WO2021231757A1 - Souches de bacillus thuringiensis et procédés de lutte contre les nuisibles - Google Patents

Souches de bacillus thuringiensis et procédés de lutte contre les nuisibles Download PDF

Info

Publication number
WO2021231757A1
WO2021231757A1 PCT/US2021/032290 US2021032290W WO2021231757A1 WO 2021231757 A1 WO2021231757 A1 WO 2021231757A1 US 2021032290 W US2021032290 W US 2021032290W WO 2021231757 A1 WO2021231757 A1 WO 2021231757A1
Authority
WO
WIPO (PCT)
Prior art keywords
spp
thuringiensis strain
strain nrrl
composition
plant
Prior art date
Application number
PCT/US2021/032290
Other languages
English (en)
Inventor
David J. Larsen
Reed Nathan Royalty
Eunice TAN
Bjorn A. TRAAG
Emily L. WHITSON-WHENNEN
Bill Hendrix
Sek Yee TAN
Original Assignee
Bayer Cropscience Lp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Cropscience Lp filed Critical Bayer Cropscience Lp
Publication of WO2021231757A1 publication Critical patent/WO2021231757A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/075—Bacillus thuringiensis

Definitions

  • the present invention relates to the field of bacterial mutant strains and their ability to control insect pests.
  • the present invention is directed to Bacillus thuringiensis strains with high levels of insecticidal activity.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a composition 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.
  • 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.
  • 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.
  • synergistic insecticidal effects include increased developmental delay or mortality of a target insect contacted with a composition provided herein.
  • the synergistic insecticidal effect occurs with Spodoptera exigua, Plutella xylostella, Tuta absoluta or Trichoplusia ni.
  • 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.
  • 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.
  • the composition comprises B.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition further comprises an agriculturally acceptable carrier, inert stabilization agent, preservative, nutrient, or physical property modifying agent.
  • the composition is a liquid formulation or a solid formulation.
  • 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.
  • 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.
  • 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.
  • a method of protecting a useful plant or a part thereof from insect pest damage 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.
  • 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
  • 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.
  • 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.
  • microorganisms of the present invention 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.
  • 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.
  • 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.
  • the method includes generating mutants and screening such mutants for increased production of zwittermicin A compared to the parent strain.
  • zwittermicin A production is increased by at least about 1.5 fold to at least about 15 -fold compared to the parent strain.
  • 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.
  • 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.
  • the production of zwittermicin A with Vip3Aa, CrylAa, and/or CrylAb results in a synergistic insecticidal effect.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition comprises a fermentation product (e.g., a broth concentrate or fermentation solid) produced from this fermentation broth.
  • 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.
  • 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.
  • 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).
  • SRM Single Reaction Monitoring
  • MS single quadrupole mass spectrometry
  • MRM Multiple Reaction Monitoring
  • UPLC- MS Ultra High Performance Liquid Chromatography/MS
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the synergistically effective amount refers to a synergistic weight ratio.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions of the present invention include fermentation products.
  • the concentrated fermentation broth is washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolites.
  • broth concentrate refers to whole broth (fermentation broth) that has been concentrated by conventional industrial methods, as described above, but remains in liquid form.
  • fermentation solid refers to the solid material that remains after the fermentation broth or broth concentrate is dried.
  • fermentation product refers to whole broth, broth concentrate and/or fermentation solids.
  • Compositions of the present invention include fermentation products.
  • 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.
  • 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.
  • 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.
  • 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.
  • the formulation ingredient is a wetting agent.
  • the fermentation product is a freeze- dried powder or a spray-dried powder.
  • 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.
  • formulation ingredients may be added individually or in combination.
  • 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.
  • 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.
  • 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.
  • a plant part such as leaves, seeds, or roots.
  • 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.
  • 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.
  • a wetting agent, or a dispersant is added to a fermentation solid, such as a freeze-dried or spray-dried powder.
  • 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 include sulfosuccinates and derivatives, such as MULTIWETTM MO-70R (Croda Inc., Edison, NJ); siloxanes such as BREAK- THRU ® (Evonik, Germany); nonionic compounds, such as ATLOXTM 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 EMULTIWETTM MO-70
  • 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.
  • the fermentation product comprises at least about 1 x 10 5 colony forming units (CFU) of the microorganism (e.g., B.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the fermentation product comprises at least about 1 xlO 10 CFU of the microorganism (e.g., B. thuringiensis strain NRRL B- 67685, B.
  • 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.
  • 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
  • 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.
  • 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.
  • the zwittermicin A is produced solely by the strain and no zwittermicin A is added to the fermentation broth.
  • 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-disp
  • the inventive compositions are liquid formulations.
  • liquid formulations include suspension concentrations and oil dispersions.
  • inventive compositions are solid formulations.
  • solid formulations include freeze-dried powders and spray-dried powders.
  • plants and plant parts can be treated in accordance with the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compositions according to the invention reach the plants via the root system.
  • 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).
  • 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).
  • 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.
  • useful plants refers to crop plants which are employed as plants for obtaining foodstuffs, feedstuffs, fuels or for industrial purposes.
  • 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
  • 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.
  • 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
  • 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.
  • 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:
  • 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,
  • 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
  • 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.;
  • Coptotermes spp. for example, Coptotermes spp., Cornitermes cumulans, Cryptotermes spp., Incisitermes spp., Kalotermes spp., Microtermes obesi, Nasutitermes spp., Odontotermes spp., Porotermes spp., Reticulitermes spp.;
  • 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.
  • Siphonaptera for example Ceratophyllus spp., Ctenocephalides spp., Pulex irritans, Tunga penetrans, Xenopsylla cheopis
  • 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.;
  • Gastropoda for example, Arion spp., Biomphalaria spp., Bulinus spp., Deroceras spp., Galba spp., Lymnaea spp., Oncomelania spp., Pomacea spp., Succinea spp.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Brassica oil seeds such as Brassica napus (e.g., canola, rapeseed), Brassica rapa, B. juncea (e.g., (field) mustard) and Brassica carinata
  • Arecaceae sp.
  • 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.
  • Rubiaceae sp. e.g., coffee
  • Theaceae sp. e.g., tea
  • Sterculiceae sp. e.g., tea
  • Rut ace ae sp. e.g., lemons, oranges, mandarins and grapefruit
  • Solanaceae sp. e.g., tomatoes, potatoes, peppers, capsicum, aubergines, tobacco
  • 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
  • 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 including turf, lawn, grass and Stevia rebaudiana ; and in each case genetically modified types of these plants.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention can also be applied to any turf grasses, including cool-season turf grasses and warm-season turf grasses.
  • 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
  • fescues 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.);
  • ryegrasses such as annual ryegrass (Folium multiflorum Lam.), perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.);
  • wheatgrasses such as fairway wheatgrass (Agropyron cristatum (L.) Gaertn.), crested wheatgrass (Agropyron desertorum (Lisch.) Schult.) and western wheatgrass (Agropyron smithii Rydb.)
  • 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.)
  • 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 Munrohack.), carpetgrass (Axonopus afflnis Chase), Bahia grass (Paspalum notatum Llugge), Kikuyu grass (Pennisetum clandestinum Hochst.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • detasseling i.e., the mechanical removal of the male reproductive organs or male flowers
  • 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.
  • 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.
  • Herbicide-tolerant plants are for example glyphosate-tolerant plants, i.e., plants made tolerant to the herbicide glyphosate or salts thereof.
  • glyphosate-tolerant plants can be obtained by transforming the plant with a gene encoding the enzyme 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS).
  • EPSPS 5-enolpyruvylshikimate- 3-phosphate synthase
  • EPSPS 5-enolpyruvylshikimate- 3-phosphate synthase
  • 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.
  • 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.
  • 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.
  • Still further herbicide resistant plants are plants that are made tolerant to acetolactate synthase (ALS) inhibitors.
  • 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
  • AHAS acetohydroxyacid synthase
  • 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.
  • 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.
  • An “insect-resistant transgenic plant”, as used herein, includes any plant containing at least one transgene comprising a coding sequence encoding:
  • 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
  • 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
  • VIP vegetative insecticidal proteins
  • 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
  • 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.
  • insect-resistant transgenic plants also include any plant comprising a combination of genes encoding the proteins of any one of the above classes 1 to 8.
  • 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.
  • Plants or plant cultivars 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.
  • 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:
  • 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.
  • 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.
  • Plants or plant cultivars 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,
  • Plants or plant cultivars 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.
  • transgenic plants which comprise one or more genes which encode one or more toxins
  • plants which comprise one or more genes which encode one or more toxins
  • KIELD 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 ® nuclear-Fi Protectet alpha ®
  • NATUREGARD ® for example maize
  • PROTECTA ® NEWLEAF ®
  • herbicide-tolerant plants examples include 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
  • CLEARFIELD ® for example maize.
  • 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.
  • 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.
  • 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.
  • 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) (CFU) per hectare
  • 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.
  • 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.
  • 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.
  • 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.
  • Examples 6-7 show that zwittermicin seems to require a route into the insect’s bloodstream to exert a toxic effect.
  • 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.
  • 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.
  • 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, Oxyopin
  • 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.
  • 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;
  • 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.
  • Example 1 Improved Insecticidal Activity by Addition of Zwittermicin A to Whole Broth Samples
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • WB whole broth
  • 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.
  • SRM Single Reaction Monitoring
  • MRM Multiple Reaction Monitoring
  • MS triple quad mass spectrometry
  • 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.
  • 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.
  • 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.
  • Ultra-high- performance liquid chromatography Waters Acquity I-Class Ultra-Performance Liquid Chromatography System
  • tandem mass spectrometry Sciex API 4000 triple quadrupole mass spectrometer
  • 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.
  • strains NRRL B-67954, NRRL B-67955, NRRL B- 68025, NRRL B-68026, and NRRL B-68027 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

Abstract

La présente invention concerne des compositions comprenant une culture biologiquement pure ou une préparation de celle-ci sans cellule de mutants insecticides de souche de Bacillus thuringiensis NRRL B-67685 ou de variants insecticides de ceux-ci. Les mutants de la présente invention peuvent produire des niveaux accrus de zwittermicine A par rapport à la souche parentale. La présente invention concerne également un procédé de lutte contre les insectes nuisibles et de protection d'une plante utile ou d'une partie de celle-ci contre les dommages dus à des nuisibles, le procédé comprenant l'application d'une souche de l'invention, ou d'une partie ou d'un extrait de celle-ci, sur la plante et/ou sur un locus de la plante.
PCT/US2021/032290 2020-05-13 2021-05-13 Souches de bacillus thuringiensis et procédés de lutte contre les nuisibles WO2021231757A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063024304P 2020-05-13 2020-05-13
US63/024,304 2020-05-13

Publications (1)

Publication Number Publication Date
WO2021231757A1 true WO2021231757A1 (fr) 2021-11-18

Family

ID=76306008

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/032290 WO2021231757A1 (fr) 2020-05-13 2021-05-13 Souches de bacillus thuringiensis et procédés de lutte contre les nuisibles

Country Status (2)

Country Link
AR (1) AR122093A1 (fr)
WO (1) WO2021231757A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010396A1 (fr) 1988-04-28 1989-11-02 Plant Genetic Systems N.V. Plantes avec cellules d'etamines modifiees
US5702703A (en) * 1994-11-16 1997-12-30 Mycogen Corporation Bacillus thuringiensis toxin enhancer
EP0828819A1 (fr) * 1995-05-30 1998-03-18 Abbott Laboratories Mutants produisant un potentialisateur de l'activite pesticide d'un bacillus
CN100999716A (zh) * 2006-11-03 2007-07-18 华中农业大学 高产ZwittermicinA和晶体蛋白的高毒力苏云金芽胞杆菌突变株D1－23及应用
WO2020102592A1 (fr) * 2018-11-14 2020-05-22 Bayer Cropscience Lp Souches de bacillus thuringiensis et procédés de lutte contre les nuisibles

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010396A1 (fr) 1988-04-28 1989-11-02 Plant Genetic Systems N.V. Plantes avec cellules d'etamines modifiees
US5702703A (en) * 1994-11-16 1997-12-30 Mycogen Corporation Bacillus thuringiensis toxin enhancer
EP0828819A1 (fr) * 1995-05-30 1998-03-18 Abbott Laboratories Mutants produisant un potentialisateur de l'activite pesticide d'un bacillus
CN100999716A (zh) * 2006-11-03 2007-07-18 华中农业大学 高产ZwittermicinA和晶体蛋白的高毒力苏云金芽胞杆菌突变株D1－23及应用
WO2020102592A1 (fr) * 2018-11-14 2020-05-22 Bayer Cropscience Lp Souches de bacillus thuringiensis et procédés de lutte contre les nuisibles

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Investigation of the Mechanism of Synergy of Bacillus thuringiensis and Zwittermicin A in Manduca sexta", YALE UNIVERSITY PROJECT; NATIONAL INSTITUTE OF FOOD AND AGRICULTURE, 31 July 2014 (2014-07-31), pages 1 - 3, XP055831369, Retrieved from the Internet <URL:https://reeis.usda.gov/web/crisprojectpages/0229643-investigation-of-the-mechanism-of-synergy-of-bacillus-thuringiensis-and-zwittermicin-a-in-manduca-sexta.html> [retrieved on 20210810] *
BRAVO, ALEJANDRASUPAPOM LIKITVIVATANAVONGSARJEET S. GILLMARIO SOBERON: "Bacillus thuringiensis: A Story of a Successful Bioinsecticide", INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 41, no. 7, 2011, pages 423 - 431, XP055281787, DOI: 10.1016/j.ibmb.2011.02.006
BRODERICK NICHOLE A. ET AL: "Synergy Between Zwittermicin A and Bacillus thuringiensis subsp. kurstaki Against Gypsy Moth (Lepidoptera: Lymantriidae)", ENVIRONMENTAL ENTOMOLOGY., vol. 29, no. 1, 1 February 2000 (2000-02-01), US, pages 101 - 107, XP055831257, ISSN: 0046-225X, Retrieved from the Internet <URL:https://www.researchgate.net/profile/Kenneth-Raffa/publication/221941838_Synergy_Between_Zwittermicin_A_and_Bacillus_thuringiensis_subsp_kurstaki_Against_Gypsy_Moth_Lepidoptera_Lymantriidae/links/0deec514b4b4fc133c000000/Synergy-Between-Zwittermicin-A-and-Bacillus-thuringiensis-subsp-kurstaki-Agains> DOI: 10.1603/0046-225X-29.1.101 *
CHANGMING ZHAO ET AL: "Identification of three Zwittermicin A biosynthesis-related genes from Bacillus thuringiensis subsp. kurstaki strain YBT-1520", ARCHIVES OF MICROBIOLOGY, SPRINGER, BERLIN, DE, vol. 187, no. 4, 16 January 2007 (2007-01-16), pages 313 - 319, XP019488365, ISSN: 1432-072X, DOI: 10.1007/S00203-006-0196-3 *
CRICKMORE ET AL., MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, vol. 62, 1998, pages 807 - 813
JING LI ET AL: "Zwittermicin A Enhances the Toxicity of Bt Cry1Ac to Spodoptera exigua Hüber", HUABEI NONGXUE BAO , ACTA AGRICULTURAE BOREALI-SINICA, vol. 23, no. 5, 28 October 2008 (2008-10-28), CN, pages 136 - 141, XP055831269, ISSN: 1000-7091, Retrieved from the Internet <URL:http://www.hbnxb.net/EN/abstract/abstract2569.shtml#> DOI: 10.7668/hbnxb.2008.05.030 *
MUELLER MGRAUSCHOPF UMAIER TGLOCKSHUBER RBAN N: "The Structure of a Cytolytic Alpha-Helical Toxin Pore Reveals its Assembly Mechanism", NATURE, vol. 459, no. 7247, June 2009 (2009-06-01), pages 726 - 30
RIVERA-DE-TORRE, ESPERANZA: "Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools.", TOXINS, vol. 11, no. 6, 25 June 2019 (2019-06-25), pages 370
TAI, WANYIXIAOHU GAO: "Functional Peptides for siRNA Delivery", ADVANCED DRUG DELIVERY REVIEWS, vol. 110-111, 2017
TAYLOR, A.G. ET AL.: "Concepts and Technologies of Selected Seed Treatments", ANNU. REV. PHYTOPATHOL., vol. 28, 1990, pages 321 - 339, XP055051009, DOI: 10.1146/annurev.py.28.090190.001541
XIAOPENG ZHANG ET AL: "Efficient screening and breeding of Bacillus thuringiensis subsp. kurstaki for high toxicity against Spodoptera exigua and Heliothis armigera", JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 36, no. 6, 1 April 2009 (2009-04-01), pages 815 - 820, XP019665619, ISSN: 1476-5535 *

Also Published As

Publication number Publication date
AR122093A1 (es) 2022-08-10

Similar Documents

Publication Publication Date Title
EP2720543B1 (fr) Utilisation d&#39;un composé énaminocarbonylé avec un agent de contrôle biologique
US20220151240A1 (en) Compositions comprising recombinant bacillus cells and an insecticide
KR101894361B1 (ko) 피리딜에틸벤즈아미드 및 기타 활성 성분을 포함하는 활성 성분 배합물
US20220007651A1 (en) Bacillus thuringiensis strains and methods for controlling pests
TR201816272T4 (tr) Bir biyolojik kontrol ajanı ve flopikolid içeren bileşim.
RU2567880C2 (ru) Комбинации активных соединений
TR201816257T4 (tr) Bir biyolojik kontrol ajanı ve trifloksistrobin içeren bileşim.
TR201816247T4 (tr) Metalaksil ve metalaksil-m&#39;den seçilen bir fungisit ve bir biyolojik kontrol ajanı içeren bileşim.
TR201808095T4 (tr) Bir biyolojik kontrol ajanı ve bir insektisit içeren bileşimler.
US9854802B2 (en) Use of an anthranilic diamide derivatives with heteroaromatic and heterocyclic substituents in combination with a biological control agent
WO2010127787A2 (fr) Combinaisons d&#39;agents actifs ayant des propriétés insecticides et acaricides
US20220030876A1 (en) Methods of controlling animal pests with paenibacillus terrae
EP2437597A2 (fr) Combinaisons d&#39;organisme utile et de flubendiamide
EP2540165A1 (fr) Utilisation d&#39;un plaguicide halogéné en combinaison avec un plaguicide biologique
WO2021231757A1 (fr) Souches de bacillus thuringiensis et procédés de lutte contre les nuisibles
EP2531030A2 (fr) Combinaison de substances actives contenant de l&#39;azadirachtine et un composé énaminocarbonyle substitué
WO2023092050A1 (fr) Combinaisons bénéfiques contenant des cellules de bacillus recombinées exprimant une sérine protéase
WO2017174430A1 (fr) Combinaison de virus de la polyédrose nucléaire et de diamides

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21730727

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21730727

Country of ref document: EP

Kind code of ref document: A1