.g., an activator of an innate immune pathway upstream of the adaptive immune response). In other examples, the adjuvant enhances presentation of the antigen on immune cells or immune moieties (e.g., MHC class 1) in the target organism. In some examples, the adjuvant is listeriolysin O (LLO). In some embodiments, the minicell comprises an antigen and one or more adjuvants.

[0059] In some embodiments, the cargo is a drug for the treatment or prevention of cancer, e.g., a drug that reduces the likelihood that a patient will develop cancer, or a drug that treats cancer (e.g., a drug that increases progression-free survival and / or overall survival in individuals with cancer). Drugs for the prevention of cancer include, but are not limited to, anti-inflammatory agents and growth inhibitory agents. Drugs for the treatment of cancer (e.g., solid tumor cancer) include, but are not limited to, anti-inflammatory agents, growth inhibitory agents, chemotherapeutic agents, immunotherapeutic agents, anti-cancer antibodies or antibody fragments (e.g., antibodies or antibody fragments that target cancer antigens (e.g., cancer neoantigens)), cancer vaccines (e.g., vaccines containing cancer neoantigens), agents that induce autophagy (e.g., activators such as listeria-lysin-o), cytotoxins, inflammasome inhibitors, immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, or CTLA-4), transcription factor inhibitors, and agents that disrupt the cytoskeleton.

[0060] In some embodiments, the cargo is RNA, such as circular RNA, mRNA, siRNA, shRNA, ASO, tRNA, dsRNA, or a combination thereof. In certain embodiments, the RNA has a stability of greater than about 1.01, 1.1, 10, 100, 1000, 10,000, 100,000, 100,000, or 10,000,000, for example, in the minicell cytoplasm. In certain embodiments, the RNA cargo can be stabilized with an added step-loop structure, such as a tRNA scaffold. For example, non-human tRNALys3 and E. coli tRNAMet (Nat. Methods, Ponchon 2007). Both are well characterized and recombinantly expressed. However, various other types, such as aptamers, lncRNAs, and ribozymes, can also be used. RNA can also be stabilized if the minicell is derived from a parent strain null (or hypomorphic) for one or more ribonucleases. In some specific embodiments, the RNA is a protein-coding mRNA. In more specific embodiments, the protein-coding mRNA, such as mRNA encoding the CMV glycoprotein gB and / or pentameric complex (PC), encodes an enzyme (e.g., an enzyme that confers liver enzymatic activity, such as human PBGD (hPBGD) mRNA) or an antigen, for example, that elicits an immune response (e.g., elicits strong and durable neutralizing antibody titers). In certain embodiments, the RNA is a small non-coding RNA, such as shRNA, ASO, tRNA, dsRNA, or a combination thereof.

[0061] Minicells containing the secretory apparatus In certain embodiments, minicells comprise a bacterial secretion apparatus (e.g., an endogenous bacterial secretion apparatus or a heterologous secretion apparatus). A "bacterial secretion apparatus" is a protein or protein complex capable of exporting cargo from the cytoplasm of a bacterial cell (or, e.g., a minicell derived therefrom). In some embodiments, the bacterial secretion apparatus functions by an active (e.g., ATP- or PMF-dependent) process, and in certain embodiments, the bacterial secretion apparatus comprises a tube or spike that extends from the host cell (or minicell) to the target cell. In other embodiments, the bacterial secretion apparatus is a transmembrane channel. Examples of bacterial secretion apparatus that can be comprised in minicells include the T3SS and T4SS (and T3 / T4SS, as defined below), which are tube-containing structures in which cargo traverses the inside of the protein tube, and the T6SS, which delivers cargo to the end of a spike. Other examples of bacterial secretion apparatus that can be comprised in minicells include the transmembrane T1SS, T2SS, T5SS, T7SS, Sec, and Tat.

[0062] In some embodiments, the parent bacterial cell comprises one or more heterologous nucleotide sequences encoding components of a T3SS. In some embodiments, the one or more nucleotide sequences encoding components of a T3SS are carried on a vector. In some embodiments, the parent bacterial cell is transiently transformed with the vector. In some embodiments, the parent bacterial cell is stably transformed with the vector. In some embodiments, the parent bacterial cell further comprises a moiety that increases the level of a T3SS in the minicell.

[0063] In some embodiments, the minicells are derived from a parent bacterial cell that contains a bacterial type 3 secretion system (T3SS) that is endogenous to the parent bacterial cell. In some embodiments, the parent bacterial cell has been modified to reduce the levels of an endogenous protein or polypeptide that can be secreted by the T3SS. In some embodiments, the parent bacterial cell has been modified by deleting a transcriptional activator of an endogenous protein or polypeptide that can be secreted by the T3SS.

[0064] Minicells lacking proteases, RNAses and / or LPS Some embodiments relate to a composition comprising a plurality of minicells, wherein the minicells have reduced protease levels or activity compared to minicells produced from a wild-type parent bacterium. In some aspects, the minicells are produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one protease.

[0065] In some embodiments, minicells have reduced RNAse levels or activity compared to minicells produced from wild-type parent bacteria. In some aspects, minicells are produced from parent bacteria that have been modified to reduce or eliminate expression of at least one RNAse. In some embodiments, the RNAse is an endoribonuclease or exoribonuclease.

[0066] In another aspect, the minicell-producing parent cells and / or the minicells that produce them are modified to reduce lipopolysaccharide (LPS). In some embodiments, the modification is a mutation in lipid A biosynthesis myristoyltransferase (msbB).

[0067] In certain embodiments, minicells lack one or more metabolically non-essential proteins. "Metabolically non-essential proteins" include, but are not limited to, pili, flagella, unwanted secretion apparatus, transposases, effectors, phage elements, or their regulatory elements, such as flhC or OmpA. In some embodiments, minicells lack one or more RNAses, proteases, or combinations thereof, and in certain embodiments, one or more endoribonucleases (e.g., RNAse A, RNAse H, RNAse III, RNAse L, RNAse PhyM) or exoribonucleases (e.g., RNAse R, RNAse PH, RNAse D); or serine proteases, cysteine ​​proteases, threonine proteases, aspartic acid proteases, glutamic acid proteases, and metalloproteases; or any combination of the above.

[0068] Minicells containing additional parts Minicells, in certain embodiments, contain a functional ATP synthase, and in some embodiments, a proton pump embedded in the membrane. Minicells can be derived from a variety of sources, including parent bacterial strains ("parent strains") engineered or induced to produce genome-free membrane apparatus, genome-excised bacteria, bacterial cell preparation extracts (e.g., by mechanical or other means), or total synthesis (optionally including fractions of bacterial cell preparations). In some embodiments, minicells have an ATP synthase concentration of 1 / 10000 nm, 1 / 5000 nm, 1 / 3500 nm, or 1 / 1000 nm.

[0069] Minicells can include a variety of additional components, including, for example, photovoltaic pumps, retinal and retinal-producing cassettes, metabolic enzymes, targeting agents, cargo, bacterial secretion apparatus, and transporters, including combinations of the foregoing, including certain embodiments described below. In certain embodiments, minicells and / or minicell-producing parent cells lack metabolically non-essential genes and / or other elements, such as certain enzymes, nucleases, or proteases.

[0070] In certain embodiments, minicells and / or minicell-producing parent cells comprise an ATP synthase that optionally lacks a regulatory domain (e.g., lacks the epsilon domain). Deletion can be achieved by various means. In certain embodiments, deletion is by inducible deletion of the native epsilon domain. In certain embodiments, deletion is achieved by flanking with LoxP sites and inducible Cre expression or CRISPR knockout, or can be inducible (placed on a plasmid under the tTa tet transactivator in an ATP synthase knockout strain).

[0071] In some embodiments, minicells and / or minicell-producing parent cells can comprise a photovoltaic proton pump. In particular embodiments, the photovoltaic proton pump is proteorhodopsin. In more particular embodiments, the proteorhodopsin comprises the amino acid sequence of proteorhodopsin from the uncultured marine bacterial clade SAR86, GenBank accession: AAS73014.1. In other embodiments, the photovoltaic proton pump is Gloeobacter rhodopsin. In certain embodiments, the photovoltaic proton pump is bacteriorhodopsin, deltarhodopsin, or halorhodopsin from Halobium salinarum, Natronomonas pharaonis, Exiguobacterium sibiricum, Haloterrigena turkmenica, or Haloarcula marismortui.

[0072] In some embodiments, the minicells and / or minicell-producing parent cells further comprise retinal. In certain embodiments, the minicells and / or minicell-producing parent cells comprise a retinal synthesis protein (or protein system) or a nucleic acid encoding the same.

[0073] In certain embodiments, minicells and / or minicell-producing parent cells comprise one or more glycolytic proteins. In some embodiments, the glycolytic protein is phosphofructokinase (Pfk-A), e.g., comprising the amino acid sequence of UniProt Accession P0A796 or a functional fragment thereof. In other embodiments, the glycolytic protein is triosephosphate isomerase (tpi), e.g., comprising the amino acid sequence of UniProt Accession P0A858 or a functional fragment thereof.

[0074] Minicell Compositions and Formulations Some embodiments relate to compositions or preparations containing minicells described herein, including, inter alia, minicell preparations in which a plurality of individual minicells lack a cell division topology specificity factor, e.g., lack the minE gene product, and optionally, the minicell preparation is substantially free of viable cells. In some embodiments, the minicell composition contains at least about 80, 81, 82, 83, 84, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% or more minicells that contain a bacterial secretion apparatus. In specific embodiments, the bacterial secretion apparatus is one of a T3SS, T4SS, T3 / 4SS, or T6SS. In some embodiments, the minicell composition contains minicells containing a T3SS, wherein the minicells comprise an average T3SS membrane density of greater than about 40,000, 35,000, 30,000, 25,000, 19,600, 15,000, 10,000, or 1 in 5,000 nm2. In certain embodiments, the minicells are derived from an Agrobacterium tumefaciens, S. typhimurium, or E. coli parent strain. In some embodiments, the minicell composition contains at least about 80, 81, 82, 83, 84, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% or more of the bacterial secretion apparatus, including T3, T4, T3 / 4SS, T6SS, and optionally one or more of exogenous carbohydrates, phosphate-producing synthases, light-responsive proteins, import proteins, enzymes, functional cargo, organism-specific effectors, and fusion proteins.

[0075] The minicell compositions can be prepared in any suitable formulation. For example, the formulation can be suitable for IP, IV, IM, oral, topical (cream, gel, ointment, transdermal patch), aerosol, or spray administration. In some embodiments, the formulation is a liquid formulation. In other embodiments, the formulation is a lyophilized formulation.

[0076] In some embodiments, the minicell compositions described herein contain less than 100 colony forming units (CFU / mL) of live bacterial cells, e.g., less than 50 CFU / mL, less than 20 CFU / mL, less than 10 CFU / mL, less than 1 CFU / mL, or less than 0.1 CFU / mL.

[0077] In some embodiments, the minicell composition comprises lyophilized, reconstituted minicells, wherein the reconstituted minicells have an ATP concentration that is at least 90% of the ATP concentration of the non-lyophilized minicells, e.g., at least 95%, 98%, or at least equal to the ATP concentration of the non-lyophilized minicells.

[0078] Some embodiments relate to minicell compositions in which the minicells are stored, e.g., stored at 4° C., and after storage, the minicells have an ATP concentration that is at least 90% of the ATP concentration of the non-stored minicells, e.g., at least 95%, 98%, or at least equal to the ATP concentration of the non-stored minicells. In some embodiments, storage is for at least 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 6 months, or at least 1 year.

[0079] In some embodiments, minicells can be stored or otherwise rendered "dormant" and then rapidly activated.

[0080] In some embodiments, the minicell compositions are formulated for delivery to an animal, for example, formulated for intraperitoneal, intravenous, intramuscular, oral, topical, aerosol, or nebulized administration.

[0081] In some embodiments, the minicell composition is formulated for delivery to plants. In some aspects, the composition includes an adjuvant, such as a surfactant (e.g., a nonionic surfactant, a surfactant plus a nitrogen source, an organosilicone surfactant, or a high-surfactant oil concentrate), a crop oil concentrate, a vegetable oil concentrate, a modified vegetable oil, a nitrogen source, a deposition (drift control) and / or retention agent (with or without ammonium sulfate and / or an antifoam agent), a compatibilizer, a buffer and / or acidifier, a water conditioner, a base blend, a spreader-sticker and / or extender, an adjuvant plus a foliar fertilizer, an antifoam agent, a foam marker, a fragrance, or a tank cleaner and / or neutralizer. In some embodiments, the adjuvant is an adjuvant described in the Compendium of Herbicide Adjuvants (Young et al. (2016). Compendium of Herbicide Adjuvants (13th ed.), Purdue University).

[0082] In some embodiments, the minicell composition is formulated for delivery to an invertebrate (e.g., an arthropod (e.g., an insect or arachnid), a nematode, a protozoan, or an annelid). In some embodiments, the minicell composition is formulated for delivery to an insect.

[0083] In some embodiments, the minicell composition is formulated as a liquid, solid, aerosol, paste, gel, or gas composition.

[0084] Preparation of minicells In some aspects, minicell production is characterized by a method for producing a composition comprising a plurality of minicells, the composition being substantially free of viable bacterial cells, the method comprising the steps of: (a) producing, providing, or obtaining a plurality of parent bacteria having a reduced level or activity of a cell division topology specificity factor; (b) exposing the parent bacteria to conditions that permit the formation of minicells; and (c) separating the minicells from the parent bacteria, thereby producing a composition that is substantially free of viable bacterial cells.

[0085] Parent bacteria include any suitable bacterial species from which minicells can be produced (e.g., species that can be modified to produce minicells using the methods described herein). The following is a non-limiting list of suitable genera from which minicells and / or minicell-producing parent cells can be derived: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, and the like. bacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas hloromonas), Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirilla Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Phyllobacterium,Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus cus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella la), Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium, Geobacillus, Lactobacillus ), Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus, Symbiobacterium or Thermoanaerobacter.

[0086] In some aspects, methods are provided for producing any of the minicell compositions described herein. For example, provided herein are methods for generating minicells and / or minicell-producing parent cells; methods for generating minicells and / or minicell-producing parent cells that lack a cell division topology specificity factor and, optionally, a Z-ring inhibitory protein (e.g., methods for generating minicells from a ΔminCDE parent bacterium); and methods for generating any of the minicells and / or minicell-producing parent cells described herein, wherein the minicells comprise cargo.

[0087] In some embodiments, minicells are generated from parent strains that are plant bacteria, such as plant symbiotic bacteria (e.g., Bacillus subtilis or Pseudomonas putida), plant pathogenic bacteria (e.g., Xanthomonas species or Pseudomonas syringae), or bacteria capable of plant rhizosphere colonization and / or nodulation, e.g., rhizobia.

[0088] In some embodiments, minicells are produced from parent strains that are symbionts of invertebrates, such as arthropods (e.g., insects or arachnids), nematodes, protozoans, or annelids. In embodiments, the invertebrate is a pest or a plant or animal pathogen.

[0089] In some embodiments, minicells are generated from a genetically transformable parent strain, such as Agrobacterium.

[0090] In some embodiments, minicells are generated from a parent strain that is a human bacterium, e.g., a commensal human bacterium (e.g., E. coli, Staphylococcus spp., Bifidobacterium spp., Micrococcus spp., Lactobacillus spp., or Actinomyces spp.), or a pathogenic human bacterium (e.g., E. coli EHEC, Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica, or Helicobacter pylori), or an extremophilic bacterium.

[0091] In some embodiments, minicells and / or minicell-producing parent strains are functional derivatives of any of the above, including, for example, functional cassettes such as functional cassettes that direct the bacterium to one or more of the following: secrete antimicrobial agents, digest plastics, secrete insecticides (e.g., insecticidal peptides such as Pir, Bt, Cry, etc.), survive in extreme environments, create nanoparticles, bind to other organisms, respond to the environment, and create reporter signals.

[0092] The parent bacterium can include any of the functional derivatives described above, for example, functional cassettes that direct the bacterium to one or more of the following: secrete antimicrobial agents, digest plastics, secrete insecticides (e.g., insecticidal peptides such as Pir, Bt, Cry, etc.), survive in extreme environments, create nanoparticles, bind to other organisms, respond to the environment, and create reporter signals.

[0093] In some embodiments, the minicells are derived from a parent strain that has been engineered or induced to overexpress ATP synthase. In some more particular embodiments, the ATP synthase is heterologous to the parent strain. In particular embodiments, the parent strain has been modified to express a functional FoF1 ATP synthase.

[0094] In some embodiments, minicells are obtained from a parent strain cultured under conditions selected from the following: applied voltage (e.g., 37 mV), extra-atmospheric oxygen concentration (e.g., 1-5% O2, 5-10% O2, 10-15% O2, 25-30% O2), low pH (about 4.5, 5.0, 5.5, 6.0, 6.5), or a combination thereof.

[0095] In some embodiments, minicells are generated from extremophilic microorganisms that contain functional derivatives of any of the foregoing, including, for example, functional cassettes that direct the bacteria to one or more of the following: secrete antimicrobials, digest plastics, secrete insecticides, survive in extreme environments, create nanoparticles, bind to other organisms, respond to the environment, and create reporter signals.

[0096] Due to bacterial diversity, minicells and / or minicell-producing parent strains can be produced with modified membranes, e.g., to improve the biodistribution of minicells upon administration to target cells. In certain embodiments, the membrane is modified to be less immunogenic or immunostimulatory in plants or animals. For example, in certain embodiments, minicells are obtained from a parent strain whose immunostimulatory capabilities are reduced or eliminated by post-production treatment with detergents, enzymes, or functionalization with PEG. In certain embodiments, minicells are produced from a parent strain whose membrane is modified by knocking out the LPS synthesis pathway in the parent strain, e.g., by knocking out msbB. In other specific embodiments, minicells are produced from a parent strain that produces cell-wall-deficient particles upon exposure to hyperosmotic conditions.

[0097] In some embodiments, the method comprises transforming the parent strain with an inducible DNAase system, such as exoI (NCBI GeneID: 946529) and sbcD (NCBI GeneID: 945049) nucleases, or I-CeuI (e.g., Swissprot: P32761.1) nuclease. In more particular embodiments, the method comprises using a single-, double-, triple-, or quadruple-auxotrophic strain and having complementary genes on a plasmid encoding the inducible nuclease.

[0098] In some embodiments, the parent strain is cultured under conditions selected from the following: applied voltage (e.g., 37 mV), extra-atmospheric oxygen concentration (e.g., 1-5% O2, 5-10% O2, 10-15% O2, 25-30% O2), low pH (about: 4.5-6.5), or a combination thereof.

[0099] In certain embodiments, the parent strain lacks flagella and unwanted secretion apparatus, and optionally, the flagella and unwanted secretion system are removed using lambda red recombination. In some embodiments, flagellar regulatory components are excised from the parent strain genome, for example, by insertion of a plasmid containing a CRISPR domain targeting flagellar regulatory genes, such as flhD and flhC.

[0100] In certain embodiments, the parent strain comprises a cargo. In some embodiments, the parent strain contains a nucleic acid sequence encoding a set of genes that synthesize a small molecule cargo.

[0101] Insecticidal composition and formulation thereof Embodiments of the present disclosure include compositions comprising a liquid carrier phase and a plurality of minicells dispersed in the carrier phase, the plurality of minicells comprising a mixture of first minicells comprising a first effective amount of a first exogenous protein toxin comprising Pir and second minicells comprising a second effective amount of a second exogenous protein toxin comprising Cry, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In further embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. The synergistic effect allows for lower effective amounts of each exogenous protein toxin to be used while maintaining their effectiveness in agricultural applications. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the first particle concentration of the first minicells is about 1 x 10 2 ~Approx. 8×10 14 and the second particle concentration in the second minicell is in the range of about 1×10 2 ~Approx. 8×10 14In some embodiments of this aspect that may be combined with any of the preceding embodiments, administering the composition to the pest includes an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the pest with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds on, or infests; or applying the composition to the plant. In further embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the composition is formulated as at least one of a Ready To Use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, and a drench application.

[0102] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the first exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, or SEQ ID NO:203; and SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO: In an additional embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 201, or SEQ ID NO: 204, or a functional component of any of these. In an additional embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 203 and a PirB comprising SEQ ID NO: 204, or the PirAB comprises a PirA comprising SEQ ID NO: 197 and a PirB comprising SEQ ID NO: 178.

[0103] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, or Cry1K, or a functional component thereof. In further embodiments of this aspect, Cry1A comprises a Cry1A selected from the group of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152, or SEQ ID NO:205; Cry1B comprises a Cry1B selected from the group of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, or SEQ ID NO:155; Cry1C comprises a Cry1C selected from the group of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, or SEQ ID NO:156; Cry1D comprises a Cry1D selected from the group of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175, or SEQ ID NO:180. In a further embodiment of this aspect, the second exogenous protein toxin comprises a Cry1D selected from the group of SEQ ID NO: 176; Cry1E comprises a Cry1E selected from the group of SEQ ID NO: 128 or SEQ ID NO: 129; Cry1F comprises a Cry1F selected from the group of SEQ ID NO: 130 or SEQ ID NO: 131; Cry1G comprises a Cry1G selected from the group of SEQ ID NO: 132 or SEQ ID NO: 133; Cry1H comprises a Cry1H selected from the group of SEQ ID NO: 134 or SEQ ID NO: 135; Cry1I comprises a Cry1I selected from the group of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139; Cry1J comprises a Cry1J selected from the group of SEQ ID NO: 140 or SEQ ID NO: 141; or Cry1K comprises SEQ ID NO: 142, or any functional component thereof. In a further embodiment of this aspect, the second exogenous protein toxin comprises a Cry1A comprising SEQ ID NO: 205.

[0104] In further embodiments of this aspect that may be combined with any of the preceding embodiments, the first minicell comprises a first vector comprising a coding sequence for a first exogenous protein toxin. In further embodiments of this aspect, the first vector comprises a CloDF origin of replication or a pMB1 origin of replication. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a first vector, the coding sequence for the first exogenous protein toxin is operably linked to a first promoter. In other embodiments of this aspect, the first promoter is an inducible promoter or a constitutive promoter. In some embodiments of this aspect, the inducible promoter comprises Ptac, Ptet, or PT7. In some embodiments of this aspect, the constitutive promoter comprises J23119 (SEQ ID NO: 200). In an additional embodiment of this aspect that may be combined with any of the preceding embodiments having a first vector, the first exogenous protein toxin comprises PirAB, which PirAB comprises PirA comprising SEQ ID NO: 203 and PirB comprising SEQ ID NO: 204, wherein the coding sequence for PirA comprises SEQ ID NO: 102, and the coding sequence for PirB comprises SEQ ID NO: 103. In a further embodiment of this aspect that may be combined with any of the preceding embodiments having a first vector and an inducible promoter, expression of the first exogenous protein toxin is induced with aTc or IPTG.

[0105] In further embodiments of this aspect that may be combined with any of the preceding embodiments, the second minicell comprises a second vector comprising a coding sequence for a second exogenous protein toxin. In further embodiments of this aspect, the second vector comprises a CloDF origin of replication or a pMB1 origin of replication. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a second vector, the coding sequence for the second exogenous protein toxin is operably linked to a second promoter. In other embodiments of this aspect, the second promoter is an inducible promoter or a constitutive promoter. In some embodiments of this aspect, the inducible promoter comprises Ptac, Ptet, or PT7. In some embodiments of this aspect, the constitutive promoter comprises J23119 (SEQ ID NO: 200). In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a second vector, the second exogenous protein toxin comprises Cry1A, wherein the Cry1A comprises SEQ ID NO: 205, and the coding sequence for Cry1A comprises SEQ ID NO: 104. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments having a second vector and an inducible promoter, expression of the second exogenous protein toxin is induced with aTc or IPTG.

[0106] Another embodiment of the present disclosure includes a composition comprising a liquid carrier phase, a plurality of minicells dispersed in the carrier phase (wherein the plurality of minicells comprises a first effective amount of an exogenous protein toxin comprising Pir); and a second effective amount of a Bt-derived AI, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In further embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. The synergistic effect allows for lower effective amounts of each active ingredient (e.g., exogenous protein toxin; Bt-derived AI) to be used while maintaining their effectiveness in agricultural applications. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the particle concentration of minicells is about 1 x 10 2 ~Approx. 8×10 14In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least one of Bacillus thuringiensis, a fermentation solid thereof, a spore thereof, and an insecticidal toxin thereof. In still further embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, a fermentation solid thereof, a spore thereof, and an insecticidal toxin thereof. In some embodiments of this aspect that may be combined with any of the preceding embodiments, administering the composition to the pest includes an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the pest with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds, or infests; or applying the composition to a plant. In further embodiments of this aspect, the composition is applied to plants as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment. In other embodiments of this aspect that may be combined with any of the preceding embodiments, the composition is formulated as at least one of a ready-to-use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, a drench application, a dust, an emulsifiable concentrate, a flowable concentrate, an intermediate formulation, a granule formulation, an impregnated material formulation, a pellet / tablet formulation, a soluble concentrate formulation, an industrial chemical formulation, a water-dispersible granule formulation, a water-soluble package formulation, or a wettable powder formulation.

[0107] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; and SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. 3, SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58, or a functional component of any of these. In additional embodiments of this aspect, PirAB comprises PirA comprising SEQ ID NO: 4 and PirB comprising SEQ ID NO: 5, or PirAB comprises PirA comprising SEQ ID NO: 6 and PirB comprising SEQ ID NO: 7.In still further embodiments of this aspect, PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:32. and a PirB selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58. The linker sequence is of sufficient structure to enable the fusion to exert insecticidal activity in the range of 0.7X to 1.5X (where X is the insecticidal activity of the same unlinked PirA and PirB). Exemplary linkers can include (Gly4Ser)3 (SEQ ID NO:59), SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:64.

[0108] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI is selected from the group consisting of Thuricide®, Thuricide® HPC-ORTU, Thuricide® HPC, Thuricide® 48LV Forestry, Thuricide® 76B, Thuricide® 76LV, Bonide Bacillus Thuringiensis (BT) moth larva (caterpillar) control, DiPel®, Security BT powder biological insecticide, Fertilome DiPel powder biological insecticide, Widestrike®, botanical pesticide BT subsp. tenebrionis Colorado potato beetle protein, SA-50 brand DiPel® dust, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 dust for vegetables, Green Light® DiPel® dust, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granule, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits® BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L WormKiller, TERRO® Larvicide, VectoMax® FG Biological Larvicide Granules, Chemsico Insect Granules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Granules, VectoBac® FG+ Biological Larvicide Granules, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE!® G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar® Bti CRG, VBC-60092 (Bactimos Briquette), Summit B.ti BRIQUETS, Dunks, Sympatico Biological Insecticide, XenTari® AS Biological Insecticide Aqueous Suspension, B402 Biological Larvicide, VectoBac® 12AS Biological Larvicide Aqueous Suspension, VectoBac® SC Biological Larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F Flowable Biological Insecticide Concentrate, Foray 76B, Safer BT Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. israelensis (Bacillus thuringiensis subsp. Israelensis slurry, Bacillus thuringiensis subsp. Kurstaki slurry, Trident® biological insecticide, VBC-60219 biological insecticide slurry, Javelin, Bacillus thuringiensis subsp. Aizawaisubsp. aizawai) Slurry, FlorBac® Slurry Biological Insecticide, DiPel® ES, DiPel® ES-NT Biological Insecticide Emulsifier, DiPel® (Worm Killer) Wettable Powder Biological Insecticide, Condor, Leap® ES Bacterial Disease Control Biological Insecticide Emulsifier, Biobit® HPWPII Biological Insecticide, VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wettable Powder, BioBit® HP Biological Insecticide Wettable Powder, DiPel® WDG Biological Insecticide, VBC -60236 Biological Insecticide Dry Flowable, Foray2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, ForayWG, ThyllombeetleGONE!® Biological Insecticide, JavelinWP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, JavelinWG, SAN420IWG, JavelinWG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW, Bonide Bacillus thuringiensis 'O', ChemsicoHPC-O Concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide® Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, or BTI Technical Powder BIOInsecticide.

[0109] In further embodiments of this aspect that may be combined with any of the preceding embodiments, the plurality of minicells comprises a vector comprising a coding sequence for an exogenous protein toxin. In further embodiments of this aspect, the vector comprises a pMB1 origin of replication. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a vector, the coding sequence for the exogenous protein toxin is operably linked to a promoter. In other embodiments of this aspect, the promoter is an inducible promoter or a constitutive promoter. In some embodiments of this aspect, the inducible promoter comprises Ptet. In additional embodiments of this aspect that may be combined with any of the preceding embodiments having a vector, the exogenous protein toxin comprises PirAB, wherein PirAB comprises PirA comprising SEQ ID NO:4 and PirB comprising SEQ ID NO:5, wherein the coding sequence for PirA comprises SEQ ID NO:2, and the coding sequence for PirB comprises SEQ ID NO:3. In further embodiments of this aspect that may be combined with any of the preceding embodiments having a vector and an inducible promoter, expression of the exogenous protein toxin is induced with aTc.

[0110] In additional embodiments of either of the two preceding aspects that can be combined with any of the preceding embodiments including control of pests, the control includes at least one of a reduction in pest numbers, a reduction in pest weight, a reduction in physical damage caused by the pests, and an increase in pest mortality. In some embodiments of this aspect, the reduction in pest numbers, the reduction in pest weight, the reduction in physical damage caused by the pests, or the increase in pest mortality is compared to pests that are not administered the composition. In further embodiments of this aspect, the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, or at least an 80% reduction; and the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, or at least an 80% reduction. a 0% reduction, a reduction in physical damage of at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, or at least an 80% reduction, and an increase in pest mortality of at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40%, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, or at least an 80% increase.In some embodiments of this aspect that may be combined with any of the preceding embodiments, control includes observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0111] In further embodiments of either of the two preceding aspects that may be combined with any of the preceding embodiments, the composition further comprises an agrochemical surfactant, which improves at least one of sprayability, spreadability, and pourability properties. In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the liquid carrier phase is aqueous or oil-based. In some embodiments of this aspect that may be combined with any of the preceding embodiments involving pest control, the at least one pest is selected from the group consisting of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species.

[0112] Additional embodiments of the present disclosure include compositions comprising a liquid carrier phase and a plurality of minicells dispersed in the carrier phase, the plurality of minicells comprising minicells comprising a first effective amount of a first exogenous protein toxin comprising Pir and a second effective amount of a second exogenous protein toxin comprising Cry1, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In some embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the combined first effective amount and second effective amount have a synergistic effect on controlling the at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling the at least one pest when the composition is administered to the pest. The synergistic effect allows for lower effective amounts of each exogenous protein toxin to be used while maintaining their effectiveness in agricultural applications.

[0113] Additional embodiments of the present disclosure include compositions comprising a liquid carrier phase and a plurality of minicells dispersed in the carrier phase, the plurality of minicells comprising minicells containing a first effective amount of an exogenous protein toxin comprising Pir and a second effective amount of a Bt-derived AI, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In some embodiments of this aspect that may be combined with any of the preceding embodiments, when the composition is administered to a pest, the combined first effective amount and second effective amount have a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the composition is administered to the pest. The synergistic effect allows for lower effective amounts of each active ingredient (e.g., exogenous protein toxin; Bt-derived AI) to be used while maintaining their effectiveness in agricultural applications.

[0114] Yet another embodiment of the present disclosure includes a wettable powder comprising a plurality of dry minicells comprising a mixture of a first effective amount of first minicells comprising a first exogenous protein toxin comprising a Pir and a second effective amount of second minicells comprising a second exogenous protein toxin comprising a Cry, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50, and the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to an insect. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In other embodiments of this aspect that can be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. The synergistic effect allows for lower effective amounts of each exogenous protein toxin to be used while maintaining their effectiveness in agricultural applications. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the first particle concentration of the first minicells is about 1 x 10 2 ~Approx. 8×10 14 and the second particle concentration in the second minicell is in the range of about 1×10 2 ~8×10 14In further embodiments of this aspect that may be combined with any of the preceding embodiments, the hydratable powder further comprises a pesticidally acceptable solid carrier component comprising at least one of a clay component, a kaolin component, a talc component, a chalk component, a calcite component, a quartz component, a pumice component, a diatomaceous earth component, a vermiculite component, a silicate component, a silicon dioxide component, a silica powder component, an aluminum component, an ammonium sulfate component, an ammonium phosphate component, a calcium carbonate component, a urea component, a sugar component, a starch component, a sawdust component, a ground coconut shell component, a ground corn cob component, and a ground tobacco stalk component. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the aqueous carrier comprises water. In further embodiments of this aspect that may be combined with any of the preceding embodiments, administering the composition to the pest includes an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the insect with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds, or infests; and applying the composition to a plant. In some embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, or a post-emergence treatment.

[0115] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the first exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, or SEQ ID NO:203; and SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO: In another embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 201, or SEQ ID NO: 204, or a functional component of any of these. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 203 and a PirB comprising SEQ ID NO: 204, or the PirAB comprises a PirA comprising SEQ ID NO: 197 and a PirB comprising SEQ ID NO: 178.

[0116] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, or Cry1K, or a functional component thereof. In further embodiments of this aspect, Cry1A comprises a Cry1A selected from the group of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152, or SEQ ID NO:205; Cry1B comprises a Cry1B selected from the group of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, or SEQ ID NO:155; Cry1C comprises a Cry1C selected from the group of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, or SEQ ID NO:156; Cry1D comprises a Cry1D selected from the group of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175, or SEQ ID NO:180. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1D selected from the group of SEQ ID NO: 176; Cry1E comprises a Cry1E selected from the group of SEQ ID NO: 128 or SEQ ID NO: 129; Cry1F comprises a Cry1F selected from the group of SEQ ID NO: 130 or SEQ ID NO: 131; Cry1G comprises a Cry1G selected from the group of SEQ ID NO: 132 or SEQ ID NO: 133; Cry1H comprises a Cry1H selected from the group of SEQ ID NO: 134 or SEQ ID NO: 135; Cry1I comprises a Cry1I selected from the group of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139; Cry1J comprises a Cry1J selected from the group of SEQ ID NO: 140 or SEQ ID NO: 141; or Cry1K comprises SEQ ID NO: 142, or any functional component thereof. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1A comprising SEQ ID NO: 205.

[0117] Yet another embodiment of the present disclosure includes a wettable powder comprising a plurality of dry minicells comprising a mixture of a first effective amount of minicells comprising an exogenous protein toxin, including Pir, and a second effective amount of an AI derived from Bt, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50, and the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to an insect. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In other embodiments of this aspect that can be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. The synergistic effect allows for lower effective amounts of each active ingredient (e.g., exogenous protein toxin; Bt-derived AI) to be used while maintaining their effectiveness in agricultural applications. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the particle concentration of minicells is about 1 x 10 2 ~8×10 1In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least one of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxin. In still further embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxin. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the wettable powder further comprises a pesticidally acceptable solid carrier component comprising at least one of a clay component, a kaolin component, a talc component, a chalk component, a calcite component, a quartz component, a pumice component, a diatomaceous earth component, a vermiculite component, a silicate component, a silicon dioxide component, a silica powder component, an aluminum component, an ammonium sulfate component, an ammonium phosphate component, a calcium carbonate component, a urea component, a sugar component, a starch component, a sawdust component, a ground coconut shell component, a ground corn cob component, and a ground tobacco stalk component. In some embodiments of this aspect that can be combined with any of the preceding embodiments, the aqueous carrier comprises water. In further embodiments of this aspect that can be combined with any of the preceding embodiments, administering the composition to the pest comprises an administration method selected from the group consisting of delivering the composition to the pest for ingestion; contacting the insect with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds, or infests; and applying the composition to a plant. In some embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, or a post-emergence treatment.

[0118] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; and SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO:4 and a PirB comprising SEQ ID NO:5, or a PirAB comprising a PirA comprising SEQ ID NO:6 and a PirB comprising SEQ ID NO:7.In another embodiment of this aspect, PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10 The PirA and PirB are linked by a linker sequence to a PirB selected from the group consisting of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58. The linker sequence is of sufficient structure to enable the fusion to exert insecticidal activity in the range of 0.7X to 1.5X (where X is the insecticidal activity of the same unlinked PirA and PirB). Exemplary linkers can include (Gly4Ser)3 (SEQ ID NO:59), SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:64.

[0119] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI is selected from the group consisting of Thuricide®, Thuricide® HPC-ORTU, Thuricide® HPC, Thuricide® 48LV Forestry, Thuricide® 76B, Thuricide® 76LV, Bonide Bacillus Thuringiensis (BT) moth larva (caterpillar) control, DiPel®, Security BT powder biological insecticide, Fertilome DiPel powder biological insecticide, Widestrike®, botanical pesticide BT subsp. tenebrionis Colorado potato beetle protein, SA-50 brand DiPel® dust, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 dust for vegetables, Green Light® DiPel® dust, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granule, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits® BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L WormKiller, TERRO® Larvicide, VectoMax® FG Biological Larvicide Granules, Chemsico Insect Granules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Granules, VectoBac® FG+ Biological Larvicide Granules, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE!® G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar® Bti CRG, VBC-60092 (Bactimos Briquette), Summit B.ti BRIQUETS, Dunks, Sympatico Biological Insecticide, XenTari® AS Biological Insecticide Aqueous Suspension, B402 Biological Larvicide, VectoBac® 12AS Biological Larvicide Aqueous Suspension, VectoBac® SC Biological Larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F Flowable Biological Insecticide Concentrate, Foray 76B, Safer BT Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. israelensis (Bacillus thuringiensis subsp. Israelensis slurry, Bacillus thuringiensis subsp. Kurstaki slurry, Trident® biological insecticide, VBC-60219 biological insecticide slurry, Javelin, Bacillus thuringiensis subsp. Aizawaisubsp. aizawai) Slurry, FlorBac® Slurry Biological Insecticide, DiPel® ES, DiPel® ES-NT Biological Insecticide Emulsifier, DiPel® (Worm Killer) Wettable Powder Biological Insecticide, Condor, Leap® ES Bacterial Disease Control Biological Insecticide Emulsifier, Biobit® HPWPII Biological Insecticide, VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wettable Powder, BioBit® HP Biological Insecticide Wettable Powder, DiPel® WDG Biological Insecticide, VBC -60236 Biological Insecticide Dry Flowable, Foray2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, ForayWG, ThyllombeetleGONE!® Biological Insecticide, JavelinWP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, JavelinWG, SAN420IWG, JavelinWG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW, Bonide Bacillus thuringiensis 'O', ChemsicoHPC-O Concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide® Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, or BTI Technical Powder BIOInsecticide.

[0120] In additional embodiments of either of the two preceding aspects that can be combined with any of the preceding embodiments, the control includes at least one of a reduction in pest numbers, a reduction in pest weight, a reduction in physical damage caused by the pests, and an increase in pest mortality. In some embodiments of this aspect, the reduction in pest numbers, the reduction in pest weight, the reduction in physical damage caused by the pests, or the increase in pest mortality is compared to pests that are not administered the composition. In further embodiments of this aspect, the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, or at least an 80% reduction; and the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, or at least an 80% reduction. % reduction, the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction or at least an 80% reduction, and the increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40%, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, and at least a 65% increase, at least a 70% increase, at least a 75% increase or at least an 80% increase.In some embodiments of this aspect that may be combined with any of the preceding embodiments, the control includes an observed pest mortality rate of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the at least one pest is selected from the group of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species.

[0121] A further embodiment of the present disclosure is a plantable composition comprising a seed and a coating overlying the seed, the coating comprising a plurality of minicells comprising a mixture of a first effective amount of first minicells comprising a first exogenous protein toxin comprising a Pir and a second effective amount of second minicells comprising a second exogenous protein toxin comprising a Cry, the first effective amount and the second effective amount being present in a ratio ranging from about 50:1 to 1:50, and the mixture comprising a composition sufficient to provide insecticidal activity against at least one pest that feeds on the seed or the seedlings produced therefrom. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. In other embodiments of this aspect that can be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on control of the at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. The synergistic effect allows for lower effective amounts of each exogenous protein toxin to be used while maintaining their effectiveness in agricultural applications. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the first particle concentration of the first minicells is about 1 x 10 2 ~Approx. 8×10 14 and the second particle concentration in the second minicell is in the range of about 1×10 2 ~Approx. 8×10 14 The range is.

[0122] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the first exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, or SEQ ID NO:203; and SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO: In another embodiment of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 172, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 201, or SEQ ID NO: 204, or a functional component of any of these. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO: 203 and a PirB comprising SEQ ID NO: 204, or the PirAB comprises a PirA comprising SEQ ID NO: 197 and a PirB comprising SEQ ID NO: 178.

[0123] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, or Cry1K, or a functional component thereof. In further embodiments of this aspect, Cry1A comprises a Cry1A selected from the group of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152, or SEQ ID NO:205; Cry1B comprises a Cry1B selected from the group of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, or SEQ ID NO:155; Cry1C comprises a Cry1C selected from the group of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, or SEQ ID NO:156; Cry1D comprises a Cry1D selected from the group of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175, or SEQ ID NO:180. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1D selected from the group of SEQ ID NO: 176; Cry1E comprises a Cry1E selected from the group of SEQ ID NO: 128 or SEQ ID NO: 129; Cry1F comprises a Cry1F selected from the group of SEQ ID NO: 130 or SEQ ID NO: 131; Cry1G comprises a Cry1G selected from the group of SEQ ID NO: 132 or SEQ ID NO: 133; Cry1H comprises a Cry1H selected from the group of SEQ ID NO: 134 or SEQ ID NO: 135; Cry1I comprises a Cry1I selected from the group of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139; Cry1J comprises a Cry1J selected from the group of SEQ ID NO: 140 or SEQ ID NO: 141; or Cry1K comprises SEQ ID NO: 142, or any functional component thereof. In another embodiment of this aspect, the second exogenous protein toxin comprises a Cry1A comprising SEQ ID NO: 205.

[0124] A further aspect of the present disclosure is a plantable composition comprising a seed and a coating overlying the seed, the coating comprising a plurality of minicells comprising a mixture of a first effective amount of minicells comprising an exogenous protein toxin including Pir and a second effective amount of a Bt-derived AI, the first effective amount and the second effective amount being present in a ratio ranging from about 50:1 to 1:50, the mixture comprising a composition sufficient to provide insecticidal activity against at least one pest that feeds on the seed or the resulting seedlings. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1. In other embodiments of this aspect that can be combined with any of the preceding embodiments, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. The synergistic effect allows for lower effective amounts of each active ingredient (e.g., exogenous protein toxin; Bt-derived AI) to be used while maintaining their effectiveness in agricultural applications. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the particle concentration of minicells is about 1 x 10 2 ~Approx. 8×10 14 The range is.

[0125] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the exogenous protein toxin comprises PirAB or a functional component thereof. In further embodiments of this aspect, PirAB is selected from the group of PirA selected from the group of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; and SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In other embodiments of this aspect, the PirAB comprises a PirA comprising SEQ ID NO:4 and a PirB comprising SEQ ID NO:5, or a PirAB comprising a PirA comprising SEQ ID NO:6 and a PirB comprising SEQ ID NO:7.In another embodiment of this aspect, PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10 The PirA and PirB are linked by a linker sequence to a PirB selected from the group consisting of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58. The linker sequence is of sufficient structure to enable the fusion to exert insecticidal activity in the range of 0.7X to 1.5X (where X is the insecticidal activity of the same unlinked PirA and PirB). Exemplary linkers can include (Gly4Ser)3 (SEQ ID NO:59), SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:64.

[0126] In some embodiments of this aspect that may be combined with any of the preceding embodiments, the Bt-derived AI is selected from the group consisting of Thuricide®, Thuricide® HPC-ORTU, Thuricide® HPC, Thuricide® 48LV Forestry, Thuricide® 76B, Thuricide® 76LV, Bonide Bacillus Thuringiensis (BT) moth larva (caterpillar) control, DiPel®, Security BT powder biological insecticide, Fertilome DiPel powder biological insecticide, Widestrike®, botanical pesticide BT subsp. tenebrionis Colorado potato beetle protein, SA-50 brand DiPel® dust, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 dust for vegetables, Green Light® DiPel® dust, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granule, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits® BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L WormKiller, TERRO® Larvicide, VectoMax® FG Biological Larvicide Granules, Chemsico Insect Granules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Granules, VectoBac® FG+ Biological Larvicide Granules, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE!® G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar® Bti CRG, VBC-60092 (Bactimos Briquette), Summit B.ti BRIQUETS, Dunks, Sympatico Biological Insecticide, XenTari® AS Biological Insecticide Aqueous Suspension, B402 Biological Larvicide, VectoBac® 12AS Biological Larvicide Aqueous Suspension, VectoBac® SC Biological Larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F Flowable Biological Insecticide Concentrate, Foray 76B, Safer BT Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. israelensis (Bacillus thuringiensis subsp. Israelensis slurry, Bacillus thuringiensis subsp. Kurstaki slurry, Trident® biological insecticide, VBC-60219 biological insecticide slurry, Javelin, Bacillus thuringiensis subsp. Aizawaisubsp. aizawai) Slurry, FlorBac® Slurry Biological Insecticide, DiPel® ES, DiPel® ES-NT Biological Insecticide Emulsifier, DiPel® (Worm Killer) Wettable Powder Biological Insecticide, Condor, Leap® ES Bacterial Disease Control Biological Insecticide Emulsifier, Biobit® HPWPII Biological Insecticide, VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wettable Powder, BioBit® HP Biological Insecticide Wettable Powder, DiPel® WDG Biological Insecticide, VBC -60236 Biological Insecticide Dry Flowable, Foray2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, ForayWG, ThyllombeetleGONE!® Biological Insecticide, JavelinWP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, JavelinWG, SAN420IWG, JavelinWG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW, Bonide Bacillus thuringiensis 'O', ChemsicoHPC-O Concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide® Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, or BTI Technical Powder BIOInsecticide.

[0127] In some embodiments of either of the two preceding aspects that can be combined with any of the preceding embodiments, the at least one pest is selected from the group consisting of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the seed is a seed of a plant selected from the group consisting of row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants, including ornamental flowers, shrubs, trees, groundcover plants, or turfgrass. In some embodiments of this aspect that can be combined with any of the preceding embodiments, the plant is a plant of any species of interest (e.g., commercially important cultivated crops, trees, and plants), including dicotyledons and monocotyledons. Examples of commercially important cultivated crops, trees, and plants include alfalfa (Medicago sativa), almonds (Prunus dulcis), apples (Malus x domestica), apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica), artichokes (Cynara cardunculus var. scolymus), asparagus (Asparagus officinalis), avocados (Persea americana), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), Brazil nuts (Bertholletia excelsa), cacao (Theobroma cacao), calamansi (Citrus x microcarpa), canola and rapeseed or oilseed rape (Brassica napus), Polish canola (Brassica rapa), and related cruciferous vegetables including broccoli, kale, cabbage, and turnip (Brassica carinata, B. juncea, B. oleracea, B.napus, B. nigra and B. rapa, and their hybrids), carnation (Dianthus caryophyllus), carrot (Daucus carota sativus), cashew (Anacardium occidentale), cassava (Manihot esculentum), celery (Apium graveolens), cherry (Prunus avium), chestnut (Castanea spp.), chickpea or garbanzo (Cicer arietinum), chicory (Cichorium intybus), chili peppers and capsicums (Capsicum annuum, C. frutescens, C. chinense, C. pubescens, C. baccatum), chrysanthemum (Chrysanthemum spp.), citron (Citrus medica), coconut (Cocos nucifera), coffee (Coffea arabica, Coffea Wild and cultivated coffee (Coffea spp.) including Coffea canephora and Coffea liberica, cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata and other Vigna spp.), broad bean (Vicia faba), cucumber (Cucumis sativus), redcurrants and gooseberries (Ribes spp.), dates (Phoenix dactylifera), duckweed (Lemnoideae), eggplant or aubergine (Solanum melongena), elderberry (Sambucus spp.), eucalyptus (Eucalyptus spp.), flax (Linum usitatissumum L.),), geranium (Pelargonium spp.), ginger (Zingiber officinale), ginseng (Panax spp.), grapefruit (Citrus x paradisi), grapes (Vitis spp.), including wine grapes (Vitis vinifera and their hybrids), guava (Psidium guajava), hazelnuts (Corylus avellana, Corylus spp.), hemp and cannabis (Cannabis sativa and Cannabis spp.), hops (Humulus lupulus), horseradish (Armoracia rusticana), iris (Iris spp.), jackfruit (Artocarpus heterophyllus), kiwifruit (Actinidia spp.), kumquat (Citrus japonica), lemon (Citrus limon), lentil (Lens culinaris), lettuce (Lactuca sativa), lime (Citrus spp.), lychee (Litchi chinensis), macadamia (Macadamia spp.), corn or corn (Zea mays L.), mandarin (Citrus reticulata), mango (Mangifera indica), mangosteen (Garcinia mangostana), melon (Cucumis melo), cereals (Setaria spp., Echinochloa spp., Eleusine spp., Panicum spp., Pennisetum spp.), oats (Avena sativa), oil palm (Ellis quineensis), okra (Abelmoschus esculentus), olive (Olea europaea), onion (Allium cepa) and other alliums (Allium spp.), orange (Citrus sinensis), papaya (Carica papaya), parsnip (Pastinaca sativa), passion fruit (Passiflora edulis, pecan (Carya illinoinensis), peaches and nectarines (Prunus persica), pears (Pyrus spp.), peas (Pisum sativum), peanuts (Arachis hypogaea), peonies (Paeonia spp.), persimmons (Diospyros kaki, Diospyros spp.), petunias (Petunia spp.), pineapples (Ananas comosus), pistachios (Pistacia vera), plantains (Musa spp.), plums (Prunus domestica), poinsettias (Euphorbia pulcherrima), pomelo (Citrus maxima), poplars (Populus spp.), potatoes (Solanum tuberosum), pumpkins and squash (Cucurbita pepo, C. maxima, C. moschata), quince (Cydonia oblonga), raspberry (Rubus idaeus, Rubus occidentalis, Rubus spp.), rhubarb (Rheum spp.), rice (Oryza sativa L.), rose (Rosa spp.), rubber (Hevea brasiliensis), rye (Secale cereale), safflower (Carthamus tinctorius L.), Satsuma mandarin (Citrus unshiu), sesame seeds (Sesame indium), sorghum (Sorghum bicolor), bitter orange (Citrus x aurantium), soursop (Annona muricata), soybean (Glycine max L.), strawberry (Fragaria spp., Fragaria x ananassa), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), sunflower (Helianthus annuus), sweet potato (Ipomoea batatas), tamarind (Tamarindus indica), tangerine (Citrus tangerina), tea (Camellia Examples of cultivated plants include (Ipomoea sinensis), tobacco (Nicotiana tabacum L.), tomatillo (Physalis philadelphica), tomato (Solanum lycopersicum or Lycopersicon esculentum), tulip (Tulipa spp.), walnut (Juglans spp. L.), watermelon (Citrulus lanatus), wheat (Triticum aestivum), and yam (Discorea spp.). Wild relatives of cultivated plants are also of interest.

[0128] A further aspect of the present disclosure includes a plantable composition comprising a seed and a coating covering the seed, the coating comprising a plurality of minicells comprising a first effective amount of first minicells comprising a first exogenous protein toxin comprising Pir, or a plurality of minicells comprising a second effective amount of second minicells comprising a second exogenous protein toxin comprising Cry, wherein the plurality of minicells comprising the second effective amount of second minicells comprising the second exogenous protein toxin comprising Cry, or the plurality of minicells comprising the first effective amount of first minicells comprising the first exogenous protein toxin comprising Pir, is applied after the composition is planted.

[0129] A further aspect of the present disclosure includes a plantable composition comprising a seed and a coating overlying the seed, the coating comprising a plurality of minicells comprising a first effective amount of an exogenous protein toxin comprising Pir or a second effective amount of a Bt-derived AI. A further embodiment of this aspect includes a plurality of minicells comprising a second effective amount of a Bt-derived AI or a first effective amount of an exogenous protein toxin comprising Pir, applied after the composition is planted.

[0130] Method for producing insecticidal composition Further aspects of the present disclosure include methods of making an insecticidal minicell mixture, comprising: a) providing first minicells generated from a first parent bacterium that contains at least one genetic mutation that causes an alteration in the cell division function of the parent bacterium and further comprises a first vector that contains a coding sequence for a first exogenous protein toxin comprising Pir; b) providing second minicells generated from a second parent bacterium that contains at least one genetic mutation that causes an alteration in the cell division function of the parent bacterium and further comprises a second vector that contains a coding sequence for a second exogenous protein toxin comprising Cry; and c) mixing a first effective amount of the first minicells and a second effective amount of the second minicells in a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are mixed at a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. The at least one genetic mutation in the parent bacterium can be a disruption of a z-ring inhibitory protein (e.g., minC or minD), a disruption of a z-ring inhibitory protein and a cell division topology specificity factor (e.g., minCDE), or overexpression of the septation machinery element FtsZ.

[0131] A further embodiment of the present disclosure includes a method of making an insecticidal minicell mixture, comprising: a) providing minicells generated from a first parent bacterium that contains at least one genetic mutation that results in altered cell division function of the parent bacterium and further contains a vector that includes a coding sequence for an exogenous protein toxin, including Pir; b) providing a Bt-derived AI; and c) mixing a first effective amount of minicells with a second effective amount of a Bt-derived AI at a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are mixed in a ratio of about 23: 1, 22: 1, 11: 1, 6: 1, 3: 1, 2: 1, or 1.5: 1. The at least one genetic mutation in the parent bacterium can be a disruption of a z-ring inhibitory protein (e.g., minC or minD), a disruption of a z-ring inhibitory protein and a cell division topology specificity factor (e.g., minCDE), or overexpression of the septation machinery component FtsZ.

[0132] An additional embodiment of the present disclosure includes a method of making an insecticidal minicell mixture, comprising the steps of: a) providing first minicells containing a first exogenous protein toxin comprising Pir; b) providing second minicells containing a second exogenous protein toxin comprising Cry; and c) mixing a first effective amount of the first minicells with a second effective amount in a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are combined in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1.

[0133] An additional embodiment of the present disclosure includes a method of making an insecticidal minicell mixture, comprising: a) providing minicells containing an exogenous protein toxin, including Pir; b) providing a Bt-derived AI; and c) mixing a first effective amount of minicells with a second effective amount of a Bt-derived AI in a ratio ranging from about 50:1 to 1:50. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are combined in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1.

[0134] Yet another aspect of the present disclosure includes a method for producing a wettable powder comprising the steps of: a) providing a first effective amount of first dry minicells comprising a first exogenous protein toxin comprising a Pir; b) a second effective amount of second dry minicells comprising a second exogenous protein toxin comprising a Cry; and c) mixing the first effective amount and the second effective amount in a ratio ranging from about 50:1 to 1:50, wherein the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to the pest. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are combined in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1.

[0135] Yet another aspect of the present disclosure includes a method of making a wettable powder, comprising the steps of: a) providing a first effective amount of first dried minicells comprising an exogenous protein toxin comprising Pir; b) a second effective amount of a dried Bt-derived AI; and c) mixing the first effective amount and the second effective amount in a ratio ranging from about 50:1 to 1:50, wherein the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to the pest. The ratio can be about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments of this aspect, the first effective amount and the second effective amount are combined in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:1.

[0136] In additional embodiments of any of the above methods of making an insecticidal composition, when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when the first effective amount or the second effective amount is administered to the pest. The synergistic effect allows for lower effective amounts of each active ingredient (e.g., exogenous protein toxin; Bt-derived AI) to be used while maintaining their effectiveness in agricultural applications.

[0137] How to control pests A further aspect of the present disclosure includes a method for controlling pests, comprising administering to the pests the composition of any one of the preceding embodiments. In some embodiments of this aspect, administering the composition to the pests includes an administration method selected from the group consisting of delivering the composition to the pests for ingestion; contacting the pests with the composition; delivering the composition to at least one habitat where the pests grow, thrive, reproduce, feed, or infest; or applying the composition to the plant. In other embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment. In further embodiments of this aspect that may be combined with any of the preceding embodiments, the composition is formulated as at least one of a ready-to-use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, or a drench application, a dust, an emulsifiable concentrate, a flowable concentrate, an intermediate formulation, a granular formulation, an impregnated material formulation, a pellet / tablet formulation, a soluble concentrate formulation, an industrial chemical formulation, a water-dispersible granular formulation, a water-soluble packaged formulation, or a wettable powder formulation. In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the control comprises at least one of a reduction in pest numbers, a reduction in pest weight, a reduction in physical damage caused by the pest, and an increase in pest mortality. In some embodiments of this aspect, the reduction in pest numbers, a reduction in pest weight, a reduction in physical damage caused by the pest, or an increase in pest mortality is compared to pests that are not administered the composition.In further embodiments of this aspect, the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, or at least an 80% reduction; and the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, or at least an 80% reduction. % reduction, the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction or at least an 80% reduction, and the increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40%, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, and at least a 65% increase, at least a 70% increase, at least a 75% increase or at least an 80% increase. In some embodiments of this aspect that may be combined with any of the preceding embodiments, control includes observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.In some embodiments of this aspect that may be combined with any of the preceding embodiments involving control of pests, the at least one pest is selected from the group of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), albatross moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, or Diptera species.

[0138] Pests of the present disclosure Pests of the present disclosure include, but are not limited to, pests from the orders Anoplura, Coleoptera, Diptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera, Orthoptera, Psocoptera, Isoptera, Blattaria, Siphonoptera, Thysanoptera, and Thysanura. Examples of pests of the order Anoplura include Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp., and Phylloxera spp.

[0139] Examples of pests from the order Coleoptera are Acalymma vittatum, Acanthoscehdes obtectus, Adoretus species, Agelastica alni, Agrilus species such as A. anxius, A. planipennis and A. sinuatus, Agriotes species such as A. fuscicollis, A. lineatus and A. obscurus, Alphitobius diaperinus, Amphimarus solstitialis, solstitialis, Anisandrus dispar, Anisoplia austriaca, Anobium punctatum, Anomala corpulenta, Anomala rufocuprea, Anoplophora species such as A. glabripennis, Anthonomus species such as A. eugenii, A. grandis, A. pomorum, Anthrenus species, Aphthona euphoridae euphoridae, Apion species, Apogonia species, Athous haemorrhoidalis, Atomaria species, e.g., A. linearis, A.linearis, Attagenus spp., Aulacophora femoralis, Blastophagus piniperda, Blitophaga undata, Bruchidius obtectus, Bruchus spp. such as B. lentis, B. pisorum and B. rufimanus, Byctiscus betulae, Callidiellum rufipenne, Callopistria floridensis, Callosobruchus chinensis, Cameraria auridera ohridella, Cassida nebulosa, Cerotoma trifurcata, Cetonia aurata, Ceuthorhynchus species such as C. assimilis and C. napi, Chaetocnema tibialis, Cleonus mendicus, Conoderus species such as C. vespertinus, Conotrachelus nenuphar, Cosmopolites species, Costelytra zealandica, Crioceris asparagi asparagi, Cryptolestes ferrugineus, Cryptorhynchus lapathi, Ctenicera species, e.g., C. destorqutldestructor, Curculio spp., Cylindrocopturus spp., Cyclocephala spp., Dactylispa balyi, Dectes texanus, Dermestes spp., Diabrotica spp. such as D. undecimpunctata, D. speciosa, D. longicornis, D. semipunctata and D. virgifera, Diaprepes abbreviates, Dichocrocis spp., Dicladispa armigera armigera, Diloboderus abderus, Diocalandra frumenti (Diocalandra stigmaticollis), Enaphalodes rufulus, Epilachna species such as E. varivestis and E. vigintioctomaculata, Epitrix species such as E. hirtipennis and E. simillaris,similaris, Eutheola humilis, Eutinobothrus brasiliensis, Faustinus cubae, Gibbium psylloides, Gnathocerus cornutus, Hellula undalis, Heteronychus arator, Hylamorpha elegans, Hylobius abietis, Hylotrupes bayurus bajulus, Hypera species such as H. brunneipennis and H. postica, Hypomeces squamosus, Hypothenemus species, Ips typographus, Lachnosterna consanguinea, Lasioderma serricorne serricorne, Latheticus oryzae, Lathridius spp., Lema spp. such as L. bilineata and L. melanopus, Leptinotarsa ​​spp. such as L. decemlineata, Leptispa pygmaea, Limonius californicus, Lissorhoptrus oryzophilus, Lixus spp., Luperodes spp., Lyctus spp. such as L. bruneus, Liogenys fuscus fuscus), Macrodactylus species, e.g., M. subspinosus,subspinosus, Maladera matrida, Megaplatypus mutates, Megascelis species, Melanotus communis, Meligethes species such as M. aeneus and Melolontha species such as M. hippocastani, M. melolontha, Metamasius hemipterus hemipterus, Microtheca spp., Migdolus spp. such as M. fryanus, Monochamus spp. such as M. alternatus, Naupactus xanthographus, Niptus hololeucus, Oberia brevis, Oemona hirta, Oryctes rhinoceros, Oryzaephilus surinamensis, Oryzaphagus oryzae, Otiorrhynchus sulcatus sulcatus, Otiorrhynchus ovatus, Otiorrhynchus sulcatus, Oulema melanopus, Oulema oryzae, Oxycetonia jucunda, Phaedon species such as P. brassicae and P. cochleariae, Phoracantha recurva, Phyllobius pyri, and Phyllopertha horticola. , Phyllophaga species such as P. helleri, Phyllotreta species such as P. chrysocephala, P. nemorum, P. striolata, P. vittula, Phyllopertha horticola, Popillia japonica, Premnotrypes species such as Psacothea hilaris, Psylliodes chrysocephala, Prostephanus torncatus truncates, Psylliodes spp., Ptinus spp., Pulga saltona, Rhizopertha dominica, Rhynchophorus spp. such as R. billineatus, R. ferrugineus, R. palmarum, R. phoenicis and R. vulneratus, Saperda candida, Scolytus schevyrewi, Scyphophorus acupunctatus, Sitona lineatus, Sitophilus species such as S. granaria, S. oryzae and S. zeamais, Sphenophorus species such as S. levis, Stegobium paniceum, Sternechus species such as S. subsignatus,subsignatus, Strophomorphus ctenotus, Symphyletes species, Tanymecus species, Tenebrio molitor, Tenebrioides mauretanicus, Tribolium species such as T. castaneum, Trogoderma species, Tychius species, Xylotrechus species such as X. pyrroderus, and Zabrus species such as Z. tenebrioides.

[0140] Examples of Diptera include Aedes species such as A. aegypti, A. albopictus, A. vexans, and Mexican fruit flies such as Anastrepha ludens, Anopheles species such as A. albimanus, A. crucians, A. freeborni, A. gambiae, A. leucosphyrus, A. maculipennis, A. minimus, A. quadrimaculatus and A. sinensis, Bactrocera invadens, Bibio hortulanus, Calliphora erythrocephala, Calliphora vicina, vicina, Ceratitis capitata, Chrysomyia species such as C. bezziana, C. hominivorax and C. macellaria, Chrysops atlanticus, Chrysops discalis, Chrysops silacea, Cochliomyia species such as C. hominivorax, Contarinia species such as C. sorghicola, Cordylobia anthropophaga, anthropophaga), Culex species such as C. nigripalpus, C. pipiens, C. quinquefasciatus, C. tarsalis and C. tritaeniorhynchus.tritaeniorhynchus, Culicoides furens, Culiseta inornata, Culiseta melanura, Cuterebra species, Dacus cucurbitae, Dacus oleae, Dasineura brassicae, Dasineura oxycoccana, Delia species such as D. antique, D. coarctata, D. platura and D. radicum, Dermatobia hominis hominis, Drosophila species such as D. suzukii, Fannia species such as F. canicularis, Gasterophilus species such as G. intestinalis, Geomyza tipunctata, Glossina species such as G. fuscipes, G. morsitans, G. palpalis and G. tachinoides, Horn flies (Haematobia irritans), Haplodiplosis equestris equestris, Hippelates spp., Hylemyia spp., for example H. platura, Hypoderma spp., for example H. lineata, Hyppobosca spp., Hydrellia philippina, Leptoconops torrens, Liriomyza spp., for example L. sativae and L. trifolii, Lucilia spp., for example L. caprina,caprina, L. cuprina and L. sericata, Lycoria pectoralis, Mansonia titillanus, Mayetiola spp. such as M. destructor, Musca spp. such as M. autumnalis and M. domestica, Muscina stabulans, Oestrus spp. such as O. ovis, Opomyza florum, Oscinella spp. such as O. frit, Orseolia oryzae oryzae, Pegomya hysocyami, Phlebotomus argentipes, Phorbia species such as P. antiqua, P. brassicae and P. coarctata, Phytomyza gymnostoma, Prosimulium mixtum, Psila rosae, Psorophora columbiae, Psorophora discala, discolor, Rhagoletis species such as R. cerasi, R. cingulate, R. indifferens, R. mendax and R. pomonella, Riverlia quadrifasciata, Sarcophaga species such as S. haemoroidalis,Examples of suitable plants include S. haemorrhoidalis, Simulium vittatum, Sitodiplosis mosellana, Stomoxys species such as S. calcitrans, Tabanus species such as T. atratus, T. bovinus, T. lineola, and T. similis, Tannia species such as Thecodiplosis japonensis, Tipula oleracea, Tipula paludosa, and Wohlfahrtia species.

[0141] Examples of pests from the order Hemiptera include Acizzia jamatonica, Acrosternum species, for example A. hilare, Acyrthosipon species, for example A. onobrychis and A. pisum, Adelges laricis, Adelges tsugae, Adelphocoris species, for example A. rapidus and A. superbus, Aeneolamia species, Agonoscena species, Aulacorthum solani, solani, Aleurocanthus woglumi, Aleurodes spp., Aleurodicus disperses, Aleurolobus barodensis, Aleurothrixus spp., Amrasca spp., Anasa tristis, Antestiopsis spp., Anuraphis cardui, Aonidiella spp., Aphanostigma piri, Aphidula nasturtii nasturtii, Aphis species such as A. craccivora, A. fabae, A. forbesi, A. gossypii, A. grossulariae, A. maidiradicis, A. pomi, A. sambuci, A. schneideri and A. spiraecola.spiraecola, Arboridia apicalis, Arilus critatus, Aspidiella spp., Aspidiotus spp., Atanus spp., Aulacaspis yasumatsui, Aulacorthum solani, Bactericera cockerelli (Paratrioza cockerelli), Bemisia spp., e.g., B. argentifolii and B. tabaci (Aleurodes tabaci), tabaci), Blissus species such as B. leucopterus, Brachycaudus species such as B. cardui, B. helichrysi, B. persicae and B. prunicola, Brachycolus species such as Brachycorynella asparagi, Brevicoryne brassicae, Cacopsylla species such as C. fulguralis, C. pyricola,pyricola (Psylla piri), Calligypona marginata, Calocoris species, Campylomma livida, Capitophorus horni, Carneocephala fulgida, Cavelerius species, Ceraplastes species, Ceratovacuna lanigera, Ceroplastes ceriferus, Cerosipha gossypii, Chaetosiphon fragaefoli fragaefolii, Chionaspis tegalensis, Chlorita onukii, Chromaphis juglandicola, Chrysomphalus ficus, Cicadulinambila, Cimex species such as C. hemipterus and C. lectularius, Coccomytilus halli, Coccus species such as C. hesperidum, C. pseudomagnorealum,pseudomagnoliarum, Corythucha arcuata, Creontiades dilutus, Cryptomyzus ribis, Chrysomphalus aonidum, Cryptomyzus ribis, Ctenarytaina spatulata, Cyrtopeltis notatus, Dalbulus species, Dasynus piperis, Dialeurodes species such as D. citrifolii, Dalbulus maidis, Diaphorina species such as D. citri, Diaspis species such as D. bromeliae, Dichelops furcatus, Diconocoris hewetti, Doralis species, Dreyfusia nordmannianae, Dreyfusia piceae, Drosicha species, Dysaphis species such as D. plantaginea, D. pyri and D. radicola, Dysaulacorthum pseudosolani. pseudosolani, Dysdercus species such as D. cingulatus and D. intermedius, Dysmicoccus species, Edessa species, Geocoris species, Empoasca species such as E. fabae and E. solana,solana, Epidiaspis leperii, Eriosoma species such as E. lanigerum and E. pyricola, Erythroneura species, Eurygaster species such as E. integriceps, Euscelis bilobatus, Euschistus species such as E. heros, E. impictiventris and E. servus, Fiorinia theae, Geococcus coffeae, Glycaspis brinblecombei brimblecombei, Halyomorpha spp., e.g., H. halys, Heliopeltis spp., Homalodisca vitripennis (=H. coagulata), Horcias nobilellus, Hyalopterus pruni, Hyperomyzus lactucae, Icerya spp., e.g., I. purchase, Idiocerus spp., Idioscopus spp., Laodelphax striatellus striatellus, Lecanium spp., Lecanoideus floccissimus, Lepidosaphes spp., e.g., L. ulmi, Leptocorisa spp., Leptoglossus phyllopus, Lipaphis erysimi, Lygus spp., e.g., L. hesperus, L. lineolaris,lineolaris and L. pratensis, Maconellicoccus hirsutus, Marchalina hellenica, Macropes excavatus, Macrosiphum species such as M. rosae, M. avenae and M. euphorbiae, Macrosteles quadrilineatus, Mahanarva fimbriolata, Megacopta cribraria, Megoura viciae, Melanaphis pyrarius pyrarius, Melanaphis sacchari, Melanocallis (=Tinocallis) caryaefoliae, Metcafiella species, Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis, etc. Myzocallis coryli, Murgantia species, Myzus species such as M. ascalonicus, M. cerasi, M. nicotianae, M. persicae and M. varians, Nasonovia ribisnigri, Neotoxoptera formosana formosana, Neomegalotomus spp., Nephotettix spp. such as N. malayanus, N. nigropictus, N. parvus and N. virescens, Nezara spp. such as N. viridula, Nilaparvata lugens, Nysius huttoni, Oebalus spp. such as O. pugnax, Oncometopia spp., Orthezia praelonga, Oxycaraenus hyalinipennis hyalinipennis, Parabemisia myricae, Parlatoria species, Parthenolecanium species such as P. corni and P. persicae, Pemphigus species such as P. bursarius and P. populivenae, Peregrinus maidis, Perkinsiella saccharicida, Phenacoccus species such as P. aceris and P. gossypii.gossypii, Phloeomyzus passerinii, Phorodon humuli, Phylloxera species such as P. devastatrix, Piesma quadrata, Piezodorus species such as P. guildinii, Pinnaspis aspidistrae, Planococcus species such as P. citri and P. ficus, Prosapia bicincta, Protopulvinaria pyriformis, Psallus seriatus, Pseudacysta persea, Pseudaulacaspis pentagona, Pseudococcus spp., e.g., P. comstocki, Psylla spp., e.g., P. mali, Pteromalus spp., Pulvinaria amygdali, Pyrilla spp., Quadraspidiotus spp., e.g., Q. perniciosus, Quesada gigas gigas, Rastrococcus spp., Reduvius senilis, Rhizoecus americanus, Rhodnius spp., Rhopalomyzus ascalonicus, Rhopalosiphum spp., e.g., R. pseudobrassicas, R. insertum, R. maidis and R. paji.padi, Sagatodes spp., Sahlbergella singularis, Saissetia spp., Sappaphis mala, Sappaphis mali, Scaptocoris spp., Scaphoides titanus, Schizaphis graminum, Schizoneura lanuginosa, Scotinophora spp., Selenaspidus articulatus, Sitobion avenae, Sogata spp., Sogatella furcifera, Solubea insularis, Spisstilus festinus (= Stictocephala festina), Stephanitis nashi, Stephanitis pyrioides, Stephanitis takeyai, Tenalapha malayensis, Tetraleurodes perseae, Therioaphis maculate), Thyanta spp. such as T. accerra and T. perditor, Tibraca spp., Tomasis spp., Toxoptera spp. such as T. aurantii, Trialeurodes spp. such as T. abutilonea, T. ricini and T. vaporariorum (T.vaporariorum, Triatoma species, Trioza species, Typhlocyba species, Unaspis species such as U. citri and U. yanonensis, and Viteus vitifolii.

[0142] Examples of pests in the Hymenoptera order include Acanthomyops interjectus, Athalia rosae, rosae, Atta species such as A. capiguara, A. cephalotes, A. cephalotes, A. laevigata, A. robusta, A. sexdens, A. texana, Bombus species, Brachymyrmex species, Camponotus species such as C. floridanus, C. pennsylvanicus and C. modoc, Cardiocondyla nuda, Cephus spp., Chalibion ​​spp., Crematogaster spp., Dasymutilla occidentalis, Diprion spp., Dolichovespula maculata, Dorymyrmex spp., Dryocosmus kuriphilus, Formica spp., Hoplocampa spp. e.g., H. minuta, H. testudinea, Iridomyrmex humilis, Lasius spp. e.g., L. niger, Argentine ant Linepithema humile, Liometopum species, Leptocybe invasa, Monomorium species, e.g., M. pharaonis,pharaonis, Neodiprion spp., Nylandria fulva, Pachycondyla chinensis, Paratrechina longicornis, Paravespula spp., for example, P. germanica, P. pennsylvanica and P. vulgaris, Pheidole spp., for example, P. megacephala, Pogonomyrmex spp., for example, P. barbatus, P. californicus, Polistes rubiginosa, rubiginosa, Prenolepis impairs, Pseudomyrmex gracilis, Schelipron species, Sirex cyaneus, Solenopsis species such as S. geminata, S. invicta, S. molesta, S. richteri and S. xyloni, Sphecius speciosus speciosus, Sphex species, Tapinoma species such as T. melanocephalum and T. sessile, Tetramorium species such as T. caespitum and T. bicarinatum, Vespa species such as V. crabro, Vespula species such as V. squamosal, Wasmannia auropunctata, and Xylocopa species.

[0143] Examples of pests from the order Isoptera include Calotermes flavicollis, Coptotermes species such as C. formosanus, C. gestroi and C. acinaciformis, Cornitermes cumulans, Cryptotermes species such as C. brevis and C. cavifrons, Globitermes sulphureus, sulfureus, Heterotermes species such as H. aureus, H. longiceps and H. tenuis, Leucotermes flavipes, Odontotermes species, Incisitermes species such as I. minor and I. Snyder, Marginitermes hubbardi hubbardi, Mastotermes species such as M. darwiniensis, Neocapritermes species such as N. opacus and N. parvus, Neotermes species, Procornitermes species, Reticuli termes species, such as R. hesperus, R. tibialis, R. speratus, R. flavipes, R. grassei, R. lucifugus, R. santonensis and R. virginicus, Termes natalensis, and Zootermopsis species, such as Z. angustichoris.angusticollis and Z. nevadensis.

[0144] Examples of pests from the order Lepidoptera include Achroia grisella, Acleris species such as A. fimbriana, A. gloverana, A. variana, Acrolepiopsis assectella, Acronicta major, Adoxophyes species such as A. cyrtosema and A. orana, Aedia leucomelas, leucomelas, Agrotis species such as A. exclamationis, A. fucosa, A. ipsilon, A. orthogoma, A. segetum and A. subterranea, Alabama argillacea, Aleurodicus dispersus, Alsophila pometaria, Ampelophaga rubiginosa, Amyelois transitella, Anacampsis sarcitella, Anagasta quaenia, kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia (= Thermesia) species, e.g., A. gemmatalis, Apamea species, Aproaerema modicella, Archips species, e.g., A. argyrospila, A. fuscocupreanus, A.A. rosana and A. xyloseanus, Argyresthia conjugella, Argyroploce species, Argyrotaenia species such as A. velutinana, Athetis mindara, Austroasca viridigrisea, Autographa gamma, Autographa nigrisigna, Barathra brassicae, Bedellia species, Bonagota salubricola, Borbo kinnara, cinnara, Bucculatrix thurberiella, Bupalus piniarius, Busseola species, Cacoecia species such as C. murinana and C. podana, Cactoblastis cactorum, Cadra cautella, Calingo braziliensis, Caloptilis theivora, Capua reticulana reticulana, Carposina species such as C. niponensis and C. sasakii, Cephus species such as Chaetocnema aridula, Cheimatobia brumata, Chilo species such as C. indicus, C. suppressalis, C. partellus, Choreutis pariana, Choristoneura species such as C. conflictana,conflictana, C. fumiferana, C. longicellana, C. murinana, C. occidentalis and C. rosaceana, Chrysodeixis (= Pseudoplusia) species such as C. eriosoma and C. includedens, Cirphis unipuncta, Clysia ambiguella, Cnaphalocerus species, Cnaphalocrocis medinalis, Cnephasia species, Cochylis hospes, Coleophora spp., Colias eurytheme, Conopomorpha spp., Conotrachelus spp., Copitarsia spp., Corcyra cephalonica, Crambus caliginosellus, Crambus toterellus teterrellus, Crocidosema (=Epinotia) aporema, Cydalima (=Diaphania) perspectalis, Cydia (=Carpocapsa) species such as C. pomonella and C. latiferreana, Dalaca noctuides, Datana integerrima, Dasychira pinicola, Dendrolimus species such as D. pini, D. spectabilis and D. sibiricus.sibiricus, Desmia funeralis, Diaphania species such as D. nitidalis and D. hyalinata, Diatraea grandiosella, Diatraea saccharalis, Diphthera festiva, Earias species such as E. insulana and E. vittella, Ecdytolopha aurantianu, Egira (=Xylomyges) curialis, Elasmopalpus lychnocerus lignosellus, Eldana saccharina, Endopiza viteana, Ennomos subsignaria, Eoreuma loftini, Ephestia species such as E. cautella, E. elutella and E. kuehniella, Epinotia aporema, Epiphyas postvittana, Erannis tiliaria, Erionota trachys thrax, Etiella species, Eulia species, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa species, Evetria bouliana, etc. Faronta albilinea, Feltia species such as F. subterranean, Galleria mellonella, Gracilaria species, Grapholita species such as G. funebrana, G. molesta and G. inopinata, Halysidota species, Harrisina americana, Hedylepta species, Helicoverpa species such as H. armigera (= Heliothis armigera) and H. zea (= Heliothis zea), Heliothis species, e.g., H. assulta, H. subflexa, H. virescens, Hellula species, e.g., H. undalis and H. logataris.rogatalis, Helocoverpa gelotopoeon, Hemileuca oliviae, Herpetogramma licarsisalis, Hibernia defoliaria, Hofmannophila pseudospretella, Homoeosoma electellum, Homona magnanima, Hypena scabra, Hyphantria cunea, Hyponomeuta padella, Hyponomeuta malinellus, Kakivoria flavofasciata, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Lamprosema indicata, Laspeyresia molesta, Leguminivora glycinivorella, Lerodea eufala, Leucinodes orbonalis, Leucoma salicis, Leucoptera species such as L. coffeella and L. citella.scitella, Leuminivora lycinivorella, Lithocolletis blancardella, Lithophane antennata, Llattia octo (= Amyna axis), Lobesia botrana, Lophocampa species, Loxagrotis albicosa albicosta, Loxostege species such as L. sticticalis and L. cereralis, Lymantria species such as L. dispar and L. monacha, peach leafminer Lyonetia clerkella, Lyonetia prunifolia prunifoliella, Malacosoma species such as M. americanum, M. californicum, M. constrictum and M. neustria, Mamestra species such as M. brassicae and M. configurata, Mamstra brassicae, Manduca species such as M. quinquemaculata and M. sexta, Marasmia species, Marmara species, Maruca testulalis, Megalopyge lanata lanata, Melanchra picta, Melanitis leda, Mocis species such as M. latipes and M. repanda.repanda, Monochroa fragariae, Mythimna separata, Nemapogon cloacella, Neoleucinodes elegantalis, Nepytia spp., Nymphula spp., Oiketicus spp., Omiodes indicata, Omphisa anastomosalis, Operophtera brumata, Orgyia pseudotsugata, Oria spp., Orthaga chilisalis thyrisalis, Ostrinia species such as O. nubilalis, Oulema oryzae, Paleacrita vernata, Panolis flammea, Parnara species such as Papaipema nebris, Papilio cresphontes, Paramyelois transitella, Paranthrene regalis, Paysandisia archon, Pectinophora species such as P. gossypiella, Peridroma saucia, Perileucoptera species such as P. coffeella, Phalera bucephala, Phryganidia californica, Phthorimaea species such as P. opelculella,operculella, Phyllocnistis citrella, Phyllonorycter species such as P. blancardella, P. crataegella, P. issikii and P. ringoniella, Pieris species such as P. brassicae, P. rapae and P. napi, Pilocrocis tripunctata, Plathypena scabra, Platynota species such as P. flavedana, P. idaeusalis and P. stultana, Platyptilia carduidactyla, Plebejus argus, Plodia interpunctella, Plusia species, Plutella maculipennis, Plutella xylostella, Pontia protodica, Prays species, Prodenia species, Proxenus lepigon lepigone, Pseudaletia species such as P. sequax and P. unipunctaunipuncta, Pyrausta nubilalis, Rachiplusia nu, Richia albicosta, Rhizobius ventralis, Rhyacionia frustrana, Sabulodes aegrotata, Schizura concinna, Schoenobius species, Schreckensteinia festaliella, Scirpophaga species such as S. incertulas and S. innotata, Scotia segementum segetum, Sesamia species such as S. inferens, Seudyra subflava, Sitotroga cerealella, Sparganothis pilleriana, Spironota recleaspis lechriaspis, S. ocellana, Spodoptera (=Lamphygma) species such as S. cosmoides, S. eridania, S. exigua, S. frugiperda, S. latisfascia, S. littoralis, S. litura and S. omithogalli, Stigmella species, Stomopteryx subsecivella, Strymon bazochii, Sylepta derogata derogata), Synanthedon species, e.g., S. exiciosa, S.exitiosa, Tecia solanivora, Telehin licus, Thaumatopoea pityocampa, Thaumatotibia (=Cryptophlebia) leucotr. eta, Thaumetopoea pityocampa, Thecla spp., Theresimima ampelophaga, Thyrinteina spp., Tildenia inconspicuella, Tinea spp. such as T. cloacella and T. pellionella, Tineola bisselliella, Tortrix spp. such as T. viridana, Trichophaga tapetum, tapetzella, Trichoplusia species such as T. ni, Tuta (=Scrobipalpula) absoluta, Udea species such as U. rubigalis, Virachola species, Yponomeuta padella, and Zeiraphera canadensis.

[0145] Examples of pests in the order Mallophaga include Damalinea species and Trichodectes species. Examples of pests from the order Orthoptera include Acheta domesticus, Calliptamus italicus, Chortoicetes terminifera, Ceuthophilus species, Diastrammena asynamora, Dociostaurus maroccanus, Gryllotalpa species such as G. africana and G. gryllotalpa, Gryllus species, Hieroglyphus daganensis, Kraussaria angulifera, Locusta species such as L. migratoria and L. pardalina, Melanoplus species such as M. bivittatus, M. femurrubrum, M. mexicanus, M. sanguinipes and M. spretus, Nomadacris septemfasciata, Oedaleus senegalensis, Scapteriscus species, Schistocerca species such as S. americana and S. gregaria.Examples of pests in the order Psocoptera include Liposcelis species. Examples of pests from the order Isoptera include Calotermes flavicollis, Coptotermes species such as C. formosanus, C. gestroi and C. acinaciformis, Cornitermes cumulans, Cryptotermes species such as C. brevis and C. cavifrons, Globitermes sulphureus, sulfureus, Heterotermes species such as H. aureus, H. longiceps and H. tenuis, Leucotermes flavipes, Odontotermes species, Incisitermes species such as I. minor and I. Snyder, Marginitermes hubbardi hubbardi, Mastotermes species such as M. darwiniensis, Neocapritermes species such as N. opacus and N. parvus, Neotermes species, Procornitermes species, Zootermopsis species such as Z. angusticollis and Z. nevadensis, Reticulitermes species such as R.These include R. hesperus, R. tibialis, R. speratus, R. flavipes, R. grassei, R. lucifugus, R. santonensis, R. virginicus, and Termes natalensis. Examples of pests of the order Blatta species, for example B. orientalis, B. lateralis, Blattella species, for example B. asahinae, B. germanica, Leucophaea maderae, Panchlora nivea, Periplaneta species, for example P. americana, P. australasiae, P. brunnea, P. fuligginosa and P. japonica, Supella longipalpa, Parcoblatta pennsylvanica pennsylvanica, Eurycotis floridana, and Pycnoscelus surinamensis.

[0146] Examples of pests from the order Siphonoptera include Cediopsylla simplex, Ceratophyllus species, Ctenocephalides species such as C. felis and C. canis, Xenopsylla cheopis, Pulex irritans, Trichodectes canis, Tunga penetrans, and Nosopsyllus fasciatus.Examples of pests from the order Thysanoptera include Baliothrips biformis, Dichromothrips corbetti, Dichromothrips ssp., Echinothrips americanus, Enneothrips flavens, Frankliniella species such as F. fusca, F. occidentalis and F. tritici, Heliothrips species, Hercinothrips femoralis, femoralis, Kakothrips spp., Microcephalothrips abdominalis, Neohydatothrips samayunkur, Pezothrips kellyanus, Rhipiphorothrips cruentatus, Scirtothrips spp., e.g., S. citri, S. dorsalis and S. perseae, Stenchaetothrips spp., Taeniothrips cardamoni, Taeniothrips inconsequence inconsequens), Thrips species such as T. imagines, T. hawaiiensis, T. oryzae, T. palmi, T. parvispinus, and T. tabaci.Examples of pests in the order Thysanura include Lepisma saccharina, Ctenolepisma urbana, and Thermobia domestica.

[0147] Additional pests of the present disclosure include non-insect pests. The mixtures and methods of the present disclosure can be used for these non-insect pests in the same way as for insect pests. Non-insect pests of the present disclosure include, but are not limited to, Arachnida pests, such as Acari pests, such as Argasidae, Ixodidae, and Sarcoptidae, such as Amblyomma species (e.g., A. americanum, A. variegatum, A. maculatum), Argas species (e.g., A. persic), Boophilus species (e.g., B. annulatus, B. decoloratus), and the like. us, B. microplus, Dermacentor species such as D. silvarum, D. andersoni and D. variabilis, Hyalomma species such as H. truncatum, Ixodes species such as I. ricinus, I. rubicundus, I. scapularis, I. holocyclus and I. pacificus, Rhipicephalus sanguineus, Ornithodorus species such as O. moubata, O. hermsi and O. turicata, Ornithonyssus bacoti, Otobius megnini, Dermanyssus gallinaegallinae, Psoroptes species such as P. ovis, Rhipicephalus species such as R. sanguineus, R. appendiculatus, Rhipicephalus evertsi evertsi, Rhizoglyphus species, Sarcoptes species such as S. scabiei, from the family Eriophyidae, for example Aceria species such as A. sheldoni, A. anthocoptes, Acallitus species, Aculops species such as A. lycopersici and A. pelekassi, Aculus species such as A. schlechtendali, Colomerus vitis, Epitrimerus pyri, Phyllocoptruta oleivora, oleivora, Eriophytes ribis and Eriophyes species, for example Eriophyes sheldoni, from the family Tarsonemidae, for example Hemitarsonemus species, Phytonemus pallidus and Polyphagotarsonemus latus, from the genus Stenotarsonemus species, Steneotarsonemus spinkispinki, from the family Tenuipalpidae, for example, Brevipalpus spp., for example, B. phoenicis, from the family Tetranychidae, for example, Eotetranychus spp., Eutetranychus spp., Oligonychus spp., Petrobia lattens, latens, Tetranychus species such as T. cinnabarinus, T. evansi, T. kanzawai, T. pacificus, T. phaseulus, T. telarius and T. urticae, Bryobia praetiosa, Panonychus species such as P. ulmi and P. citri, Metatetranychus species, Oligonychus species such as O. pratensis, O. perseae, Vasates lycopersici lycopersici, Raoiella indica, from the family Carpoglyphidae, for example Carpoglyphus spp., Penthaleidae spp., for example Halotydeus destructor, from the family Demodicidae and species thereof, for example Demodex spp., from the family Trombicidea, for example Trombicula spp., from the family Macronyssidae, for example Ornothonyssus spp., from the family Pyemotidae, for example Pyemotes tritici, Tyrophagus putresentiaeputrescentiae, from the family Acaridae, for example Acarus siro, from the order Araneida, for example Latrodectus mactans, Tegenaria agrestis, the genera Chiracanthium spp., Lycosa spp., Achaearanea tepidariorum and Loxosceles reclusa, pests from the class Chilopoda, for example Geophilus spp., Scutigera spp., for example Scutigera coleoptrata coleoptrata, Diplopoda pests such as Blaniulus guttulatus, Julus spp., Narceus spp., Symphyla pests such as Scutigerella immaculata, Isopoda pests such as Armadillidium vulgare, Oniscus asellus, Porcellio scaber.

[0148] definition As used herein, the term "Bt-derived active ingredient" or "Bt-derived AI" means at least one of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxin. Bacillus thuringiensis is classified into the following strains: Bacillus thuringiensis subsp. kurstaki strain SA-11, Bacillus thuringiensis subsp. kurstaki strain SA-12, Bacillus thuringiensis subsp. kurstaki strain ABTS-351, Bacillus thuringiensis subsp. kurstaki strain BMP123, Bacillus thuringiensis subsp. kurstaki strain EG2348 ...EG2348, Bacillus thuringiensis subsp. kurstaki strain EG2348, Bacillus thuringiensis subsp. kurstaki strain EG2348, Bacillus thuringiensis subsp. kurstaki strain EG2348, Bacillus thuringiensis subsp. kurstaki strain subsp. kurstaki) strain EG2371, Bacillus thuringiensis subsp. kurstaki) strain EG7841, Bacillus thuringiensis subsp. kurstaki) strain EG7826, Bacillus thuringiensis subsp. kurstaki) strain EVB-113-19, Bacillus thuringiensis subsp. kurstaki) strain VBTS2546, Bacillus thuringiensis subsp. israelensis, Bacillus thuringiensis subsp.israelensis strain AM65-52, Bacillus thuringiensis subsp. israelensis strain BMP144, Bacillus thuringiensis subsp. israelensis strain EG2215, Bacillus thuringiensis subsp. tenebrionis, Bacillus thuringiensis subsp. tenebrionis strain SA-10, Bacillus thuringiensis subsp. tenebrionis strain NB-176, Bacillus thuringiensis var. aizawai aizawai), Bacillus thuringiensis var. aizawai strain GC-91, Bacillus thuringiensis var. aizawai strain ABTS1857, Bacillus thuringiensis var. aizawai strain NB200, Bacillus thuringiensis subsp. Galleriea, or Bacillus thuringiensis subsp. galleriea strain SDS-502. Bt-derived AIs can be formulated as dusts, emulsifiable concentrates, flowable concentrates, intermediate formulations, granules, impregnated materials, pellets / tablets, soluble concentrates, ready-to-use solutions, industrial chemicals, water-dispersible granules, water-soluble packages, or wettable powders. In end-use applications, Bt-derived biological control products (containing Bt-derived AIs) can contain about 0.0005-100% Bt-derived AIs and about 0-99.9995% inert materials. Specifically, end-use products contain about 0.0005%, 0.001%,0.0012%,0.006%,0.0183%,0.049%,0.064%,0.1%,0.1466%,0.2%,0.41%,0.4365%,0.47%,0.49%,0.7%,0.72%,1%,1.16%,1.6%,1.7%,1.94%,2.15%,2.19%,2 .3%, 2.5%, 2.6%, 2.8%, 2.86%, 3%, 3.5%, 4.5%, 4.95%, 5%, 5.35%, 5.71%, 6.07%, 6.38%, 6.4%, 6.6%, 7%, 8%, 9%, 9.5%, 9.83%, 10%, 10.3%, 10.31%, 10.4%, 10.8%, 11.61% ,12%,12.3%,12.65%,12.74%,14%,14.32%,14.4%,14.49%,14.6%,15%,16%,17%,17.19%,17.6%,17.8%,18%,18.44%,19%,21%,22%,23.7%,24.5%,25.5%,34.11%,3 It may contain 7.4%, 40%, 43%, 45%, 48.1%, 50%, 54%, 58.2%, 63.5%, 67%, 70%, 76.2%, 76.5%, 80%, 83%, 84%, 85%, 89%, 90%, 91%, 92.6%, 98%, 98.35%, 99%, 99.3% or 100% Bt-derived AIs.

[0149] As used herein, the term "insecticidal toxin" refers to a peptide or polypeptide that is capable of controlling pests. Insecticidal toxins of the present disclosure include Cry and Pir toxins.

[0150] As used herein, the term "effective amount" means a pest-controlling amount.

[0151] As used herein, the term "control" refers to the killing, reduction in numbers, and / or reduction in growth, feeding, or normal physiological development of any or all life stages of a plant pest, and / or a reduction in the effects of plant pest infestation and / or infestation. An effective amount is an amount that can significantly reduce pest growth, feeding, root penetration, root maturation, and / or general normal physiological development, and / or symptoms resulting from plant pest infestation. In some embodiments, symptoms resulting from plant pest infestation and / or the number of plant pest particles are reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to an untreated control.

[0152] With respect to pests, the term "control," as used herein, means the death, reduction in numbers, and / or reduction in growth, feeding, or normal physiological development of any or all life stages of insects, the reduction in the effects of insect feeding and / or infestation, the resistance of plants to insect feeding and / or infestation, the resistance of plants to the effects of insect feeding and / or infestation, or any combination thereof. An effective amount is an amount that can significantly reduce insect survival, growth, feeding, infestation, and / or general normal physiological development, and symptoms resulting from insect feeding and / or infestation. In some embodiments, symptoms and / or insects are reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to untreated controls.

[0153] The term "minicell" or ADAS refers to a chromosomal, non-replicating, enclosed membrane system comprising at least one membrane and having an interior volume suitable for containing cargo (e.g., one or more of a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immunomodulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP)). Minicells are the product of aberrant cell division and are chromosomal cells that contain RNA and protein but little or no chromosomal DNA. Minicells are capable of plasmid-induced synthesis. Minicells are preferably derived from parent bacterial cells (e.g., gram-negative or gram-positive bacterial cells) using, for example, genetic manipulation of the parent cell to disrupt the parent cell's cell division machinery. In some embodiments, minicells can include one or more endogenous or heterologous features of the surface of a parent cell, e.g., a cell wall, a cell wall modification, a flagellum or pili, and / or one or more endogenous or heterologous features of the interior volume of the parent cell, e.g., a nucleic acid, a plasmid, a protein, a small molecule, the transcription machinery, or the translation machinery. In other embodiments, minicells can lack one or more characteristics of the parent cell. In still other embodiments, minicells can be equipped with or modified with features not included in the parent cell.

[0154] As used herein, the term "parent bacterial cell" is used interchangeably with "chassis" and refers to a cell (e.g., a Gram-negative or Gram-positive bacterial cell) from which minicells are derived. Parent bacterial cells are typically viable bacterial cells. The term "viable bacterial cell" refers to a bacterial cell that contains a genome and is capable of cell division. The parent bacterial cell may be from any of the following genera: Escherichia, Acinetobacter, Agrobacterium, Anabaena, Anaplasma, Aquifex, Azoarcus, Azospirillum, Azotobacter, Bartonella, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Coxiella, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter er), Legionella, Magnetospirillum, Mesorhizobium, Methylobacterium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium,Photorhabdus, Phyllobacterium, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia thia), Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Exiguobacterium Exiguobacterium, Geobacillus, Lactobacillus, Listeria, Leuconostoc, Moorella, Oceanobacillus, Rhizobium, Rickettsia, Staphylococcus, Streptococcus,Symbiobacterium and Thermoanaerobacter. The parent bacterial cell contains at least one genetic mutation that causes an alteration in the cell division function of the parent bacterium.

[0155] A composition or preparation that is "substantially free" of parental and / or live bacterial cells is defined herein as a composition that has 500 or fewer colony forming units (CFU) per mL, e.g., 400, 300, 200, 150, 100 or fewer. A composition that is substantially free of parental or live bacterial cells may contain less than 50 CFU / mL, less than 25 CFU / mL, less than 10 CFU / mL, less than 5 CFU / mL, less than 1 CFU / mL, less than 0.1 CFU / mL, or less than 0.001 CFU / mL.

[0156] The term "cell division topology specificity factor" refers to a component of the cell division machinery in bacterial species that is involved in determining the site of septation and functions by restricting the location of other components of the cell division machinery, such as by restricting the location of one or more Z-ring inhibitory proteins. An exemplary cell division topology specificity factor is minE, which was first discovered in Escherichia coli and has since been identified in a wide range of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). minE functions by restricting the Z-ring inhibitory proteins minC and minD to the cell poles. A second exemplary cell division topology specificity factor is DivIVA, first discovered in Bacillus subtilis (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005).

[0157] The term "Z-ring inhibitory protein" refers to a component of the cell division machinery in bacterial species that is involved in determining the site of septation and functions by inhibiting the formation of a stable FtsZ ring or by anchoring such components to the membrane. The localization of Z-ring inhibitory proteins can be regulated by cell division topology specificity factors, such as MinE and DivIVA. Exemplary Z-ring inhibitory proteins include minC and minD, which were first discovered in E. coli and have since been identified in a wide range of Gram-negative and Gram-positive bacterial species (Rothfield et al., Nature Reviews Microbiology, 3:959-968, 2005). In E. coli and other species, minC, minD, and minE reside at the same locus, which may be referred to as the "min operon," the minCDE operon, or the min or minCDE locus. The term "Δmin" refers to disruption of the minCDE operon.

[0158] As used herein, the term "decreased level or activity of a cell topology-specific factor" refers to an overall 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more decrease in the level or activity of a cell topology-specific factor (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) as detected by standard methods, compared to the level in a reference sample (e.g., a minicell produced from a wild-type cell or a cell harboring a wild-type minCDE operon or a wild-type divIVA gene), a reference cell (e.g., a wild-type cell or a cell harboring a wild-type minC, minD, minE, divIVA, or minCDE gene or operon), a control sample, or a control cell. In some embodiments, a decrease in level or activity refers to a decrease in the level or activity in a sample of at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold compared to the level or activity of the cell division topology specificity factor in a reference sample, reference cell, control sample, or control cell.

[0159] As used herein, the term "endogenous type 3 secretion system" or "endogenous T3SS" refers to a T3SS present in a cell (e.g., a parent cell) or a minicell derived therefrom that is naturally encoded by the cell (e.g., encoded by a wild-type version of the cell). The T3SS may be expressed by an endogenous gene of the cell and / or may be encoded and expressed by a synthetic construct in the cell. The expression or abundance of an endogenous T3SS can be increased, for example, by adding a moiety that increases the abundance of the T3SS (e.g., a transcriptional activator of the T3SS) or by reducing or removing a negative regulator of T3SS expression.

[0160] As used herein, the term "heterologous type 3 secretion system" or "heterologous T3SS" refers to a T3SS present in a cell (e.g., a parent cell) or a minicell derived therefrom that is not naturally encoded by the cell (e.g., not encoded by the wild-type version of the cell).

[0161] As used herein, an "endogenous effector" of the secretion apparatus (e.g., a T3SS, T4SS, or T6SS) is a portion (e.g., a protein or polypeptide) that is naturally encoded by the cell from which the secretion apparatus (e.g., a T3SS) is derived (e.g., encoded by a wild-type version of the cell) and that can be secreted by the secretion apparatus. The secretion apparatus and one or more of its endogenous effectors can be expressed in the cell in which they naturally occur, or can be heterologously expressed, e.g., expressed by a cell that does not naturally encode the endogenous effector or secretion system.

[0162] As used herein, an effector that is heterologous with respect to the secretion apparatus ("heterologous effector") is a moiety (e.g., a protein or polypeptide) that is not naturally encoded by the cell from which the secretion apparatus (e.g., T3SS) is derived (e.g., not encoded by a wild-type version of the cell) and that can be secreted by the secretion system of the cell from which the heterologous effector is derived. The effector can be secreted by the secretion apparatus to which it is heterologous, or can be engineered such that it is secreted by the secretion apparatus to which it is heterologous. In some embodiments, the heterologous effector is an effector of a T4SS or T6SS that is secreted by a T3SS.

[0163] As used herein, the term "heterologous" means not naturally occurring in a cell or composition. In some embodiments, "heterologous" refers to a molecule, e.g., a cargo or payload (e.g., a nucleic acid such as a polypeptide, protein-encoding RNA or tRNA, or a small molecule) or structure (e.g., a plasmid or gene editing system) that is not naturally found in the ADAS or parent bacterium (e.g., a gram-negative or gram-positive bacterial cell) in which it is produced.

[0164] As used herein, the term "genes involved in DNA repair" refers to genes encoding polypeptides that play a role in cellular processes that are set up after DNA damage occurs in cells. DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can then disrupt the regular helical structure of the molecule by introducing unnatural chemical bonds or bulky adducts that are incompatible with the standard double helix. There are several types of DNA damage that can occur through normal cellular processes or environmental exposure of cells to DNA-damaging agents. DNA bases can be damaged by (1) oxidation processes, (2) base alkylation, (3) base loss caused by base hydrolysis, (4) bulky adduct formation, (5) DNA cross-linking, and (6) DNA strand breaks, including single- and double-strand breaks.

[0165] DNA mismatch repair (MMR) is a highly conserved DNA repair system that contributes significantly to maintaining genome stability by correcting mismatched base pairs and small modifications of DNA bases, such as alkylation. Mismatched base pairs are primarily caused by replication errors, but can also arise from other biological processes. In Escherichia coli (E. coli), DNA methylation is a common post-replicative modification. In the mismatched DNA repair system, strand discrimination is achieved, for example, by nicking endonucleases. In E. coli, MutH nicks the unmethylated strand of the duplex to create an entry point for excision. After excision of the damaged or mismatched strand, helicases (e.g., UvrD) and exonucleases (e.g., ExoI) excise a short stretch of nucleotides from the damaged strand. DNA pol III or DNA pol δ fills the gap, and DNA ligase repairs the backbone. Furthermore, RecJ, ExoI, ExoVII, and ExoX are exonucleases involved in this process. RecA is required for homologous recombination and catalyzes the ATP-driven homologous pairing and strand exchange of DNA molecules required for DNA recombinational repair. Therefore, MutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD, and DNA polymerase III are exemplary genes involved in DNA repair.

[0166] As referred to herein, a T7 family polymerase refers to a member of the single-subunit DNA-dependent RNAP (ssRNAP) family. In some embodiments, the T7 family polymerase is T7, T3, K11, or SP6 polymerase.

[0167] T7 polymerase is commonly used to transcribe DNA cloned into a vector containing a gene under the control of two different promoters in opposing directions. In some embodiments, an expression vector incorporating a T7 promoter and a gene of interest downstream of this promoter is used. In some embodiments, an expression vector containing the T7 promoter is transformed into one of several related strains of E. coli, and the strain name is given the extension "(DE3)". E. coli naturally contains a gene encoding T7 RNA polymerase. T7 RNA polymerase is responsible for initiating transcription at the T7 promoter. To initiate transcription, an inducer binds to the lac repressor, thereby preventing it from inhibiting gene expression of the T7 gene. The T7 gene is then typically transcribed to produce T7 RNA polymerase. T7 RNA polymerase then binds to the T7 promoter on the expression vector, thereby initiating transcription of the downstream gene of interest. In some embodiments, IPTG is used as an inducer in this process.

[0168] The term "about" as used herein refers to a normal error range for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0169] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a reference to a "minicell" is a reference to one to many minicells, such as a particle concentration, and includes equivalents thereof known to those skilled in the art, and so forth.

[0170] It is understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments. [Example]

[0171] The presently disclosed subject matter will be more fully understood by reference to the following examples, which are offered by way of illustration and not by way of limitation.

[0172] Example 1: Production of Minicells and Insecticidal Toxins This example demonstrates that minicells carrying and expressing insecticidal toxins can be produced from parent bacterial cells, e.g., E. coli strains, by various genetic alterations. In this example, disruption of one or more genes involved in regulating parent cell division functions, e.g., disruption of the z-ring inhibitor protein and cell division topology specificity factor (e.g., minCDE), allows the production of toxin-bearing minicells (listed in Table 1).

[0173] Materials and Methods Generation of minicells by deletion of the minCDE operon In the absence of the min system, proper localization of the septum machinery component FtsZ is dysregulated. This leads to the formation of closely spaced cell division planes, generating minicells. Disruption of the min operon was achieved by lambda Red recombination (Datsenko and Wanner, PNAS, 97(12):6640-6645, 2000). Briefly, the camR resistance cassette was amplified from pKD3 by standard PCR using primers encoding 40 bp of homology upstream and downstream of the min operon. The purified PCR product was then transformed by electroporation into E. coli cells harboring pKD46, which encodes the lambda Red system. Mutants were then selected by plating cells on LB plates containing 35 μg / mL chloramphenicol at 37°C, and min-disrupted mutants were verified by PCR.

[0174] To remove the antibiotic marker, cells were first transformed with the pCP20 plasmid and plated on LB plates containing 100 μg / mL carbenicillin at 30°C. Individual colonies from these plates were inoculated onto fresh LB plates, and the cells were grown at 42°C to allow flippase expression and loss of the pCP20 plasmid. Individual colonies were patched onto selective and non-selective LB plates to confirm antibiotic resistance and loss of the pCP20 plasmid. The resulting strain contained an FRT scar at the min locus. This strain was then grown in LB and frozen as a glycerol stock.

[0175] Minicell isolation and characterization To generate minicells, a seed culture was grown overnight from a glycerol stock in LB. The next day, the seed culture was diluted 1:200 in LB and grown at 37°C. The following day, the culture was processed to collect minicells generated overnight after differential centrifugation. Briefly, the bacterial culture was diluted to OD = 10 and centrifuged in a 1-liter bottle at 4000 × g (Sorval Lynx 6000) using the slowest acceleration speed for 40 minutes. The minicell-rich supernatant was then centrifuged at 17,000 g for 1 hour to pellet the minicells. The resulting pellet was then resuspended in 50 mL of fresh LB containing 200 μg / mL ceftriaxone and 20 μg / mL ciprofloxacin, and the culture was placed at 37°C for 2 hours to remove any remaining parent bacteria. The solution was centrifuged at 4,000 × g for 15 minutes in a swinging bucket rotor (Beckman Coulter) to remove dead parent bacterial cells and large debris. The minicells were then pelleted at 20,000 × g (Sorval Lynx 6000) for 20 minutes and resuspended in an equal volume of 0.2 μm-filtered PBS. This process was repeated for a total of two washes, and the resulting minicell pellet was resuspended in 0.2 μm-filtered PBS to a final volume of 10 mL.

[0176] Isolated minicells were examined by microscopy, particle size and distribution, and concentration, which was measured by counting with a Spectradyne nCS1. Particle concentrations of greater than 1e10 / liter, 1e11 / liter, and 1e12 / liter were collected from a final volume of 10 mL (average size 450 nm).

[0177] Generation of minicells containing exogenous insecticidal toxins Additionally, the gene sequences of insecticidal proteins (Table 1 below), such as the Cry1Ac toxin from Bacillus thuringensis or the PirAB toxin from Photorhabdus luminescens, were cloned into expression vectors. The vectors contained CloDF or pMB1 origins of replication behind an inducible promoter (e.g., Ptac, Ptet, or PT7) or a constitutive promoter. Expression of the insecticidal proteins was induced by aTc or IPTG at an OD600 of 0.5, and the proteins were produced and loaded into minicells. Isolation and characterization were performed as described above.

[0178] Protein concentrations in each isolated minicell sample were quantified by Western blot. 10, 1, and 0.1 μL of minicells and a standard curve of purified BAP-FLAG (Sigma: P7457) at 20, 10, 5, and 2.5 ng or Cry1Ac (Sino Biological: 12008-A07E) at 100, 50, 25, and 12.5 ng were run on a 4-12% BIS-Tris SDS gel (Thermo Fisher: NW04125BOX), transferred to nitrocellulose using iBlot (Thermo Fisher), and blotted with rabbit anti-FLAG (Sigma: F7425) or anti-Cry1A antibody (Abcam: ab51586) and goat anti-rabbit HRP conjugate (Sigma: AP307P). Western blots were imaged using iBright (Thermo Fisher), and band intensities were quantified using ImageJ. For protein concentrations, see Tables 2A-2B below.

[0179] result Table 1 below lists the names of minicells that load and express insecticidal toxins, the insecticidal toxins, the parental cells, and the vectors that load and express multiple insecticidal toxins in E. coli minicell strain chassis 1. Minicells AD1, AD2, and AD3 were tested as described in Example 2. Minicells AD4 through AD14 are expected, based on structural similarities, to exhibit similar levels of Pir and Cry activity as the tested minicells AD1, AD2, and AD3. Minicells AD1 and AD2 were tested as described in Example 3. Minicells AD3 through AD6 are expected to exhibit similar levels of Pir activity as the tested minicells AD1 and AD2.

[0180] [Table 1]

[0181] [Table 2]

[0182] Example 2: Synergistic effect of minicell PirAB and minicell Cry1Ac against the lepidopteran diamondback moth (DBM) This example demonstrates the unexpected synergistic effect of a mixture of minicells expressing the insecticidal toxins PirAB and Cry1Ac in a feeding assay using diamondback moth (DBM).

[0183] Materials and Methods Mini cell installed Minicells expressing PirAB and Cry1AC were used in the assay, and these minicells corresponded to AD1, AD2, and AD3 in Table 1 above.

[0184] Artificial diet feeding assay The synergistic effect of PirAB and Cry1Ac was evaluated using an artificial diet feeding assay. Diamondback moth (DBM) eggs (purchased from Benzon Research) were placed in a container filled with common noctuid artificial diet (purchased as a dry powder from Southland Products Inc.). Water was boiled at 80°C, and 33.4 g of diet was mixed per 200 ml of water. The mixture was then cooled to 70°C and 1.07 ml of linseed oil was added per 200 ml of water. The diet was poured into a container (Sistema KLIP IT 33.8 oz container, Amazon catalog number B00284AG7S) and allowed to cool and solidify.

[0185] The eggs in the diet were incubated at 25°C for 4 days until the last first-instar larvae emerged. The day before the start of the feeding assay, 48-well plates were prepared and each well was filled with 0.38 ml of the same artificial diet. On the day of the feeding assay, solutions containing the active substances were prepared at the application rates shown in Tables 2A-2B.

[0186] Table 2A shows the results of the first setup (Experiment 1), in which the combination of AD1 and AD2 (see Table 1 above) was tested. Table 2B shows the results of the second setup (Experiment 2), in which the combination of AD3 and AD2 (see Table 1 above) was tested. Thirty microliters of these solutions were applied per well to 48-well plates containing diet. After the solutions dried, late first-instar DBM larvae were placed, one per well, and the plates were sealed with Breathe Easier sealing film from Diversified Biotech to allow gas exchange. The plates were then placed in an incubator at 25°C with a 16-hour light, 8-hour dark cycle. After three days, each plate was opened, and the number of live and dead larvae in each plate was recorded to assess mortality.

[0187] result Mortality data for the solutions tested and their application rates are shown in Tables 2A-2B below. Reported values ​​represent percent mortality. The Colby formula was used to determine the expected insecticidal activity of the mixtures (Colby, SR (1967). Calculating Synergistic and Antagonistic Responses of Herbicide Combinations. Weeds, 15(1), 20-22. doi.org / 10.2307 / 4041058). Briefly, the following formula was used to calculate the expected activity of a mixture containing two active proteins, A and B: Expected value = A + B - (A × B / 100) (1) where A is the observed efficacy of active ingredient A at the same concentration as used in the mixture, and B is the observed efficacy of active ingredient B at the same concentration as used in the mixture.

[0188] Tables 2A-2B below show the results of synergy experiments using a mixture of PirAB-containing minicells and Cry1Ac-containing minicells. In both tables, the values ​​in parentheses indicate the efficacy (% mortality) of that AD alone at that particular concentration.

[0189] [Table 3]

[0190] [Table 4]

[0191] As can be seen from Tables 2A-2B, the observed mortality of the combination exceeded the predicted mortality of the combination at almost all concentrations. In some cases, such as 0.44 ng / μl AD1 (PirAB) and 0.154 ng / μl AD2 (Cry1A) (approximately a 3:1 ratio), the observed mortality was approximately twice the predicted mortality (Table 2A). Thus, these results demonstrate that the minicell-PirAB and minicell-Cry1Ac mixtures have a synergistic effect on DBM.

[0192] Example 3: Synergistic effect of minicell PirAB and Bt-derived AI against Bt-resistant diamondback moth (DBM) This example demonstrates the unexpected synergistic effect of a mixture of minicells expressing the insecticidal toxin PirAB and a Bt-derived AI (obtained in this example from Thuricide®) in a feeding assay using Bt-resistant diamondback moth (DBM). The Thuricide® insecticide used in this example contained 15% Bt-derived AI.

[0193] Materials and Methods On-board minicell and mixture Minicells expressing PirAB were used in the assay. These minicells corresponded to AD1 and AD2 in Table 1 above. The minicells were then mixed with Bt-derived AIs (obtained from Thuricide®) as described in Tables 3A-3B below.

[0194] Artificial diet feeding assay The synergistic effect of PirAB-expressing minicells and Bt-derived AIs was evaluated using an artificial diet feeding assay. Bt-resistant diamondback moth (DBM) eggs (purchased from Benzon Research) were placed in a container filled with common noctuid artificial diet (purchased as a dry powder from Southland Products Inc.). Water was boiled at 80°C, and 33.4 g of diet was mixed per 200 ml of water. The mixture was then cooled to 70°C and 1.07 ml of linseed oil was added per 200 ml of water. The diet was poured into a container (Sistema KLIP IT 33.8 oz container, Amazon catalog number B00284AG7S) and allowed to cool and solidify.

[0195] The eggs in the diet were incubated at 25°C for 4 days until the last first-instar larvae emerged. The day before the start of the feeding assay, 48-well plates were prepared and each well was filled with 0.38 ml of the same artificial diet. On the day of the feeding assay, solutions containing the active substances were prepared at the application rates shown in Tables 3A-3B.

[0196] Table 3A shows the results of the first setup (Experiment 1), which tested the combination of AD1 (see Table 1 above) with a Bt-derived AI (obtained from Thuricide®). Table 3B shows the results of the second setup (Experiment 2), which tested the combination of AD2 (see Table 1 above) with a Bt-derived AI (obtained from Thuricide®). Thirty microliters of these solutions were applied per well to 48-well plates containing diet. After the solutions dried, late first-instar DBM larvae were placed, one per well, and the plates were sealed with Breathe Easier sealing film from Diversified Biotech to allow gas exchange. The plates were then placed in an incubator at 25°C with a 16-hour light, 8-hour dark cycle. After three days, each plate was opened, and the number of live and dead larvae in each plate was recorded to assess mortality.

[0197] result Mortality data for the solutions tested and their application rates are shown in Tables 3A-3B below. Reported values ​​represent percent mortality. The Colby formula was used to determine the expected insecticidal activity of the mixtures (Colby, SR (1967). Calculating Synergistic and Antagonistic Responses of Herbicide Combinations. Weeds, 15(1), 20-22. doi.org / 10.2307 / 4041058). Briefly, the following formula was used to calculate the expected activity of a mixture containing two active proteins, A and B: Expected value = A + B - (A × B / 100) (1) (where A is the observed efficacy of active ingredient A at the same concentration as used in the mixture, and B is the observed efficacy of active ingredient B at the same concentration as used in the mixture).

[0198] Tables 3A-3B below show the results of synergy experiments using a mixture of PirAB-containing minicells and a Bt-derived AI (added at 2 μl / ml as Thuricide® containing 15% Bt-derived AI). In both tables, the values ​​in parentheses indicate the efficacy (% mortality) of that AD alone at that particular concentration.

[0199] [Table 5]

[0200] [Table 6]

[0201] As can be seen from Tables 3A-3B, the observed mortality of the combinations was equal to or exceeded the predicted mortality of the combinations at all concentrations. In some cases, such as 0.8775 ng / μl AD1 (PirAB) and 0.3 μl / ml BT-derived AI (approximately a 3:1 ratio), the observed mortality was approximately twice the predicted mortality (Table 3A). Thus, these results demonstrate that the mixture of minicell-PirAB and BT-derived AI has a synergistic effect against Bt-resistant DBM.

[0202] Example 4: Generation of high-yielding minicell strains The minCDE locus of E. coli strains BW25113 and DH10B was disrupted using Lambda Red recombination to generate BW25113ΔminCDE and DH10BΔminCDE. The camR resistance cassette from pKD3 was amplified with 40 bp of homology to the 5' and 3' regions of the minCDE locus. Five mL of DH10BΔminCDE cells harboring plasmid pKD46 were grown in LB + 0.2% (w / v) arabinose at 30°C to an OD600 of 0.5. Cells were washed five times in ice-cold 10% (v / v) glycerol and electroporated in a 0.2 mm cuvette with a Bio-Rad GenePulser Xcell using standard E. coli settings. Cells were resuspended in 1 mL of SOC and allowed to recover for 1 hour at 37°C. The cells were then plated on LB agar containing 20 μg / mL chloramphenicol at 37° C. to select for mutants. The resulting strain was then transformed with the pCP20 plasmid and plated on LB agar containing 50 μg / mL carbenicillin at 37° C.

[0203] Minicell isolation and characterization To generate minicells, a seed culture was grown overnight from a glycerol stock in LB. The next day, the seed was diluted 1:200 in LB and grown at 37°C. The following day, the culture was processed to collect minicells generated overnight after differential centrifugation. Briefly, the bacterial culture was diluted to OD = 10 and centrifuged in a 1-liter bottle at 4000 × g (Sorval Lynx 6000) using the slowest acceleration speed for 40 minutes. The minicell-rich supernatant was then centrifuged at 17,000 g for 1 hour to pellet the minicells. The resulting pellet was then resuspended in 50 mL of fresh LB containing 200 μg / mL ceftriaxone and 20 μg / mL ciprofloxacin, and the culture was placed at 37°C for 2 hours to remove any remaining parent bacteria. The solution was centrifuged at 4,000 × g for 15 minutes in a swinging bucket rotor (Beckman Coulter) to remove dead parent bacterial cells and large debris. The minicells were then pelleted at 20,000 × g (Sorval Lynx 6000) for 20 minutes and resuspended in an equal volume of 0.2 μm-filtered PBS. This process was repeated for a total of two washes, and the resulting minicell pellet was resuspended in 0.2 μm-filtered PBS to a final volume of 10 mL.

[0204] Isolated minicells were examined microscopically, and particle size and distribution, as well as concentration, were measured by counting using a Spectradyne nCS1. Minicells were harvested from 1 L cultures (average size 350 nm) (Fig. 1A).

[0205] Example 5: Generation of high-yielding minicells and high-yielding protein strains Generation of DH10BΔminCDE harboring the λDE3 prophage A strain of DH10BΔminCDE carrying the λDE3 prophage was generated to enable expression of cargo expressed by T7 RNA polymerase (RNAP) via a T7 promoter (pT7). DE3 encodes a mutant λ phage unable to enter the lytic cycle, encoding the T7 RNAP gene behind a LacUV (lac-inducible promoter). Addition of IPTG to cells harboring the DE prophage produces T7 RNAP, which then transcribes mRNA for the gene behind pT7. λDE3 was introduced into the DH10BΔminCDE chromosome using a λDE3 lysogenization kit (Millipore Sigma 69734). DH10BΔminCDE cells were grown in LB containing 0.2% maltose and 10 mM MgSO4 to an OD600 of 0.5. Ten μL of cells were mixed with 10 pfu λDE3, 10 pfu helper phage, and 10 pfu selection phage and incubated at 37°C for 20 minutes. The entire culture was then plated onto LB agar plates and incubated overnight at 37°C. Colonies were screened for T7 RNAP induction by plaque assay on LB plates + / IPTG and T7 tester phage. Clones showing a small number of plaques on -IPTG plates and a large number of plaques on +IPTG plates were selected and frozen as glycerol stocks. Minicells were isolated as described in Example 4. The particle size distribution is shown in Figure 1B.

[0206] PirAB expression vector To determine which minicell chassis strain would maximize both protein expression and minicell numbers, two expression vectors encoding inducible expression of the entomotoxin PirAB were constructed: one containing a tetracycline (tet)-dependent promoter, and one containing a T7 promoter (both plasmids encode a kanamycin resistance gene and the pMB1 origin of replication). These plasmids were then transformed into the previously described minicell chassis strains BW25113, DH10BΔminCDE, and DH10B(DE3)ΔminCDE.

[0207] PirAB was cloned into the Ptet_pMB1_kan plasmid by Golden Gate cloning. A gene block (IDT) encoding PirAB codons optimized for expression in E. coli (de novo DNA) with flanking BsaI sites was synthesized and used to insert the PirAB gene behind the Tet promoter. To track PirAB expression, a 3xFLAG tag was added by PCR and blunt-end ligation. Briefly, the entire plasmid was amplified using primers containing the coding sequence for the 3xFLAG epitope tag and homology to the PirAB plasmid. The PCR product containing the PirAB plasmid and the 3xFLAG tag was then blunt-end ligated with T4 ligase and transformed into DH10BΔminCDE E. coli. Expression from this promoter is repressed by the gene product of the TetR gene, which is encoded on the plasmid behind a constitutive promoter. When anhydrotetracycline (aTc) is added to the medium, aTc binds to TetR and relieves the repression of expression from Ptet.

[0208] 3xFLAG PirAB was cloned into pET28a by Gibson Assembly. 3xFLAG PirAB was amplified using primers containing homology to the NdeI and HindIII cleavage sites of pET28a, and the pET28a vector was linearized. The PCR product was assembled into pET28a_PirAB by Gibson Assembly using NEB HiFi DNA Assembly Mix according to the manufacturer's guidelines. The assembly was transformed into chemically competent E. coli and selected on LB agar containing μg / mL kanamycin.

[0209] Expression of PirAB in E. coli strains BW, DH10B, and DH10B(DE3) PirAB expression in Escherichia coli (E. coli) strains BW25113, DH10B, and DH10B(DE3) (all ΔminCDE) was examined by Western blot. Cells were grown in LB at 37°C with shaking to an OD of 0.5, and PirAB protein expression was induced for 16 hours with 100 ng / μL aTc or 1 mM IPTG for the Tet- and T7-inducible systems, respectively. PirAB expression levels were quantified by Western blot. 10, 1, and 0.1 μL of induced cells were loaded onto a 4-12% Bis-Tris SDS-PAGE gel (Thermo) along with 2.5, 5, 10, and 20 ng of pure BAP-FLAG fusion (Sigma) or GFP protein standard and run at 200 V for 25 minutes. Proteins were transferred to a nitrocellulose membrane using the iBlot2 system (Thermo) with program V0. Subsequently, membranes were blotted for 3xFLAG PirAB using rabbit anti-FLAG polyclonal antibody (Sigma) and goat anti-rabbit IgG HRP-conjugated secondary antibody (Sigma). Blots were imaged with iBright (Thermo), and band intensities were quantified with ImageJ. Calculated protein levels were then normalized to minicell numbers (Table 4).

[0210] Immunoblotting results showed that DH10BΔminCDE (strain AD16) produced approximately 10 times more minicells per liter than BW25113ΔminCDE (strain AD15; Table 4). DH10B(DE3)ΔminCDE (strain AD19) produced 10 times more minicells than AD15 and also produced approximately 10 times more protein than AD16.

[0211] [Table 7]

[0212] Further optimization of Pir protein production by testing multiple expression cassettes Additional expression cassettes (Table 4) were generated by standard molecular biology techniques. FLAG-PirAB was PCR-amplified from the original expression cassette pMB1_Ptet-FLAG-pirAB using primers oJK225 (SEQ ID NO: 211) and oJK226 (SEQ ID NO: 212). The PCR product was then cloned into a vector containing the Ptac promoter (SEQ ID NO: 218) by TIIS cloning with BsaI using standard techniques known to those skilled in the art, resulting in the expression cassette CloDF_ampR_Ptac_pir_mutantLacI. Due to a spontaneous mutation in the LacI coding sequence, this expression cassette does not express a functional LacI protein, resulting in constitutive expression of FLAG-Pir from the tac promoter. The pUC_camR_pTac_pirFLAG_newLacI expression cassette was generated by replacing the mutated copy of the LacI gene with a functional copy from pET28a by Gibson Assembly. Briefly, the LacI gene was amplified by PCR using primers oJK316 (SEQ ID NO: 215) and oJK317 (SEQ ID NO: 216), and the Pir-containing plasmid backbone was amplified by PCR using primers oJK314 (SEQ ID NO: 213) and oJK315 (SEQ ID NO: 214). These two PCR products were then used for Gibson assembly using methods known in the art. The expression cassette pET28a(+)_pirFLAG was generated by Gibson assembly. FLAG-Pir was amplified by PCR using primers oJK161 (SEQ ID NO: 209) and oJK162 (SEQ ID NO: 210), and pET28a(+) was amplified using primers oJK159 (SEQ ID NO: 207) and oJK160 (SEQ ID NO: 208). These PCR products were then used in a standard Gibson assembly reaction to generate pET28a(+)_pirFLAG. The promoter and terminator sequences used are shown in Table 5.

[0213] [Table 8]

[0214] Example 6: Generation of high yield Cry1Ac and Vip3Aa19 minicell strains Expression cassettes for Cry1Ac and Vip3Aa19 were generated using standard molecular biology techniques. The Cry1Ac gene was obtained from strain JM103(pOS4201) (Bacillus Genetic Stock Center) and cloned into the same expression cassette library as the PirAB toxin described above, with or without a 3xFLAG tag. The Vip3Aa19 gene was codon-optimized for expression in E. coli using De Novo DNA Opener Calc and a gene synthesized by Twist Biosciences. This codon-optimized gene was then cloned into the same expression cassette library described above, with or without a 3xFLAG tag.

[0215] Expression of the insecticidal protein was induced by aTc or IPTG at OD600=0.5, and the protein was produced and loaded into minicells as in Example 4.

[0216] Protein concentrations in each isolated minicell sample were measured by quantitative Western blot (Table 4). Aliquots of 10, 1, and 0.1 μL of minicells, along with standard curves of 20, 10, 5, and 2.5 ng of purified BAP-FLAG (Sigma: P7457) or 100, 50, 25, and 12.5 ng of Cry1Ac (Sino Biological: 12008-A07E), were run on a 4-12% BIS-Tris SDS gel (Thermo Fisher: NW04125BOX), transferred to nitrocellulose using iBlot (Thermo Fisher), and blotted with rabbit anti-FLAG (Sigma: F7425) or anti-Cry1Ac antibody (Abcam: ab51586) and goat anti-rabbit HRP conjugate (Sigma: AP307P). Western blots were imaged using iBright (Thermo Fisher), and band intensities were quantified using ImageJ. The results showed that at least Cry1AC was successfully expressed in three different strains (AD30, AD31 and AD32).

[0217] Example 7: Biological efficacy of minicell-PirAB and minicell-Cry1Ac Artificial diet feeding assay The efficacy of four minicell strains was evaluated in the artificial diet assay: PirAB-containing strains AD16 and AD17 (DH10B chassis), Cry1Ac-containing strain AD28 (DH10B chassis), and PirAB-containing strain AD19 (DH10B(DE3) chassis). Fresh batches of cells were used for these experiments.

[0218] Diamondback moth (DBM) eggs (purchased from Benzon Research) were placed in a container filled with common noctuid artificial diet (purchased as a dry powder from Southland Products Inc.). The DBM eggs were incubated on the artificial diet for 4 days at 25°C until the last first-instar larvae emerged. The day before the start of the feeding assay, a 48-well plate was prepared, and each well was filled with 0.38 ml of the same artificial diet. On the day of the feeding assay, solutions containing the active substances were prepared as follows: PirAB-minicells were diluted in four two-fold serial dilutions starting from an expressed protein concentration of 16–18 ng / μl (Figure 2). Cry1Ac-minicells (strain AD28) were diluted in five four-fold serial dilutions starting from an expressed protein concentration of 8.4 ng / μl (Figure 3). PirAB-minicells (strain AD19) were diluted in five two-fold serial dilutions starting from a concentration of 27.8 ng / μl (Figure 4). Aliquots of these solutions, 30 μl per well, were applied to 48-well plates containing artificial diet. After the solutions dried, late-first instar DBM larvae were placed, one per well, and the plates were sealed with Breathe Easier sealing film (Diversified Biotech) to allow gas exchange. The plates were then placed in an incubator at 25°C with a 16-hour light, 8-hour dark cycle. After 3 days, each plate was opened, and the live and dead larvae in each plate were recorded to assess mortality.

[0219] Greenhouse feeding assay The efficacy of minicell-PirAB and minicell-Cry1Ac against DBM was evaluated using a whole-plant assay in a greenhouse environment. Cabbage (Tiara variety) was grown in ProMix General Purpose soil and fertilized with Multicote controlled-release fertilizer until it reached the four-leaf stage. Approximately 300 diamondback moth (DBM) eggs (purchased from Benzon Research) were placed in a 1-mL tube. On the day of the efficacy assay, 15 mL of a solution of minicell-PirAB and minicell-Cry1Ac was sprayed on each cabbage plant. Once the plants had dried, they were placed in individual cages. The 1-mL tubes containing DBM eggs were then opened and placed horizontally, one at the base of each plant. After approximately 7 days, the plants were cut just above the cotyledons and individually placed in paper bags. The entire plant was weighed, and sample leaves were harvested to determine the percentage of leaves consumed. The number of larvae per plant was then counted and recorded.

[0220] Translational Field Testing Assays To evaluate the field efficacy of MiniCell-PirAB and MiniCell-Cry1Ac in cabbage, greenhouse-grown cabbage (Blue Lagoon) seedling plugs were transferred to the field and grown for an additional 5 weeks. The field experiment design incorporated a completely randomized block design with four treatments and four replicates. PirAB was tested at three concentrations: 0.5x (27.5 mg / L), 1.0x (55 mg / L), and 2.0x (110 mg / L), and Cry1AC was tested at three concentrations: 0.5x (3.75 mg / L), 1.0x (7.5 mg / L), and 2.0x (15 mg / L), each in a 1-liter spray volume. Applications were made using a hollow-cone nozzle and a CO2-powered backpack sprayer at 40 gal / Ac / min and 22 psi operating pressure. After application, 10 leaf discs per treatment (40 leaf discs per treatment) were collected from field plants. Each leaf disc was then subjected to a leaf disc assay using second-instar DBM larvae. Leaf discs were placed on moistened filter paper discs, infested with two larvae, and incubated for 48 hours before evaluation. Twenty larvae were collected from each round and classified as dead, moribund, alive, or missing. For each classification, the average of four replicates was calculated. The percentage of dead and moribund larvae was added together to evaluate the effectiveness of the treatment.

[0221] As shown in the artificial diet assay, minicell-PirAB and minicell-Cry1AC produced on the DH10B or DH10B(DE3) chassis are effective in controlling the lepidopteran insect DBM in a concentration-dependent manner (Figures 2-4). Figure 4 further confirms the efficacy of minicell-PirAB derived from strains AD18 and AD16 in the whole leaf assay. Figures 5A-5C confirm the efficacy of minicell-PirAB and minicell-Cry1Ac against DBM in the greenhouse assay, and Figure 6 confirms the efficacy of minicell-PirAB and minicell-Cry1Ac against DBM in the translational field test assay.

[0222] Taken together, these results demonstrate that minicells derived from DH10B and DH10B(DE) cells, which produce high minicell numbers, can be used to express high yields of bioactive substances (e.g., toxic peptides) with concentration-dependent efficacy for the control of agricultural pests.

[0223] Example 8: Biological efficacy of minicell-Vip3Aa19 Artificial diet feeding assay The efficacy of Vip3Aa19-minicells produced in DH10B and DH10B(DE) chassis (strains AD34-AD39) was evaluated using an artificial diet feeding assay. Diamondback moth (DBM) eggs (available from Benzon Research) were placed in a container filled with general noctuid artificial diet (available as a dry powder from Southland Products Inc.). The eggs were incubated on the artificial diet for 4 days at 25°C until the last instar larvae emerged. The day before the start of the feeding assay, a 48-well plate was prepared and each well was filled with 0.38 ml of artificial diet. On the day of the feeding assay, a solution containing the active substance was prepared. Vip3Aa19-minicells were diluted in four 2-fold serial dilutions, starting at a concentration of approximately 15 ng / μl of expressed protein. 30 μl aliquots of these solutions were applied per well to a 48-well plate containing diet. The solution is allowed to dry, and late first instar DBM larvae are placed, one per well, and the plates are sealed with Diversified Biotech's Breathe Easier sealing film to allow gas exchange. The plates are then placed in an incubator at 25°C on a 16-hour light, 8-hour dark cycle. After 3 days, each plate is opened and the live and dead larvae on each plate are recorded to assess mortality.

[0224] Greenhouse feeding assay The greenhouse efficacy of Vip3Aa19 in minicells was evaluated using a whole-plant assay in a greenhouse environment. Cabbage (Tiara variety) was grown in ProMix General Purpose soil and fertilized with Multicote controlled-release fertilizer until the four-leaf stage was reached. Approximately 300 diamondback moth (DBM) eggs (available from Benzon Research) were placed in a 1-mL tube. On the day of the efficacy assay, each cabbage plant was sprayed with 15 mL of a minicell solution containing Vip3Aa19. Once the plants had dried, they were placed in individual cages. 1-mL tubes containing DBM eggs were then opened and placed horizontally, one at the base of each plant. After approximately 7 days, the plants were cut just above the cotyledons and individually placed in paper bags. The entire plant was weighed, and sample leaves were harvested to determine the percentage of leaves consumed. The number of larvae per plant was then counted and recorded.

[0225] Translational Field Testing Assays To evaluate the field efficacy of Vip3Aa19 on cabbage, greenhouse-grown cabbage (Blue Lagoon) seedling plugs were transferred to the field and grown for an additional five weeks. The field trial design incorporated a completely randomized block design with four treatments and four replicates. Vip3Aa19 in minicells was tested at three concentrations: 0.5x (27.5 mg / L), 1.0x (55 mg / L), and 2.0x (110 mg / L), each with a spray volume of 1 liter. It was applied using a hollow cone nozzle and a CO2-powered backpack sprayer at 40 gal / Ac / min and 22 psi operating pressure. After application, 10 leaf discs per treatment (40 leaf discs per treatment) were collected from field plants. Each leaf disc was placed on a moist filter paper disc, infested with two second-instar DMB larvae, and incubated for 48 hours before evaluation. Twenty larvae are collected from each run and classified as dead, moribund, alive, or missing. For each run, the mean of four replicates is calculated. The percentage of dead and moribund larvae is summed to assess the effectiveness of the treatment.

[0226] Minicells containing Vip3Aa19 are predicted to be highly effective in controlling lepidopteran insects in a concentration-dependent manner. The strain is also effective in whole leaf and translational field trial assays against DBM.

[0227] Example 9: Preparation of storage-stable insecticidal minicells This example demonstrates the ability to create storage-stable insecticidal minicells that maintain activity.

[0228] In this example, minicells are lyophilized to produce storage-stable insecticidal minicells. Isolated minicells are prepared as described above, and 1 mL of minicells is placed in a 1.5 mL plastic tube and pelleted by centrifugation at 21,000 g for 15 minutes. The pellet is resuspended in an equal volume of Microbial Lyophilization Buffer (OPS Diagnostics), transferred to a 15 mL conical tube, and flash-frozen in liquid nitrogen. The insecticidal minicells are then lyophilized for 16 hours using a FreeZone benchtop freeze dryer (Labconco) with an automatic collection setting. Tubes of lyophilized minicells are sealed with parafilm and stored at room temperature in the dark until use.

[0229] Lyophilized minicells are stored for 1, 2, 6, 12, or 24 months. Activity is measured after hydration. Briefly, powdered minicells are rehydrated with 1 mL of PBS. Maintenance of particle number is confirmed by concentration measurement on a Spectradyne nCS1. ATP content of minicells is also measured to confirm stability.

[0230] Example 10: Preparation of a wettable powder (WP) insecticidal composition The freeze-dried insecticidal minicells prepared in the previous examples are used to produce wettable powders (WP). Wettable powders include finely divided particles that disperse readily in water or other liquid carriers. The particles contain insecticidal minicells, typically in freeze-dried form, held in a solid matrix. Typical solid matrices include fuller's earth, kaolin clay, silica, and other readily wet organic or inorganic solids. Wettable powders can contain from about 5% to about 95% insecticidal minicells plus small amounts of wetting agents, dispersing agents, or emulsifying agents. Examples of wettable powders include those listed in Table 6.

[0231] [Table 9]

[0232] Those skilled in the art can also prepare water dispersible granules (WDG) using the teachings contained herein.

[0233] Example 11: Preparation of a Suspension Concentrate (SC) Pesticidal Composition The minicells produced in the previous examples can be used to make suspension concentrates (SC). Suspension concentrates include aqueous formulations in which solid particles of micronized insecticidal minicells are stably suspended. Such formulations contain anti-settling and dispersing agents, and may further include wetting agents to enhance activity, as well as anti-foaming agents and crystal growth inhibitors. Suspension concentrates are diluted with water and applied as a spray to the area to be treated. The amount of insecticidal minicells can range from about 0.5% to about 95% of the concentrate. Examples of suspension concentrate formulations are listed in Table 7.

[0234] [Table 10]

[0235] Example 12: Methods of Making Seed Treatment and Plantable Compositions The minicells produced in the preceding examples can be used to prepare seed treatment and plantable compositions. In such seed treatment compositions, the compositions can include, in addition to the insecticidal minicells, other insecticides, surfactants, film-forming polymers, carriers, antifreeze agents, and other formulation additives that, when used together, provide a composition that is storage stable and suitable for use in conventional seed treatment devices such as slurry seed treaters, direct treaters, farm hopper boxes, planter boxes, and the like.

[0236] Examples of seed treatment compositions are listed in Table 8.

[0237] [Table 11]

[0238] Plantable compositions may be produced by coating seeds (e.g., corn seeds, soybean seeds, canola seeds, rice seeds, wheat seeds, etc.) with the seed treatment composition, thereby producing novel compositions with improved plantability.

[0239] Enumeration of Embodiments 1. A method for producing an agricultural ADAS composition comprising a plurality of minicells that are substantially free of viable parent bacterial cells, comprising: (a) exposing a population of parental bacterial cells that exhibit a reduced level or activity of at least one cell division topology specificity factor to conditions that allow for the formation of minicells, wherein the parental bacterial cells are deficient in a gene involved in DNA repair; (b) separating the minicells from the parent bacterial cells, thereby producing a composition comprising a plurality of minicells substantially free of live parent bacterial cells; A method comprising: 2. The method of embodiment 1, wherein the cell division topology specificity factor is selected from the group consisting of minC, minD, minE, and ftsZ. 3. The method of embodiment 1, wherein the genes involved in DNA repair are selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD, and DNA polymerase III. 4. The method of embodiment 1, wherein the gene involved in DNA repair is RecA. 5. The method of embodiment 4, wherein the bacterial cell is E. coli DH10BΔminCDE. 6. The method of embodiment 1, wherein the parent bacterial cell comprises a T3, T7, K11, or SP6 polymerase, or a nucleic acid encoding a T3, T7, K11, or SP6 polymerase. 7. The method of embodiment 1, wherein the parent bacterial cell comprises T7 polymerase or a nucleic acid encoding T7 polymerase. 8. The method of embodiment 7, wherein the parent bacterial cell is E. coli DH10B(DE3)ΔminCDE. 9. The composition contains at least 10 11 The method of any of embodiments 1-8, comprising minicells. 10. A method for producing an agricultural ADAS composition comprising a plurality of protein-loaded minicells, the minicells being substantially free of live parent bacterial cells, comprising: (a) exposing parental bacterial cells that exhibit a reduced level or activity of at least one cell division topology specificity factor to conditions that allow protein expression, wherein the parental bacterial cells are deficient in a gene involved in DNA repair; (b) exposing the parent bacterial cells to conditions that allow for the formation of minicells; (c) separating the minicells from the parent bacterial cells, thereby producing a composition comprising a plurality of protein-loaded minicells substantially free of live parent bacterial cells; A method comprising: 11. The method of embodiment 10, wherein the cell division topology specificity factor is selected from the group consisting of minC, minD, minE, and ftsZ. 12. The method of embodiment 10, wherein the genes involved in DNA repair are selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD and DNA polymerase III. 13. The method of embodiment 10, wherein the gene involved in DNA repair is RecA. 14. The method of embodiment 13, wherein the bacterial cell is E. coli DH10BΔminCDE. 15. The method of embodiment 10, wherein the parent bacterial cell comprises a T3, T7, K11, or SP6 polymerase, or a nucleic acid encoding a T3, T7, K11, or SP6 polymerase. 16. The method of embodiment 10, wherein the parent bacterial cell comprises T7 polymerase or a nucleic acid encoding T7 polymerase. 17. The method of embodiment 16, wherein the parent bacterial cell is E. coli DH10B(DE3)ΔminCDE. 18. The method of any of embodiments 10-17, wherein the parent bacterial cell comprises an expression vector. 19. The method of embodiment 18, wherein the expression vector comprises a gene encoding a protein. 20. The method of embodiment 19, wherein the gene is under the control of a T7, Ptac, Ptet, or constitutive promoter. 21. The method of any of embodiments 18-20, wherein the protein is an insecticidal, herbicidal, or antimicrobial protein. 22. The method of embodiment 21, wherein the protein is selected from the group consisting of PirAB, Cry1AC, and Vip3Aa19. 23. The method of embodiment 21, wherein the protein is PirAB. 24. The method of embodiment 21, wherein the protein is Cry1Ac. 25. The method of embodiment 22, wherein the protein is Vip3Aa19. 26. The composition contains at least 10 per liter of parent bacterial cell culture. 11 23. The method of any of embodiments 10-22, comprising minicells. 27. The composition is 10 923. The method of any of embodiments 10-22, comprising at least 100 ng of protein per minicell. 28. (a) a plurality of minicells, comprising minicells derived from a plurality of parent bacterial cells that exhibit a reduced level or activity of at least one cell division topology specificity factor; (b) the plurality of minicells comprises at least 100 ng of protein per 109 minicells; Agricultural ADAS composition. 29. The composition of embodiment 28, wherein the cell division topology specificity factor is selected from the group consisting of minC, minD, minE, and ftsZ. 30. The composition of embodiment 28, wherein the parent bacterial cell is defective in a gene involved in DNA repair. 31. The composition of embodiment 28, wherein the gene involved in DNA repair is selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD and DNA polymerase III. 32. The composition of embodiment 28, wherein the gene involved in DNA repair is RecA. 33. The composition of embodiment 32, wherein the bacterial cell is E. coli DH10BΔminCDE. 34. The composition of embodiment 28, wherein the parent bacterial cell comprises a T3, T7, K11, or SP6 polymerase, or a nucleic acid encoding a T3, T7, K11, or SP6 polymerase. 35. The composition of embodiment 28, wherein the parent bacterial cell comprises T7 polymerase or a nucleic acid encoding T7 polymerase. 36. The composition of embodiment 34, wherein the parent bacterial cell is E. coli DH10B(DE3)ΔminCDE. 37. The composition of any of embodiments 29-37, wherein the parent bacterial cell comprises an expression vector. 38. The composition of embodiment 37, wherein the expression vector comprises a gene encoding a protein. 39. The composition of embodiment 38, wherein the gene is under the control of a T7, Ptac, Ptet, or constitutive promoter. 40. The composition of embodiment 28, wherein the protein is an insecticidal, herbicidal, or antimicrobial protein. 41. The composition of embodiment 40, wherein the protein is selected from the group consisting of pirAB, Cry, and Vip3Aa19. 42. The composition of embodiments 28-41, wherein the plurality of minicells comprises a mixture of first minicells comprising a first effective amount of a first exogenous protein toxin comprising Pir and second minicells comprising a second effective amount of a second exogenous protein toxin comprising Cry. 43. The composition of embodiment 42, wherein the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50. 44. The composition of embodiment 43, wherein the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:1. 45. The composition of embodiments 42-44, wherein when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of the at least one pest when either the first effective amount or the second effective amount is administered to the pest. 46. ​​The first particle concentration in the first minicell is about 1 x 10 2 ~Approx. 8×10 14 and the second particle concentration in the second minicell is in the range of about 1×10 2 ~Approx. 8×10 14 46. ​​The composition of embodiments 42-45, wherein the 47. The composition of embodiments 42-46, formulated as at least one of a Ready To Use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, and a drench application. 48. The composition of embodiments 42-47, wherein the first exogenous protein toxin comprises PirAB or a functional component thereof. 49. PirAB is selected from the group consisting of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, and SEQ ID NO:203; and PirA selected from the group consisting of SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151 3, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NO:172, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:201, and SEQ ID NO:204, or a functional component of any of these. 50. The composition of embodiment 49, wherein PirAB comprises PirA comprising SEQ ID NO: 203 and PirB comprising SEQ ID NO: 204, or wherein PirAB comprises PirA comprising SEQ ID NO: 197 and PirB comprising SEQ ID NO: 178. 51. The composition of embodiments 42 to 50, wherein the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J or Cry1K, or a functional component thereof. 52. Cry1A includes Cry1A selected from the group consisting of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:205; Cry1B includes Cry1B selected from the group consisting of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:155; Cry1C includes Cry1C selected from the group consisting of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125 and SEQ ID NO:156; Cry1D includes Cry1A selected from the group consisting of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175 and SEQ ID NO:176. The composition of embodiment 51, wherein Cry1D comprises Cry1E selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 129; Cry1F comprises Cry1F selected from the group consisting of SEQ ID NO: 130 and SEQ ID NO: 131; Cry1G comprises Cry1G selected from the group consisting of SEQ ID NO: 132 and SEQ ID NO: 133; Cry1H comprises Cry1H selected from the group consisting of SEQ ID NO: 134 and SEQ ID NO: 135; Cry1I comprises Cry1I selected from the group consisting of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 and SEQ ID NO: 139; Cry1J comprises Cry1J selected from the group consisting of SEQ ID NO: 140 and SEQ ID NO: 141; or Cry1K comprises SEQ ID NO: 142, or any functional component thereof. 53. The composition of embodiments 50 and 51, wherein the second exogenous protein toxin comprises Cry1A comprising SEQ ID NO: 205. 54. An agricultural composition comprising a plurality of parent bacterial cells, wherein the parent bacterial cells are: (a) having a reduced level or activity of at least one cell division topology specificity factor; (b) defective in genes involved in DNA repair; The parent bacterial cells were at least 10 per liter of parent bacterial cell culture. 11 A composition that produces individual minicells. 55. The composition of embodiment 54, wherein the cell division topology specificity factor is selected from the group consisting of minC, minD, minE, and ftsZ. 56. The composition of embodiment 54, wherein the genes involved in DNA repair are selected from the group consisting of mutS, MutH, MutL, SSB, RecA, RecJ, ExoI, ExoVII, ExoX, UvrD and DNA polymerase III. 57. The composition of embodiment 54, wherein the gene involved in DNA repair is RecA. 58. The composition of embodiment 57, wherein the bacterial cell is E. coli DH10BΔminCDE. 59. The composition of embodiment 54, wherein the parent bacterial cell comprises a T3, T7, K11, or SP6 polymerase, or a nucleic acid encoding a T3, T7, K11, or SP6 polymerase. 60. The composition of embodiment 54, wherein the parent bacterial cell comprises T7 polymerase or a nucleic acid encoding T7 polymerase. 61. The composition of embodiment 60, wherein the parent bacterial cell is E. coli DH10B(DE3)ΔminCDE. 62. The composition of any of embodiments 54-60, wherein the parent bacterial cell comprises an expression vector. 63. The composition of embodiment 62, wherein the expression vector comprises a gene encoding a protein. 64. The composition of embodiment 62, wherein the gene is under the control of a T7, Ptac, Ptet, or constitutive promoter. 65. The composition of embodiment 63 or 64, wherein the protein is an insecticidal, herbicidal or antimicrobial protein. 66. The composition of embodiment 65, wherein the protein is selected from the group consisting of pirAB, Cry, and Vip3Aa19. 67. The composition of any of embodiments 54-66, wherein the parent bacterial cells are capable of producing minicells having a protein concentration of greater than 100 ng per 10 9 minicells. 68. A method for reducing the survival rate of DBM larvae, comprising contacting DBM larvae with any of the compositions of embodiments 28-68, wherein the survival rate of the larvae contacted with the composition is lower than the survival rate of the larvae not contacted with the composition. 69. A hydratable powder comprising a plurality of dry minicells comprising a mixture of a first effective amount of first minicells comprising a first exogenous protein toxin comprising Pir and a second effective amount of second minicells comprising a second exogenous protein toxin comprising Cry; the first effective amount and the second effective amount are present in a ratio ranging from about 50:1 to 1:50; A wettab...

Claims

1. a liquid carrier phase, and A plurality of minicells dispersed in said carrier phase. Including, the plurality of minicells comprises a mixture of first minicells comprising a first effective amount of a first exogenous protein toxin comprising Pir and second minicells comprising a second effective amount of a second exogenous protein toxin comprising Cry; The composition, wherein said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:

50.

2. 2. The composition of claim 1, wherein the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1 or 1:

1.

3. 3. The composition of claim 1 or 2, wherein when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on controlling at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling at least one pest when the first effective amount or the second effective amount is administered to the pest.

4. The first particle concentration in the first minicell is about 1×10 ２ ~Approx. 8×10 １４ and the second particle concentration in the second minicell is in the range of about 1×10 ２ ~Approx. 8×10 １４ The composition according to any one of claims 1 to 3, wherein

5. 5. The composition of any one of claims 1 to 4, wherein administering the composition to the pest comprises an administration method selected from the group consisting of: delivering the composition to the pest for ingestion; contacting the pest with the pest composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds or infests; and applying the composition to a plant.

6. 6. The composition of claim 5, applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment.

7. 7. The composition according to any one of claims 1 to 6, formulated as at least one of a ready-to-use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, and a drench application.

8. The composition of any one of claims 1 to 7, wherein the first exogenous protein toxin comprises PirAB or a functional component thereof.

9. PirAB is selected from the group consisting of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, and SEQ ID NO:203; and PirA selected from the group consisting of SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:153, 9. The composition of claim 8, comprising PirB selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NO:172, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:201, and SEQ ID NO:204, or a functional component of any of these.

10. The composition of claim 9, wherein PirAB comprises PirA comprising SEQ ID NO: 203 and PirB comprising SEQ ID NO: 204, or wherein PirAB comprises PirA comprising SEQ ID NO: 197 and PirB comprising SEQ ID NO:

178.

11. 11. The composition of any one of claims 1 to 10, wherein the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J or Cry1K, or a functional component thereof.

12. The Cry1A includes Cry1A selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 205; the Cry1B includes Cry1B selected from the group consisting of SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 155; the Cry1C includes Cry1C selected from the group consisting of SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 156; the Cry1D includes Cry1D selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 175 and SEQ ID NO: 176 12. The composition of claim 11, wherein the Cry1E comprises Cry1E selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 129; the Cry1F comprises Cry1F selected from the group consisting of SEQ ID NO: 130 and SEQ ID NO: 131; the Cry1G comprises Cry1G selected from the group consisting of SEQ ID NO: 132 and SEQ ID NO: 133; the Cry1H comprises Cry1H selected from the group consisting of SEQ ID NO: 134 and SEQ ID NO: 135; the Cry1I comprises Cry1I selected from the group consisting of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 and SEQ ID NO: 139; the Cry1J comprises Cry1J selected from the group consisting of SEQ ID NO: 140 and SEQ ID NO: 141; or the Cry1K comprises SEQ ID NO: 142, or any functional component thereof.

13. 13. The composition of claim 11 or 12, wherein the second exogenous protein toxin comprises Cry1A comprising SEQ ID NO:

205.

14. The composition of any one of claims 1 to 13, wherein the first minicell comprises a first vector comprising a coding sequence for the first exogenous protein toxin.

15. 15. The composition of claim 14, wherein the first vector comprises a CloDF origin of replication or a pMB1 origin of replication.

16. 16. The composition of claim 14 or 15, wherein the coding sequence for the first exogenous protein toxin is operably linked to a first promoter.

17. 17. The composition of claim 16, wherein the first promoter is an inducible promoter or a constitutive promoter.

18. The composition of claim 17 , wherein the inducible promoter comprises Ptac, Ptet, or PT7.

19. 18. The composition of claim 17, wherein the constitutive promoter comprises J23119 (SEQ ID NO: 200).

20. 20. The composition of any one of claims 14 to 19, wherein the first exogenous protein toxin comprises PirAB, PirAB comprises PirA comprising SEQ ID NO:203 and PirB comprising SEQ ID NO:204, the coding sequence of PirA comprises SEQ ID NO:102 and the coding sequence of PirB comprises SEQ ID NO:

103.

21. The composition of any one of claims 14 to 18 and 20, wherein expression of the first exogenous protein toxin is induced with aTc or IPTG.

22. The composition of any one of claims 1 to 21, wherein the second minicell comprises a second vector comprising a coding sequence for the second exogenous protein toxin.

23. 23. The composition of claim 22, wherein the second vector comprises a CloDF origin of replication or a pMB1 origin of replication.

24. 24. The composition of claim 22 or 23, wherein the coding sequence for the second exogenous protein toxin is operably linked to a second promoter.

25. 25. The composition of claim 24, wherein the second promoter is an inducible promoter or a constitutive promoter.

26. 26. The composition of claim 25, wherein the inducible promoter comprises Ptac, Ptet, or PT7.

27. 26. The composition of claim 25, wherein the constitutive promoter comprises J23119 (SEQ ID NO: 200).

28. 28. The composition of any one of claims 22 to 27, wherein the second exogenous protein toxin comprises Cry1A, the Cry1A comprising SEQ ID NO: 205, and the coding sequence of the Cry1A comprising SEQ ID NO:

104.

29. The composition of any one of claims 22 to 26 and 28, wherein expression of the second exogenous protein toxin is induced with aTc or IPTG.

30. Prevention is Reduction in pest population Reduction in pest weight, A reduction in the physical damage caused by said pests; and Increased pest mortality The composition according to any one of claims 3 to 29, comprising at least one of:

31. the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; The increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase.

31. The composition of claim 30.

32. 32. The composition of any one of claims 3 to 31, wherein control comprises observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

33. 33. The composition of any one of claims 1 to 32, further comprising an agrochemical surfactant, said agrochemical surfactant improving at least one of the following properties: sprayability, spreadability, and pourability.

34. A composition according to any one of claims 1 to 33, wherein the liquid carrier phase is aqueous or oil-based.

35. 35. The composition of any one of claims 3 to 34, wherein the at least one pest is selected from the group consisting of: Diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species and Diptera species.

36. a liquid carrier phase, and A plurality of minicells dispersed in said carrier phase. Including, the plurality of minicells includes minicells comprising a first effective amount of a first exogenous protein toxin comprising Pir and a second effective amount of a second exogenous protein toxin comprising Cry1; The composition, wherein said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:

50.

37. 37. The composition of claim 36, wherein the first effective amount and the second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1 or 1:

1.

38. 38. The composition of claim 36 or 37, wherein the first effective amount and the second effective amount in combination have a synergistic effect on controlling at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling at least one pest when the composition is administered to the pest.

39. 1. A method for controlling pests comprising: A method comprising administering to a pest a composition according to any one of claims 1 to 38.

40. 40. The method of claim 39, wherein administering the composition to the pest includes an administration method selected from the group consisting of: delivering the composition to the pest for ingestion; contacting the pest with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds or infests; and applying the composition to a plant.

41. 41. The method of claim 40, wherein the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment.

42. 42. The method of any one of claims 39 to 41, wherein the composition is formulated as at least one of a ready-to-use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, or a drench application.

43. Prevention is Reduction in pest population Reduction in pest weight, A reduction in the physical damage caused by said pests; and Increased pest mortality The method according to any one of claims 39 to 42, comprising at least one of the following:

44. the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; The increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase.

44. The method of claim 43.

45. 45. The method of any one of claims 39 to 44, wherein the control comprises observing about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% pest mortality on the plant.

46. 46. ​​The method of any one of claims 39 to 45, wherein the pest is selected from the group consisting of: Diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species and Diptera species.

47. 1. A hydratable powder comprising a plurality of dry minicells comprising a mixture of a first effective amount of first minicells comprising a first exogenous protein toxin comprising Pir and a second effective amount of second minicells comprising a second exogenous protein toxin comprising Cry, said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:50; A wettable powder that is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to an insect.

48. 48. The wettable powder of claim 47, wherein said first effective amount and said second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1 or 1:

1.

49. 50. The wettable powder of claim 47 or 48, wherein when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of at least one pest when the first effective amount or the second effective amount is administered to the pest.

50. The first particle concentration in the first minicell is about 1×10 ２ ~Approx. 8×10 １４ and the second particle concentration in the second minicell is in the range of about 1×10 ２ ~Approx. 8×10 １４ The hydratable powder according to any one of claims 47 to 49, wherein the hydratable powder is in the range of

51. 51. The wettable powder of any one of claims 47 to 50, further comprising a pesticidally acceptable solid carrier component comprising at least one of a clay component, a kaolin component, a talc component, a chalk component, a calcite component, a quartz component, a pumice component, a diatomaceous earth component, a vermiculite component, a silicate component, a silicon dioxide component, a silica powder component, an aluminum component, an ammonium sulfate component, an ammonium phosphate component, a calcium carbonate component, a urea component, a sugar component, a starch component, a sawdust component, a ground coconut shell component, a ground corn cob component, and a ground tobacco stem component.

52. 52. The wettable powder of any one of claims 47 to 51, wherein the aqueous carrier comprises water.

53. 53. The wettable powder of any one of claims 47 to 52, wherein administering the composition to the pest comprises an administration method selected from the group consisting of: delivering the composition to the pest for ingestion; contacting the insect with the composition; delivering the composition to at least one habitat where the pest grows, breeds, reproduces, feeds or infests; and applying the composition to a plant.

54. 54. The composition of claim 53, applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment.

55. 55. The composition of any one of claims 47 to 54, wherein the first exogenous protein toxin comprises PirAB or a functional component thereof.

56. PirAB is selected from the group consisting of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, and SEQ ID NO:203; and PirA selected from the group consisting of SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:153, 56. The composition of claim 55, comprising PirB selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NO:172, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:201, and SEQ ID NO:204, or a functional component of any of these.

57. 57. The composition of claim 56, wherein PirAB comprises PirA comprising SEQ ID NO: 203 and PirB comprising SEQ ID NO: 204, or wherein PirAB comprises PirA comprising SEQ ID NO: 197 and PirB comprising SEQ ID NO:

178.

58. 58. The composition of any one of claims 47 to 57, wherein the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J or Cry1K, or a functional component thereof.

59. The Cry1A includes Cry1A selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 14, SEQ ID NO: 115, SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 205; the Cry1B includes Cry1B selected from the group consisting of SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 155; the Cry1C includes Cry1C selected from the group consisting of SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 156; the Cry1D includes Cry1D selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 175 and SEQ ID NO:

176.

59. The composition of claim 58, wherein the Cry1E comprises Cry1E selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 129; the Cry1F comprises Cry1F selected from the group consisting of SEQ ID NO: 130 and SEQ ID NO: 131; the Cry1G comprises Cry1G selected from the group consisting of SEQ ID NO: 132 and SEQ ID NO: 133; the Cry1H comprises Cry1H selected from the group consisting of SEQ ID NO: 134 and SEQ ID NO: 135; the Cry1I comprises Cry1I selected from the group consisting of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 and SEQ ID NO: 139; the Cry1J comprises Cry1J selected from the group consisting of SEQ ID NO: 140 and SEQ ID NO: 141; or the Cry1K comprises SEQ ID NO: 142, or any functional component thereof.

60. 60. The composition of claim 59, wherein the second exogenous protein toxin comprises Cry1A comprising SEQ ID NO:

205.

61. Prevention is Reduction in pest population Reduction in pest weight, A reduction in the physical damage caused by said pests; and Increased pest mortality The composition according to any one of claims 47 to 60, comprising at least one of:

62. the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; The increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase.

62. The composition of claim 61.

63. 63. The composition of any one of claims 47 to 62, wherein control comprises observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

64. 64. The composition of any one of claims 47 to 63, wherein the at least one pest is selected from the group consisting of: Diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, and Diptera species.

65. a) providing a first effective amount of first dried minicells comprising a first exogenous protein toxin comprising Pir; b) a second effective amount of a second dried minicell comprising a second exogenous protein toxin comprising Cry; and c) mixing said first effective amount and said second effective amount in a ratio ranging from about 50:1 to 1:

50.

1. A method for producing a hydratable powder comprising: The method wherein the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to a pest.

66. 66. The method of claim 65, wherein the first effective amount and the second effective amount are mixed in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1, or 1:

1.

67. Seeds, and a coating covering the seed, the coating comprising a plurality of minicells comprising a mixture of a first effective amount of first minicells comprising a first exogenous protein toxin comprising Pir and a second effective amount of second minicells comprising a second exogenous protein toxin comprising Cry. Including, said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:50; the mixture is sufficient to provide insecticidal activity against at least one pest that feeds on the seeds or the seedlings produced therefrom; Plantable composition.

68. 68. The plantable composition of claim 67, wherein said first effective amount and said second effective amount are present in a ratio of about 46:1, 23:1, 11:1, 9:1, 6:1, 5:1, 3:1 or 1:

1.

69. 69. The plantable composition of claim 67 or 68, wherein when the composition is administered to the pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on controlling at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling at least one pest when the first effective amount or the second effective amount is administered to the pest.

70. The first particle concentration in the first minicell is about 1×10 ２ ~Approx. 8×10 １４ and the second particle concentration in the second minicell is in the range of about 1×10 ２ ~Approx. 8×10 １４ 70. The plantable composition of any one of claims 67 to 69, wherein

71. 71. The plantable composition of any one of claims 67-70, wherein the first exogenous protein toxin comprises PirAB or a functional component thereof.

72. PirAB is selected from the group consisting of SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:174, SEQ ID NO:174, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:202, and SEQ ID NO:203; and PirA is selected from the group consisting of SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:153, SEQ ID NO:

72. The plantable composition of claim 71, comprising a PirB selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NO:172, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:201, and SEQ ID NO:204, or a functional component of any of these.

73. 73. The plantable composition of claim 72, wherein PirAB comprises PirA comprising SEQ ID NO:203 and PirB comprising SEQ ID NO:204, or wherein PirAB comprises PirA comprising SEQ ID NO:197 and PirB comprising SEQ ID NO:

178.

74. 74. The plantable composition of any one of claims 67-73, wherein the second exogenous protein toxin comprises Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J or Cry1K, or a functional component thereof.

75. The Cry1A comprises Cry1A selected from the group consisting of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:205; the Cry1B comprises Cry1B selected from the group consisting of SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:155; the Cry1C comprises Cry1C selected from the group consisting of SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125 and SEQ ID NO:156; the Cry1D comprises Cry1D selected from the group consisting of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:175 and SEQ ID NO:176; The plantable composition of claim 74, wherein the Cry1E comprises Cry1E selected from the group consisting of SEQ ID NO:128 and SEQ ID NO:129; the Cry1F comprises Cry1F selected from the group consisting of SEQ ID NO:130 and SEQ ID NO:131; the Cry1G comprises Cry1G selected from the group consisting of SEQ ID NO:132 and SEQ ID NO:133; the Cry1H comprises Cry1H selected from the group consisting of SEQ ID NO:134 and SEQ ID NO:135; the Cry1I comprises Cry1I selected from the group consisting of SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 and SEQ ID NO:139; the Cry1J comprises Cry1J selected from the group consisting of SEQ ID NO:140 and SEQ ID NO:141; or the Cry1K comprises SEQ ID NO:142, or any functional component thereof.

76. 76. The plantable composition of claim 75, wherein the second exogenous protein toxin comprises Cry1A comprising SEQ ID NO:

205.

77. 77. The plantable composition of any one of claims 67-76, wherein the at least one pest is selected from the group consisting of: Diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species and Diptera species.

78. The seeds include, but are not limited to, alfalfa, almonds, apples, apricots, artichokes, asparagus, avocados, bananas, barley, beans, blueberries, cranberries, Brazil nuts, cacao, calamansi, canola, Polish canola, broccoli, kale, cabbage, turnips, carnations, carrots, cashews, cassava, celery, cherries, chestnuts, chickpeas, chicory, chilli peppers, chrysanthemums, citrons, coconuts, coffee, cotton, cowpeas, broad beans, cucumbers, redcurrants, gooseberries, dates, duckweed, eggplant, elderberries, eucalyptus, flax, geraniums, ginger, ginseng, grapefruit, grapes, guavas, hazelnuts, hemp, cannabis, hops, horseradish, irises, jackfruit, kiwifruit, kumquats, lemons, lentils, lettuce, limes, litchis, maca Damia, corn, mandarin, mango, mangosteen, melon, millet, oats, palm, okra, olive, onion, orange, papaya, parsley, passion fruit, pecan, peach, nectarine, pear, pea, peanut, peony, persimmon, petunia, pineapple, pistachio, plantain, plum, poinsettia, pomelo, poplar, potato, pumpkin, squash, quince, raspberry 78. The plantable composition of any one of claims 67-77, wherein the plant is derived from a plant selected from the group consisting of: rye, safflower, satsuma mandarin, sesame, sorghum, bitter orange, soursop, soybean, strawberry, sugar beet, sugar cane, sunflower, sweet potato, tamarind, tangerine, tea, tobacco, tomatillo, tomato, tulip, walnut, watermelon, wheat, and yam.

79. A liquid carrier phase; a plurality of minicells dispersed in the carrier phase, said plurality of minicells comprising a first effective amount of an exogenous protein toxin comprising Pir; and a second effective amount of a Bt-derived active ingredient ("AI") Including; The composition, wherein said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:

50.

80. 80. The composition of claim 79, wherein the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:

1.

81. 81. The composition of claim 79 or 80, wherein when the composition is administered to a pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on controlling at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling at least one pest when the first effective amount or the second effective amount is administered to the pest.

82. The particle concentration of the minicell is about 1×10 ２ ~Approx. 8×10 １４ The composition according to any one of claims 79 to 81, wherein

83. 83. The composition of any one of claims 79-82, wherein the Bt-derived AI comprises at least one of Bacillus thuringiensis, a fermentation solid thereof, a spore thereof, and an insecticidal toxin thereof.

84. 84. The composition of claim 83, wherein the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxins.

85. 85. The composition of any one of claims 79 to 84, wherein administering the composition to the pest comprises an administration method selected from the group consisting of: delivering the composition to the pest for ingestion; contacting the pest with a pest composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds or infests; and applying the composition to a plant.

86. 86. The composition of claim 85, applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment.

87. 87. The composition of any one of claims 79 to 86, formulated as at least one of a ready to use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, a drench treatment, a dust, an emulsifiable concentrate, a flowable concentrate, an intermediate formulation, a granular formulation, an impregnated material formulation, a pellet / tablet formulation, a soluble concentrate formulation, an industrial chemical formulation, a water dispersible granular formulation, a water soluble packaged formulation, or a wettable powder formulation.

88. 88. The composition of any one of claims 79 to 87, wherein the exogenous protein toxin comprises PirAB or a functional component thereof.

89. PirAB is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, 3. The composition of claim 88, comprising PirB selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, or a functional component of any of these.

90. 90. The composition of claim 89, wherein PirAB comprises PirA comprising SEQ ID NO:4 and PirB comprising SEQ ID NO:5, or wherein PirAB comprises PirA comprising SEQ ID NO:6 and PirB comprising SEQ ID NO:

7.

91. PirAB comprises PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA comprises PirA selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 , SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:

58.

92. The Bt-derived AIs include Thuricide, Thuricide HPC-ORTU, Thuricide HPC, Thuricide 48LV Forestry, Thuricide 76B, Thuricide 76LV, Bonide Bacillus Thuringiensis (BT) moth larvae (caterpillar) control, DiPel, Security BT powdered biological insecticide, Fertilome Dipel powdered biological insecticide, Widestrike, botanical pesticide B.T. B.T. subsp. tenebrionis Colorado Potato Beetle Protein, SA-50 Brand DiPel® Powder, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 Powder for Vegetables, Green Light® DiPel® Powder, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granules, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits®, BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L Worm Killer, TERRO® Larvicide, VectoMax® FG Biological Larvicide Microparticles, Chemsico InsectGranules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Microparticles, VectoBac® FG+ Biological Larvicide Microparticles, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE! (R) G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar (R) Bti CRG, VBC-60092 (Bactimos Briquette), Summit B.t.i. BRIQUETS, Dunks, Sympatico biological insecticide, Xentari® AS biological insecticide aqueous suspension, B402 biological larvicide, VectoBac® 12 AS biological larvicide aqueous suspension, VectoBac® SC biological larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F flowable biological insecticide concentrate, Foray 76B, Safer B.T. Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. Israelensis Slurry, Bacillus thuringiensis subsp. Kurstaki Slurry, Trident® Biological Insecticide, VBC-60219 Biological Insecticide Slurry, Javelin, Bacillus thuringiensis subsp. Aizawaisubsp. aizawai slurry, FlorBac® slurry biological insecticide, DiPel® ES, DiPel® ES-NT biological insecticide emulsified suspension, DiPel® (Worm Killer) wettable powder biological insecticide, Condor, Leap® ES bacterial disease control biological insecticide emulsified suspension, Biobit® HPWPII biological insecticide, Vec VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wet Powder, BioBit® HP Biological Insecticide Wet Powder, DiPel® WDG Biological Insecticide, VBC-60236 Biological Insecticide Dry Flowable, Foray 2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, Foray WG, ThyllombeetleGONE! (R) Biological Insecticide, Javelin WP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, Javelin WG, SAN420IWG, Javelin WG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW, Bonide Bacillus thuringiensis 'O', ChemsicoHPC-O concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec 92. The composition of any one of claims 79 to 91, comprising a Bt-derived biological control formulation selected from the group consisting of: Bt Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, and BTI Technical Powder BIOInsecticide.

93. 93. The composition of any one of claims 79-92, wherein the plurality of minicells comprises a vector comprising a coding sequence for the exogenous protein toxin.

94. 94. The composition of claim 93, wherein the vector comprises a pMB1 origin of replication.

95. 95. The composition of claim 93 or 94, wherein the coding sequence for the exogenous protein toxin is operably linked to a promoter.

96. 96. The composition of claim 95, wherein the first promoter is an inducible promoter or a constitutive promoter.

97. 97. The composition of claim 96, wherein the inducible promoter comprises Ptet.

98. 98. The composition of any one of claims 93 to 97, wherein the first exogenous protein toxin comprises PirAB, wherein PirAB comprises PirA comprising SEQ ID NO:4 and PirB comprising SEQ ID NO:5, wherein the coding sequence of PirA comprises SEQ ID NO:2 and the coding sequence of PirB comprises SEQ ID NO:

3.

99. The composition of any one of claims 93 to 98, wherein expression of the exogenous protein toxin is induced with aTc.

100. Prevention is Reduction in pest population Reduction in pest weight, A reduction in the physical damage caused by said pests; and Increased pest mortality The composition according to any one of claims 81 to 99, comprising at least one of:

101. the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; The increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase. The composition of claim 100.

102. 102. The composition of any one of claims 81-101, wherein control comprises observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

103. 103. The composition of any one of claims 79 to 102, further comprising an agrochemical surfactant, said agrochemical surfactant improving at least one of the following properties: sprayability, spreadability, and pourability.

104. A composition according to any one of claims 79 to 103, wherein the liquid carrier phase is aqueous or oil-based.

105. 105. The composition of any one of claims 81 to 104, wherein the at least one pest is selected from the group consisting of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, and Diptera species.

106. a liquid carrier phase, and A plurality of minicells dispersed in said carrier phase. Including, the plurality of minicells comprising minicells containing a first effective amount of an exogenous protein toxin comprising Pir and a second effective amount of a Bt-derived AI; The composition, wherein said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:

50.

107. 107. The composition of claim 106, wherein the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:

1.

108. 108. The composition of claim 106 or 107, wherein the combined first effective amount and the second effective amount have a synergistic effect on controlling at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling at least one pest when the composition is administered to the pest.

109. 1. A method for controlling pests comprising: A method comprising administering to a pest a composition according to any one of claims 79 to 108.

110. The method of claim 109, wherein administering the composition to the pest includes an administration method selected from the group consisting of: delivering the composition to the pest for ingestion; contacting the pest with the composition; delivering the composition to at least one habitat where the pest grows, develops, reproduces, feeds or infests; and applying the composition to a plant.

111. 111. The method of claim 110, wherein the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment.

112. 112. The method of any one of claims 109 to 111, wherein the composition is formulated as at least one of a ready-to-use (RTU) formulation, a suspension concentrate, a tank mix, an aerosol, a seed treatment, a root dip, a soil treatment, an irrigation formulation, a sprinkler formulation, a drench treatment, a dust, an emulsifiable concentrate, a flowable concentrate, an intermediate formulation, a granular formulation, an impregnated material formulation, a pellet / tablet formulation, a soluble concentrate formulation, an industrial chemical formulation, a water dispersible granular formulation, a water soluble packaged formulation, or a wettable powder formulation.

113. Prevention is Reduction in pest population Reduction in pest weight, A reduction in the physical damage caused by said pests; and Increased pest mortality The method according to any one of claims 109 to 112, comprising at least one of the following:

114. the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; The increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase. The method of claim 113.

115. 115. The method of any one of claims 109 to 114, wherein the control comprises observing about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% pest mortality on the plant.

116. 116. The method of any one of claims 109 to 115, wherein the pest is selected from the group consisting of: Diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species and Diptera species.

117. a plurality of dried minicells comprising a mixture of a first effective amount of minicells containing an exogenous protein toxin including Pir and a second effective amount of a Bt-derived AI; said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:50; A wettable powder that is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to an insect.

118. 118. The wettable powder of claim 117, wherein the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:

1.

119. 119. The wettable powder of claim 117 or 118, wherein when the composition is administered to the pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on the control of at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on the control of at least one pest when the first effective amount or the second effective amount is administered to the pest.

120. The particle concentration of the minicell is about 1×10 ２ ~Approx. 8×10 １４ The hydratable powder according to any one of claims 117 to 119, wherein the hydratable powder is in the range of

121. 121. The wettable powder of any one of claims 117-120, wherein the Bt-derived AI comprises at least one of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxins.

122. 122. The wettable powder of any one of claims 117-121, wherein the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxins.

123. 123. The wettable powder of any one of claims 117 to 122, further comprising a pesticidally acceptable solid carrier component comprising at least one of a clay component, a kaolin component, a talc component, a chalk component, a calcite component, a quartz component, a pumice component, a diatomaceous earth component, a vermiculite component, a silicate component, a silicon dioxide component, a silica powder component, an aluminum component, an ammonium sulfate component, an ammonium phosphate component, a calcium carbonate component, a urea component, a sugar component, a starch component, a sawdust component, a ground coconut shell component, a ground corn cob component, and a ground tobacco stem component.

124. 124. The wettable powder of any one of claims 117 to 123, wherein the aqueous carrier comprises water.

125. 125. The wettable powder of any one of claims 117 to 124, wherein administering the composition to the pest comprises an administration method selected from the group consisting of: delivering the composition to the pest for ingestion; contacting the insect with the composition; delivering the composition to at least one habitat where the pest grows, breeds, reproduces, feeds or infests; and applying the composition to a plant.

126. 126. The wettable powder of claim 125, wherein the composition is applied to the plant as at least one of a foliar treatment, an injection treatment, a pre-emergence treatment, and a post-emergence treatment.

127. 127. The wettable powder of any one of claims 117 to 126, wherein the exogenous protein toxin comprises PirAB or a functional component thereof.

128. PirAB is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, 128. The wettable powder of claim 127, comprising PirB selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, or a functional component of any of these.

129. 129. The wettable powder of claim 128, wherein PirAB comprises PirA comprising SEQ ID NO:4 and PirB comprising SEQ ID NO:5, or wherein PirAB comprises PirA comprising SEQ ID NO:6 and PirB comprising SEQ ID NO:

7.

130. PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA comprises a PirA selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, 128. The wettable powder of claim 127, wherein the PirB is linked by a linker sequence to a PirB selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:

58.

131. The Bt-derived AIs include Thuricide, Thuricide HPC-ORTU, Thuricide HPC, Thuricide 48LV Forestry, Thuricide 76B, Thuricide 76LV, Bonide Bacillus Thuringiensis (BT) moth larvae (caterpillar) control, DiPel, Security BT powdered biological insecticide, Fertilome Dipel powdered biological insecticide, Widestrike, botanical pesticide B.T. B.T. subsp. tenebrionis Colorado Potato Beetle Protein, SA-50 Brand DiPel® Powder, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 Powder for Vegetables, Green Light® DiPel® Powder, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granules, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits®, BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L Worm Killer, TERRO® Larvicide, VectoMax® FG Biological Larvicide Microparticles, Chemsico InsectGranules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Microparticles, VectoBac® FG+ Biological Larvicide Microparticles, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE! (R) G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar (R) Bti CRG, VBC-60092 (Bactimos Briquette), Summit B.t.i. BRIQUETS, Dunks, Sympatico biological insecticide, Xentari® AS biological insecticide aqueous suspension, B402 biological larvicide, VectoBac® 12 AS biological larvicide aqueous suspension, VectoBac® SC biological larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F flowable biological insecticide concentrate, Foray 76B, Safer B.T. Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. Israelensis Slurry, Bacillus thuringiensis subsp. Kurstaki Slurry, Trident® Biological Insecticide, VBC-60219 Biological Insecticide Slurry, Javelin, Bacillus thuringiensis subsp. Aizawaisubsp. aizawai slurry, FlorBac® slurry biological insecticide, DiPel® ES, DiPel® ES-NT biological insecticide emulsified suspension, DiPel® (Worm Killer) wettable powder biological insecticide, Condor, Leap® ES bacterial disease control biological insecticide emulsified suspension, Biobit® HPWPII biological insecticide, Vec VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wet Powder, BioBit® HP Biological Insecticide Wet Powder, DiPel® WDG Biological Insecticide, VBC-60236 Biological Insecticide Dry Flowable, Foray 2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, Foray WG, ThyllombeetleGONE! (R) Biological Insecticide, Javelin WP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, Javelin WG, SAN420IWG, Javelin WG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW, Bonide Bacillus thuringiensis 'O', ChemsicoHPC-O concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec 131. The wettable powder of any one of claims 117 to 130, comprising a Bt-derived biological control formulation selected from the group consisting of: Bt Technical, Summit BTIMP, BMP144 Primary Powder, BMP123 Primary Powder, BMP144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide Technical Concentrate, SAN420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, and BTI Technical Powder BIOInsecticide.

132. Prevention is Reduction in pest population Reduction in pest weight, A reduction in the physical damage caused by said pests; and Increased pest mortality The hydratable powder according to any one of claims 117 to 131, comprising at least one of:

133. the reduction in pest numbers is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in pest weight is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; the reduction in physical damage is at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, or at least an 80% reduction; The increase in pest mortality is at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, or at least an 80% increase.

133. The hydratable powder of claim 132.

134. 134. The wettable powder of any one of claims 117-133, wherein the control comprises observed pest mortality of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

135. The wettable powder of any one of claims 117 to 134, wherein the at least one pest is selected from the group consisting of diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species, and Diptera species.

136. a) providing a first effective amount of dried minicells comprising an exogenous protein toxin comprising Pir; b) a second effective amount of a dried Bt-derived AI; and c) mixing said first effective amount and said second effective amount in a ratio ranging from about 50:1 to 1:

50.

1. A method for producing a hydratable powder comprising: The method wherein the wettable powder is configured to be dispersed in an aqueous carrier to produce a composition that controls at least one pest when the composition is administered to a pest.

137. 137. The method of claim 136, wherein the first effective amount and the second effective amount are mixed in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:

1.

138. Seeds, and a coating covering the seeds, the coating comprising a plurality of minicells comprising a mixture of a first effective amount of minicells comprising an exogenous protein toxin including Pir and a second effective amount of a Bt-derived AI. Including, said first effective amount and said second effective amount are present in a ratio ranging from about 50:1 to 1:50; the mixture is sufficient to provide insecticidal activity against at least one pest that feeds on the seeds or seedlings produced therefrom; Plantable composition.

139. 139. The plantable composition of claim 138, wherein the first effective amount and the second effective amount are present in a ratio of about 23:1, 22:1, 11:1, 6:1, 3:1, 2:1, or 1.5:

1.

140. 140. The plantable composition of claim 138 or 139, wherein when the composition is administered to the pest, the mixture of the first effective amount and the second effective amount has a synergistic effect on controlling at least one pest that exceeds the effect of the first effective amount alone and the effect of the second effective amount alone on controlling at least one pest when the first effective amount or the second effective amount is administered to the pest.

141. The particle concentration of the minicell is about 1×10 ２ ~Approx. 8×10 １４ 141. The plantable composition of any one of claims 138 to 140, wherein

142. 142. The plantable composition of any one of claims 138-141, wherein the Bt-derived AI comprises at least one of Bacillus thuringiensis, a fermentation solid thereof, a spore thereof, and an insecticidal toxin thereof.

143. 143. The plantable composition of claim 142, wherein the Bt-derived AI comprises at least two, at least three, or all of Bacillus thuringiensis, its fermentation solids, its spores, and its insecticidal toxins.

144. 144. The plantable composition of any one of claims 138-143, wherein the exogenous protein toxin comprises PirAB or a functional component thereof.

145. PirAB is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; and PirA is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:33, SEQ ID NO:

145. The plantable composition of claim 144, comprising PirB selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, or a functional component of any of these.

146. 146. The plantable composition of claim 145, wherein PirAB comprises PirA comprising SEQ ID NO:4 and PirB comprising SEQ ID NO:5, or wherein PirAB comprises PirA comprising SEQ ID NO:6 and PirB comprising SEQ ID NO:

7.

147. PirAB comprises a PirA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32; and PirA comprises a PirA selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:

145. The plantable composition of claim 144, wherein the PirB is linked by a linker sequence to a PirB selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:

58.

148. The Bt-derived AIs include Thuricide, Thuricide HPC-ORTU, Thuricide HPC, Thuricide 48LV Forestry, Thuricide 76B, Thuricide 76LV, Bonide Bacillus Thuringiensis (BT) moth larvae (caterpillar) control, DiPel, Security BT powdered biological insecticide, Fertilome Dipel powdered biological insecticide, Widestrike, botanical pesticide B.T. B.T. subsp. tenebrionis Colorado Potato Beetle Protein, SA-50 Brand DiPel® Powder, Ringer® Vegetable Insect Attack®, Bonide DiPel® 150 Powder for Vegetables, Green Light® DiPel® Powder, Green Light® BT Worm Killer, BT Sulfur 15-50 Powder, NEU1701I, FourStar® Briquettes, FourStar® CRG, Britz Bt 25 Sulfur Powder, Thuricide® 32LV, Bonide Mosquito Beater® Water Soluble Pouches, Britz BT Powder, Summit Fallout Bti Granules, DiPel® 10G Sweet Corn Granules, DiPel® 10G Biological Insecticide Granules, DiPel® SG Plus Biological Insecticide Sand Granules, DiPel® 6L WormKiller, VectoBac® G Biological Larvicide Granules, VectoBac® GR, Summit Mosquito Bits®, Mosquito Bits®, BMP144 (200G), Bti Granular Larvicide, FourStar® MBG, DiPel® 8L Worm Killer, TERRO® Larvicide, VectoMax® FG Biological Larvicide Microparticles, Chemsico InsectGranules ML, VectoBac® FG Biological Larvicide Granules, Bti Granules, RF2230D-Granules, RF2248U-Granules, AllPro Sustain BCG, BMP144 (400G), VectoPrime® FG Biological Larvicide Microparticles, VectoBac® FG+ Biological Larvicide Microparticles, VectoBac® AS, DiPel® WP Home & Garden Insecticide, DiPel® Bio Garden Spray, WideStrike®, Meridian-02, BMP144 (2X), grubGONE! (R) G, Bioprotec, Bioprotec Plus, Bioprotec HP, Bioprotec Caterpillar Insecticide Concentrate, BES0531, Novodor Flowable Biological Insecticide Concentrate, FourStar (R) Bti CRG, VBC-60092 (Bactimos Briquette), Summit B.t.i. BRIQUETS, Dunks, Sympatico biological insecticide, Xentari® AS biological insecticide aqueous suspension, B402 biological larvicide, VectoBac® 12 AS biological larvicide aqueous suspension, VectoBac® SC biological larvicide, BMP 144 (3X), CMP 123 (48LC), Bactimos PT, Foray 48B, Foray 48BC, Foray 48F flowable biological insecticide concentrate, Foray 76B, Safer B.T. Caterpillar Killer Concentrate, Bacillus thuringiensis subsp. Israelensis Slurry, Bacillus thuringiensis subsp. Kurstaki Slurry, Trident® Biological Insecticide, VBC-60219 Biological Insecticide Slurry, Javelin, Bacillus thuringiensis subsp. Aizawaisubsp. aizawai slurry, FlorBac® slurry biological insecticide, DiPel® ES, DiPel® ES-NT biological insecticide emulsified suspension, DiPel® (Worm Killer) wettable powder biological insecticide, Condor, Leap® ES bacterial disease control biological insecticide emulsified suspension, Biobit® HPWPII biological insecticide, Vec VectoBac® WDG Biological Larvicide Water Dispersible Granules, VectoBac® DT Biological Larvicide, Bactimos® WG, VectoBac® WG, BMP123 (2XWP), Cutlass, Crymax®, Lepinox™ WDG, Crymax® WP, BMP144DF3000, CondorWP, BTI WDG, Condor XL, XenTari® Biological Insecticide Water Dispersible Granules, Agree® WG, Agree® 50WP, DiPel® DF Biological Insecticide Dry Flowable, XenTari® Biological Insecticide Dry Flowable, DiPel® DF Biological Insecticide, DiPel® 2X Biological Insecticide Wet Powder, BioBit® HP Biological Insecticide Wet Powder, DiPel® WDG Biological Insecticide, VBC-60236 Biological Insecticide Dry Flowable, Foray 2WG Biological Insecticide (Water Dispersible Granules), Thuricide® HPWP, Foray WG, ThyllombeetleGONE! (R) Biological Insecticide, Javelin WP, ABG-6346 Biological Insecticide, ABG-6345 Technical Powder, Javelin WG, SAN420IWG, Javelin WG2, BTK32, XenTari® Biological Insecticide Technical Powder, Phyllom SDS-502MP, DiPel® Biological Insecticide Technical Powder, Novodor Technical, VBC-60225 Biological Insecticide Technical Active Ingredient POW, Bonide Bacillus thuringiensis 'O', ChemsicoHPC-O concentrate, Thuricide® HPC-O Home & Garden, VectoBac® Technical Powder, VectoBac® Biological Larvicide Primary Powder, Bioprotec 148. The plantable composition of any one of claims 138-147, comprising a Bt derived biological control agent selected from the group of: Bt Technical, Summit BTIMP, BMP 144 Primary Powder, BMP 123 Primary Powder, BMP 144 Primary Powder OSF, Technical CGA-237218, Javelin Technical Concentrate, Thuricide® Technical Concentrate, SAN 420I Technical Concentrate, CONDOR Technical Powder, Cutlass Technical Powder, Lepinox, BTTSDTC, BTT Technical Concentrate, and BTI Technical Powder BIOInsecticide.

149. 149. The plantable composition of any one of claims 138-148, wherein the at least one pest is selected from the group consisting of: Diamondback moth (DBM), armyworm, fall armyworm (FAW), red flour beetle (RFB), Colorado potato beetle (CPB), alder moth, Helicoverpa armigera, Lepidoptera species, Coleoptera species and Diptera species.

150. The seeds include, but are not limited to, alfalfa, almonds, apples, apricots, artichokes, asparagus, avocados, bananas, barley, beans, blueberries, cranberries, Brazil nuts, cacao, calamansi, canola, Polish canola, broccoli, kale, cabbage, turnips, carnations, carrots, cashews, cassava, celery, cherries, chestnuts, chickpeas, chicory, chilli peppers, chrysanthemums, citrons, coconuts, coffee, cotton, cowpeas, broad beans, cucumbers, redcurrants, gooseberries, dates, duckweed, eggplant, elderberries, eucalyptus, flax, geraniums, ginger, ginseng, grapefruit, grapes, guavas, hazelnuts, hemp, cannabis, hops, horseradish, irises, jackfruit, kiwifruit, kumquats, lemons, lentils, lettuce, limes, litchis, macadamia. Mia, corn, mandarin, mango, mangosteen, melon, millet, oats, palm, okra, olive, onion, orange, papaya, parsley, passion fruit, pecan, peach, nectarine, pear, pea, peanut, peony, persimmon, petunia, pineapple, pistachio, plantain, plum, poinsettia, pomelo, poplar, potato, pumpkin, squash, quince, raspberry 150. The plantable composition of any one of claims 138-149, wherein the plant is derived from a plant selected from the group consisting of: rhubarb, rice, rose, rubber, rye, safflower, satsuma mandarin, sesame, sorghum, bitter orange, soursop, soybean, strawberry, sugar beet, sugar cane, sunflower, sweet potato, tamarind, tangerine, tea, tobacco, tomatillo, tomato, tulip, walnut, watermelon, wheat, and yam.

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