CROSS REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 63/024,304, filed May 13, 2020, the contents of which are incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to the field of bacterial mutant strains and their ability to control insect pests. In particular, the present invention is directed to Bacillus thuringiensis strains with high levels of insecticidal activity.

BACKGROUND

[0003] Synthetic pesticides may be non-specific and therefore can act on an overly broad spectrum of organisms, including targeting other naturally occurring beneficial organisms. Because of their chemical nature, pesticides may also be toxic and non-biodegradable. Consumers worldwide are increasingly conscious of the potential environmental and health problems associated with the residuals of chemicals, particularly in food products. This has resulted in growing consumer pressure to reduce the use or at least the quantity of chemical (i.e., synthetic) pesticides. Thus, there is a need to manage food chain requirements while still allowing for effective pest control.

[0004] A further problem arising with the use of synthetic insecticides is that the repeated and exclusive application of an insecticide often leads to selection of resistant insects. Normally, such insects are also cross-resistant against other active ingredients having the same mode of action. Effective control of the insects with said active ingredients is then no longer possible, and active ingredients having new mechanisms of action are difficult and expensive to develop.

[0005] The risk of resistance development in insect populations as well as environmental and human health concerns have fostered interest in identifying alternatives to synthetic insecticides for managing plant and crop damage from insects. The use of biological insect control agents is one alternative.

[0006] B. thuringiensis is a Gram-positive spore forming soil bacterium characterized by its ability to produce crystalline inclusions that are specifically toxic to certain orders and species of plant pests, including insects, but are harmless to plants and other non-target organisms. For this reason, compositions comprising B. thuringiensis strains or their insecticidal proteins can be used as environmentally acceptable insecticides to control agricultural insect pests or insect vectors of a variety of human or animal diseases.

[0007] There is a need for effective biological control agents with insecticidal activity to complement the use of traditional, synthetic insecticides and to address the growing challenge of insect resistance.

SUMMARY OF THE INVENTION

[0008] The present invention is directed to a strategy for enhancing the insecticidal activity of a B. thuringiensis strain and mutant derivatives thereof. A strain improvement strategy was devised to enhance the production of zwittermicin A through sequential rounds of chemical treatment and high throughput screening. Several B. thuringiensis strains with improved insecticidal characteristics were generated and characterized.

[0009] The present invention is directed to a composition comprising mutants of a biologically pure culture of a B. thuringiensis strain NRRL B-67685, such as B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, capable of producing zwittermicin A in an amount greater than that produced by the parent strain. The present invention is also directed to a composition comprising a biologically pure culture of B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, B. thuringiensis strain NRRL B-68027, or a mutant strain derived from one or more of these strains, wherein the strain is capable of producing zwittermicin A in an amount greater than that produced by the parent strain. In some aspects, the composition comprises a fermentation product of a zwittermicin A producing mutant of B. thuringiensis strain NRRL B-67685, such as B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B- 67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain derived therefrom.

[0010] In one aspect, a composition is provided herein comprising a biologically pure culture of a B. thuringiensis strain or a cell-free preparation thereof, wherein the B. thuringiensis strain is capable of producing zwittermicin A in an amount at least 2-fold greater than that produced by a biologically pure culture of B. thuringiensis strain NRRL B-67685. In one embodiment, the B. thuringiensis is capable of producing zwittermicin A, Vip3Aa, CrylAa, and CrylAb and the production of zwittermicin A with Vip3Aa, CrylAa, or CrylAb results in a synergistic insecticidal effect. In another embodiment the B. thuringiensis strain is further capable of producing CrylCa and CrylDa, and the production of zwittermicin A with Vip3Aa, CrylAa, CrylAb, CrylCa or CrylDa results in a synergistic insecticidal effect. Non-limiting examples of synergistic insecticidal effects include increased developmental delay or mortality of a target insect contacted with a composition provided herein. In other embodiments, the synergistic insecticidal effect occurs with Spodoptera exigua, Plutella xylostella, Tuta absoluta or Trichoplusia ni.

[0011] In another aspect, the composition comprises B. thuringiensis strain NRRL B- 67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, B. thuringiensis strain NRRL B-68027or an insecticidal variant thereof that has all of the identifying characteristics of the respective deposited strain. In one embodiment, the composition comprises B. thuringiensis strain NRRL B-67954, or an insecticidal variant thereof that has all of the identifying characteristics of said deposited strain. In another embodiment, the composition comprises B. thuringiensis strain NRRL B-67955, or an insecticidal variant thereof that has all of the identifying characteristics of said deposited strain. In another embodiment, the composition comprises B. thuringiensis strain NRRL B-68025, or an insecticidal variant thereof that has all of the identifying characteristics of said deposited strain. In another embodiment, the composition comprises B. thuringiensis strain NRRL B-68026, or an insecticidal variant thereof that has all of the identifying characteristics of said deposited strain. In another embodiment, the composition comprises B. thuringiensis strain NRRL B-68027, or an insecticidal variant thereof that has all of the identifying characteristics of said deposited strain. In further embodiments, the insecticidal variant strain is defined as having a genomic sequence with greater than about 90% sequence identity to B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027.

[0012] In yet another aspect, the composition further comprises an agriculturally acceptable carrier, inert stabilization agent, preservative, nutrient, or physical property modifying agent. In some embodiments, the composition is a liquid formulation or a solid formulation. In particular embodiments, the composition is a liquid formulation that comprises at least about 1 x 10 ⁴ colony forming units (CFU) of the B. thuringiensis strain/m L.

[0013] In still yet another aspect, a fermentation product of B. thuringiensis strain NRRL B-67954, B. thuringiensis NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, B. thuringiensis strain NRRL B-68027, or an insecticidal variant thereof that has all of the identifying characteristics of the respective deposited strain is provided.

[0014] In one aspect, the present disclosure provides a method of controlling an insect pest comprising applying an effective amount of a composition as described herein to the insect pest or an environment thereof. [0015] In another aspect, a method of protecting a useful plant or a part thereof from insect pest damage is provided comprising contacting an insect pest, a plant, a plant part, a plant propagule, a seed of a plant, or a locus where a plant is growing or is intended to grow with an effective amount of a composition as described herein. In some embodiments, the composition is applied at about 1 x 10 ⁴ to about 1 x 10 ¹⁴ CFU per hectare or at about 0.1 kg to about 20 kg fermentation solids per hectare. Non-limiting examples of insect pests include insect pests of the order of Lepidoptera, including Acronicta major, Aedia leucomelas, Agrotis spp., Alabama argillacea, Anticarsia spp., Barathra brassicae, Bucculatrix thurberiella, Bupalus piniarius, Cacoecia podana, Capua reticulana, Carpocapsa pomonella, Cheimatobia brumata, Chile spp., Choristoneura fumiferana, Clysia ambiguella, Cnaphalocerus spp., Earias insulana, Ephestia kuehniella, Euproctis chrysorrhoea, Euxoa spp., Feltia spp., Galleria mellonella, Helicoverpa spp., Heliothis spp., Hofmannophila pseudospretella, Homona magnanima, Hyponomeuta padella, Laphygma spp., Lithocolletis blancardella, Lithophane antennata, Loxagrotis albicosta, Lymantria spp., Malacosoma neustria, Mamestra brassicae, Mods repanda, Mythimna separata, Oria spp., Oulema oryzae, Panolis flammea, Pectinophora gossypiella, Phyllocnistis citrella, Pieris spp., Plutella xylostella, Prodenia spp., Pseudaletia spp., Pseudoplusia includens, Pyrausta nubilalis, Spodoptera spp., Thermesia gemmatalis, Tinea pellionella, Tineola bisselliella, Tortrix viridana, Trichoplusia spp., Spodoptera exigua, Plutella xylostella, and Trichoplusia ni. In particular embodiments, useful plants include, but are not limited to, soybean, com, wheat, triticale, barley, oat, rye, rape, millet, rice, sunflower, cotton, sugar beet, pome fruit, stone fruit, citrus, banana, strawberry, blueberry, almond, grape, mango, papaya, peanut, potato, tomato, pepper, cucurbit, cucumber, melon, watermelon, garlic, onion, broccoli, carrot, cabbage, bean, dry bean, canola, pea, lentil, alfalfa, trefoil, clover, flax, elephant grass, grass, lettuce, sugarcane, tea, tobacco and coffee; each of which may be in a natural or genetically modified form.

[0016] In yet another aspect, the present disclosure provides the use of a composition as described herein for controlling insect pests. In still yet another aspect, the present disclosure provides the use of a composition as described herein for protecting a useful plant or part of a useful plant in need of protection from insect pest damage.

DETAILED DESCRIPTION

[0017] The microorganisms of the present invention, unless specifically noted otherwise, are separated from nature and produced under artificial conditions in a biologically pure culture. Such microorganisms may be produced, for example, in shake flask cultures or by scaled-up manufacturing processes. In specific embodiments, the microorganisms of the present invention may be produced in bioreactors to maximize bioactive metabolite production. Growth under such conditions leads to strain “domestication.” A “domesticated” strain differs from counterpart strains found in nature in that it is cultured as a homogenous population and is subject to artificial selection pressures rather than the selection pressures of the natural environment.

[0018] The verb “comprise” and its conjugations are used herein in the non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

[0019] To “control” insects means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, or reproduce, or to limit insect-related damage or loss in crop plants or to protect the yield potential of a crop when grown in the presence of insect pests. To “control” insects may or may not include killing insects, or may include inhibition of insect growth or reproduction.

[0020] In one aspect, the present invention provides methods for generating mutants of B. thuringiensis strain NRRL B-67685, a sample of which was deposited with the Agricultural Research Service Culture Collection located at the National Center for Agricultural Utilization Research, Agricultural Research Service on September 26, 2018. In one embodiment, the method includes generating mutants and screening such mutants for increased production of zwittermicin A compared to the parent strain. In some embodiments, zwittermicin A production is increased by at least about 1.5 fold to at least about 15 -fold compared to the parent strain. For example, zwittermicin A production may be increased by at least about 15-fold, at least about 14-fold, at least about 13- fold, at least about 12-fold, at least about 11-fold, at least about 10-fold, at least about 9-fold, at least about 8-fold, at least about 7-fold, at least about 6-fold, at least about 5 -fold, at least about 4-fold, at least about 3-fold, at least about 2-fold, or at least about 1.5-fold compared to the parent strain.

[0021] The compositions of the present invention comprise a biologically pure culture of a B. thuringiensis strain or a cell-free preparation thereof that is capable of producing zwittermicin A and Vip3Aa, CrylAa, and/or CrylAb, wherein the B. thuringiensis strain is capable of producing increased levels of zwittermicin A compared to the B. thuringiensis strain NRRL B-67685. In one embodiment, the production of zwittermicin A with Vip3Aa, CrylAa, and/or CrylAb results in a synergistic insecticidal effect. In some embodiments, the compositions may further comprise CrylCa and/or CrylDa and the production of zwittermicin A with Vip3Aa, CrylAa, CrylCa, Cry 1 Da and/or CrylAb may result in a synergistic insecticidal effect. [0022] In some embodiments, the zwittermicin A may be produced by the B. thuringiensis strain in an amount between 1.5-fold and 5-fold, between 5-fold and 10-fold, or between 10-fold and 15 -fold greater than the amount that is produced by a biologically pure culture of B. thuringiensis strain NRRL B-67685. Zwittermicin A may, for example, be produced in an amount between 1.5-fold and 2-fold, between 1.5-fold and 3-fold, between 1.5-fold and 4-fold, between 1.5-fold and 5-fold, between 1.5-fold and 6-fold, between 1.5-fold and 7-fold, between 1.5- fold and 8-fold, between 1.5-fold and 9-fold, between 1.5-fold and 10-fold, between 1.5-fold and 11- fold between 1.5-fold and 12-fold, between 1.5-fold and 13-fold, between 1.5-fold and 14-fold, or between 1.5-fold and 15-fold greater than the amount that is produced by a biologically pure culture of B. thuringiensis strain NRRL B-67685.

[0023] In other embodiments, zwittermicin A may be produced in an amount at least about 15-fold, at least about 14-fold, at least about 13-fold, at least about 12-fold, at least about 11- fold, at least about 10-fold, at least about 9-fold, at least about 8-fold, at least about 7-fold, at least about 6-fold, at least about 5-fold, at least about 4-fold, at least about 3-fold, at least about 2-fold, or at least about 1.5-fold greater than that produced by a biologically pure culture of B. thuringiensis strain NRRL B-67685.

[0024] In another embodiment, the compositions of the present invention comprise a fermentation broth of a B. thuringiensis strain that is capable of producing zwittermicin A and Vip3Aa, CrylAa, CrylAb, CrylCa and/or CrylDa, wherein the zwittermicin is present in the fermentation broth at a concentration of at least about 0.75 mg/g fermentation broth, at least about 1.0 mg/g fermentation broth, at least about 1.5 mg/g fermentation broth, at least about 2.0 mg/g fermentation broth, at least about 2.5 mg/g fermentation broth, at least about 3.0 mg/g fermentation broth, at least about 3.5 mg/g fermentation broth, at least about 4.0 mg/g fermentation broth. In this embodiment, the zwittermicin A in the fermentation broth is solely the zwittermicin produced by the B. thuringiensis strain. In other words, zwittermicin A from a source other than the strain is not added to the fermentation broth. In another aspect of this embodiment, the strain is an insecticidal mutant strain of B. thuringiensis strain NRRL B-67685, such as B. thuringiensis strain NRRL B- 67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027. In another aspect of this embodiment, the composition comprises a fermentation product (e.g., a broth concentrate or fermentation solid) produced from this fermentation broth.

[0025] In another aspect, the present invention relates to a method of identifying a B. thuringiensis mutant derivative strain capable of producing zwittermicin A an amount greater than that produced by the B. thuringiensis parental strain. In one embodiment, the method may comprise: a) mutagenizing the B. thuringiensis parental strain to produce mutant isolates; b) culturing the mutant isolates and the B. thuringiensis parental strain in liquid medium; c) collecting the liquid medium from each strain; d) measuring the amount of zwittermicin A; and e) identifying a B. thuringiensis mutant derivative strain with increased production of zwittermicin A compared to the B. thuringiensis parental strain. In specific embodiments, zwittermicin A may be measured using Single Reaction Monitoring (SRM) single quadrupole mass spectrometry (MS), Multiple Reaction Monitoring (MRM) triple quad MS, or Ultra High Performance Liquid Chromatography/MS (UPLC- MS).

[0026] B. thuringiensis strain NRRL B-67685 was previously identified as a strain capable of producing increased amounts of zwittermicin A (PCT/US2019/061554) compared to a biologically pure culture of B. thuringiensis subsp. kurstaki strain EG7841.

[0027] In one aspect, an insecticidal mutant strain of the B. thuringiensis strain NRRL B- 67685, B. thuringiensis strain NRRL B-67954, or B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, is provided. The term “mutant” refers to a genetic variant derived from the B. thuringiensis strain. The mutant strain may have, for example, one or more or all of the identifying, functional characteristics of the B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027. In a particular instance, the mutant or a fermentation product thereof controls, as an identifying functional characteristic, insects at least as well as the parent B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027. Such mutants may be genetic variants having a genomic sequence that has greater than about 85%, greater than about 90%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% sequence identity to B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027. As used herein, the term “sequence identity” refers to the extent to which two optimally aligned polynucleotide sequences are identical. An optimal sequence alignment is created by manually aligning two sequences, e.g., a reference sequence and another sequence, to maximize the number of nucleotide matches in the sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term “reference sequence” refers to the genomic sequence of B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027. In particular embodiments, insecticidal mutant strains of the B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027 may be genetic variants having greater than about 90%, 95%, 96%, 97%, 98% or 99% sequence identity over the full length of the genomic sequence of B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027. Mutants may be obtained by treating cells of B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027 with chemicals or irradiation or by selecting spontaneous mutants from a population of B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027 cells (such as phage resistant or antibiotic resistant mutants), by genome shuffling, as described below, or by other means well known to those practiced in the art. Such mutants may then be screened for improved production of zwittermicin A compared to the parent strains. Multiple rounds of mutagenesis, with and without screening between rounds, may be used to generate and screen mutants. Fermentation products of mutants having increased production of zwittermicin A may be produced and applied to plants to control pests.

[0028] Genome shuffling among B. thuringiensis strains can be facilitated through the use of a process called protoplast fusion. The process begins with the formation of protoplasts from vegetative bacillary cells. The removal of peptidoglycan cell wall, typically using lysozyme and an osmotic stabilizer, results in the formation of a protoplast. This process is visible under a light microscope with the appearance of spherical cells. Addition of polyethylene glycol (PEG) then induces fusion among protoplasts, allowing genetic contents of two or more cells to come in contact facilitating recombination and genome shuffling. Fused cells then repartition and are recovered on a solid growth medium. During recovery, protoplasts rebuild peptidoglycan cell walls, transitioning back to bacillary shape. See Schaeffer, et a , (1976) PNAS USA, vol. 73, 6:2151-2155.

[0029] Specific examples of generating B. thuringiensis mutants are described below in the Examples section.

[0030] In another aspect, the present invention provides a composition comprising a) zwittermicin A and b) a Vip3A, Cryl Aa, Cryl Ab, CrylCa and/or CrylDa protein in a synergistically effective amount. The composition may comprise a fermentation product of a bacterial strain capable of producing the zwittermicin A and Vip3A, Cryl Aa, Cryl Ab, Cry ICa and/or CrylDa. The bacterial strain may be, for example, an Escherichia coli strain or a Bacillus sp. strain (e.g., B. thuringiensis). The Vip3A, CrylAa, CrylAb, CrylCa and/or CrylDa may be provided, for example, as a fermentation product of an E. coli strain that expresses Vip3A, CrylAa, CrylAb, CrylCa and/or CrylDa or a cell-free preparation of such E. coli strain. The zwittermicin A may be provided separately as a purified compound, a fermentation product of a B. thuringiensis strain that expresses zwittermicin A, or as a purified or partially purified extract of such fermentation product. The zwittermicin A may be provided, for example, as a fermentation product of a B. thuringiensis strain that has increased production of zwittermicin A compared to B. thuringiensis strain NRRL B-67685 or as a purified or partially purified extract of such fermentation product. Surprisingly, zwittermicin A is shown in the instant application to exhibit a synergistic insecticidal effect when produced together with VIP3a, CrylAa, CrylAb, CrylCa and/or CrylDa or combinations thereof even when total Cry protein content is reduced.

[0031] In one aspect, the synergistically effective amount refers to a synergistic weight ratio. In one aspect, the synergistic weight ratio of a) zwittermicin A and b) a Vip3A, CrylAa, CrylAb, CrylCa and/or CrylDa protein lies in the range of 1:500 to 1000:1, preferably in the range of 1:500 to 500:1, more preferably in the range of 1:500 to 300:1. In other aspects, the synergistic weight ratio of a) zwittermicin A; and b) a Vip3A, CrylAa, CrylAb, CrylCa and/or CrylDa protein lies in the range of 1:1000 to 1000:1, 1:100 to 100:1, 1:50 to 50:1, 1:25 to 25:1, 1:10 to 10:1, 1:5 to 5:1, or 1:2 to 2:1.

[0032] The present invention also provides methods of treating a plant to control insect pests by administering to a plant or a plant part, such as a leaf, stem, flowers, fruit, root, or seed or by applying to a locus on which plant or plant parts grow, such as soil, the disclosed B. thuringiensis strains or mutants thereof, or cell-free preparations thereof or metabolites thereof.

[0033] In a method according to the invention a composition containing a disclosed B. thuringiensis strain or an insecticidal mutant thereof can be applied to any plant or any part of any plant grown in any type of media used to grow plants (e.g., soil, vermiculite, shredded cardboard, and water) or applied to plants or the parts of plants grown aerially, such as orchids or staghorn ferns. The composition may for instance be applied by spraying, atomizing, vaporizing, scattering, dusting, watering, squirting, sprinkling, pouring or fumigating. Application may be carried out at any desired location where the plant of interest is positioned, such as agricultural, horticultural, forest, plantation, orchard, nursery, organically grown crops, turfgrass and urban environments. [0034] Compositions of the present invention can be obtained by culturing the disclosed B. thuringiensis strains or an insecticidal mutant strain derived therefrom according to methods well known in the art, including by using the media and other methods described in the examples below. Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, cells begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of B. thuringiensis and is generally initiated by the cell in response to nutrient limitation. Fermentation is configured to obtain high levels of colony forming units and to promote sporulation. The bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation.

[0035] Compositions of the present invention include fermentation products. In some embodiments, the concentrated fermentation broth is washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolites. The term “broth concentrate,” as used herein, refers to whole broth (fermentation broth) that has been concentrated by conventional industrial methods, as described above, but remains in liquid form. The term “fermentation solid,” as used herein, refers to the solid material that remains after the fermentation broth or broth concentrate is dried. The term “fermentation product,” as used herein, refers to whole broth, broth concentrate and/or fermentation solids. Compositions of the present invention include fermentation products.

[0036] The fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation.

[0037] The resulting dry products may be further processed, such as by milling or granulation, to achieve a specific particle size or physical format. Carriers, described below, may also be added post-drying.

[0038] Cell-free preparations of fermentation broth of the strains of the present invention can be obtained by any means known in the art, such as extraction, centrifugation and/or filtration of fermentation broth. Those of skill in the art will appreciate that so-called cell-free preparations may not be devoid of cells but rather are largely cell-free or essentially cell-free, depending on the technique used (e.g., speed of centrifugation) to remove the cells. The resulting cell-free preparation may be dried and/or formulated with components that aid in its application to plants or to plant growth media. Concentration methods and drying techniques described above for fermentation broth are also applicable to cell-free preparations. [0039] In certain aspects, the fermentation product further comprises a formulation ingredient. The formulation ingredient may be a wetting agent, extender, solvent, spontaneity promoter, emulsifier, dispersant, frost protectant, thickener, and/or an adjuvant. In one embodiment, the formulation ingredient is a wetting agent. In other aspects, the fermentation product is a freeze- dried powder or a spray-dried powder.

[0040] Compositions of the present invention may include formulation ingredients added to compositions of the present invention to improve recovery, efficacy, or physical properties and/or to aid in processing, packaging and administration. Such formulation ingredients may be added individually or in combination.

[0041] The formulation ingredients may be added to compositions comprising cells, cell- free preparations, isolated compounds, and/or metabolites to improve efficacy, stability, and physical properties, usability and/or to facilitate processing, packaging and end-use application. Such formulation ingredients may include agriculturally acceptable carriers, inerts, stabilization agents, UV protectants, preservatives, nutrients, or physical property modifying agents, which may be added individually or in combination. In some embodiments, the carriers may include liquid materials such as water, oil, and other organic or inorganic solvents and solid materials such as minerals, polymers, or polymer complexes derived biologically or by chemical synthesis. In some embodiments, the formulation ingredient is a binder, adjuvant, or adhesive that facilitates adherence of the composition to a plant part, such as leaves, seeds, or roots. See, for example, Taylor, A.G., et a , “Concepts and Technologies of Selected Seed Treatments,” Annu. Rev. Phytopathol. , 28: 321-339 (1990). The stabilization agents may include anti-caking agents, anti-oxidation agents, anti-settling agents, antifoaming agents, desiccants, protectants or preservatives. The nutrients may include carbon, nitrogen, and phosphorus sources such as sugars, polysaccharides, oil, proteins, amino acids, fatty acids and phosphates. The physical property modifiers may include bulking agents, wetting agents, thickeners, pH modifiers, rheology modifiers, dispersants, adjuvants, surfactants, film-formers, hydrotropes, builders, antifreeze agents or colorants. In some embodiments, the composition comprising cells, cell-free preparation and/or metabolites produced by fermentation can be used directly with or without water as the diluent without any other formulation preparation. In a particular embodiment, a wetting agent, or a dispersant, is added to a fermentation solid, such as a freeze-dried or spray-dried powder. In some embodiments, the formulation inerts are added after concentrating fermentation broth and/or during and/or after drying. A wetting agent increases the spreading and penetrating properties, or a dispersant increases the dispersability and solubility of the active ingredient (once diluted) when it is applied to surfaces. Exemplary wetting agents are known to those of skill in the art and include sulfosuccinates and derivatives, such as MULTIWET™ MO-70R (Croda Inc., Edison, NJ); siloxanes such as BREAK- THRU ^® (Evonik, Germany); nonionic compounds, such as ATLOX™ 4894 (Croda Inc., Edison, NJ); alkyl polyglucosides, such as TERWET ^® 3001 (Huntsman International LLC, The Woodlands, Texas); C12-C14 alcohol ethoxylate, such as TERGITOL ^® 15-S-15 (The Dow Chemical Company, Midland, Michigan); phosphate esters, such as RHODAFAC ^® BG-510 (Rhodia, Inc.); and alkyl ether carboxylates, such as EMULSOGEN™ LS (Clariant Corporation, North Carolina).

[0042] In one embodiment, the fermentation product comprises at least about 1 x 10 ⁴ colony forming units (CFU) of the microorganism (e.g., B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth. In another embodiment, the fermentation product comprises at least about 1 x 10 ⁵ colony forming units (CFU) of the microorganism (e.g., B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth. In another embodiment, the fermentation product comprises at least about 1 x 10 ⁶ CFU of the microorganism (e.g., B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B- 68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth. In yet another embodiment, the fermentation product comprises at least about 1 x 10 ⁷ CFU of the microorganism (e.g., B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth. In another embodiment, the fermentation product comprises at least about 1 x 10 ⁸ CFU of the microorganism (e.g., B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth. In another embodiment, the fermentation product comprises at least about 1 x 10 ⁹ CFU of the microorganism (e.g., B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth. In another embodiment, the fermentation product comprises at least about 1 xlO ¹⁰ CFU of the microorganism (e.g., B. thuringiensis strain NRRL B- 67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth. In another embodiment, the fermentation product comprises at least about 1 x 10 ¹¹ CFU of the microorganism (e.g., B. thuringiensis strain NRRL B-67685, B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain thereof)/mL broth.

[0043] In another aspect, the zwittermicin A is present in the fermentation broth of a B. thuringiensis strain, such as B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B- 67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, or B. thuringiensis strain NRRL B-68027, or an insecticidal mutant strain of any of these strains at a concentration of at least about 0.75 mg/g fermentation broth, at least about 1.0 mg/g fermentation broth, at least about 1.5 mg/g fermentation broth, at least about 2.0 mg/g fermentation broth, at least about 2.5 mg/g fermentation broth, or at least about 3.0 mg/g fermentation broth. In one version of this aspect, the zwittermicin A is produced solely by the strain and no zwittermicin A is added to the fermentation broth.

[0044] The inventive compositions can be used as such or, depending on their particular physical and/or chemical properties, in the form of their formulations or the use forms prepared therefrom, such as aerosols, capsule suspensions, cold-fogging concentrates, warm-fogging concentrates, encapsulated granules, fine granules, flowable concentrates for the treatment of seed, ready-to-use solutions, dustable powders, emulsifiable concentrates, oil-in-water emulsions, water- in-oil emulsions, macrogranules, microgranules, oil-dispersible powders, oil-miscible flowable concentrates, oil-miscible liquids, gas (under pressure), gas generating product, foams, pastes, pesticide coated seed, suspension concentrates, oil dispersion, suspo-emulsion concentrates, soluble concentrates, suspensions, wettable powders, soluble powders, dusts and granules, water-soluble and water-dispersible granules or tablets, water-soluble and water-dispersible powders for the treatment of seed, wettable powders, natural products and synthetic substances impregnated with active ingredient, and also microencapsulations in polymeric substances and in coating materials for seed, and also ULV cold-fogging and warm-fogging formulations.

[0045] In some embodiments, the inventive compositions are liquid formulations. Non limiting examples of liquid formulations include suspension concentrations and oil dispersions. In other embodiments, the inventive compositions are solid formulations. Non-limiting examples of solid formulations include freeze-dried powders and spray-dried powders.

[0046] All plants and plant parts can be treated in accordance with the invention. In the present context, plants are understood as meaning all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants which can be obtained by traditional breeding and optimization methods or by biotechnological and recombinant methods, or combinations of these methods, including the transgenic plants and including the plant varieties capable or not of being protected by Plant Breeders’ Rights. Plant parts are understood as meaning all aerial and subterranean parts and organs of the plants, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruiting bodies, fruits and seeds, and also roots, tubers and rhizomes. The plant parts also include crop material and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, slips and seeds.

[0047] In a preferred embodiment, plant species and plant varieties, and their parts, which grow wild or which are obtained by traditional biological breeding methods such as hybridization or protoplast fusion are treated. In a further preferred embodiment, transgenic plants and plant varieties which have been obtained by recombinant methods, if appropriate in combination with traditional methods (genetically modified organisms), and their parts are treated. The term “parts” or “parts of plants” or “plant parts” has been explained herein above. Plants of the plant varieties which are in each case commercially available or in use are especially preferably treated in accordance with the invention. Plant varieties are understood as meaning plants with novel traits which have been bred both by traditional breeding, by mutagenesis or by recombinant DNA techniques. They may take the form of varieties, races, biotypes and genotypes.

[0048] The treatment of the plants and plant parts with the compositions according to the invention is carried out directly or by acting on the environment, habitat or storage space using customary treatment methods, for example by dipping, spraying, atomizing, misting, evaporating, dusting, fogging, scattering, foaming, painting on, spreading, injecting, drenching, trickle irrigation and, in the case of propagation material, in particular in the case of seed, furthermore by the dry seed treatment method, the wet seed treatment method, the slurry treatment method, by encrusting, by coating with one or more coats and the like. It is furthermore possible to apply the active substances by the ultra-low volume method or to inject the active substance preparation or the active substance itself into the soil.

[0049] A preferred direct treatment of the plants is the leaf application treatment, i.e., compositions according to the invention are applied to the foliage, it being possible for the treatment frequency and the application rate to be matched to the infection pressure of the pathogen in question.

[0050] In the case of systemically active agents, the compositions according to the invention reach the plants via the root system. In this case, the treatment of the plants is effected by allowing the compositions according to the invention to act on the environment of the plant. This can be done for example by drenching, incorporating in the soil or into the nutrient solution, i.e., the location of the plant (for example the soil or hydroponic systems) is impregnated with a liquid form of the compositions according to the invention, or by soil application, i.e., the compositions according to the invention are incorporated into the location of the plants in solid form (for example in the form of granules). In the case of paddy rice cultures, this may also be done by metering the compositions according to the invention into a flooded paddy field in a solid use form (for example in the form of granules).

[0051] Preferred plants are those from the group of the useful plants, ornamentals, turfs, generally used trees which are employed as ornamentals in the public and domestic sectors, and forestry trees. Forestry trees comprise trees for the production of timber, cellulose, paper and products made from parts of the trees.

[0052] The term “useful plants” as used in the present context refers to crop plants which are employed as plants for obtaining foodstuffs, feedstuffs, fuels or for industrial purposes.

[0053] The useful plants which can be treated and/or improved with the compositions and methods of the present invention include for example the following types of plants: turf, vines, cereals, for example wheat, barley, rye, oats, rice, maize and millet/sorghum; beet, for example sugar beet and fodder beet; fruits, for example pome fruit, stone fmit and soft fruit, for example apples, pears, plums, peaches, almonds, cherries and berries, for example strawberries, raspberries, blackberries; legumes, for example beans, lentils, peas and soybeans; oil crops, for example oilseed rape, mustard, poppies, olives, sunflowers, coconuts, castor oil plants, cacao and peanuts; cucurbits, for example pumpkin/squash, cucumbers and melons; fibre plants, for example cotton, flax, hemp and jute; citrus fruit, for example oranges, lemons, grapefruit and tangerines; vegetables, for example spinach, lettuce, asparagus, cabbage species, carrots, onions, tomatoes, potatoes and bell peppers; Lauraceae, for example avocado, Cinnamomum, camphor, or else plants such as tobacco, nuts, coffee, aubergine, sugar cane, tea, pepper, grapevines, hops, bananas, latex plants and ornamentals, for example flowers, shrubs, deciduous trees and coniferous trees. This enumeration is no limitation.

[0054] The following plants are considered to be particularly suitable target crops for applying compositions and methods of the present invention: cotton, aubergine, turf, pome fruit, stone fruit, soft fmit, maize, wheat, barley, cucumber, tobacco, vines, rice, cereals, pear, beans, soybeans, oilseed rape, tomato, bell pepper, melons, cabbage, potato and apple.

[0055] Additional useful plants include cereals, for example wheat, rye, barley, triticale, oats or rice; beet, for example sugar beet or fodder beet; fruits, such as pomes, stone fruits or soft fruits, for example apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries; leguminous plants, such as lentils, peas, alfalfa or soybeans; oil plants, such as rape, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans; cucurbits, such as squashes, cucumber or melons; fiber plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruits or mandarins; vegetables, such as broccoli, spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika; lauraceous plants, such as avocados, cinnamon or camphor; energy and raw material plants, such as com, soybean, rape, sugar cane or oil palm; com; tobacco; nuts; coffee; tea; bananas; vines (table grapes and grape juice grape vines); hop; turf; natural rubber plants or ornamental and forestry plants, such as flowers, shmbs, broad-leaved trees or evergreens, for example conifers; and on the plant propagation material, such as seeds, and the crop material of these plants.

[0056] In a preferred embodiment of the invention, the useful plant is selected from soybean, com, wheat, triticale, barley, oat, rye, rape, millet, rice, sunflower, cotton, sugar beet, pome fruit, stone fmit, citms, banana, strawberry, blueberry, almond, grape, mango, papaya, peanut, potato, tomato, pepper, cucurbit, cucumber, melon, watermelon, garlic, onion, broccoli, carrot, cabbage, bean, dry bean, canola, pea, lentil, alfalfa, trefoil, clover, flax, elephant grass, grass, lettuce, sugarcane, tea, tobacco and coffee; each in its natural or genetically modified form.

[0057] The B. thuringiensis strains according to the invention, in combination with good plant tolerance and favorable toxicity to warm-blooded animals and being tolerated well by the environment, are suitable for protecting plants and plant organs, for increasing harvest yields, for improving the quality of the harvested material and for controlling insect pests, which are encountered in agriculture, in horticulture, in animal husbandry, in forests, in gardens and leisure facilities, in protection of stored products and of materials, and in the hygiene sector. They can be preferably employed as plant protection agents. They are active against normally sensitive and resistant species and against all or some stages of development. The abovementioned pests include:

[0058] pests from the phylum Arthropoda, especially from the class Arachnida, for example Acarus spp., Aceria kuko, Aceria sheldoni, Aculops spp., Aculus spp., Amblyomma spp., Amphitetranychus viennensis, Argas spp., Boophilus spp., Brevipalpus spp., Bryobia graminum, Bryobia praetiosa, Centruroides spp., Chorioptes spp., Dermanyssus gallinae, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Dermacentor spp., Eotetranychus spp., Epitrimerus pyri, Eutetranychus spp., Eriophyes spp., Glycyphagus domesticus, Halotydeus destructor, Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus spp., Loxosceles spp., Metatetranychus spp., Neutrombicula autumnalis, Nuphersa spp., Oligonychus spp., Ornithodorus spp., Ornithonyssus spp., Panonychus spp., Phyllocoptruta oleivora, Platytetranychus multidigituli, Polyphagotarsonemus latus, Psoroptes spp., Rhipicephalus spp., Rhizoglyphus spp., Sarcoptes spp., Scorpio maurus, Steneotarsonemus spp., Steneotarsonemus spinki, Tarsonemus spp., Tetranychus spp., Trombicula alfreddugesi, Vaejovis spp., Vasates lycopersicv,

[0059] from the class Chilopoda, for example, Geophilus spp., Scutigera spp.;

[0060] from the order or the class Collembola, for example, Onychiurus armatus, Sminthurus viridis,

[0061] from the class Diplopoda, for example, Blaniulus guttulatus,

[0062] from the class Insecta, e.g., from the order Blattodea, for example Blatta orientalis, Blattella asahinai, Blattella germanica, Leucophaea maderae, Loboptera decipiens, Neostylopyga rhombifolia, Panchlora spp., Parcoblatta spp., Periplaneta spp., Pycnoscelus surinamensis, Supella longipalpcv,

[0063] from the order Coleoptera, for example, Acalymma vittatum, Acanthoscelides obtectus, Adoretus spp., Aethina tumida, Agelastica alni, Agriotes spp., Alphitobius diaperinus, Amphimallon solstitialis , Anobium punctatum, Anoplophora spp., Anthonomus spp., Anthrenus spp., Apion spp., Apogonia spp., Atomaria spp., Attagenus spp., Baris caerulescens, Bruchidius obtectus, Bruchus spp., Cassida spp., Cerotoma trifurcata, Ceutorrhynchus spp., Chaetocnema spp., Cleonus mendicus, Conoderus spp., Cosmopolites spp., Costelytra zealandica, Ctenicera spp., Curculio spp., Cryptolestes ferrugineus, Cryptorhynchus lapathi, Cylindrocopturus spp., Dermestes spp., Diabrotica spp., Dichocrocis spp., Dicladispa armigera, Diloboderus spp., Epicaerus spp., Epilachna spp., Epitrix spp., Faustinus spp., Gibbium psylloides, Gnathocerus cornutus, Hellula undalis, Heteronychus arator, Heteronyx spp., Hylamorpha elegans, Hylotrupes bajulus, Hypera postica, Hypomeces squamosus, Hypothenemus spp., Lachnosterna consanguinea, Lasioderma serricorne, Latheticus oryzae, Lathridius spp., Lema spp., Leptinotarsa decemlineata, Leucoptera spp., Lissorhoptrus oryzophilus, Listronotus (Hyperodes) spp., Lixus spp., Luperodes spp., Luperomorpha xanthodera, Lyctus spp., Megascelis spp., Melanotus spp., Meligethes aeneus, Melolontha spp., Migdolus spp., Monochamus spp., Naupactus xanthographus, Necrobia spp., Neogalerucella spp., Niptus hololeucus, Oryctes rhinoceros, Oryzaephilus surinamensis, Oryzaphagus oryzae, Otiorrhynchus spp., Oulema spp., Oulema melanopus, Oulema oryzae, Oxycetonia jucunda, Phaedon cochleariae, Phyllophaga spp., Phyllophaga helleri, Phyllotreta spp., Popillia japonica, Premnotrypes spp., Prostephanus truncatus, Psy Modes spp., Ptinus spp., Rhizobius ventralis, Rhizopertha dominica, Rhynchophorus spp., Rhynchophorus ferrugineus, Rhynchophorus palmarum, Sinoxylon perforans, Sitophilus spp., Sitophilus oryzae, Sphenophorus spp., Stegobium paniceum, Sternechus spp., Symphyletes spp., Tanymecus spp., Tenebrio molitor, Tenebrioides mauretanicus, Tribolium spp., Trogoderma spp., Tychius spp., Xylotrechus spp., Zabrus spp.; [0064] from the order Dermaptera, for example Anisolabis maritime, Forficula auricularia, Labidura riparia,

[0065] from the order Diptera, for example Aedes spp., Agromyza spp., Anastrepha spp., Anopheles spp., Asphondylia spp., Bactrocera spp., Bibio hortulanus, Calliphora erythrocephala, Calliphora vicina, Ceratitis capitata, Chironomus spp., Chrysomya spp., Chrysops spp., Chrysozona pluvialis, Cochliomyia spp., Contarinia spp., Cordylobia anthropophaga, Cricotopus sylvestris, Culex spp., Culicoides spp., Culiseta spp., Cuterebra spp., Dacus oleae, Dasineura spp., Delia spp., Dermatobia hominis, Drosophila spp., Echinocnemus spp., Euleia heraclei, Fannia spp., Gasterophilus spp., Glossina spp., Elaematopota spp., Etydrellia spp., Etydrellia griseola, Elylemya spp., Elippobosca spp., Elypoderma spp., Liriomyza spp., Lucilia spp., Lutzomyia spp., Mansonia spp., Musca spp., Oestrus spp., Oscinella frit, Paratanytarsus spp., Paralauterborniella subcincta, Pegomyia or Pegomya spp., Phlebotomus spp., Phorbia spp., Phormia spp., Piophila casei, Platyparea poeciloptera, Prodiplosis spp., Psila rosae, Rhagoletis spp., Sarcophaga spp., Simulium spp., Stomoxys spp., Tabanus spp., Tetanops spp., Tipula spp., Toxotrypana curvicauda,

[0066] from the order Hemiptera, for example, Acizzia acaciaebaileyanae, Acizzia dodonaeae, Acizzia uncatoides, Acrida turrita, Acyrthosiphon spp., Acrogonia spp., Aeneolamia spp., Agonoscena spp., Aleurocanthus spp., Aleyrodes proletella, Aleurolobus barodensis, Aleurothrixus floccosus, Allocaridara malayensis, Amrasca spp., Anur aphis cardui, Aonidiella spp., Aphanostigma piri, Aphis spp., Arboridia apicalis, Arytainilla spp., Aspidiella spp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia tabaci, Blastopsylla occidentalis, Boreioglycaspis melaleucae, Brachycaudus helichrysi, Brachycolus spp., Brevicoryne brassicae, Cacopsylla spp., Calligypona marginata, Capulinia spp., Carneocephala fulgida, Ceratovacuna lanigera, Cercopidae, Ceroplastes spp., Chaetosiphon fragaefolii, Chionaspis tegalensis, Chlorita onukii, Chondracris rosea, Chromaphis juglandicola, Chrysomphalus aonidum, Chrysomphalus ficus, Cicadulina mbila, Coccomytilus halli, Coccus spp., Cryptomyzus ribis, Cryptoneossa spp., Ctenarytaina spp., Dalbulus spp., Dialeurodes chittendeni, Dialeurodes citri, Diaphorina citri, Diaspis spp., Diuraphis spp., Doralis spp., Drosicha spp., Dysaphis spp., Dysmicoccus spp., Empoasca spp., Eriosoma spp., Erythroneura spp., Eucalyptolyma spp., Euphyllura spp., Euscelis bilobatus, Ferrisia spp., Fiorinia spp., Furcaspis oceanica, Geococcus coffeae, Glycaspis spp., Eleteropsylla cubana, Eleteropsylla spinulosa, Elomalodisca coagulata, Hyalopterus arundinis, Hyalopterus pruni, Icerya spp., Idiocerus spp., Idioscopus spp., Laodelphax striatellus, Lecanium spp., Lepidosaphes spp., Lipaphis erysimi, Lopholeucaspis japonica, Lycorma delicatula, Macrosiphum spp., Macrosteles facifrons, Mahanarva spp., Melanaphis sacchari, Metcalfiella spp., Metcalfa pruinosa, Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis, Myzus spp., Nasonovia ribisnigri, Neomaskellia spp., Nephotettix spp., Nettigoniclla spectra, Nilaparvata lugens, Oncometopia spp., Orthezia praelonga, Oxya chinensis, Pachypsylla spp., Parabemisia myricae, Paratrioza spp., Parlatoria spp., Pemphigus spp., Peregrinus maidis, Perkinsiella spp., Phenacoccus spp., Phloeomyzus passerinii, Phorodon humuli, Phylloxera spp., Pinnaspis aspidistrae, Planococcus spp., Prosopidopsylla flava, Protopulvinaria pyriformis, Pseudaulacaspis pentagona, Pseudococcus spp., Psyllopsis spp., Psylla spp., Pteromalus spp., Pulvinaria spp., Pyrilla spp., Quadraspidiotus spp., Quesada gigas, Rastrococcus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus titanus, Schizaphis graminum, Selenaspidus articulatus, Sitobion avenae, Sogata spp., Sogatella furcifera, Sogatodes spp., Stictocephala festina, Siphoninus phillyreae, Tenalaphara malayensis, Tetragonocephela spp., Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp., Trialeurodes vaporariorum, Trioza spp., Typhlocyba spp., Unaspis spp., Viteus vitifolii, Zygina spp.;

[0067] from the suborder Heteroptera, for example, Aelia spp., Anasa tristis, Antestiopsis spp., Boisea spp., Blissus spp., Calocoris spp., Campylomma livida, Cavelerius spp., Cimex spp., Collaria spp., Creontiades dilutus, Dasynus piperis, Dichelops furcatus, Diconocoris hewetti, Dysdercus spp., Euschistus spp., Eurydema spp., Eurygaster spp., Halyomorpha halys, Heliopeltis spp., Elorcias nobilellus, Leptocorisa spp., Leptocorisa varicornis, Leptoglossus occidentalis, Leptoglossus phyllopus, Lygocoris spp., Lygus spp., Macropes excavatus, Megacopta cribraria, Miridae, Monalonion atratum, Nezara spp., Nysius spp., Oebalus spp., Pentomidae, Piesma quadrata, Piezodorus spp., Psallus spp., Pseudacysta persea, Rhodnius spp., Sahlbergella singularis, Scaptocoris castanea, Scotinophora spp., Stephanitis nashi, Tibraca spp., Triatoma spp.;

[0068] from the order Hymenoptera, for example, Acromyrmex spp., Athalia spp., Atta spp., Camponotus spp., Dolichovespula spp., Diprion spp., Hoplocampa spp., Lasius spp., Linepithema (Iridiomyrmex) humile, Monomorium pharaonis, Paratrechina spp., Paravespula spp., Plagiolepis spp., Sirex spp., Solenopsis invicta, Tapinoma spp., Technomyrmex albipes, Urocerus spp., Vespa spp., Wasmannia auropunctata, Xeris spp.;

[0069] from the order Isopoda, for example, Armadillidium vulgare, Oniscus asellus, Porcellio scaber,

[0070] from the order Isoptera, for example, Coptotermes spp., Cornitermes cumulans, Cryptotermes spp., Incisitermes spp., Kalotermes spp., Microtermes obesi, Nasutitermes spp., Odontotermes spp., Porotermes spp., Reticulitermes spp.;

[0071] from the order Lepidoptera, for example, Achroia grisella, Acronicta major, Adoxophyes spp., Aedia leucomelas, Agrotis spp., Alabama spp., Alabama argillacea, Amyelois transitella, Anarsia spp., Anticarsia spp., Argyroploce spp. Autographa spp., Barathra brassicae, Blastodacna atra, Borbo cinnara, Bucculatrix thurberiella, Bupalus piniarius, Busseola spp., Cacoecia spp., Cacoecia podana, Caloptilia theivora, Capua reticulana, Carpocapsa pomonella, Carposina niponensis, Cheimatobia brumata, Chile spp., Choreutis pariana, Choristoneura spp., Choristoneura fumiferana, Chrysodeixis chalcites, Clysia ambiguella, Cnaphalocerus spp., Cnaphalocrocis medinalis, Cnephasia spp., Conopomorpha spp., Conotrachelus spp., Copitarsia spp., Cydia spp., Dalaca noctuides, Diaphania spp., Diparopsis spp., Diatraea saccharalis, Earias spp., Earias insulana, Ecdytolopha aurantium, Elasmopalpus lignosellus, Eldana saccharina, Ephestia spp., Ephestia kuehniella, Epinotia spp., Epiphyas postvittana, Erannis spp., Erschoviella musculana, Etiella spp., Eudocima spp., Eulia spp., Eupoecilia ambiguella, Euproctis spp., Euproctis chrysorrhoea, Euxoa spp., Feltia spp., Galleria mellonella, Gracillaria spp., Grapholitha spp., Hedylepta spp., Helicoverpa spp., Heliothis spp., Hofmannophila pseudospretella, Homoeosoma spp., Homona spp., Homona magnanima, Hyponomeuta padella, Kakivoria flavofasciata, Lampides spp., Laphygma spp., Laspeyresia molesta, Leucinodes orbonalis, Leucoptera spp., Lithocolletis spp., Lithocolletis blancardella, Lithophane antennata, Lobesia spp., Loxagrotis albicosta, Lymantria spp., Lyonetia spp., Malacosoma neustria, Maruca testulalis, Mamestra brassicae, Melanitis leda, Mods spp., Mods repanda, Monopis obviella, Mythimna separata, Nemapogon cloacellus, Nymphula spp., Oiketicus spp., Omphisa spp., Operophtera spp., Oria spp., Orthaga spp., Ostrinia spp., Panolis flammea, Parnara spp., Pectinophora spp., Pectinophora gossypiella, Perileucoptera spp., Phthorimaea spp., Phyllocnistis dtrella, Phyllonorycter spp., Pieris spp., Platynota stultana, Plodia interpunctella, Plusia spp., Plutella xylostella (Plutella maculipennis), Prays spp., Prodenia spp., Protoparce spp., Pseudaletia spp., Pseudaletia unipuncta, Pseudoplusia indudens, Pyrausta nubilalis, Rachiplusia nu, Schoenobius spp., Scirpophaga spp., Scirpophaga innotata, Scotia segetum, Sesamia spp., Sesamia inferens, Sparganothis spp., Spodoptera spp., Spodoptera exigua, Spodoptera praefica, Stathmopoda spp., Stenoma spp., Stomopteryx subsecivella, Synanthedon spp., Tecia solanivora, Thaumetopoea spp., Thermesia gemmatalis, Tinea cloacella, Tinea pellionella, Tineola bisselliella, Tortrix spp., Tortrix viridana, Trichophaga tapetzella, Trichoplusia spp., Trichoplusia ni, Tryporyza incertulas, Tuta absoluta, Virachola spp.;

[0072] from the order Orthoptera or Saltatoria, for example, Acheta domesticus, Dichroplus spp., Gryllotalpa spp., Hieroglyphus spp., Locusta spp., Melanoplus spp., Paratlanticus ussuriensis, Schistocerca gregariw,

[0073] from the order Phthiraptera, for example Damalinia spp., Haematopinus spp., Linognathus spp., Pediculus spp., Phylloxera vastatrix, Phthirus pubis, Trichodectes spp.;

[0074] from the order Psocoptera, for example Lepinotus spp., Liposcelis spp.;

[0075] from the order Siphonaptera, for example Ceratophyllus spp., Ctenocephalides spp., Pulex irritans, Tunga penetrans, Xenopsylla cheopis, [0076] from the order Thysanoptera, for example Anaphothrips obscurus, Baliothrips biformis, Chaetanaphothrips leeuweni, Drepanothrips reuteri, Enneothrips flavens, Frankliniella spp., Haplothrips spp., Heliothrips spp., Hercinothrips femoralis, Kakothrips spp., Rhipiphorothrips cruentatus, Scirtothrips spp., Taeniothrips cardamomi, Thrips spp.;

[0077] from the order Zygentoma (Thysanura), for example Ctenolepisma spp., Lepisma saccharina, Lepismodes inquilinus, Thermobia domesticcv,

[0078] from the class Symphyla, for example, Scutigerella spp.;

[0079] pests from the phylum Mollusca, especially from the class Bivalvia, for example, Dreissena spp.,

[0080] and from the class Gastropoda, for example, Arion spp., Biomphalaria spp., Bulinus spp., Deroceras spp., Galba spp., Lymnaea spp., Oncomelania spp., Pomacea spp., Succinea spp.

[0081] Some insects, such as Plodia interpunctella, Plutella xylostella, T. ni, H. zea, S. frugiperda, Busseolafusca, and P. gossypiella include strains that are resistant to certain Cry proteins. See Bravo, Alejandra, Supapom Likitvivatanavong, Sarjeet S. Gill, and Mario Soberon, “ Bacillus thuringiensis A Story of a Successful Bioinsecticide,” Insect Biochemistry and Molecular Biology 41, No. 7 (2011): 423 -431. The compositions of the present invention are effective against resistant insect strains, as described in Example 8, below.

[0082] The fact that the compositions are well tolerated by plants at the concentrations required for controlling plant diseases and pests allows the treatment of above-ground parts of plants, of propagation stock and seeds, and of the soil.

[0083] According to the invention all plants and plant parts can be treated including cultivars and plant varieties (whether or not protectable by plant variety or plant breeder’s rights). Cultivars and plant varieties can be plants obtained by conventional propagation and breeding methods which can be assisted or supplemented by one or more biotechnological methods such as by use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers or by bioengineering and genetic engineering methods.

[0084] The inventive compositions, when they are well tolerated by plants, have favorable homeotherm toxicity and are well tolerated by the environment, are suitable for protecting plants and plant organs, for enhancing harvest yields, for improving the quality of the harvested material. They can preferably be used as crop protection compositions. They are active against normally sensitive and resistant species and against all or some stages of development.

[0085] Plants which can be treated in accordance with the invention include the following main crop plants: maize, soya bean, alfalfa, cotton, sunflower, Brassica oil seeds such as Brassica napus (e.g., canola, rapeseed), Brassica rapa, B. juncea (e.g., (field) mustard) and Brassica carinata, Arecaceae sp. (e.g., oilpalm, coconut), rice, wheat, sugar beet, sugar cane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vine and various fruit and vegetables from various botanic taxa, e.g., Rosaceae sp. (e.g., pome fruits such as apples and pears, but also stone fruits such as apricots, cherries, almonds, plums and peaches, and berry fruits such as strawberries, raspberries, red and black currant and gooseberry), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (e.g., olive bee), Actinidaceae sp., Lauraceae sp. (e.g., avocado, cinnamon, camphor), Musaceae sp. (e.g., banana bees and plantations), Rubiaceae sp. (e.g., coffee), Theaceae sp. (e.g., tea), Sterculiceae sp. , Rut ace ae sp. (e.g., lemons, oranges, mandarins and grapefruit); Solanaceae sp. (e.g., tomatoes, potatoes, peppers, capsicum, aubergines, tobacco), Liliaceae sp., Compositae sp. (e.g., lettuce, artichokes and chicory - including root chicory, endive or common chicory), Umbelliferae sp. (e.g., carrots, parsley, celery and celeriac), Cucurbitaceae sp. (e.g., cucumbers - including gherkins, pumpkins, watermelons, calabashes and melons), Alliaceae sp. (e.g., leeks and onions), Cruciferae sp. (e.g., white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, pak choi, kohlrabi, radishes, horseradish, cress and Chinese cabbage), Leguminosae sp. (e.g., peanuts, peas, lentils and beans - e.g., common beans and broad beans), Chenopodiaceae sp. (e.g., Swiss chard, fodder beet, spinach, beeboot), Linaceae sp. (e.g., hemp), Cannabeacea sp. (e.g., cannabis), Malvaceae sp. (e.g., okra, cocoa), Papaveraceae (e.g., poppy), Asparagaceae (e.g., asparagus); useful plants and ornamental plants in the garden and woods including turf, lawn, grass and Stevia rebaudiana ; and in each case genetically modified types of these plants.

[0086] Examples of trees which can be improved in accordance with the method according to the invention are: Abies sp., Eucalyptus sp., Picea sp., Pinus sp., Aesculus sp., Platanus sp., Tilia sp., Acer sp., Tsuga sp., Fraxinus sp., Sorbus sp., Betula sp., Crataegus sp., Ulmus sp., Quercus sp., Fagus sp., Salix sp., Populus sp.

[0087] Preferred bees which can be improved in accordance with the method according to the invention are: from the tree species Aesculus: A. hippocastanum, A. pariflora, A. carnew, from the tree species Platanus: P. aceriflora, P. occidentalis, P. racemosw, from the tree species Picea: P. abies, from the tree species Pinus: P. radiata, P. ponderosa, P. contorta, P. sylvestre, P. elliottii, P. montecola, P. albicaulis, P. resinosa, P. palustris, P. taeda, P. flexilis, P. jeffregi, P. baksiana, P. strobus, from the bee species Eucalyptus: E. grandis, E. globulus, E. camadentis, E. nitens, E. obliqua, E. regnans, E. pilularus.

[0088] Especially preferred trees which can be improved in accordance with the method according to the invention are: from the tree species Pinus: P. radiata, P. ponderosa, P. contorta, P. sylvestre, P. strobus, from the tree species Eucalyptus: E. grandis, E. globulus, E. camadentis. [0089] Very particularly preferred trees which can be improved in accordance with the method according to the invention are: horse chestnut, Platanaceae, linden tree, maple tree.

[0090] The present invention can also be applied to any turf grasses, including cool-season turf grasses and warm-season turf grasses. Examples of cold-season turf grasses are bluegrasses (. Poa spp.), such as Kentucky bluegrass (Poa pratensis L.), rough bluegrass (Poa trivialis L.), Canada bluegrass (Poa compressa L.), annual bluegrass (Poa annua L.), upland bluegrass ( Poa glaucantha Gaudin), wood bluegrass (Poa nemoralis L.) and bulbous bluegrass (Poa bulbosa L.); bentgrasses (Agrostis spp.) such as creeping bentgrass (Agrostis palustris Huds.), colonial bentgrass (Agrostis tenuis Sibth.), velvet bentgrass (Agrostis canina L.), South German mixed bentgrass (Agrostis spp. including Agrostis tenuis Sibth Agrostis canina L., and Agrostis palustris Huds.), andredtop (Agrostis alba L.);

[0091] fescues (Festuca spp.), such as red fescue (Festuca rubra L. spp. rubra), creeping fescue (Festuca rubra L.), chewings fescue (Festuca rubra commutata Gaud.), sheep fescue (Festuca ovina L.), hard fescue (Festuca longifolia ThuilL), hair fescue (Festucu capillata Lam.), tall fescue (Festuca arundinacea Schreb.) and meadow fescue (Festuca elanor L.);

[0092] ryegrasses (Lolium spp.), such as annual ryegrass (Folium multiflorum Lam.), perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.);

[0093] and wheatgrasses (Agropyron spp.), such as fairway wheatgrass (Agropyron cristatum (L.) Gaertn.), crested wheatgrass (Agropyron desertorum (Lisch.) Schult.) and western wheatgrass (Agropyron smithii Rydb.)

[0094] Examples of further cool-season turf grasses are beachgrass (Ammophila breviligulata Lem.), smooth bromegrass (Bromus inermis Leyss.), cattails such as timothy (Phleum pratense L.), sand cattail (Phleum subulatum L.), orchardgrass (Dactylis glomerata L.), weeping alkaligrass (Puccinellia distans (L.) Pari.) and crested dog’s-tail (Cynosurus cristatus L.)

[0095] Examples of warm-season turf grasses are Bermuda grass (Cynodon spp. L. C. Rich), zoysia grass (Zoysia spp. Willd.), St. Augustine grass (Stenotaphrum secundatum Walt Kuntze), centipede grass (Eremochloa ophiuroides Munro Hack.), carpetgrass (Axonopus afflnis Chase), Bahia grass (Paspalum notatum Llugge), Kikuyu grass (Pennisetum clandestinum Hochst. ex Chiov.), buffalo grass (Buchloe dactyloids (Nutt.) Engelm.), blue grama (Bouteloua gracilis (H.B.K.) Lag. ex Griffiths), seashore paspalum (Paspalum vaginatum Swartz) and sideoats grama (Bouteloua curtipendula (Michx. Torn). Cool-season turf grasses are generally preferred for the use according to the invention. Especially preferred are bluegrass, benchgrass and redtop, fescues and ryegrasses. Bentgrass is especially preferred. [0096] Plants and plant cultivars which are preferably to be treated according to the invention include all plants which have genetic material which impart particularly advantageous, useful traits to these plants (whether obtained by breeding and/or biotechnological means).

[0097] Plants and plant cultivars which are also preferably to be treated according to the invention are resistant against one or more biotic stresses, i.e., said plants have a better defense against insect and microbial pests, such as against nematodes, insects, mites, phytopathogenic fungi, bacteria, viruses and/or viroids.

[0098] Plants and plant cultivars which may also be treated according to the invention are those plants which are resistant to one or more abiotic stresses. Abiotic stress conditions may include, for example, drought, cold temperature exposure, heat exposure, osmotic stress, flooding, increased soil salinity, increased mineral exposure, ozone exposure, high light exposure, limited availability of nitrogen nutrients, limited availability of phosphorus nutrients or shade avoidance.

[0099] Plants and plant cultivars which may also be treated according to the invention, are those plants characterized by enhanced yield characteristics. Increased yield in said plants can be the result of, for example, improved plant physiology, growth and development, such as water use efficiency, water retention efficiency, improved nitrogen use, enhanced carbon assimilation, improved photosynthesis, increased germination efficiency and accelerated maturation. Yield can furthermore by affected by improved plant architecture (under stress and non-stress conditions), including early flowering, flowering control for hybrid seed production, seedling vigor, plant size, intemode number and distance, root growth, seed size, fruit size, pod size, pod or ear number, seed number per pod or ear, seed mass, enhanced seed filling, reduced seed dispersal, reduced pod dehiscence and lodging resistance. Further yield traits include seed composition, such as carbohydrate content, protein content, oil content and composition, nutritional value, reduction in anti-nutritional compounds, improved processability and better storage stability.

[0100] Plants that may be treated according to the invention are hybrid plants that already express the characteristic of heterosis or hybrid vigor which results in generally higher yield, vigor, health and resistance towards biotic and abiotic stress factors. Such plants are typically made by crossing an inbred male-sterile parent line (the female parent) with another inbred male-fertile parent line (the male parent). Hybrid seed is typically harvested from the male sterile plants and sold to growers. Male sterile plants can sometimes (e.g., in corn) be produced by detasseling, (i.e., the mechanical removal of the male reproductive organs or male flowers) but, more typically, male sterility is the result of genetic determinants in the plant genome. In that case, and especially when seed is the desired product to be harvested from the hybrid plants, it is typically useful to ensure that male fertility in the hybrid plants, which contain the genetic determinants responsible for male sterility, is fully restored. This can be accomplished by ensuring that the male parents have appropriate fertility restorer genes which are capable of restoring the male fertility in hybrid plants that contain the genetic determinants responsible for male sterility. Genetic determinants for male sterility may be located in the cytoplasm. Examples of cytoplasmic male sterility (CMS) were for instance described in Brassica species. However, genetic determinants for male sterility can also be located in the nuclear genome. Male sterile plants can also be obtained by plant biotechnology methods such as genetic engineering. A particularly useful means of obtaining male sterile plants is described in WO 89/10396 in which, for example, a ribonuclease such as barnase is selectively expressed in the tapetum cells in the stamens. Fertility can then be restored by expression in the tapetum cells of a ribonuclease inhibitor such as barstar.

[0101] Plants or plant cultivars (obtained by plant biotechnology methods such as genetic engineering) which may be treated according to the invention are herbicide-tolerant plants, i.e., plants made tolerant to one or more given herbicides. Such plants can be obtained either by genetic transformation, or by selection of plants containing a mutation imparting such herbicide tolerance.

[0102] Herbicide-tolerant plants are for example glyphosate-tolerant plants, i.e., plants made tolerant to the herbicide glyphosate or salts thereof. For example, glyphosate-tolerant plants can be obtained by transforming the plant with a gene encoding the enzyme 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS). Examples of such EPSPS genes are the AroA gene (mutant CT7) of the bacterium Salmonella typhimurium, the CP4 gene of the bacterium Agrobacterium sp., the genes encoding a petunia EPSPS, a tomato EPSPS, or an Eleusine EPSPS. It can also be a mutated EPSPS. Glyphosate-tolerant plants can also be obtained by expressing a gene that encodes a glyphosate oxido-reductase enzyme. Glyphosate-tolerant plants can also be obtained by expressing a gene that encodes a glyphosate acetyl transferase enzyme. Glyphosate-tolerant plants can also be obtained by selecting plants containing naturally-occurring mutations of the above-mentioned genes.

[0103] Other herbicide resistant plants are for example plants that are made tolerant to herbicides inhibiting the enzyme glutamine synthase, such as bialaphos, phosphinothricin or glufosinate. Such plants can be obtained by expressing an enzyme detoxifying the herbicide or a mutant glutamine synthase enzyme that is resistant to inhibition. One such efficient detoxifying enzyme is, for example, an enzyme encoding a phosphinothricin acetyltransferase (such as the bar or pat protein from Streptomyces species). Plants expressing an exogenous phosphinothricin acetyltransferase.

[0104] Further herbicide-tolerant plants are also plants that are made tolerant to the herbicides inhibiting the enzyme hydroxyphenylpyruvatedioxygenase (HPPD). Hydroxyphenylpyruvatedioxygenases are enzymes that catalyze the reaction in which parahydroxyphenylpyruvate (HPP) is transformed into homogentisate. Plants tolerant to HPPD- inhibitors can be transformed with a gene encoding a naturally-occurring resistant HPPD enzyme, or a gene encoding a mutated HPPD enzyme. Tolerance to HPPD-inhibitors can also be obtained by transforming plants with genes encoding certain enzymes enabling the formation of homogentisate despite the inhibition of the native HPPD enzyme by the HPPD-inhibitor. Tolerance of plants to HPPD inhibitors can also be improved by transforming plants with a gene encoding an enzyme prephenate dehydrogenase in addition to a gene encoding an HPPD-tolerant enzyme.

[0105] Still further herbicide resistant plants are plants that are made tolerant to acetolactate synthase (ALS) inhibitors. Known ALS-inhibitors include, for example, sulfonylurea, imidazolinone, triazolopyrimidines, pyrimidinyloxy(thio)benzoates, and/or sulfonylaminocarbonyltriazolinone herbicides. Different mutations in the ALS enzyme (also known as acetohydroxyacid synthase, AHAS) are known to confer tolerance to different herbicides and groups of herbicides. The production of sulfonylurea-tolerant plants and imidazolinone-tolerant plants has been described. Other imidazolinone-tolerant plants have also been described. Further sulfonylurea- and imidazolinone-tolerant plants have also been described.

[0106] Other plants tolerant to imidazolinone and/or sulfonylurea can be obtained by induced mutagenesis, selection in cell cultures in the presence of the herbicide or mutation breeding as described for example for soya beans, for rice, for sugar beet, for lettuce or for sunflower.

[0107] Plants or plant cultivars (obtained by plant biotechnology methods such as genetic engineering) which may also be treated according to the invention are insect-resistant transgenic plants, i.e., plants made resistant to attack by certain target insects. Such plants can be obtained by genetic transformation, or by selection of plants containing a mutation imparting such insect resistance.

[0108] An “insect-resistant transgenic plant”, as used herein, includes any plant containing at least one transgene comprising a coding sequence encoding:

1) an insecticidal crystal protein from B. thuringiensis or an insecticidal portion thereof, such as the insecticidal crystal proteins listed by Crickmore et ah, Microbiology and Molecular Biology Reviews (1998), 62, 807-813, updated by Crickmore et a , (2005) in the B. thuringiensis toxin nomenclature, online at: http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/), or insecticidal portions thereof, e.g., proteins of the Cry protein classes CrylAb, CrylAc, CrylF, Cry2Ab, Cry3Ae, or Cry3Bb or insecticidal portions thereof; or

2) a crystal protein from B. thuringiensis or a portion thereof which is insecticidal in the presence of a second other crystal protein from B. thuringiensis or a portion thereof, such as the binary toxin made up of the Cy34 and Cy35 crystal proteins; or 3) a hybrid insecticidal protein comprising parts of two different insecticidal crystal proteins from B. thuringiensis, such as a hybrid of the proteins of 1) above or a hybrid of the proteins of 2) above, e.g., the Cry 1 A.105 protein produced by com event MON98034; or

4) a protein of any one of 1) to 3) above wherein some, particularly 1 to 10, amino acids have been replaced by another amino acid to obtain a higher insecticidal activity to a target insect species, and/or to expand the range of target insect species affected, and/or because of changes induced into the encoding DNA during cloning or transformation, such as the Cry3Bbl protein in corn events MON863 or MON88017, or the Cry3A protein in corn event MIR604; or

5) an insecticidal secreted protein from B. thuringiensis or B. cereus, or an insecticidal portion thereof, such as the vegetative insecticidal proteins (VIP) listed at: http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.h tml, e.g., proteins from the VIP3Aa protein class; or

6) a secreted protein from B. thuringiensis or B. cereus which is insecticidal in the presence of a second secreted protein from B. thuringiensis or B. cereus, such as the binary toxin made up of the VIPla and VIP2A proteins; or

7) a hybrid insecticidal protein comprising parts from different secreted proteins from B. thuringiensis or B. cereus, such as a hybrid of the proteins in 1) above or a hybrid of the proteins in 2) above; or

8) a protein of any one of 1) to 3) above wherein some, particularly 1 to 10, amino acids have been replaced by another amino acid to obtain a higher insecticidal activity to a target insect species, and/or to expand the range of target insect species affected, and/or because of changes induced into the encoding DNA during cloning or transformation (while still encoding an insecticidal protein), such as the VIP3Aa protein in cotton event COT102.

[0109] Of course, insect-resistant transgenic plants, as used herein, also include any plant comprising a combination of genes encoding the proteins of any one of the above classes 1 to 8. In one embodiment, an insect-resistant plant contains more than one transgene encoding a protein of any one of the above classes 1 to 8, to expand the range of target insect species affected or to delay insect resistance development to the plants, by using different proteins insecticidal to the same target insect species but having a different mode of action, such as binding to different receptor binding sites in the insect.

[0110] Plants or plant cultivars (obtained by plant biotechnology methods such as genetic engineering) which may also be treated according to the invention are tolerant to abiotic stresses. Such plants can be obtained by genetic transformation, or by selection of plants containing a mutation imparting such stress resistance. Particularly useful stress tolerance plants include: a. plants which contain a transgene capable of reducing the expression and/or the activity of poly(ADP-ribose)polymerase (PARP) gene in the plant cells or plants. b. plants which contain a stress tolerance enhancing transgene capable of reducing the expression and/or the activity of the PARG encoding genes of the plants or plants cells. c. plants which contain a stress tolerance enhancing transgene coding for a plant- functional enzyme of the nicotinamide adenine dinucleotide salvage biosynthesis pathway, including nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase, nicotinamide adenine dinucleotide synthetase or nicotine amide phosphoribosyltransferase.

[0111] Plants or plant cultivars (obtained by plant biotechnology methods such as genetic engineering) which may also be treated according to the invention show altered quantity, quality and/or storage-stability of the harvested product and/or altered properties of specific ingredients of the harvested product such as:

1) transgenic plants which synthesize a modified starch, which in its physical-chemical characteristics, in particular the amylose content or the amylose/amylopectin ratio, the degree of branching, the average chain length, the side chain distribution, the viscosity behavior, the gelling strength, the starch grain size and/or the starch grain morphology, is changed in comparison with the synthesized starch in wild type plant cells or plants, so that this modified starch is better suited for special applications. Said transgenic plants synthesizing a modified starch have been described.

2) transgenic plants which synthesize non-starch carbohydrate polymers or which synthesize non starch carbohydrate polymers with altered properties in comparison to wild type plants without genetic modification. Examples are plants which produce polyfructose, especially of the inulin and levan-type, plants which produce alpha- 1,4-glucans, plants which produce alpha- 1,6 branched alpha- 1,4-glucans, and plants producing alternan.

3) transgenic plants which produce hyaluronan.

[0112] Plants or plant cultivars (that can be obtained by plant biotechnology methods such as genetic engineering) which may also be treated according to the invention are plants, such as cotton plants, with altered fiber characteristics. Such plants can be obtained by genetic transformation, or by selection of plants containing a mutation imparting such altered fiber characteristics and include: a) plants, such as cotton plants which contain an altered form of cellulose synthase genes; b) plants, such as cotton plants which contain an altered form of rsw2 or rsw3 homologous nucleic acids; c) plants, such as cotton plants, with an increased expression of sucrose phosphate synthase; d) plants, such as cotton plants, with an increased expression of sucrose synthase; e) plants, such as cotton plants, wherein the timing of the plasmodesmatal gating at the basis of the fiber cell is altered, e.g., through downregulation of fiber-selective b- 1 ,3-glucanase; f) plants, such as cotton plants, which have fibers with altered reactivity, e.g., through the expression of N-acetylglucosaminetransferase gene including nodC and chitin synthase genes.

[0113] Plants or plant cultivars (that can be obtained by plant biotechnology methods such as genetic engineering) which may also be treated according to the invention are plants, such as oilseed rape or related Brassica plants, with altered oil profile characteristics. Such plants can be obtained by genetic transformation or by selection of plants containing a mutation imparting such altered oil characteristics and include: a) plants, such as oilseed rape plants, which produce oil having a high oleic acid content; b) plants, such as oilseed rape plants, which produce oil having a low linolenic acid content; c) plant such as oilseed rape plants, which produce oil having a low level of saturated fatty acids.

[0114] Particularly useful transgenic plants which may be treated according to the invention are plants which comprise one or more genes which encode one or more toxins, are the following which are sold under the trade names YIELD GARD ^® (for example maize, cotton, soya beans), KNOCKOUT ^® (for example maize), BITEGARD ^® (for example maize), BT-XTRA ^® (for example maize), STARLINK ^® (for example maize), BOLLGARD ^® (cotton), NUCOTN ^® (cotton), NUCOTN 33B ^® (cotton), NATUREGARD ^® (for example maize), PROTECTA ^® and NEWLEAF ^® (potato). Examples of herbicide-tolerant plants which may be mentioned are maize varieties, cotton varieties and soya bean varieties which are sold under the trade names ROUNDUP READY ^® (tolerance to glyphosate, for example maize, cotton, soya beans), LIBERTY LINK ^® (tolerance to phosphinothricin, for example oilseed rape), IMI ^® (tolerance to imidazolinone) and SCS ^® (tolerance to sulphonylurea, for example maize). Herbicide -resistant plants (plants bred in a conventional manner for herbicide tolerance) which may be mentioned include the varieties sold under the name CLEARFIELD ^® (for example maize).

[0115] Particularly useful transgenic plants which may be treated according to the invention are plants containing transformation events, or a combination of transformation events, that are listed for example in the databases for various national or regional regulatory agencies. [0116] The compositions according to the invention are particularly suitable for the treatment of seed. The combinations according to the invention which have been mentioned above as being preferred or especially preferred must be mentioned by preference in this context. Thus, a large proportion of the damage to crop plants which is caused by pests is already generated by infestation of the seed while the seed is stored and after the seed is introduced into the soil, and during and immediately after germination of the plants. This phase is particularly critical since the roots and shoots of the growing plant are particularly sensitive and even a small amount of damage can lead to the death of the whole plant. There is therefore in particular a great interest in protecting the seed and the germinating plant by using suitable compositions.

[0117] The control of pests by treating the seed of plants has been known for a long time and is the subject of continuous improvements. However, the treatment of seed poses a series of problems which cannot always be solved in a satisfactory manner. Thus, it is desirable to develop methods of protecting the seed and the germinating plant which dispense with the additional application of plant protection compositions after sowing or after the emergence of the plants. It is furthermore desirable to optimize the amount of the compositions employed in such a way as to provide the best possible protection for the seed and the germinating plant against attack by pests. In particular, methods for the treatment of seed should also include the intrinsic fungicidal and/or insecticidal properties of transgenic plants in order to achieve an optimal protection of the seed and of the germinating plant while keeping the application rate of plant protection compositions as low as possible.

[0118] The present invention therefore particularly also relates to a method of protecting seed and germinating plants from attack by pests by treating the seed with a composition according to the invention.

[0119] In certain aspects, the compositions of the present invention are applied at about 1 x 10 ⁴ to about 1 x 10 ¹⁴ colony forming units (CFU) per hectare, at about 1 x 10 ⁴ to about 1 x 10 ¹² colony forming units (CFU) per hectare, at about 1 x 10 ⁴ to about 1 x 10 ¹⁰ colony forming units (CFU) per hectare, at about 1 x 10 ⁴ to about 1 x 10 ⁸ colony forming units (CFU) per hectare, at about 1 x 10 ⁶ to about 1 x 10 ¹⁴ colony forming units (CFU) per hectare, at about 1 x 10 ⁶ to about 1 x 10 ¹² colony forming units (CFU) per hectare, at about 1 x 10 ⁶ to about 1 x 10 ¹⁰ colony forming units (CFU) per hectare, at about 1 x 10 ⁶ to about 1 x 10 ⁸ colony forming units (CFU) per hectare, at about 1 x 10 ⁸ to about 1 x 10 ¹⁴ colony forming units (CFU) per hectare, at about 1 x 10 ⁸ to about 1 x 10 ¹² colony forming units (CFU) per hectare, or at about 1 x 10 ⁸ to about 1 x 10 ¹⁰ colony forming units (CFU) per hectare. [0120] In other aspects, the compositions of the present invention are applied at about 1 x 10 ⁶ to about 1 x 10 ¹⁴ colony forming units (CFU) per hectare, at about 1 x 10 ⁶ to about 1 x 10 ¹² colony forming units (CFU) per hectare, at about 1 x 10 ⁶ to about 1 x 10 ¹⁰ colony forming units (CFU) per hectare, at about 1 x 10 ⁶ to about 1 x 10 ⁸ colony forming units (CFU) per hectare. In yet other aspects, the compositions of the present invention are applied at about 1 x 10 ⁹ to about 1 x 10 ¹³ colony forming units (CFU) per hectare. In one aspect, the compositions of the present invention are applied at about 1 x 10 ¹⁰ to about 1 x 10 ¹² colony forming units (CFU) per hectare.

[0121] In certain embodiments, the compositions of the present invention are applied at about 0.1 kg to about 20 kg fermentation solids per hectare. In some embodiments, the compositions of the present invention are applied at about 0.1 kg to about 10 kg fermentation solids per hectare. In other embodiments, the compositions of the present invention are applied at about 0.25 kg to about 7.5 kg fermentation solids per hectare. In yet other embodiments, the compositions of the present invention are applied at about 0.5 kg to about 5 kg fermentation solids per hectare. The compositions of the present invention may also be applied at about 1 kg or about 2 kg fermentation solids per hectare.

[0122] Examples 6-7 show that zwittermicin seems to require a route into the insect’s bloodstream to exert a toxic effect. Without wishing to be bound by any particular theory, the mode of action study in Example 6 and the results showing synergy between zwittermicin and Cry proteins suggest that any pore-forming protein, and not only Cry proteins, would have a synergistic insecticidal effect when combined with zwittermicin by providing a route for zwittermicin A to enter the insect’s bloodstream. Thus, the present invention encompasses compositions containing zwittermicin and any pore-forming protein, except Cry and Vip proteins, or zwittermicin and any pore-forming protein in addition to Cry or Vip proteins. In one embodiment, such pore-forming proteins include alpha-pore-forming toxins: Haemolysin E, Actinoporins, Corynebacterial porin B, and Cytolysin A; beta-barrel pore-forming toxins: alpha-hemolysin, Panton- Valentine leucocidin, aerolysin, clostridial epsilon-toxin, Mtx2, Mtx3; large beta-barrel pore-forming toxins: cholesterol- dependent cytolysins, and gasdermin; binary toxins: Pleurotolysin; small pore-forming toxins: Gramicidin A; and additional cell-penetrating, pore forming, or membrane destabilizing peptides including spider toxin peptides: Lycotoxin I, Lycotoxin II, Cupiennin la, Cupiennin lb, Cupiennin lc, Cupiennin Id, Oxyopinin 1, Oxyopinin 2a, Oxyopinin 2b, Oxyopinin 2c, Oxyopinin 2d, Lycocitin 1, Lycocitin 2, Lycocitin 3, Gomesin; cell penetrating peptides: KALA, CADY, Penetratin, Tat, Poly Arginine oligomers, R8, Transportan, Xentry, HRSV, AIP6, MPG, Pep-1, PFVYLI, Pep-7, and MAP17. The addition of zwittermicin A to any of these pore-forming proteins may result in a synergistic insecticidal effect. For more details on pore-forming proteins that may be used in the compositions described above, see Mueller M, Grauschopf U, Maier T, Glockshuber R, Ban N (June 2009), “The Structure of a Cytolytic Alpha-Helical Toxin Pore Reveals its Assembly Mechanism, ^* ’ Nature 459 (7247): 726-30. Bibcode:2009Natur. Rivera-de-Torre, Esperanza et al. “Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools. ^' Toxins vol. 11,6370. 25 Jun. 2019 and Tai, Wanyi, and Xiaohu Gao. “Functional Peptides for siRNA Delivery.” Advanced Drug Delivery Reviews vol. 110-111 (2017)

[0123] The synergistic compositions described above, comprising pore-forming proteins, other than Cry and Vip, and zwittermicin A, may be used in any of the insect control methods described in this application and to treat all plants and plant parts, as described above.

DEPOSIT INFORMATION

[0124] Samples of the B. thuringiensis strains of the invention have been deposited with the Agricultural Research Service Culture Collection located at the National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture (NRRL), 1815 North University Street, Peoria, IL 61604, U.S.A., under the Budapest Treaty. B. thuringiensis strain NRRL B-67685 was deposited on September 26, 2018; B. thuringiensis strain NRRL B-67954 and B. thuringiensis strain NRRL B-67955 were deposited on April 23, 2020; and B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, and B. thuringiensis strain NRRL B-68027 were deposited on April 14, 2021.

[0125] The B. thuringiensis strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

[0126] The following examples are given for purely illustrative and non-limiting purposes of the present invention.

EXAMPLES

Example 1. Improved Insecticidal Activity by Addition of Zwittermicin A to Whole Broth Samples

[0127] B. thuringiensis strain NRRL B-67685 was previously identified as a strain capable of producing increased amounts of zwittermicin A (PCT/US2019/061554) compared to other commercially available B. thuringiensis insecticidal strains. To determine whether addition of zwittermicin A to whole broth cultures of strain NRRL B-67685 could further enhance insecticidal activity, zwittermicin A was partially purified and added to whole broth cultures of the strain.

[0128] B. thuringiensis strain NRRL B-67685 was grown in a soy-based medium for 48 hours at 30° C to obtain a whole broth culture. Zwittermicin A was then purified from strain NRRL B-67685 whole broth by either filtration or high-performance liquid chromatography (HPLC).

[0129] In one example, the whole broth was centrifuged, and the supernatant was removed and passed through a 3 kDa filter to separate the zwittermicin A from larger molecules, including any Cry toxins. Zwittermicin A levels were measured in the supernatant, which was then concentrated and added to strain NRRL B-67685 whole broth at levels 20-fold higher than the initial zwittermicin A concentration. This modified broth is referred to in Table 1 as WT 3kDa Filtrate.

[0130] Alternatively, zwittermicin A was purified from the supernatant using HPLC, and semi-pure fractions were added to strain NRRL B-67685 whole broth at levels 20-fold higher than the initial zwittermicin A concentration. This modified broth is referred to as WT Zwa Frxn in Table 4. B. thuringiensis strain NRRL B-67685 whole broth with and without addition of exogenous zwittermicin A, a commercial standard (XenTari), and an untreated control (UTC) were used in feeding assays targeting the 3 ^rd instar of Helicoverpa zea (Table 1). The experiment was performed in duplicate and the means reported for the average leaf consumption of 6 replicates, each replicate comprising 12 larvae.

Table 1. Insecticidal Activity of B. thuringiensis Strain NRRL B-67685 Whole Cell Broth with and without the Addition of Exogenous Zwittermicin A.

Strain Spike Leaf Consumption (%)

UTC None 87.08

NRRL B-67685 None 45.09 NRRL B-67685 WT 3kDa Filtrate 20.56 NRRL B-67685 WT Zwa Frxn 14.17 XenTari None 29.72

[0131] Exogenous addition of zwittermicin A significantly improved the insecticidal activity of B. thuringiensis strain NRRL B-67685. Example 2. Mutagenesis to Generate Improved Mutants

[0132] With the goal of generating mutants capable of producing increased zwittermicin A, mutants were created from B. thuringiensis NRRL B-67685 and insecticidal variants thereof. The parent strains were grown in a 250 mL baffled flask containing 50 mL of Tryptic Soy Broth and incubated at 30° C while agitating. The parent strains were then subjected to mutagenesis using N- methyl-N’-nitro-N-nitrosoguanidine (“NTG”). NTG treatments were used at concentrations suitable to yield acceptable kill percentages of actively dividing B. thuringiensis. Kill percentages were assessed through CFU analysis of pre- and post-treatment samples. Mutant strains were screened for enhanced zwittermicin A production, as described in Example 3, below. Based on screening results certain isolates were further mutagenized in the same manner as described above.

Example 3. Screening for Increased Zwittermicin A Production in Mutants

[0133] Mutants generated from B. thuringiensis strain NRRL B-67685 and insecticidal variants thereof were screened for increased production of zwittermicin A. A mutant library of strain NRRL B-67685 and insecticidal variants thereof was diluted and plated onto nutrient agar. Individual isolates were then grown in 96-well deep well blocks containing a soy-based medium. To measure zwittermicin A, each sample along with a whole broth (WB) standard containing zwittermicin A (i.e., a whole broth culture of the parent strain, NRRL B-67685) was derivatized using o-phthalaldehyde and chemically quenched with ethanolamine. Following derivatization, zwittermicin A levels were analyzed by Single Reaction Monitoring (SRM) single quadrupole or Multiple Reaction Monitoring (MRM) triple quad mass spectrometry (MS), and each sample was compared to the WB standard to establish a relative value of zwittermicin A. Selected isolates were grown in a soy-based fermentation media in a microreactor and in 20-L stir tank fermenters to assess strain scalability. At larger fermentation scales, strains were also further analyzed to assess zwittermicin A production, spore CFU counts, bioactivity against early and late instar lepidopteran species, and Cry protein expression. Cry protein expression was measured using SDS PAGE. Insoluble crystals were solubilized using sodium hydroxide and ran on a polyacrylamide gel alongside protein standards of known concentration. Gels were stained and bands associated with known Cry toxins were then quantified using the loaded protein standard.

[0134] Mutant strains identified as having increased zwittermicin A production and acceptable spore CFU counts, bioactivity against early and late instar lepidopteran species, and Cry protein expression were further mutagenized and screened as described above. Five strains were identified using the mutagenesis and screening procedures described herein: B. thuringiensis strain NRRL B-67954, B. thuringiensis strain NRRL B-67955, B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, and B. thuringiensis strain NRRL B-68027. B. thuringiensis strain NRRL B-67954 was developed from mutagenesis of the parent strain, NRRL B-67685, whereas B. thuringiensis strain NRRL B-67955 was developed from mutagenesis of B. thuringiensis strain NRRL B-67954. B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B- 68026, and B. thuringiensis strain NRRL B-68027 were developed from mutagenesis of B. thuringiensis strain NRRL B-67955. Table 2 describes the zwittermicin A and Cry protein levels produced by the improved strains compared to the parent strain. Samples from B. thuringiensis mutant strains NRRL B-67954 and NRRL B-67955 as well as parent strain NRRL B-67685 were obtained from 20-L fermentations. Samples from B. thuringiensis strain NRRL B-68025, B. thuringiensis strain NRRL B-68026, and B. thuringiensis strain NRRL B-68027 were obtained from microreactor fermentations.

Table 2. Relative Zwittermicin A Production and Cry Protein Expression of B. thuringiensis Mutant Strains Compared to Parent Strain NRRL B-67685.

Example 4. Quantifying Zwittermicin A in a Whole Cell Broth

[0135] Zwittermicin A was quantified in a representative NRRL B-67685 whole broth sample. Approximately 1 mL of whole broth with a mass of 0.9232 g was diluted in 100 mL of MilliQ water and mixed. An aliquot of this mixture was centrifuged at 17000 xg for 5 minutes at room temperature to form a pellet of insoluble solids. The supernatant was analyzed via ultra-high- performance liquid chromatography (Waters Acquity I-Class Ultra-Performance Liquid Chromatography System) coupled to tandem mass spectrometry (Sciex API 4000 triple quadrupole mass spectrometer) using hydrophobic interaction liquid chromatography as the mode of separation. The strong and weak solvents were water and acetonitrile, respectively, each modified to include 0.1 % (v/v) formic acid and a linear gradient was applied with a flow rate of 0.4 mL/min. A Waters BEH Amide 2.1 c 100 mm column packed with 1.7-micron silica was used for separation at a column temperature of 60 °C. Multiple reaction monitoring was used for selective detection of zwittermicin A by tandem mass spectrometry, with a transition of 397.2 m/z to fragment ion 130.2 m/z. The results were compared to an external standard calibration of purified zwittermicin A, yielding a value of 0.75 mg of zwittermicin A per gram of whole broth.

[0136] Integration and quantification of zwittermicin A was performed in SCIEX MultiQuant software. The concentration as injected on the instrument was converted in Microsoft Excel to the mass fraction of the delivered amount of NRRL B-67685 whole broth, yielding a value of 0.75 mg/g of whole broth. Zwittermicin A concentrations from the mutant strains NRRL B-67954, NRRL B-67955, NRRL B-68025, NRRL B-68026, NRRL B-68027 were extrapolated from the relative concentrations in Table 2 by multiplying by a factor of 0.75.

Table 3. Absolute Zwittermicin A Production of B. thuringiensis Mutant Strains Compared to Parent Strain NRRL B-67685.

Example 5. In vitro Biological Efficacy of B. thuringiensis Mutants

[0137] To evaluate the insecticidal activity of whole broth cultures of B. thuringiensis parental strain NRRL B-67685 and its mutants, strains NRRL B-67954, NRRL B-67955, NRRL B- 68025, NRRL B-68026, and NRRL B-68027, first instar corn earworm, Heliothis zea, were exposed to whole broths in a 24-well microtiter plate diet overlay bioassay. The whole broth was prepared by growing each strain in soy-based media, as described above, and the diet surface of each well was topically treated with 120 pL of sample and air dried prior to insect exposure. Six to eight concentrations of whole broth from each strain, ranging from 0% to 1%, were tested. Water was used as the diluent and as the negative control sample. The individual neonate enclosed in each test well was allowed to feed on the treated diet surface for 5 days at 28° C and 60% relative humidity. Dead and live counts were collected, and the results were fitted into a 2-parameter logistic model to estimate the lethal concentration 50% (LC50) and 95% confidence interval (Cl). The experiment was conducted in triplicate with twelve neonates tested per replicate. The LC50 is the concentration of whole broth at which 50% of the insects are killed, such that a lower LC50 is desired. The negative control delivered < 20% larval mortality. B. thuringiensis strain NRRL B-67954 showed significantly increased insecticidal activity against the first instar of corn earworm, Heliothis zea, compared to B. thuringiensis strain NRRL B-67685 (Table 4). In the table below, samples from B. thuringiensis mutant strains NRRL B-67954 and NRRL B-67955 as well as parent strain NRRL B- 67685 were obtained from 20-L fermentations. Samples from B. thuringiensis strain NRRL B- 68025, B. thuringiensis strain NRRL B-68026, and B. thuringiensis strain NRRL B-68027 were obtained from microreactor fermentations.

Table 4. In vitro Biological Efficacy of B. thuringiensis Mutants.

[0138] The observation of improved biological efficacy in the mutant strains was particularly unexpected given the reduced total Cry protein content, especially for NRRL B-67955. Specifically, it was not expected that Cry protein content would decrease in the mutagenized strains nor that the synergistic activity of zwittermicin A could more than off-set such reduction, resulting in enhanced efficacy compared to a strain with higher Cry content, such as parent strain NRRL B-67685.

Example 6. Zwittermicin A Injection Study in Spodoptera frurgiperda Larva

[0139] Appropriate amounts of purified zwittermicin A were dissolved in 3 pL water and injected into fifth instar larva of Spodoptera frugiperda. Five larvae were tested per concentration. Symptomology was observed daily, and mortality was recorded 7 days following injection. Doses and results are shown in Table 5. Table 5. Spodoptera frurgiperda Larval Mortality Following Injection of Purified Zwittermicin

A.

[0140] The injection study showed zwittermicin A has insecticidal activity against Spodoptera frurgiperda upon injection into the bloodstream.

Example 7. Zwittermicin A and CrylAc Feeding Study in Resistant and Susceptible Diamondback Moth Larva

[0141] A feeding study was performed on second instar larvae of a resistant (rDBM) and susceptible (DBM) diamondback moth strains supplied by Benzon Research (Carlisle, Pennsylvania). The resistant diamondback moth colony exhibits 500 to 1000-fold resistance to a B. thuringiensis subspecies kurstaki standard compared to the susceptible colony. Both susceptible and resistant diamondback moth strains were exposed to 6-concentrations of truncated CrylAc, with and without the addition of purified zwittermicin A, or NRRL B-67685 permeate collected from a 3kDa membrane filtration, which, as described in Example 1 above, contained zwittermicin A but not the larger Cry proteins. The truncated CrylAc protein was prepared from trypsinized full length CrylAc protein from a fermentation of recombinant Escherichia coli containing an expression plasmid with sequence for the CrylAc protein.

[0142] CrylAc was fed to the susceptible and resistant diamondback moth strains in a 24-well diet bioassay format, using a 6-concentration dosing scheme starting at top concentrations of 10 and 500 ppm, with 10 and 2-fold dilution factors, respectively. In the mixtures, purified zwittermicin A or NRRL B-67685 permeate was tested at 0.0025 mg/mL in each concentration of the CrylAc dosing scheme. Each sample was tested twice with 12 larvae per replication. Dead and live counts were collected, and the results were fitted into a 2-parameter logistic model to estimate the lethal concentration at 50% (LC50) and the lower and upper 95% confidence intervals (LCL, UCL). Results are shown in Table 6. Table 6. LC50s of CrylAc in Combination with Zwittermicin A or NRRL B-67685 Permeate in DBM and rDBM Larva Feeding Study.

[0143] The combination of zwittermicin A or NRRL B-67685 permeate with CrylAc showed a 4 to 10-fold increase in insecticidal activity in susceptible DBM over CrylAc alone. In the resistant insect strain, the addition of purified zwittermicin A did not impact toxicity.

[0144] The CrylAc protein has been extensively studied and has been shown to form pores in susceptible insect lepidopteran midguts which eventually cause cell lysis, septicemia, and insect death. Without being bound to any particular theory, these results suggest zwittermicin A requires a route into the bloodstream to exert its toxic effect, and the CrylAc protein forms pores in an insect’s midgut that allow zwittermicin A to enter the bloodstream. The CrylAc protein does not form midgut pores in the resistant strains, preventing zwittermicin A to enter the bloodstream which results in no insecticidal activity in the feeding assay.

Example 8. NRRL B-67685 and NRRL B-67955 Strain Feeding Study in Resistant and Susceptible Diamondback Moth Larvae

[0145] Both B. thuringiensis strains NRRL B-67685 and NRRL B-67955 were bioassayed with second instar larvae of a resistant (rDBM) and susceptible (DBM) diamondback moth larvae in a 24-well diet bioassay format. Whole broth was prepared by growing each strain in soy-based media and the diet surface of each well was topically treated with 120 pL of sample and air-dried prior to insect exposure. Six to eight concentrations of whole broth from each strain, ranging from 0% to 1%, were tested. Water was used as the diluent and as the negative control sample. Each sample was tested twice with 12 larvae per replication. Dead and live counts were collected, and the results were fitted into a 2-parameter logistic model to estimate the lethal concentration at 50% (LC50) and the lower and upper 95% confidence intervals (LCL, UCL). Results are shown in Table 7. Table 7. Potency of NRRL B-67685 and NRRL B-67955 Strains against the Resistant and Susceptible Diamondback Moth Larvae.

[0146] Both strains showed potent insecticidal activity against both the susceptible and resistant diamondback moth larvae in the feeding assay. Additionally, the resistant diamondback moth larvae were only 3 to 4-fold more tolerant as compared to the susceptible diamondback moth larvae (Table 7). This was considerably less than the resistance level observed against the CrylAc protein in Example 7 (Table 6), showing a greater than 16100-fold resistance. Without being bound to any particular theory, these results indicate the NRRL B-67685 and NRRL B-67955 strains contain different Cry proteins or other insecticidal components that allow zwittermicin A to enter the bloodstream and exert its toxic effect in controlling the resistant DBM.