Patent Application
20250228243
The present disclosure relates to the fields of agriculture, plant biotechnology, and molecular biology. More specifically, the disclosure relates to the use of certain substituted phenyl uracils, such as those comprising a cyclopropylcarboxylic acid-based side chain, or an agrochemically acceptable salt thereof, for controlling or preventing weed growth in plant growth areas of transgenic crop plants that are tolerant to protoporphyrinogen oxidase (PPO) inhibiting herbicides.
A computer readable form of a sequence listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The sequence listing is contained in the file named MONS576US_ST26.xml, which is 670 kilobytes in size (measured in operating system MS Windows) and was created on Dec. 4, 2024.
Chemical herbicides are often used to control the growth and spread of weeds or other plants that are unwanted in a particular environment. These chemicals are active at one or more target sites within a plant where they interrupt normal plant functions. Herbicides vary in their modes of action, in their effects on weeds and crop plants, and how they are used. While herbicides are very effective in controlling growth of undesirable vegetation, their use may also cause incidental damage to desired plants located in the same vicinity, such as crop plants. In order to minimize crop damage, extensive research has been directed toward the development of herbicide tolerant plants, especially through use of transgenic traits. Examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. With the increase of weed species resistant to the commonly used herbicides, especially glyphosate, growers have turned to use of herbicides having different modes of action and thus new herbicide tolerance traits are needed in the field. Herbicides of particular interest include herbicides that inhibit protoporphyrinogen oxidase (PPO, EC 1.3.3.4), referred to as PPO inhibitor herbicides. PPO inhibitor herbicides provide control of a spectrum of herbicide-resistant weeds, thus making a trait conferring tolerance to these herbicides particularly useful in a cropping system combined with one or more other herbicide-tolerance trait(s). Substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain that is linked via an oxygen atom and that belong to the class of PPO inhibitor herbicides have been described in U.S. Pat. No. 6,403,534 and in WO2023228935 A1, which are hereby incorporated by reference in their entirety.
Provided herein is a method for controlling or preventing weed growth in a plant growth area, wherein the method comprises the steps of: (a) providing in said plant growth area a plant or a seed that when grown produces said plant, wherein the plant comprises a recombinant DNA molecule comprising a DNA sequence encoding a heterologous HemG protein, wherein said protein confers tolerance in said plant to an herbicidally active compound corresponding to a compound selected from the group consisting of A1, A2, A3, A4, A5, A6 and A7, or an agrochemically acceptable salt thereof, wherein:
A1 corresponds to:
In some embodiments of the methods provided herein, the heterologous HemG protein has herbicide-insensitive protoporphyrinogen oxidase activity. In some embodiments, the heterologous HemG protein has at least 85% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1-20 and 65-193. In some embodiments, the DNA sequence encoding the heterologous HemG protein is selected from the group consisting of SEQ ID NOs: 22-64 and 194-322. In further embodiments, the heterologous HemG protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-20 and 65-193. In some embodiments, the DNA sequence encoding a heterologous HemG protein is operably linked to a DNA sequence encoding a chloroplast transit peptide (CTP). In some embodiments, the CTP comprises an amino acid sequence with at least 97% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 323-328, 340, and 342-407. In further embodiments, the DNA sequence encoding the CTP comprises at least 97% identity to a sequence selected from the group consisting of SEQ ID NOs: 329-339, 341, and 408-483. In additional embodiments, said recombinant DNA molecule further comprises a heterologous promoter operably linked to the DNA sequence encoding said HemG protein. In some embodiments, the plant comprising the recombinant DNA molecule is a monocotyledonous plant. In other embodiments, the plant comprising the recombinant DNA molecule is a dicotyledonous plant.
In some embodiments of the methods provided herein, the herbicidally active compound is applied to the area at a rate of about 0.02 g a.i./ha to about 750 g a.i./ha, about 0.05 g a.i./ha to about 400 g a.i./ha, about 0.25 g a.i./ha to about 300 g a.i./ha, about 0.3 g a.i./ha to about 250 g a.i./ha, about 0.4 g a.i./ha to about 150 g a.i./ha, or about 0.5 g a.i./ha to about 120 g a.i./ha. In some embodiments, the method is further defined as comprising applying said compound to said area at least twice. In some embodiments, the herbicidally active compound is applied in an amount that does not damage said plant comprising the recombinant DNA molecule. In some embodiments, said applying of the compound is carried out pre-emergence. In some embodiments, said applying of the compound is carried out post-emergence. In some embodiments, said applying of the compound comprises contacting said plant with the compound. In further embodiments, said applying of the compound comprises an over the top application of said compound. In additional embodiments, said applying of the compound results in an increase in the growth or yield of said plant relative to a plant of the same genotype cultivated in a growth area in which said compound has not been applied.
In some embodiments of the methods provided herein, the method further comprises applying to said area an effective amount of at least a second herbicide. In further embodiments, the second herbicide is selected from the group consisting of: an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, a HPPD inhibitor, a PPO inhibitor, and a long-chain fatty acid inhibitor. In even further embodiments, the ACCase inhibitor is an aryloxyphenoxy propionate or a cyclohexanedione; the ALS inhibitor is a sulfonylurea, imidazolinone, triazoloyrimidine, or a triazolinone; the EPSPS inhibitor is glyphosate; the synthetic auxin is a phenoxy herbicide, a benzoic acid, a carboxylic acid, or a semicarbazone; the photosynthesis inhibitor is a triazine, a triazinone, a nitrile, a benzothiadiazole, or a urea; the glutamine synthesis inhibitor is glufosinate; the HPPD inhibitor is an isoxazole, a pyrazolone, or a triketone; the PPO inhibitor is a diphenylether, a N-phenylphthalimide, an aryl triazinone, or a pyrimidinedione; or the long-chain fatty acid inhibitor is a chloroacetamide, an oxyacetamide, or a pyrazole.
SEQ ID NO:1 is the amino acid sequence of the HemG PPO H_N90.
SEQ ID NO:2 is the amino acid sequence of the HemG PPO H_N20.
SEQ ID NO:3 is the amino acid sequence of the HemG PPO H_N60.
SEQ ID NO:4 is the amino acid sequence of H_N10, which is the E. coli wild-type HemG protoporphyrinogen oxidase (NCBI GenBank Accession No. WP_021498199).
SEQ ID NO:5 is the amino acid sequence of the HemG PPO H_N30.
SEQ ID NO:6 is the amino acid sequence of the HemG PPO H_N40.
SEQ ID NO:7 is the amino acid sequence of the HemG PPO H_N50.
SEQ ID NO:8 is the amino acid sequence of the HemG PPO H_N70.
SEQ ID NO:9 is the amino acid sequence of the HemG PPO H_N100.
SEQ ID NO:10 is the amino acid sequence of the HemG PPO H_N110.
SEQ ID NOs: 11-17 are amino acid sequences lacking the start methionine and corresponding to SEQ ID NOs: 1, 2, 4, 5, 6, 7, and 9, respectively.
SEQ ID NOs: 18-19 are amino acid sequences of two variants of SEQ ID NO:11.
SEQ ID NO:20 is the amino acid sequence of a variant of SEQ ID NO:17.
SEQ ID NO:21 is the amino acid sequence of the wild-type PPO from Amaranthus tuberculatus (waterhemp).
SEQ ID NOs: 22-31 are nucleotide sequences encoding SEQ ID NOs: 1-10, respectively, codon optimized for E. coli expression.
SEQ ID NOs: 32-41 are nucleotide sequences encoding SEQ ID NOs: 1-10, respectively, codon optimized for dicot expression.
SEQ ID NOs: 42-48 are nucleotide sequences encoding SEQ ID NOs: 11-17, respectively, codon optimized for dicot expression.
SEQ ID NOs: 49-50 are recombinant nucleotide sequences encoding SEQ ID NO:11.
SEQ ID NO:51 is a recombinant nucleotide sequence encoding SEQ ID NO:12.
SEQ ID NOs: 52-54 are recombinant nucleotide sequences encoding SEQ ID NOs: 18-20.
SEQ ID NOs: 55-64 are nucleotide sequences encoding SEQ ID NOs: 1-10, respectively, codon optimized for monocot expression.
SEQ ID NOs: 65-77 are amino acid sequences of HemG PPOs from different species with variations in the long chain insert loop.
SEQ ID NOs: 78-193 are amino acid sequences of recombinant HemG PPO variants, each incorporating a mutation to the long chain insert loop.
SEQ ID NOs: 194-206 are nucleotide sequences encoding the HemG PPOs of SEQ ID NOs: 65-77.
SEQ ID NOs: 207-322 are nucleotide sequences encoding the recombinant HemG PPO variants of SEQ ID NOs: 78-193.
SEQ ID NO:323 is the amino acid sequence of the Arabidopsis thaliana albino and pale green (APG6) chloroplast transit peptide (CTP).
SEQ ID NO:324 is the amino acid sequence of an amino-terminal optimized variant of the APG6 CTP.
SEQ ID NO:325 is the amino acid sequence of the Arabidopsis thaliana 90 kDa heat shock protein (CR88) CTP.
SEQ ID NO:326 is the amino acid sequence of the petunia ShkG-EPSPS CTP.
SEQ ID NO:327 is the amino acid sequence of the pea rbcS-3C CTP.
SEQ ID NO:328 is the amino acid sequence of the rice Waxy CTP.
SEQ ID NOs: 329-333 are the nucleotide sequences encoding APG6 CTP of SEQ ID NO: 323, optimized for plant expression.
SEQ ID NO:334 is the nucleotide sequence encoding APG6 CTP of SEQ ID NO:324.
SEQ ID NOs: 335-336 are nucleotide sequences encoding AtCR88 CTP optimized for dicot and monocot expression, respectively.
SEQ ID NOs: 337-339 are nucleotide sequences encoding SEQ ID NOs: 326-328.
SEQ ID NO:340 is the amino acid sequence of the cotton 12G088600TP CTP.
SEQ ID NO:341 is the nucleotide sequence encoding the cotton 12G088600TP CTP, optimized for dicot expression.
SEQ ID NOs: 342-407 are amino acid sequences of transit peptides from different species.
SEQ ID NOs: 408-483 are nucleotide sequences encoding SEQ ID NOs: 342-407.
The following descriptions and definitions are provided to better define the invention and to guide those of ordinary skill in the art in the practice of the invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
The present disclosure provides for the use of certain substituted phenyl uracils, specifically those comprising a cyclopropylcarboxylic acid-based side chain or an agrochemically acceptable salt thereof, in controlling or preventing weed growth in plant growth areas of transgenic crop plants that express herbicide insensitive PPOs (protoporphyrinogen oxidases), and that are therefore tolerant to PPO inhibiting herbicides. PPO is an essential enzyme in plants that catalyzes the dehydrogenation of protoporphyrinogen IX to form protoporphyrin IX, which is the precursor to heme and chlorophyll. PPO inhibition in plant cells causes accumulation of intermediate tetrapyrroles and the formation of reactive oxygen species, resulting in membrane disruption and ultimately cell death. There are several herbicide families that are classified as PPO inhibitors, such as diphenyl ethers, aryl triazolinones, pyrimidinediones, and N-phenylphthalimides.
Certain uracils, specifically those comprising a substituted phenyl cyclopropylcarboxylic acid-based side chain, have been identified as having herbicidal activity and can be used for controlling monocotyledonous and dicotyledonous weeds. These compounds are effective against a broad spectrum of harmful plants when applied both preemergence and postemergence, with the possibility of non-selective use for control of unwanted plant growth or selective use in plant crops. The present application shows that certain substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, or agrochemically acceptable salts thereof, can be applied to transgenic crop plants that comprise one or more genes conferring tolerance to PPO inhibitor herbicides. The herbicide tolerance trait described herein provides tolerance to one of more of the herbicidally active compounds described herein or agrochemically acceptable salts thereof.
Provided herein are uses for specific substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain or agrochemically acceptable salts thereof for controlling or preventing weed growth in plant growth areas of transgenic crop plants that are tolerant to PPO inhibiting herbicides wherein the plants comprise a recombinant DNA molecule comprising a DNA sequence encoding a heterologous HemG protein that has herbicide-insensitive PPO activity.
In preferred embodiments, the specific substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain are referenced herein as compounds A1, A2, A3, A4, A5, A6 and A7 and are further characterized as shown in Table 1 below.
As used herein, a “herbicide” is any molecule that is used to control, prevent, or interfere with the growth of one or more plants. Illustrative herbicides include acetyl-CoA carboxylase (ACCase) inhibitors (for example, aryloxyphenoxy propionates and cyclohexanediones); acetolactate synthase (ALS) inhibitors (for example, sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones); 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors (for example, glyphosate), synthetic auxins (for example, phenoxys, benzoic acids, carboxylic acids, and semicarbazones), photosynthesis (photosystem II) inhibitors (for example, triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthetase (GS) inhibitors (for example, glufosinate and bialaphos), 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors (for example, isoxazoles, pyrazolones, and triketones), protoporphyrinogen oxidase (PPO) inhibitors (for example, diphenylethers, N-phenylphthalimide, aryl triazinones, and pyrimidinediones), very long-chain fatty acid inhibitors (for example, chloroacetamides, oxyacetamides, and pyrazoles), cellulose biosynthesis inhibitors (for example, indaziflam), photosystem I inhibitors (for example, paraquat), microtubule assembly inhibitors (for example, pendimethalin), and phytoene desaturase (PDS) inhibitors (for example, norflurazone), among others.
Specific PPO inhibiting herbicides are known in the art and commercially available. Examples of PPO inhibiting herbicides include, but are not limited to, diphenylethers (such as acifluorfen, its salts and esters, bifenox, its salts and esters, ethoxyfen, its salts and esters, fluoronitrofen, furyloxyfen, halosafen, chlomethoxyfen, fluoroglycofen, its salts and esters, lactofen, its salts and esters, oxyfluorfen, and fomesafen, its salts and esters); thiadiazoles (such as fluthiacet-methyl and thidiazimin); pyrimidinediones or phenyluracils (such as benzfendizone, butafenacil, ethyl [3-2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl) phenoxy]-2-pyridyloxylacetate (CAS Registry Number 353292-31-6, epyrifenacil), flupropacil, saflufenacil, and tiafenacil); phenylpyrazoles (such as fluazolate, pyraflufen, and pyraflufen-ethyl); oxadiazoles (such as oxadiargyl and oxadiazon); triazolinones (such as azafenidin, bencarbazone, carfentrazone, its salts and esters, and sulfentrazone); oxazolidinediones (such as pentoxazone); N-phenylphthalimides (such as cinidon-ethyl, flumiclorac, flumiclorac-pentyl, and flumioxazin); benzoxazinone derivatives (such as 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazinane-2,4-dione (trifludimoxazin)); flufenpyr and flufenpyr-ethyl; pyraclonil; and profluazol.
As used herein, “herbicide-tolerant” or “herbicide-tolerance” means the ability to be wholly or partially unaffected by the presence or application of one or more herbicide(s), for example to resist the toxic effects of an herbicide when applied. A cell or organism is “herbicide-tolerant” if it is able to maintain at least some normal growth or phenotype in the presence of one or more herbicide(s). A trait is an herbicide-tolerance trait if its presence can confer improved tolerance to an herbicide upon a cell, plant, or seed as compared to the wild-type or control cell, plant, or seed. Crops comprising a herbicide-tolerance trait can continue to grow and are minimally affected by the presence of the herbicide. A target enzyme is “herbicide-tolerant” if it exhibits improved enzyme activity relative to a wild-type or control enzyme in the presence of the herbicide. Herbicide-tolerance may be complete or partial insensitivity to a particular herbicide, and may be expressed as a percent (%) tolerance or insensitivity to a particular herbicide.
Contemplated plants which might be produced with an herbicide tolerance trait of the present disclosure could include, for instance, any plant susceptible to a PPO inhibitor herbicide, including crop plants such as soybean (Glycine max), maize (Zea mays), cotton (Gossypium sp.), Brassica plants, alfalfa, barley, beans, beet, broccoli, cabbage, carrot, canola, cauliflower, celery, Chinese cabbage, cucumber, eggplant, leek, lettuce, melon, oat, onion, pea, pepper, peanut, potato, pumpkin, radish, rice, sweet corn, sorghum, spinach, squash, sugar beet, sugar cane, sunflower, tomato, watermelon, and wheat, among others.
Herbicides may be applied to a plant growth area comprising the plants and seeds provided by the disclosure as a method for controlling weeds. Plants and seeds provided by the disclosure comprise an herbicide tolerance trait and as such are tolerant to the application of one or more PPO inhibiting herbicides. The herbicide application may be the recommended commercial rate (1×) or any fraction or multiple thereof, such as twice the recommended commercial rate (2×). Herbicide rates may be expressed as acid equivalent per pound per acre (lb ae/acre) or acid equivalent per gram per hectare (g ae/ha) or as pounds active ingredient per acre (lb ai/acre) or grams active ingredient per hectare (g ai/ha), depending on the herbicide and the formulation. The herbicide application comprises at least one PPO inhibiting herbicide. The plant growth area may or may not comprise weed plants at the time of herbicide application. A herbicidally-effective dose of PPO inhibiting herbicide(s) for use in an area for controlling weeds may consist of a range from about 0.1× to about 30× label rate(s) over a growing season. One (1) hectare is equivalent to 2.47105 acres and one (1) pound is equivalent to 453.592 grams. Herbicide rates can be converted between English and metric as: (lb ai/ac) multiplied by 1.12=(kg ai/ha) and (kg ai/ha) multiplied by 0.89=(lb ai/ac).
The desired application rate of the compounds A1, A2, A3, A4, A5, A6 and A7 and/or their salts is generally impacted to a certain extent by external conditions such as temperature, humidity, etc. The application rate may therefore vary within wide limits, and can be determined empirically by one of skill in the art in view of the present disclosure. For the application as a herbicide for controlling weeds or other undesirable plants, the total amount of the compound A1, A2, A3, A4, A5, A6 or A7 and/or their salts, is often desirably in the range from about 0.02 g a.i./ha to about 750 g a.i./ha, about 0.05 g a.i./ha to about 400 g a.i./ha, or about 0.25 g a.i./ha to about 300 g a.i./ha, but preferably from about 0.3 g a.i./ha to about 250 g a.i./ha, especially from about 0.4 g a.i./ha to about 150 g a.i./ha, and most preferably from about 0.5 g a.i./ha to about 120 g a.i./ha. This applies both to the pre-emergence or the post-emergence application.
Herbicide applications may be sequentially or tank mixed with one, two, or a combination of several PPO inhibiting herbicides or any other compatible herbicide. Multiple applications of one herbicide or of two or more herbicides, in combination or alone, may be used over a growing season to areas comprising transgenic plants of the disclosure for the control of a broad spectrum of dicot weeds, monocot weeds, or both, for example, two applications (such as a pre-planting application and a post-emergence application or a pre-emergence application and a post-emergence application) or three applications (such as a pre-planting application, a pre-emergence application, and a post-emergence application or a pre-emergence application and two post-emergence applications).
Certain substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as compounds A1, A2, A3, A4, A5, A6 or A7 as defined above, to be used according to the disclosure and its salts, have excellent herbicidal efficacy against a broad spectrum of economically important monocotyledonous and dicotyledonous annual harmful plants. The present disclosure therefore provides methods for controlling weeds, in areas of transgenic crop plants being tolerant to PPO inhibitor herbicides wherein the plants comprises a recombinant DNA molecule comprising a DNA sequence encoding a heterologous HemG protein, wherein the protein confers tolerance to such herbicides, comprising the application of the compound of A1, A2, A3, A4, A5, A6 or A7 and/or salts as defined above, to the plants (for example harmful plants such as monocotyledonous or dicotyledonous weeds or undesired crop plants), to the seed (for example grains, seeds or vegetative propagules such as tubers or shoot parts with buds) or to the area on which the plants grow (for example the area under cultivation), including any possible combinations thereof. Specific examples may be mentioned of some representatives of the monocotyledonous and dicotyledonous weed flora which can be controlled by the compounds and methods as described herein, without the enumeration being restricted to certain species. Among the monocotyledonous weed species, for example, Aegilops, Agropyron, Agrostis, Alopecurus, Apera, Avena, Brachicaria, Bromus, Cynodon, Dactyloctenium, Digitaria, Echinochloa, Eleocharis, Eleusine, Eragrostis, Eriochloa, Festuca, Fimbristylis, Imperata, Ischaemum, Heteranthera, Imperata, Leptochloa, Lolium, Monochoria, Panicum, Paspalum, Phalaris, Phleum, Poa, Rottboellia, Sagittaria, Scirpus, Setaria, Sorghum, Sphenoclea, and Cyperus species are covered by the annual group. In the case of dicotyledonous weed species, the spectrum of action extends to species such as, for example, Abutilon, Amaranthus, Ambrosia, Anoda, Anthemis, Aphanes, Artemisia, Atriplex, Bellis, Bidens, Capsella, Carduus, Cassia, Centaurea, Chenopodium, Cirsium, Convolvulus, Datura, Desmodium, Emex, Erodium, Erysimum, Euphorbia, Galeopsis, Galinsoga, Galium, Geranium, Hibiscus, Ipomoea, Kochia, Lamium, Lepidium, Lindernia, Matricaria, Mentha, Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago, Polygonum, Portulaca, Ranunculus, Raphanus, Rorippa, Rotala, Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis, Solanum, Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi, Trifolium, Urtica, Veronica, Viola, and Xanthium.
Although the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein display outstanding herbicidal activity against monocotyledonous and dicotyledonous weeds, many economically important crop plants, depending on the structure of the respective active ingredients and the application rate thereof, are damaged only insignificantly, if at all. Economically important crops here are, for example, dicotyledonous crops from the genera of Arachis, Beta, Brassica, Cucumis, Cucurbita, Helianthus, Daucus, Glycine, Gossypium, Ipomoea, Lactuca, Linum, Lycopersicon, Nicotiana, Phaseolus, Pisum, Solanum, and Vicia, or monocotyledonous crops from the genera of Allium, Ananas, Asparagus, Avena, Hordeum, Oryza, Panicum, Saccharum, Secale, Sorghum, Triticale, Triticum, and Zea.
The substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain and/or salts thereof can be formulated in various ways according to which biological and/or physicochemical parameters are required. Examples of general formulation options are: wettable powders (WP), water-soluble powders (SP), emulsifiable concentrates (EC), water-soluble concentrates, aqueous solutions (SL), emulsions (EW), such as oil-in-water and water-in-oil emulsions, sprayable solutions or emulsions, dispersions based on oil or water, oil dispersions (OD), suspoemulsions (SE), suspension concentrates (SC), oil-miscible solutions, capsule suspensions (CS), dusting products (DP), dressings, granules for soil application or scattering, granules (GR) in the form of microgranules, spray granules, absorption and adsorption granules, water-dispersible granules (WG), water-soluble granules (SG), ULV formulations, microcapsules, or waxes.
The individual formulation types are known in principle and are described in, for example, Winnacker-Küchler, “Chemische Technologie,” Vol. 7, 4th ed., Carl Hanser Verlag, Munich, 1986; Van Valkenburg, “Pesticide Formulations,” Marcel Dekker Inc., New York, NY, 1973; and Masters, “Spray Drying Handbook,” 3rd ed., George Goodwin Ltd. London, 1979. The necessary formulation auxiliaries such as inert materials, surfactants, solvents and further additives are likewise known and are described in, for example, Watkins, “Handbook of Insecticide Dust Diluents and Carriers,” 2nd ed., Dorland Books, Caldwell, NJ, 1955; Van Olphen, “An Introduction to Clay Colloid Chemistry,” 2nd ed., J. Wiley & Sons, New York, NY, 1974; Marsden, “Solvents Guide,” 2nd ed., Interscience Publishers Inc., New York, NY, 1963; Mccutcheon's “Detergents and Emulsifiers Annual,” MC Publishing Corp., Ridgewood, NJ, 1998; Sisley and Wood, “Encyclopedia of Surface-Active Agents,” Chemical Publishing Company, New York, NY, 1964; Schönfeldt, “Grenzflächenaktive Äthylenoxid-Addukte,” Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, 1976; and Winnacker-Küchler, “Chemische Technologie,” Vol. 7, 4th ed., Carl Hanser Verlag, Munich, 1986.
Wettable powders are preparations which can be dispersed uniformly in water and, in addition to the active ingredient, apart from a diluent or inert substance, also comprise surfactants of the ionic and/or nonionic type (wetting agents, dispersants), for example polyoxyethylated alkylphenols, polyoxyethylated fatty alcohols, polyoxyethylated fatty amines, fatty alcohol polyglycol ether sulfates, alkanesulfonates, alkylbenzene sulfonates, sodium lignosulfonate, sodium 2,2′-dinaphthylmethane-6,6′-disulfonate, sodium dibutylnaphthalenesulfonate, or sodium oleoyl methyltaurate. To produce the wettable powders, the active herbicidal ingredients are finely ground, for example, in customary apparatuses such as hammer mills, blower mills, and air-jet mills, and simultaneously or subsequently mixed with the formulation auxiliaries.
Emulsifiable concentrates are produced by dissolving the active ingredient in an organic solvent, for example butanol, cyclohexanone, dimethylformamide, xylene, or else relatively high-boiling aromatics or hydrocarbons or mixtures of the organic solvents, with addition of one or more ionic and/or nonionic surfactants (emulsifiers). Examples of emulsifiers which may be used are: calcium alkyl aryl sulfonate salts, such as calcium dodecylbenzenesulfonate, or nonionic emulsifiers such as fatty acid polyglycol esters, alkyl aryl polyglycol ethers, fatty alcohol polyglycol ethers, propylene oxide-ethylene oxide condensation products, alkyl polyethers, sorbitan esters, for example sorbitan fatty acid esters, or for example polyoxyethylene sorbitan fatty acid esters.
Dusting products are obtained by grinding the active ingredient with finely distributed solids, for example talc, natural clays, such as kaolin, bentonite and pyrophyllite, or diatomaceous earth.
Suspension concentrates may be water- or oil-based. They may be produced, for example, by wet-grinding by means of commercial bead mills and optional addition of surfactants as already listed above, for example, for the other formulation types.
Emulsions, for example oil-in-water emulsions (EW), can be produced, for example, by means of stirrers, colloid mills and/or static mixers using aqueous organic solvents and optionally surfactants as already listed above, for example, for the other formulation types.
Active compounds that can be employed in combination with substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein in compositions described herein (for example in mixed formulations or in the tank mix) are, for example, known active compounds which are based on inhibition of, for example, acetolactate synthase, acetyl-CoA carboxylase, cellulose synthase, enolpyruvylshikimate-3-phosphate synthase, glutamine synthetase, p-hydroxyphenylpyruvate dioxygenase, phytoene desaturase, photosystem I, photosystem II, or protoporphyrinogen oxidase, as are described in, for example, “Glossary of Common Names and Abbreviations of Herbicides,” Weed Research 26:441-445, 1986 or MacBean, “The Pesticide Manual,” 16th ed., British Crop Protection Council, Alton, U K, 2012, and the literature cited therein. Known herbicides or plant growth regulators which can be combined with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein are, for example, the following, where said active compounds are designated either with their “common name” in accordance with the International Organization for Standardization (ISO) or with the chemical name or with the code number. They always encompass all the use forms, for example acids, salts, esters and also all isomeric forms such as stereoisomers and optical isomers, even if they are not mentioned explicitly.
Examples of such herbicidal mixing partners include one or more of the following: Acetochlor, Acifluorfen, Acifluorfen-methyl, Acifluorfen-Sodium, Aclonifen, Alachlor, Allidochlor, Alloxydim, Alloxydim-Sodium, Ametryn, Amicarbazone, Amidochlor, Amidosulfuron, 4-Amino-3-chloro-6-(4-chloro-2-fluoro-3-methylphenyl)-5-fluoropyridin-2-carboxylic acid, Aminocyclopyrachlor, Aminocyclopyrachlor-potassium, Aminocyclopyrachlor-methyl, Aminopyralid, Aminopyralid-dimethylammonium, Aminopyralid-tripromine, Amitrol, Ammoniumsulfamate, Anilofos, Asulam, Asulam-potassium, Asulam-sodium, Atrazin, Azafenidin, Azimsulfuron, Beflubutamide, (S)-(−)-Beflubutamide, Beflubutamide-M, Benazolin, Benazolin-ethyl, Benazolin-dimethylammonium, Benazolin-Potassium, Benfluralin, Benfuresate, Bensulfuron, Bensulfuron-methyl, Bensulid, Bentazone, Bentazone-Sodium, Benzobicyclon, Benzofenap, Bicyclopyrone, Bifenox, Bilanafos, Bilanafos-Sodium, Bipyrazone, Bispyribac, Bispyribac-Natium, Bixlozone, Bromacil, Bromacil-lithium, Bromacil-Sodium, Bromobutid, Bromofenoxim, Bromoxynil, Bromoxynilbutyrate, Bromoxynil-Potassium, Bromoxynil-heptanoate and Bromoxynil-octanoate, Busoxinone, Butachlor, Butafenacil, Butamifos, Butenachlor, Butralin, Butroxydim, Butylate, Cafenstrole, Cambendichlor, Carbetamide, Carfentrazone, Carfentrazone-Ethyl, Chloramben, Chloramben-ammonium, Chloramben-diolamin, Chlroamben-methyl, Chloramben-methylammonium, Chloramben-Sodium, Chlorbromuron, Chlorfenac, Chlorfenac-ammonium, Chlorfenac-Sodium, Chlorfenprop, Chlorfenprop-methyl, Chlorflurenol, Chlorflurenol-methyl, Chloridazon, Chlorimuron, Chlorimuron-ethyl, Chlorophthalim, Chlorotolurone, Chlorsulfuron, Chlorthal, Chlorthal-dimethyl, Chlorthal-monomethyl, Cinidon, Cinidon-ethyl, Cinmethylin, exo-(+)-Cinmethylin, i.e. (1R,2S,4S)-4-isopropyl-1-methyl-2-[(2-methylbenzyl)oxy]-7-oxabicyclo[2.2.1]heptan, exo-(−)-Cinmethylin, i.e. (1R,2S,4S)-4-isopropyl-1-methyl-2-[(2-methylbenzyl)oxy]-7-oxabicyclo[2.2.1]heptan, Cinosulfuron, Clacyfos, Clethodim, Clodinafop, Clodinafop-ethyl, Clodinafop-propargyl, Clomazone, Clomeprop, Clopyralid, Clopyralid-methyl, Clopyralid-olamin, Clopyralid-Potassium, Clopyralid-tripomin, Cloransulam, Cloransulam-methyl, Cumylurone, Cyanamide, Cyanazine, Cycloate, Cyclopyranil, Cyclopyrimorat, Cyclosulfamuron, Cycloxydim, Cyhalofop, Cyhalofop-butyl, Cyprazin, 2,4-D (including the Ammonium, Butotyl, Butyl, Choline, Diethylammonium, Dimethylammonium, Diolamin, Doboxyl, Dodecylammonium, Etexyl, Ethyl, 2-Ethylhexyl, Heptylammonium, Isobutyl, Isooctyl, Isopropyl, Isopropylammonium, Lithium, Meptyl, Methyl, Potassium, Sodium, Tetradecylammonium, Triethylammonium, Triisopropanolammonium, Tripromin and Trolamin salts thereof), 2,4-DB, 2,4-DB-butyl, 2,4-DB-Dimethylammonium, 2,4-DB-isooctyl, 2,4-DB-Potassium and 2,4-DB-Sodium, Daimurone (Dymron), Dalapon, Dalapon-Calcium, Dalapon-Magnesium, Dalapon-Natium, Dazomet, Dazomet-Sodium, n-Decanol, 7-Deoxy-D-sedoheptulose, Desmedipham, Detosyl-pyrazolat (DTP), Dicamba and its salts (e.g. Dicamba-biproamine, Dicamba-N,N-Bis(3-aminopropyl)methylamine, Dicamba-butotyl, Dicamba-choline, Dicamba-Diglycolamine, Dicamba-Dimethylammonium, Dicamba-Diethanolaminemmonium, Dicamba-Dicamba-Diethylammonium, Dicamba-isopropylammonium, Dicamba-methyl, monoethanolamine, Dicamba-olamine, Dicamba-Potassium, Dicamba-Sodium, Dicamba-Triethanolamin), Dichlobenil, 2-(2,4-Dichlorobenzyl)-4,4-dimethyl-1,2-oxazolidin-3-one, 2-(2,5-Dichlorobenzyl)-4,4-dimethyl-1,2-oxazolidin-3-one, Dichlorprop, Dichlorprop-butotyl, Dichlorprop-Dimethylammonium, Dichhlorprop-etexyl, Dichlorprop-ethylammonium, Dichlorprop-isoctyl, Dichlorprop-methyl, Dichlorprop-Potassium, Dichlorprop-Sodium, Dichlorprop-P, Dichlorprop-P-Dimethylammonium, Dichlorprop-P-etexyl, Dichlorprop-P-Potassium, Dichlorprop-Sodium, Diclofop, Diclofop-methyl, Diclofop-P, Diclofop-P-methyl, Diclosulam, Difenzoquat, Difenzoquat-metilsulfate, Diflufenican, Diflufenzopyr, Diflufenzopyr-Sodium, Dimefuron, Dimepiperate, Dimesulfazet, Dimethachlor, Dimethametryn, Dimethenamid, Dimethenamid-P, Dimetrasulfuron, Dinitramine, Dinoterb, Dinoterb-Acetate, Diphenamid, Diquat, Diquat-Dibromide, Diquat-Dichloride, Dithiopyr, Diuron, DNOC, DNOC-Ammonium, DNOC-Potassium, DNOC-Sodium, Endothal, Endothal-Diammonium, Endothal-Dipotassium, Endothal-Disodium, Epyrifenacil, EPTC, Esprocarb, Ethalfluralin, Ethametsulfuron, Ethamet-sulfuron-Methyl, Ethiozin, Ethofumesate, Ethoxyfen, Ethoxyfen-Ethyl, Ethoxysulfuron, Etobenzanid, F-5231, d.h. N-[2-Chloro-4-fluoro-5-[4-(3-fluoropropyl)-4,5-dihydro-5-oxo-1H-tetrazol-1-yl]-phenyl]-ethansulfonamide, F-7967, i.e. 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)pyrimidin-2,4 (1H,3H)-dione, Fenoxaprop, Fenoxaprop-P, Fenoxaprop-Ethyl, Fenoxaprop-P-Ethyl, Fenoxasulfone, Fenpyrazone, Fenquinotrione, Fentrazamide, Flamprop, Flamprop-Isoproyl, Flamprop-Methyl, Flamprop-M-Isopropyl, Flamprop-M-Methyl, Flazasulfuron, Florasulam, Florpyrauxifen, Florpyrauxifen-benzyl, Fluazifop, Fluazifop-Butyl, Fluazifop-Methyl, Fluazifop-P, Fluazifop-P-Butyl, Flucarbazone, Flucarbazone-Sodium, Flucetosulfuron, Fluchloralin, Flufenacet, Flufenpyr, Flufenpyr-Ethyl, Flumetsulam, Flumiclorac, Flumiclorac-Pentyl, Flumioxazin, Fluometuron, Flurenol, Flurenol-Butyl,-Dimethylammonium and-Methyl, Fluoroglycofen, Fluoroglycofen-Ethyl, Flupropanat, Flupropanat-Sodium, Flupyrsulfuron, Flupyrsulfuron-Methyl, Flupyrsulfuron-Methyl-Sodium, Fluridon, Flurochloridon, Fluroxypyr, Fluroxypyr-Butometyl, Fluroxypyr-Meptyl, Flurtamon, Fluthiacet, Fluthiacet-Methyl, Fomesafen, Fomesafen-Sodium, Foramsulfuron, Foramsulfuron-Sodium, Fosamine, Fosamine-Ammonium, Glufosinate, Glufosinate-Ammonium, Glufosinate-Sodium, L-Glufosinate-Ammonium, L-Glufosinate-Sodium, Glufosinate-P-Sodium, Glufosinate-P-Ammonium, Glyphosate, Glyphosate-Ammonium, Glyphosate-Isopropylammonium, Glyphosate-Diammonium, Glyphosate-Dimethylammonium, Glyphosate-Potassium, Glyphosate-Sodium, Glyphosate-Sesquisodium and Glyphosate-Trimesium, H-9201, i.e. O-(2,4-Dimethyl-6-nitrophenyl)-O-ethyl-isopropylphosphoramidothioate, Halauxifen, Halauxifen-methyl, Halosafen, Halosulfuron, Halosulfuron-Methyl, Haloxyfop, Haloxyfop-P, Haloxyfop-Ethoxyethyl, Haloxyfop-P-Ethoxyethyl, Haloxyfop-Methyl, Haloxyfop-P-Methyl, Haloxifop-Sodium, Hexazinon, HNPC-A8169, i.e. Prop-2-yn-1-yl (2S)-2-{3-[(5-tert-butylpyridin-2-yl)oxy]phenoxy}propanoate, HW-02, i.e. 1-(Dimethoxyphosphoryl)-ethyl-(2,4-dichlorophenoxy)acetate, Hydantocidine, Icafolin, Icafolin-Methyl, Imazamethabenz, Imazamethabenz-Methyl, Imazamox, Imazamox-Ammonium, Imazapic, Imazapic-Ammonium, Imazapyr, Imazapyr-Isopropylammonium, Imazaquin, Imazaquin-Ammonium, Imazaquin-Methyl, Imazethapyr, Imazethapyr-Ammonium, Imazosulfuron, Indanofan, Indaziflam, Indolauxipyr, Iodosulfuron, Iodosulfuron-Methyl, Iodosulfuron-Methyl-Sodium, oxynil, Ioxynil-Lithium,-Octanoate,-Potassium and Sodium, Ipfencarbazone, Iptriazopyrid, i.e. 3-[(Isopropylsulfonyl)methyl]-N-(5-methyl-1,3,4-oxadiazol-2-yl)-5-(trifluoromethyl) [1,2,4]triazolo-[4,3-a]pyridin-8-carboxamide, Isoproturon, Isouron, Isoxaben, Isoxaflutole, Karbutilate, KUH-043, i.e. 3-({[5-(Difluoromethyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]methyl}sulfonyl)-5,5-dimethyl-4,5-dihydro-1,2-oxazole, Ketospiradox, Ketospiradox-Potassium, Lactofen, Lenacil, Linuron, MCPA, MCPA-Butotyl,-Butyl,-Dimethylammonium,-Diolamin,-2-Ethylhexyl,-Ethyl,-Isobutyl, Isoctyl,-Isopropyl,-Isopropylammonium,-Methyl, Olamin,-Potassium,-Sodium and-Trolamin, MCPB, MCPB-Methyl,-Ethyl and-Sodium, Mecoprop, Mecoprop-Butotyl, Mecoprop-dimethylammonium, Mecoprop-Diolamin, Mecoprop-Etexyl, Mecoprop-Ethadyl, Mecoprop-Isoctyl, Mecoprop-Methyl, Mecoprop-Potassium, Mecoprop-Sodium, and Mecoprop-Trolamin, Mecoprop-P, Mecoprop-P-Butotyl,-Dimethylammonium,-2-Ethylhexyl and-Potassium, Mefenacet, Mefluidid, Mefluidid-Diolamine, Mefluidid-Potassium, Mesosulfuron, Mesosulfuron-Methyl, Mesosulfuron-Sodium, Mesotrion, Methabenzthiazuron, Metam, Metamifop, Metamitron, Metazachlor, Metazosulfuron, Methabenzthiazuron, Methiopyrsulfuron, Methiozoline, Methyl isothiocyanate, Metobromuron, Metolachlor, S-Metolachlor, Metosulam, Metoxuron, Metproxybicyclone, Metribuzine, Metsulfuron, Metsulfuron-Methyl, Molinat, Monolinuron, Monosulfuron, Monosulfuron-Methyl, MT-5950, i.e. N-[3-Chloro-4-(1-methylethyl)-phenyl]-2-methylpentanamide, NGGC-011, Napropamid, NC-310, i.e. 4-(2,4-Dichlorobenzoyl)-1-methyl-5-benzyloxypyrazole, Neburone, Nicosulfuron, Nonanoic acid (Pelargonic acid), Norflurazone, Orbencarb, Orthosulfamuron, Oryzalin, Oxadiargyl, Oxadiazone, Oxasulfuron, Oxaziclomefone, Oxyfluorfen, Paraquat, Paraquat-dichloride, Paraquat-Dimethylsulfate, Pebulat, Pendimethaline, Penoxsulam, Pentachlorphenol, Pentoxazone, Pethoxamid, Petroleum oil, Phenmedipham, Phenmedipham-Ethyl, Picloram, Picloram-dimethylammonium, Picloram-Etexyl, Picloram-Isoctyl, Picloram-Methyl, Picloram-Olamin, Picloram-Potassium, Picloram-Triethylammonium, Picloram-Tripromin, Picloram-Trolamin, Picolinafen, Pinoxaden, Piperophos, Pretilachlor, Primisulfuron, Primisulfuron-Methyl, Prodiamine, Profoxydim, Prometon, Prometryn, Propachlor, Propanil, Propaquizafop, Propazine, Propham, Propisochlor, Propoxycarbazone, Propoxycarbazone-Sodium, Propyrisulfuron, Propyzamid, Prosulfocarb, Prosulfuron, Pyraclonil, Pyraflufen, Pyraflufen-Ethyl, Pyraquinate, Pyrasulfotol, Pyrazolynat (Pyrazolat), Pyrazosulfuron, Pyrazosulfuron-Ethyl, Pyrazoxyfen, Pyribambenz, Pyribambenz-Isopropyl, Pyribambenz-Propyl, Pyribenzoxim, Pyributicarb, Pyridafol, Pyridat, Pyriftalid, Pyriminobac, Pyriminobac-Methyl, Pyrimisulfan, Pyrithiobac, Pyrithiobac-Sodium, Pyroxasulfon, Pyroxsulam, Quinclorac, Quinclorac-Dimethylammonium, Quinclorac-Methyl, Quinmerac, Quinoclamin, Quizalofop, Quizalofop-Ethyl, Quizalofop-P, Quizalofop-P-Ethyl, Quizalofop-P-Tefuryl, QYM201, i.e. 1-{2-Chloro-3-[(3-cyclopropyl-5-hydroxy-1-methyl-1H-pyrazol-4-yl) carbonyl]-6-(trifluoromethyl)phenyl}piperidin-2-on, Rimisoxafen, Rimsulfuron, Saflufenacil, Sethoxydim, Siduron, Simazine, Simetryn, SL-261, Sulcotrione, Sulfentrazone, Sulfometuron, Sulfometuron-Methyl, Sulfosulfuron, SYP-249, i.e. 1-Ethoxy-3-methyl-1-oxobut-3-en-2-yl-5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrobenzoate, SYP-300, i.e. 1-[7-Fluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-3-propyl-2-thioxoimidazolidin-4,5-dione, 2,3,6-TBA, TCA (Trichloroacetic acid) and its salts, z.B. TCA-ammonium, TCA-Calcium, TCA-Ethyl, TCA-Magnesium, TCA-Sodium, Tebuthiuron, Tefuryltrione, Tembotrione, Tepraloxydim, Terbacil, Terbucarb, Terbumeton, Terbuthylazine, Terbutryn, Tetflupyrolimet, Thaxtomin, Thenylchlor, Thiazopyr, Thiencarbazone, Thiencarbazone-Methyl, Thifensulfuron, Thifensulfuron-Methyl, Thiobencarb, Tiafenacil, Tolpyralat, Topramezone, Tralkoxydim, Triafamon, Tri-allate, Triasulfuron, Triaziflam, Tribenuron, Tribenuron-Methyl, Triclopyr, Triclopyr-Butotyl, Triclopyr-Choline, Triclopyr-Ethyl, Triclopyr-Triethylammonium, Trietazine, Trifloxysulfuron, Trifloxysulfuron-Sodium, Trifludimoxazin, Trifluralin, Triflusulfuron, Triflusulfuron-Methyl, Tritosulfuron, Harnstoffsulfat, Vernolat, XDE-848, ZJ-0862, i.e. 3,4-Dichloro-N-{2-[(4,6-dimethoxypyrimidin-2-yl)oxy]benzyl}aniline, Methyl 3-(2-Chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydropyrimidin-1 (2H)-yl)phenyl)-5-methyl-4,5-dihydroisoxazol-5-carboxylate, Ethyl 3-(2-Chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydropyrimidin-1 (2H)-yl)phenyl)-5-methyl-4,5-dihydroisoxazol-5-carboxylate, 3-(2-Chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydropyrimidin-1 (2H)-yl)phenyl)-5-methyl-4,5-dihydroisoxazol-5-carboxylic acid, Ethyl-[(3-{2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenoxy}pyridin-2-yl)oxy]acetate, 3-Chloro-2-[3-(difluoromethyl) isoxazolyl-5-yl]phenyl-5-chloropyrimidin-2-ylether, 2-(3,4-Dimethoxyphenyl)-4-[(2-hydroxy-6-oxocyclohex-1-en-1-yl) carbonyl]-6-methylpyridazin-3 (2H)-one, 2-({2-[(2-Methoxyethoxy)methyl]-6-methylpyridin-3-yl}carbonyl)cyclohexane-1,3-dione, (5-Hydroxy-1-methyl-1H-pyrazol-4-yl) (3,3,4-trimethyl-1,1-dioxido-2,3-dihydro-1-benzothiophen-5-yl) methanone, 1-Methyl-4-[(3,3,4-trimethyl-1,1-dioxido-2,3-dihydro-1-benzothiophen-5-yl) carbonyl]-1H-pyrazol-5-yl propan-1-sulfonate, 4-{2-Chloro-3-[(3,5-dimethyl-1H-pyrazol-1-yl)methyl]-4-(methylsulfonyl)benzoyl}-1-methyl-1H-pyrazol-5-yl-1,3-dimethyl-1H-pyrazol-4-carboxylate; Cyanomethyl-4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, Prop-2-yn-1-yl 4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, Methyl-4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, Benzyl-4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, Ethyl-4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, Methyl-4-amino-3-chloro-5-fluoro-6-(7-fluoro-1-isobutyryl-1H-indol-6-yl) pyridin-2-carboxylate, Methyl 6-(1-acetyl-7-fluoro-1H-indol-6-yl)-4-amino-3-chloro-5-fluoropyridin-2-carboxylate, Methyl-4-amino-3-chloro-6-[1-(2,2-dimethylpropanoyl)-7-fluoro-1H-indol-6-yl]-5-fluoropyridin-2-carboxylate, Methyl-4-amino-3-chloro-5-fluoro-6-[7-fluoro-1-(methoxyacetyl)-1H-indol-6-yl]pyridin-2-carboxylate, Potassium 4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, Sodium-4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, Butyl-4-amino-3-chloro-5-fluoro-6-(7-fluoro-1H-indol-6-yl) pyridin-2-carboxylate, 4-Hydroxy-1-methyl-3-[4-(trifluoromethyl)pyridin-2-yl]imidazolidin-2-one, 3-(5-tert-butyl-1,2-oxazol-3-yl)-4-hydroxy-1-methylimidazolidin-2-one, 3-[5-Chloro-4-(trifluoromethyl)pyridin-2-yl]-4-hydroxy-1-methylimidazolidin-2-one, 4-Hydroxy-1-methoxy-5-methyl-3-[4-(trifluoromethyl)pyridin-2-yl]imidazolidin-2-one, 6-[(2-Hydroxy-6-oxocyclohex-1-en-1-yl) carbonyl]-1,5-dimethyl-3-(2-methylphenyl) quinazolin-2,4 (1H,3H)-dione, 3-(2,6-Dimethylphenyl)-6-[(2-hydroxy-6-oxocyclohex-1-en-1-yl) carbonyl]-1-methylquinazolin-2,4 (1H,3H)-dione, 2-[2-chloro-4-(methylsulfonyl)-3-(morpholin-4-ylmethyl)benzoyl]-3-hydroxycyclohex-2-en-1-one, 1-(2-carboxyethyl)-4-(pyrimidin-2-yl)pyridazin-1-ium salt (combined with suitable anions such as chloride, acetate or trifluoroacetate), 1-(2-Carboxyethyl)-4-(pyridazin-3-yl)pyridazin-1-ium salt (combined with suitable anions such as chloride, acetate or trifluoroacetate), 4-(Pyrimidin-2-yl)-1-(2-sulfoethyl)pyridazin-1-ium salt (combined with suitable anions such as chloride, acetate or trifluoroacetate), 4-(Pyridazin-3-yl)-1-(2-sulfoethyl)pyridazin-1-ium salt (combined with suitable anions such as chloride, acetate or trifluoroacetate), 1-(2-Carboxyethyl)-4-(1,3-thiazol-2-yl)pyridazin-1-ium salt (combined with suitable anions such as chloride, acetate or trifluoroacetate), 1-(2-Carboxyethyl)-4-(1,3,4-thiadiazol-2-yl)pyridazin-1-ium salt (combined with suitable anions such as chloride, acetate or trifluoroacetate), Methyl (2R)-2-{[(E)-({2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}methyliden)amino]oxy}propanoate, Methyl (2S)-2-{[(E)-({2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}methyliden)amino]oxy}propanoate, Methyl (2R/S)-2-{[(E)-({2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}methyliden)amino]oxy}propanoate, (E)-2-(Trifluoromethyl)benzaldehyde-O-{2,6-bis [(4,6-dimethoxypyrimidin-2-yl)oxy]benzoyl}oxim, 2-Fluoro-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-[(R)-propylsulfinyl]-4-(trifluoromethyl)benzamide, (2R)-2-[(4-Amino-3,5-dichloro-6-fluoro-2-pyridyl)oxy]propanecarboxylic acid, 2-(2-Bromo-4-chlorobenzyl)-4,4-dimethyl-1,2-oxazolidin-3-one, Methyl 3-{2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}-3a,4,5,6-tetrahydro-6aH-cyclopenta[d][1,2]oxazol-6a-carboxylate, Ethyl 3-{2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}-3a,4,5,6-tetrahydro-6aH-cyclopenta[d][1,2]oxazol-6a-carboxylate, Methyl-3-{2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}-6-methyl-3a,4,5,6-tetrahydro-6aH-cyclopenta[d][1,2]oxazol-6a-carboxylate, 3-{2-Chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}-6-methyl-3a,4,5,6-tetrahydro-6aH-cyclopenta[d][1,2]oxazol-6a-carboxylic acid, 3-{2-Chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1 (2H)-yl]phenyl}-3a,4,5,6-tetrahydro-6aH-cyclopenta[d][1,2]oxazol-6a-carboxylic acid.
Examples of plant growth regulators as possible mixing partners are: Abscisic acid and related analogs [e.g. (2Z,4E)-5-[6-Ethynyl-1-hydroxy-2,6-dimethyl-4-oxocyclohex-2-en-1-yl]-3-methylpenta-2,4-dienoic acid, methyl-(2Z,4E)-5-[6-ethynyl-1-hydroxy-2,6-dimethyl-4-oxocyclohex-2-en-1-yl]-3-methylpenta-2,4-dienoate, (2Z,4E)-3-ethyl-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl) penta-2,4-dienoic acid, (2E,4E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl)-3-(trifluoromethyl) penta-2,4-dienoic acid, methyl (2E,4E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl)-3-(trifluoromethyl) penta-2,4-dienoate, (2Z,4E)-5-(2-hydroxy-1,3-dimethyl-5-oxobicyclo[4.1.0]hept-3-en-2-yl)-3-methylpenta-2,4-dienoic acid], acibenzolar, acibenzolar-S-methyl, S-adenosylhomocysteine, allantoin, 2-Aminoethoxyvinylglycine (AVG), aminooxyacetic acid and related esters [e.g. (Isopropylidene)-aminooxyacetic acid-2-(methoxy)-2-oxoethylester, (Isopropylidene)-aminooxyacetic acid-2-(hexyloxy)-2-oxoethylester, (Cyclohexylidene)-aminooxyacetic acid-2-(isopropyloxy)-2-oxoethylester], 1-aminocycloprop-1-ylcarboxylic N-Methyl-1-aminocyclopropyl-1-carboxylic acid, 1-aminocyclopropyl-1-carboxamide, 1-aminocycloprop-1-yl carboxylic acid and derivatives thereof, e.g. disclosed in DE3335514, EP30287, DE2906507 or U.S. Pat. No. 5,123,951, 1-aminocyclopropyl-1-hydroxamic acid, 5-aminolevulinic acid, ancymidol, 6-benzylaminopurine, bikinin, brassinolide, brassinolide-ethyl, L-canaline, catechin and catechines (e.g. (2S,3R)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-2H-chromen-3,5,7-triol), chitooligosaccharides (CO; COs differ from LCOs in that they lack the pendant fatty acid chain that is characteristic of LCOs. COs, sometimes referred to as N-acetylchitooligosaccharides, are also composed of GlcNAc residues but have side chain decorations that make them different from chitin molecules [(C8H13NO5)n, CAS No. 1398-61-4] and chitosan molecules [(C5H11NO4)n, CAS No. 9012-76-4]), chitinous compounds, chlormequat chloride, cloprop, cyclanilide, 3-(Cycloprop-1-enyl) propionic acid, 1-[2-(4-cyano-3,5-dicyclopropylphenyl) acetamido]cyclohexanecarboxylic acid, 1-[2-(4-cyano-3-cyclopropylphenyl) acetamido]cyclohexanecarboxylic acid, daminozide, dazomet, dazomet-sodium, n-decanol, dikegulac, dikegulac-sodium, endothal, endothal-dipotassium,-disodium, and mono(N,N-dimethylalkylammonium), ethephon, flumetralin, flurenol, flurenol-butyl, flurenol-methyl, flurprimidol, forchlorfenuron, gibberellic acid, inabenfide, indol-3-acetic acid (IAA), 4-indol-3-ylbutyric acid, isoprothiolane, probenazole, jasmonic acid, Jasmonic acid or derivatives thereof (e.g. jasmonic acid methyl ester, jasmonic acid ethyl ester), lipo-chitooligosaccharides (LCO, sometimes referred to as symbiotic nodulation (Nod) signals (or Nod factors) or as Myc factors, consist of an oligosaccharide backbone of β-1,4-linked N-acetyl-D-glucosamine (“GlcNAc”) residues with an N-linked fatty acyl chain condensed at the non-reducing end. As understood in the art, LCOs differ in the number of GlcNAc residues in the backbone, in the length and degree of saturation of the fatty acyl chain and in the substitutions of reducing and non-reducing sugar residues), linoleic acid or derivatives thereof, linolenic acid or derivatives thereof, maleic hydrazide, mepiquat chloride, mepiquat pentaborate, 1-methylcyclopropene, 3-methylcyclopropene, 1-ethylcyclopropene, 1-n-propylcyclopropene, 1-cyclopropenylmethanol, methoxyvinylglycin (MVG), 3′-methyl abscisic acid, 1-(4-methylphenyl)-N-(2-oxo-1-propyl-1,2,3,4-tetrahydroquinolin-6-yl) methanesulfonamide and related substituted tetrahydroquinolin-6-yl) methanesulfonamides, (3E,3aR,8bS)-3-({[(2R)-4-Methyl-5-oxo-2,5-dihydrofuran-2-yl]oxy}methylen)-3,3a,4,8b-tetrahydro-2H-indeno[1,2-b]furan-2-one and related lactones as outlined in EP2248421, 2-(1-naphthyl) acetamide, 1-naphthylacetic acid, 2-naphthyloxyacetic acid, nitrophenolate-mixture, 4-Oxo-4 [(2-phenylethyl)amino]butyric acid, paclobutrazol, 4-phenylbutyric acid and its related salts (e.g. sodium-4-phenylbutanoate, potassium-4-phenylbutanoate), phenylalanine, N-phenylphthalamic acid, prohexadione, prohexadione-calcium, putrescine, prohydrojasmon, rhizobitoxin, salicylic acid, salicylic acid methyl ester, sarcosine, sodium cycloprop-1-en-1-yl acetate, sodium cycloprop-2-en-1-yl acetate, sodium-3-(cycloprop-2-en-1-yl) propanoate, sodium-3-(cycloprop-1-en-1-yl) propanoate, sidefungin, spermidine, spermine, strigolactone, tecnazene, thidiazuron, triacontanol, trinexapac, trinexapac-ethyl, tryptophan, tsitodef, uniconazole, uniconazole-P, 2-fluoro-N-(3-methoxyphenyl)-9H-purin-6-amine, 2-chloro-N-(3-methoxyphenyl)-9H-purin-6-amine
Active compounds which can be employed in combination with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain according to the present disclosure in compositions according to the present disclosure (for example in mixed formulations or in the tank mix) are, for example, the following safeners:
Safeners that may be used in combination with the herbicidal compounds described herein include, but are not limited to, cloquintocet-mexyl, cyprosulfamide, fenchlorazole ethyl ester, isoxadifen-ethyl, mefenpyr-diethyl, fenclorim, cumyluron, S4-1, and S4-5. Preferred safeners include cloquintocet-mexyl, cyprosulfamide, isoxadifen-ethyl, and mefenpyr-diethyl.
The herbicide combinations described herein may comprise further components, for example plant growth regulators or compounds that prevent or eliminate unwanted species. Such compounds include, but are not limited to herbicides, fungicides, insecticides, acaricides, nematicides, miticides, and related substances.
Examples of plant growth regulators that may be used include, but are not limited to, acibenzolar, acibenzolar-S-methyl, 5-aminolevulinic acid, ancymidol, 6-benzylaminopurine, brassinolide, catechol, chlormequat chloride, cloprop, cyclanilide, 3-(cycloprop-1-enyl) propionic acid, daminozide, dazomet, n-decanol, dikegulac, dikegulac-sodium, endothal, endothal-dipotassium,-disodium, and mono (N,N-dimethylalkylammonium), ethephon, flumetralin, flurenol, flurenol-butyl, flurprimidol, forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid (IAA), 4-indol-3-ylbutyric acid, isoprothiolane, probenazole, jasmonic acid, jasmonic acid methyl ester, maleic hydrazide, mepiquat chloride, 1-methylcyclopropene, 2-(1-naphthyl) acetamide, 1-naphthylacetic acid, 2-naphthyloxyacetic acid, nitrophenolate mixture, 4-oxo-4 [(2-phenylethyl)amino]butyric acid, paclobutrazole, N-phenylphthalamic acid, prohexadione, prohexadione-calcium, prohydrojasmone, salicylic acid, strigolactone, tecnazene, thidiazuron, triacontanol, trinexapac, trinexapac-ethyl, tsitodef, uniconazole, and uniconazole-P.
Active compounds that may be used in combination with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain described herein (in, for example, mixed formulations or a tank mix) are, for example, fungicidally active compounds. The preferred fungicidally active compounds comprise at least one standard commercial active ingredient, and include, but are not limited to:
Preferred fungicides are selected from the group consisting of benalaxyl, bitertanol, bromuconazole, captafol, carbendazim, carpropamid, cyazofamid, cyproconazole, diethofencarb, edifenphos, fenpropimorph, fentin acetate, fluquinconazole, fosetyl, fluoroimide, folpet, iminoctadine, iprodione, iprovalicarb, kasugamycin, maneb, nabam, pencycuron, prochloraz, propamocarb, propineb, prothioconazole, pyrimethanil, spiroxamine, quintozene, tebuconazole, tolylfluanid, triadimefon, triadimenol, trifloxystrobin, and zineb.
Active compounds which can be employed in combination with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain according to the present disclosure in compositions described herein (for example in mixed formulations or in the tank mix) are, for example, insecticidal, acaricidal, nematicidal, miticidal and related active ingredients are, for example (the compounds are, if possible, referred to by their common names):
Insecticides that can preferably be used together with the substituted phenyl uracils carrying a cyclopropylcarboxylic acid-based side chain are, for example, as follows: acetamiprid, acrinathrin, aldicarb, amitraz, acinphos-methyl, cyfluthrin, carbaryl, cypermethrin, deltamethrin, endosulfan, ethoprophos, fenamiphos, fenthion, fipronil, imidacloprid, methamidophos, methiocarb, niclosamide, oxydemeton-methyl, prothiophos, silafluofen, thiacloprid, thiodicarb, tralomethrin, triazophos, trichlorfon, triflumuron, terbufos, fonofos, phorate, chlorpyriphos, carbofuran, and tefluthrin.
The disclosure relates, in certain embodiments, to recombinant DNA molecules that encode herbicide-insensitive protoporphyrinogen oxidases (PPOs) and the proteins encoded thereby. As used herein, the term “engineered” refers to a non-natural DNA, protein, cell, or organism that would not normally be found in nature and was created by human intervention. An “engineered protein,” “engineered enzyme,” or “engineered PPO,” refers to a protein, enzyme, or PPO whose amino acid sequence was conceived of and created in the laboratory using one or more of the techniques of biotechnology, protein design, or protein engineering, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site-directed mutagenesis, directed evolution using random mutagenesis, genome editing, gene editing, gene cloning, DNA ligation, DNA synthesis, protein synthesis, and DNA shuffling. For example, an engineered protein may have one or more deletions, insertions, or substitutions relative to the coding sequence of the wild-type protein and each deletion, insertion, or substitution may consist of one or more amino acids. Genetic engineering can be used to create a DNA molecule encoding an engineered protein, such as an engineered PPO that is herbicide tolerant and comprises at least a first amino acid substitution relative to a wild-type PPO protein as described herein.
In one embodiment, proteins provided herein have herbicide-tolerant protoporphyrinogen oxidase activity. As used herein, “herbicide-tolerant protoporphyrinogen oxidase” means the ability of a protoporphyrinogen oxidase to maintain at least some of its protoporphyrinogen oxidase activity in the presence of one or more PPO inhibiting herbicide(s). The term “protoporphyrinogen oxidase activity” means the ability to catalyze the six-electron oxidation (removal of electrons) of protoporphyrinogen IX to form protoporphyrin IX, that is, to catalyze the dehydrogenation of protoporphyrinogen to form protoporphyrin. Enzymatic activity of a protoporphyrinogen oxidase can be measured by any means known in the art, for example, by an enzymatic assay in which the production of the product of protoporphyrinogen oxidase or the consumption of the substrate of protoporphyrinogen oxidase in the presence of one or more PPO inhibiting herbicide(s) is measured via fluorescence, high performance liquid chromatography (HPLC), or mass spectrometry (MS). Another example of an assay for measuring enzymatic activity of a protoporphyrinogen oxidase is a bacterial assay, such as the assays described herein, whereby a recombinant protoporphyrinogen oxidase is expressed in a bacterial cell otherwise lacking PPO activity and the ability of the recombinant protoporphyrinogen oxidase to complement this knockout phenotype is measured. As used herein, a “hemG knockout strain” means an organism or cell of an organism, such as E. coli, that lacks HemG activity to the extent that it is unable to grow on heme-free growth medium, or such that its growth is detectably impaired in the absence of heme relative to an otherwise isogenic strain comprising a functional HemG. A hemG knockout strain of, for instance, E. coli may be prepared in view of knowledge in the art, for instance in view of the E. coli HemG PPO sequence (Ecogene Accession No. EG11485; Sasarman et al., “Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of E. coli K12” Can. J. Microbiol. 39:1155-1161, 1993).
As used herein, the term “recombinant” refers to a non-naturally occurring DNA, protein, cell, seed, or organism that is the result of genetic engineering and was created by human intervention. A “recombinant DNA molecule” is a DNA molecule comprising a DNA sequence that does not naturally occur and as such is the result of human intervention, such as a DNA molecule comprising at least two DNA molecules heterologous to each other. An example of a recombinant DNA molecule is a DNA molecule provided herein encoding an herbicide-tolerant protoporphyrinogen oxidase operably linked to a heterologous promoter. A “recombinant protein” is a protein comprising an amino acid sequence that does not naturally occur and as such is the result of human intervention, such as an engineered protein. A recombinant cell, seed, or organism is a cell, seed, or organism comprising transgenic or heterologous DNA or protein, for example a transgenic plant cell, seed, or plant comprising a DNA construct or engineered protein described herein.
As used herein, “wild-type” means a naturally occurring. A “wild-type DNA molecule,” “wild-type protein” is a naturally occurring version of a DNA molecule or protein, that is, a version of a DNA molecule or protein pre-existing in nature. A wild-type version of a DNA molecule or protein may be useful for comparison with a recombinant or engineered DNA molecule or protein. An example of a wild-type protein useful for comparison with the engineered proteins provided by the present disclosure is the PPO enzyme from E. cloacae (H_N90) provided as SEQ ID NO:1.
A “wild-type plant” is a naturally occurring plant. Such wild-type plants may also be useful for comparison with a plant comprising a recombinant or engineered DNA molecule or protein. An example of a wild-type plant useful for comparison with plants comprising a recombinant or engineered DNA molecule or protein may be a plant of the same type as the plant comprising the engineered DNA molecule or protein, such as a protein conferring an herbicide tolerance trait, and as such is genetically distinct from the plant comprising the herbicide tolerance trait.
In certain embodiments, wild-type plants may also be used or referred to as “control plants.” As used herein, “control” means an experimental control designed for comparison purposes. For example, a control plant in a transgenic plant analysis is a plant of the same type as the experimental plant (that is, the plant to be tested) but does not contain the transgenic insert, recombinant DNA molecule, or DNA construct of the experimental plant. Examples of control plants useful for comparison with transgenic plants include: for maize plants, non-transgenic LH244 maize (U.S. Pat. No. 6,252,148) or non-transgenic 01DKD2 maize (U.S. Pat. No. 7,166,779); for comparison with soybean plants, non-transgenic A3555 soybean (ATCC deposit number PTA-10207); for comparison with cotton plants, non-transgenic DP393 (U.S. Pat. No. 6,930,228, PVP 200400266); for comparison with canola or Brassica napus plants, non-transgenic Brassica napus variety 65037 Restorer line (Canada Plant Breeders' Rights Application 06-5517); for comparison with wheat plants, non-transgenic wheat variety Samson germplasm (PVP 1994).
As used herein, the term “DNA” or “DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin (that is, a polymer of deoxyribonucleotide bases or a polynucleotide molecule) read from the 5′ (upstream) end to the 3′ (downstream) end. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of by Title 37 of the United States Code of Federal Regulations § 1.822, and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
As used herein, the term “protein-coding DNA molecule” refers to a DNA molecule comprising a DNA sequence that encodes a protein. As used herein, the term “protein” refers to a chain of amino acids linked by peptide (amide) bonds and includes both polypeptide chains that are folded or arranged in a biologically functional way and polypeptide chains that are not. As used herein, a “protein-coding sequence” means a DNA sequence that encodes a protein. As used herein, a “sequence” means a sequential arrangement of nucleotides or amino acids. A “DNA sequence” may refer to a sequence of nucleotides or to the DNA molecule comprising of a sequence of nucleotides; a “protein sequence” may refer to a sequence of amino acids or to the protein comprising a sequence of amino acids. The boundaries of a protein-coding sequence are usually determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus.
As used herein, the term “isolated” refers to at least partially separating a molecule from other molecules typically associated with it in its natural state. In one embodiment, the term “isolated” refers to a DNA molecule that is separated from the nucleic acids that normally flank the DNA molecule in its natural state. For example, a DNA molecule encoding a protein that is naturally present in a bacterium would be an isolated DNA molecule if it was not within the DNA of the bacterium from which the DNA molecule encoding the protein is naturally found. Thus, a DNA molecule fused to or operably linked to one or more other DNA molecule(s) with which it would not be associated in nature, for example as the result of recombinant DNA or plant transformation techniques, is considered isolated herein. Such molecules are considered isolated even when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules.
Any number of methods well known to those skilled in the art can be used to isolate and manipulate a DNA molecule, or fragment thereof, as disclosed herein. For example, polymerase chain reaction (PCR) technology can be used to amplify a particular starting DNA molecule or to produce variants of the original molecule. DNA molecules, or fragment thereof, can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.
Because of the degeneracy of the genetic code, a variety of different DNA sequences can encode proteins, such as the altered or engineered proteins disclosed herein. It is well within the capability of one of skill in the art to create alternative DNA sequences encoding the same, or essentially the same, altered or engineered proteins as described herein. These variant or alternative DNA sequences are within the scope of the embodiments described herein. As used herein, references to “essentially the same” sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions that do not materially alter the functional activity of the protein encoded by the DNA molecule of the embodiments described herein. Allelic variants of the nucleotide sequences encoding a wild-type or engineered protein are also encompassed within the scope of the embodiments described herein. Substitution of amino acids other than those specifically exemplified or naturally present in a wild-type or engineered PPO enzyme are also contemplated within the scope of the embodiments described herein, so long as the PPO enzyme having the substitution still retains substantially the same functional activity described herein.
Recombinant DNA molecules of the present disclosure may be synthesized and modified by methods known in the art, either completely or in part, where it is desirable to provide sequences useful for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant-codon usage or Kozak consensus sequences), or sequences useful for DNA construct design (such as spacer or linker sequences). The present disclosure includes recombinant DNA molecules and engineered proteins having at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, and at least 99% sequence identity to any of the recombinant DNA molecule or amino acid sequences provided herein, and having herbicide-tolerant protoporphyrinogen oxidase activity. As used herein, the term “percent sequence identity” or “% sequence identity” refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or amino acid sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, WI 53715), and MUSCLE (version 3.6) (Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput,” Nucleic Acids Research 32 (5): 1792-7, 2004) for instance with default parameters. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the portion of the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.
As used herein, a “DNA construct” is a recombinant DNA molecule comprising two or more heterologous DNA sequences. DNA constructs are useful for transgene expression and may be comprised in vectors and plasmids. DNA constructs may be used in vectors for transformation (that is, the introduction of heterologous DNA into a host cell) to produce recombinant bacteria or transgenic plants and cells (and as such may also be contained in the plastid DNA or genomic DNA of a transgenic plant, seed, cell, or plant part). As used herein, a “vector” means any recombinant DNA molecule that may be used for bacterial or plant transformation. DNA molecules provided by the present disclosure can, for example, be inserted into a vector as part of a DNA construct having the DNA molecule operably linked to a heterologous gene expression element that functions in a plant to affect expression of the engineered protein encoded by the DNA molecule. Methods for making and using DNA constructs and vectors are well known in the art and described in detail in, for example, handbooks and laboratory manuals including Green and Sambrook, “Molecular Cloning: A Laboratory Manual” Vol. 1, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012. The components for a DNA construct, or a vector comprising a DNA construct, include one or more gene expression elements operably linked to a transcribable nucleic acid sequence, such as the following: a promoter for the expression of an operably linked DNA, an operably linked protein-coding DNA molecule, and an operably linked 3′ untranslated region (UTR). Gene expression elements useful in practicing the present disclosure include, but are not limited to, one or more of the following type of elements: promoter, 5′ UTR, enhancer, leader, cis-acting element, intron, transit sequence, 3′ UTR, and one or more selectable marker transgenes.
The term “transgene” refers to a DNA molecule artificially incorporated into the genome of an organism as a result of human intervention, such as by plant transformation methods. As used herein, the term “transgenic” means comprising a transgene, for example a “transgenic plant” refers to a plant comprising a transgene in its genome and a “transgenic trait” refers to a characteristic or phenotype conveyed or conferred by the presence of a transgene incorporated into the plant genome. As a result of such genomic alteration, the transgenic plant is something distinctly different from the related wild-type plant and the transgenic trait is a trait not naturally found in the wild-type plant. Transgenic plants of the present disclosure comprise the recombinant DNA molecules and proteins described herein.
As used herein, the term “heterologous” refers to the relationship between two or more things not normally associated in nature, for instance that are derived from different sources or not normally found in nature together in any other manner. For example, a DNA molecule or protein may be heterologous with respect to another DNA molecule, protein, cell, plant, seed, or organism if not normally found in nature together or in the same context. In certain embodiments, a first DNA molecule is heterologous to a second DNA molecule if the two DNA molecules are not normally found in nature together in the same context. For instance, a protein-coding recombinant DNA molecule is heterologous with respect to an operably linked promoter if such a combination is not normally found in nature. Similarly, a protein is heterologous with respect to a second operably linked protein, such as a transit peptide, if such combination is not normally found in nature. In another embodiment, a recombinant DNA molecule encoding a PPO enzyme is heterologous with respect to an operably linked promoter that is functional in a plant cell if such combination is not normally found in nature. A recombinant DNA molecule also may be heterologous with respect to a cell, seed, or organism into which it is inserted when it would not naturally occur in that cell, seed, or organism.
A “heterologous protein” is a protein present in a plant, seed, cell, tissue, or organism in which it does not naturally occur or operably linked to a protein with which it is not naturally linked. Examples of heterologous proteins are the PPO enzymes described herein that is expressed in any plant, seed, cell, tissue, or organism. Another example is a protein operably linked to a second protein, such as a transit peptide or herbicide-tolerant protein, with which it is not naturally linked, or a protein introduced into a plant cell in which it does not naturally occur using the techniques of genetic engineering.
As used herein, “operably linked” means two or more DNA molecules or two or more proteins linked in manner so that one may affect the function of the other. Operably linked DNA molecules or operably linked proteins may be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked with a protein-coding DNA molecule in a DNA construct where the two DNA molecules are so arranged that the promoter may affect the expression of the transgene.
The DNA constructs described herein may include a promoter operably linked to a protein-coding DNA molecule provided herein, whereby the promoter drives expression of the protein. Useful promoters include those that function in a cell for expression of an operably linked DNA molecule, such as a bacterial or plant promoter. Plant promoters are varied and well known in the art and include, for instance, those that are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, or spatio-temporally regulated.
In one embodiment, a DNA construct provided herein includes a DNA sequence encoding a transit sequence that is operably linked to a heterologous DNA sequence encoding a PPO enzyme, whereby the transit sequence facilitates localizing the protein molecule within the cell. Transit sequences are known in the art as signal sequences, targeting peptides, targeting sequences, localization sequences, and transit peptides. An example of a transit sequence is a chloroplast transit peptide (CTP), a mitochondrial transit sequence (MTS), or a dual chloroplast and mitochondrial transit peptide. By facilitating protein localization within the cell, the transit sequence may increase the accumulation of recombinant protein, protect the protein from proteolytic degradation, or enhance the level of herbicide tolerance, and thereby reduce levels of injury in the cell, seed, or organism after herbicide application. CTPs and other targeting molecules that may be used in connection with the present disclosure are well known in the art.
As used herein, “transgene expression,” “expressing a transgene,” “protein expression,” and “expressing a protein,” mean the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into polypeptide chains, which are ultimately folded into proteins. A protein-coding DNA molecule may be operably linked to a heterologous promoter in a DNA construct for use in expressing the protein in a cell transformed with the recombinant DNA molecule.
In one aspect, cells, tissues, plants, and seeds that comprising the recombinant DNA molecules or proteins are provided herein. These cells, tissues, plants, and seeds comprising the recombinant DNA molecules or proteins exhibit tolerance to one or more PPO inhibiting herbicide(s).
In the commercial production of crops, it is desirable to eliminate under reliable pesticidal management unwanted plants (i.e., “weeds”) from a field of crop plants. An ideal treatment would be one which could be applied to an entire field but which would eliminate only the unwanted plants while leaving the crop plants unaffected. One such treatment system would involve the use of crop plants which are tolerant to an herbicide so that when the herbicide is sprayed on a field of herbicide-tolerant crop plants, the crop plants would continue to thrive while non-herbicide-tolerant weeds are killed or severely damaged. Ideally, such treatment systems would take advantage of varying herbicide properties so that weed control could provide the best possible combination of flexibility and economy. For example, individual herbicides have different longevities in the field, and some herbicides persist and are effective for a relatively long time after they are applied to a field while other herbicides are quickly broken down into other and/or non-active compounds. An ideal treatment system would allow the use of different herbicides so that growers could tailor the choice of herbicides for a particular situation.
While a number of herbicide-tolerant crop plants are presently commercially available, one issue that has arisen for many commercial herbicides and herbicide/crop combinations is that individual herbicides typically have incomplete spectrum of activity against common weed species. For most individual herbicides which have been in use for some time, populations of herbicide resistant weed species and biotypes have become more prevalent (see, e.g., Tranel and Wright, Weed Science 50:700-712, 2002; Owen and Zelaya, Pest Manag. Sci. 61:301-311, 2005). Transgenic plants which are resistant to more than one herbicide have been described (see, e.g., WO 2005/012515). However, improvements in every aspect of crop production, weed control options, extension of residual weed control, and improvement in crop yield are continuously in demand.
One method of producing such cells, tissues, plants, and seeds is through plant transformation. Suitable methods for transformation of host plant cells for use with the current disclosure include any method by which DNA can be introduced into a cell (for example, where a recombinant DNA construct is stably integrated into a plant chromosome) and are well known in the art. Two effective, and widely utilized, methods for cell transformation are Agrobacterium-mediated transformation and microprojectile bombardment-mediated transformation. Microprojectile bombardment methods are illustrated, for example, in U.S. Pat. Nos. 5,550,318; 5,538,880; 6,160,208; and 6,399,861. Agrobacterium-mediated transformation methods are described, for example in U.S. Pat. No. 5,591,616. A cell with a recombinant DNA molecule or protein of the present disclosure may be selected for the presence of the recombinant DNA molecule or protein, for instance through its encoded enzymatic activity, before or after regenerating such a cell into a plant.
Another method of producing the cells, plants, and seeds of the present disclosure is through genome modification using site-specific integration or genome editing. Targeted modification of plant genomes through the use of genome editing methods can be used to create improved plant lines through modification of plant genomic DNA. As used herein “site-directed integration” refers to genome editing methods the enable targeted insertion of one or more nucleic acids of interest into a plant genome. Suitable methods for altering a wild-type DNA sequence or a preexisting transgenic sequence or for inserting DNA into a plant genome at a pre-determined chromosomal site include any method known in the art. Exemplary methods include the use of sequence specific nucleases, such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cascade system). Several embodiments relate to methods of genome editing by using single-stranded oligonucleotides to introduce precise base pair modifications in a plant genome, as described by Sauer et al., Plant Physiology 170 (4): 1917-1928, 2016. Methods of genome editing to modify, delete, or insert nucleic acid sequences into genomic DNA are known in the art.
In certain embodiments, the present disclosure provides modification or replacement of an existing coding sequence, such as a PPO coding sequence or another existing transgenic insert, within a plant genome with a sequence encoding a protein, such as a PPO coding sequence of the present disclosure, or an expression cassette comprising such a protein. Several embodiments relate to the use of a known genome editing methods, such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cascade system).
Several embodiments may therefore relate to a recombinant DNA construct comprising an expression cassette(s) encoding a site-specific nuclease and, optionally, any associated protein(s) to carry out genome modification. These nuclease-expressing cassette(s) may be present in the same molecule or vector as a donor template for templated editing or an expression cassette comprising nucleic acid sequence encoding a PPO protein as described herein (in cis) or on a separate molecule or vector (in trans). Several methods for site-directed integration are known in the art involving different sequence-specific nucleases (or complexes of proteins or guide RNA or both) that cut the genomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus. As understood in the art, during the process of repairing the DSB or nick introduced by the nuclease enzyme, the donor template DNA, transgene, or expression cassette may become integrated into the genome at the site of the DSB or nick. The presence of the homology arm(s) in the DNA to be integrated may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process through homologous recombination, although an insertion event may occur through non-homologous end joining (NHEJ).
As used herein, the term “double-strand break inducing agent” refers to any agent that can induce a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break inducing agent is a site-specific genome modification enzyme.
As used herein, the term “site-specific genome modification enzyme” refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a single-strand break. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a double-strand break. In some embodiments, a site-specific genome modification enzyme comprises a cytidine deaminase. In some embodiments, a site-specific genome modification enzyme comprises an adenine deaminase. In the present disclosure, site-specific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, helicases and any combination thereof. In some embodiments, the site-specific genome modification enzyme is a sequence-specific nuclease.
In one aspect, the endonuclease is selected from a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), an Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), an RNA-guided nuclease, such as a CRISPR associated nuclease (non-limiting examples of CRISPR associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, homologs thereof, or modified versions thereof).
In some embodiments, the site-specific genome modification enzyme is a recombinase. Non-limiting examples of recombinases include a tyrosine recombinase attached to a DNA recognition motif and is selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnp1 recombinase. In an aspect, a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc-finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, a serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase. In another aspect, a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.
Any of the DNA of interest provided herein can be integrated into a target site of a chromosome sequence by introducing the DNA of interest and the provided site-specific genome modification enzymes. Any method provided herein can utilize any site-specific genome modification enzyme provided herein.
As used herein, a “weed” is any undesired plant. A plant may be considered generally undesirable for agriculture or horticulture purposes (for example, Amaranthus species) or may be considered undesirable in a particular situation (for example, a crop plant of one species in a field of a different species, also known as a volunteer plant).
The transgenic plants, progeny, seeds, plant cells, and plant parts described herein may also contain one or more additional traits. Additional traits may be introduced by crossing a plant containing a transgene comprising the recombinant DNA molecules provided herein with another plant containing one or more additional trait(s). As used herein, “crossing” means breeding two individual plants to produce a progeny plant. Two plants may thus be crossed to produce progeny that contain the desirable traits from each parent. As used herein “progeny” means the offspring of any generation of a parent plant, and transgenic progeny comprise a DNA construct provided herein and inherited from at least one parent plant.
Additional trait(s) also may be introduced by co-transforming a DNA construct for that additional transgenic trait(s) with a DNA construct comprising the recombinant DNA molecules provided herein (for example, with all the DNA constructs present as part of the same vector used for plant transformation) or by inserting the additional trait(s) into a transgenic plant comprising a DNA construct provided by the herein or vice versa (for example, by using any of the methods of plant transformation or genome editing on a transgenic plant or plant cell). Such additional traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, in which the trait is measured with respect to a wild-type plant. Illustrative additional herbicide-tolerance traits may include transgenic or non-transgenic tolerance to one or more herbicides such as ACCase inhibitors (for example aryloxyphenoxy propionates and cyclohexanediones), ALS inhibitors (for example sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones) EPSPS inhibitors (for example glyphosate), synthetic auxins (for example phenoxys, benzoic acids, carboxylic acids, semicarbazones), photosynthesis inhibitors (for example triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthesis inhibitors (for example glufosinate), HPPD inhibitors (for example isoxazoles, pyrazolones, and triketones), PPO inhibitors (for example diphenylethers, N-phenylphthalimide, aryl triazinones, and pyrimidinediones), and long-chain fatty acid inhibitors (for example chloroacetamindes, oxyacetamides, and pyrazoles), among others. Examples of herbicide-tolerance proteins useful for producing additional herbicide-tolerance traits are well known in the art and include, but are not limited to, glyphosate-tolerant 5-enolypyruvyl shikimate 3-phosphate synthases (e.g., CP4 EPSPS, 2mEPSPS), glyphosate oxidoreductases (GOX), glyphosate N-acetyltransferases (GAT), herbicide-tolerant acetolactate synthases (ALS)/acetohydroxyacid synthases (AHAS), herbicide-tolerant 4-hydroxyphenylpyruvate dioxygenases (HPPD), dicamba monooxygenases (DMO), phosphinothricin acetyl transferases (PAT), herbicide-tolerant glutamine synthetases (GS), 2,4-dichlorophenoxyproprionate dioxygenases (TfdA), R-2,4-dichlorophenoxypropionate dioxygenases (RdpA), S-2,4-dichlorophenoxypropionate dioxygenases (SdpA), herbicide-tolerant protoporphyrinogen oxidases (PPO), and cytochrome P450 monooxygenases. Exemplary insect resistance traits may include resistance to one or more insect members within one or more of the orders of Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, Diptera, Hymenoptera, and Orthoptera, among others. Such additional traits are well known to one of skill in the art; for example, and a list of such transgenic traits is provided by the United States Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS).
Transgenic plants and progeny that are tolerant to PPO inhibiting herbicides may be used with any breeding methods that are known in the art. In plant lines comprising two or more traits, the traits may be independently segregating, linked, or a combination of both in plant lines comprising three or more transgenic traits. Backcrossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of breeding methods that are commonly used for different traits and crops are well known to those of skill in the art. To confirm the presence of the transgene(s) in a particular plant or seed, a variety of assays may be performed. Such assays include, for example, molecular biology assays, such as Southern and Northern blotting, PCR, and DNA sequencing; biochemical assays, such as detecting the presence of a protein product, for example, by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole plant.
Introgression of a transgenic trait into a plant genotype is achieved as the result of the process of backcross conversion. A plant genotype into which a transgenic trait has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly, a plant genotype lacking the desired transgenic trait may be referred to as an unconverted genotype, line, inbred, or hybrid.
As used herein, the term “comprising” means “including but not limited to.”
Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that the examples in the present disclosure are provided as non-limiting examples.
Novel microbial HemG protoporphyrinogen oxidases that are tolerant to PPO inhibitor herbicides were previously identified from microbial sequence databases using bioinformatic methods and an herbicide bacterial screening system and are provided as SEQ ID NOs: 1-20 and recombinant variants of these microbial HemG protoporphyrinogen oxidases are provided as SEQ ID NOs: 65-193. DNA sequences encoding microbial HemG protoporphyrinogen oxidases and their variants, along with DNA sequences that are optimized for expression in a monocot or dicot can optionally be synthesized and are provided as SEQ ID NOs: 22-64 and 194-322.
At the 5′ end of the DNA sequence encoding a protoporphyrinogen oxidase, a codon for a methionine, commonly known as a start codon, may be present. Alternatively, this codon (and optionally a few amino-terminal amino acids, for example 2 to 7), can be eliminated to facilitate operable linkage of a transit peptide sequence to the 5′ end of the coding sequence. Novel transit peptides were previously identified by using bioinformatic methods and tools, such as hidden Markov models (HMM), the Pfam database, and basic local alignment search tool (BLAST), to identify thousands of EST and genomic sequences predicted to encode proteins known to be localized to the chloroplast and mitochondria in plant cells and are provided herein as SEQ ID NOs: 323-328, 340, and 342-407, along with their corresponding nucleotide sequences, provided herein as SEQ ID NOs: 329-339, 341, and 408-483.
Protoporphyrinogen oxidases operably linked to transit peptides were tested in transgenic soybean, corn, and cotton plants for tolerance to PPO inhibiting herbicides and for weed control in the field.
Plant transformation vectors were constructed for expressing a chloroplast transit peptide operably linked to the PPO H_N90 (SEQ ID NO:1) in transgenic soybean, inbred corn, hybrid corn, and cotton plants, and introduced into seed-derived explants of soybean, inbred and hybrid corn, and cotton, respectively, through Agrobacterium tumefaciens-mediated transformation using standard methods known in the art. The regenerated R0 plants were analyzed to select for events with a single copy insertion for advancement to R1 nursery for R1 seed production.
Seeds of the regenerated transgenic plants described above were sown in 12-cm tall plastic pots containing standard soil (14.7% sand, 19.9% clay, 65.4% silt, and 1.8% organic matter). Transgenic cotton and corn seed were sown at a density of one seed per pot, whereas soybean was sown at a density of three seeds per pot. Plants were grown in a greenhouse with 60% relative humidity in a light cycle of 13 hour day and 11 hour night. The temperature was kept at 23° C. during the day and 12° C. at night.
Herbicide tolerance of transgenic soybean, inbred corn, hybrid corn, and cotton plants, to herbicidal formulations comprising any of A1 to A3 and A7 was measured by applying respective formulations to plants at the 2-4 leaf stage (corresponding to BBCH 12-14) at dose rates of 100 or 200 g ai/ha. Methylated rapeseed oil (Mero) is used as an adjuvant and added to the spray mix for each herbicide at 0.5% v/v.
Plants were visually assessed for herbicide injury at the following time points: 8 days, 15 days, 22 days after application. Unsprayed transgenic plants were used for phenotypic comparison with unsprayed wild-type plants. Injury rating was determined as the percentage of leaf area of a plant exhibiting damage such as necrosis (brown or dead tissue), chlorosis (yellow tissue or yellow spotting), and malformation (misshapen leaves or plant structures, epinasty or twisting of stem, cupping of leaves) caused by herbicide application and was measured on a scale of 0-100, with zero being no injury and 100 being complete crop death. The data obtained at 15 days after application are shown in Table 2 below.
Protoporphyrinogen oxidases operably linked to transit peptides are tested in transgenic soybean, corn, and cotton plants for tolerance to PPO inhibiting herbicides and for weed control in the field.
Plant transformation vectors are constructed for expressing a chloroplast transit peptide operably linked to a HemG PPO enzyme, such as H_N90 (SEQ ID NO:1) in transgenic soybean, corn, and cotton plants, and introduced into seed-derived explants of soybean, corn, and cotton, respectively, through Agrobacterium tumefaciens-mediated transformation using standard methods known in the art. The regenerated R0 plants are analyzed to select for events with a single copy insertion for advancement to R1 nursery for R1 seed production. Homozygous R1 or later generation events are tested under field conditions to confirm their tolerance to PPO inhibitor herbicides.
To confirm herbicide tolerance of transgenic soybean plants under field conditions, substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of A1, A2, A3, A4, A5, A6 and A7 as described herein, and a commercial PPO inhibitor herbicide (such as saflufenacil) are applied at emergence (VE), V3, and R1 developmental stages at one of two rates. Plants are visually assessed for herbicide injury 14 days after treatment for VE, and 7 days after treatment for V3 and R1 stages. Unsprayed transgenic plants are used for phenotypic comparison with unsprayed wild-type plants.
To confirm herbicide tolerance of transgenic corn plants under field conditions, substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of A1, A2, A3, A4, A5, A6 and A7 as described herein, and a commercial PPO inhibitor herbicide (such as fomesafen) are applied to transgenic inbred or hybrid plants at emergence (VE), V2, V6, and VT developmental stages at one of two rates. Plants are visually assessed for herbicide injury 10-14 days after herbicide treatment. Unsprayed transgenic plants are used for phenotypic comparison with unsprayed wild-type plants.
To confirm herbicide tolerance of transgenic cotton plants under field conditions, substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of A1, A2, A3, A4, A5, A6 and A7 as described herein, and a commercial PPO inhibitor herbicide (such as fomesafen) are applied at pre-emergence (PRE), V4, and V8 developmental stages at one of two rates. Plants are visually assessed for herbicide injury 10-14 days after herbicide treatment. Unsprayed transgenic plants are used for phenotypic comparison with unsprayed wild-type plants.
For soybean, corn, and cotton, the injury rating is determined as the percentage of leaf area of a plant exhibiting damage such as necrosis (brown or dead tissue), chlorosis (yellow tissue or yellow spotting), and malformation (misshapen leaves or plant structures, epinasty or twisting of stem, cupping of leaves) caused by herbicide application and is measured on a scale of 0-100, with zero being no injury and 100 being complete crop death.
Transgenic crop seeds conferring PPO inhibiting herbicide tolerance prepared as described above are planted in a field or crop growing area. PPO inhibiting herbicide formulations, such as substituted phenyl uracils comprising a cyclopropylcarboxylic acid-based side chain, such as any of the compounds of A1, A2, A3, A4, A5, A6 and A7 as described herein, are applied to the field or crop growing area before or/and after planting the seeds to control weed growth. The herbicide application comprises an effective amount of at least one PPO inhibiting herbicide that prevents or controls the growth of weeds, but does not damage or injure the transgenic soybean, corn, or cotton crop plants comprising a recombinant DNA molecule encoding a chloroplast-targeted heterologous HemG protein. The PPO inhibiting herbicides can be applied, once or more than once, at about 1X application rate. However, the rate may be adjusted or varied depending on environmental conditions (such as temperature and humidity) and the type of weeds being controlled, as is known in the art. Therefore, the application rate may vary within wide limits, and may consist of a range from about 0.02 g a.i./ha to about 750 g a.i./ha, about 0.05 g a.i./ha to about 400 g a.i./ha, about 0.25 g a.i./ha to about 300 g a.i./ha, about 0.3 g a.i./ha to about 250 g a.i./ha, about 0.4 g a.i./ha to about 150 g a.i./ha, or about 0.5 g a.i./ha to about 120 g a.i./ha.
A desired application rate in any particular environment or in the context of a particular weed can be determined empirically by one of skill in the art in view of the present disclosure. The herbicide rate is carefully selected to avoid 1) over use than what is needed, which could lead to injury to the herbicide tolerant crop(s); 2) under use, resulting in poor weed control, which could lead to development of herbicide tolerant weeds.
The PPO inhibiting herbicides may be applied pre-emergence and/or post emergence. In the case of post emergence application, the PPO inhibiting herbicides may be applied over the top of the crop growing area.
In addition to a PPO inhibiting herbicide, an effective amount of at least a second herbicide can be applied for weed control. Examples of such a second herbicide include, but are not limited to, an ACCase inhibitor (such as an aryloxyphenoxy propionate or a cyclohexanedione), an ALS inhibitor (such as sulfonylurea, imidazolinone, triazoloyrimidine, or a triazolinone), an EPSPS inhibitor (such as glyphosate), a synthetic auxin (such as a phenoxy herbicide, a benzoic acid, a carboxylic acid, or a semicarbazone), a photosynthesis inhibitor (such as a triazine, a triazinone, a nitrile, a benzothiadiazole, or a urea), a glutamine synthetase inhibitor (such as glufosinate), a HPPD inhibitor (such as an isoxazole, a pyrazolone, or a triketone), a PPO inhibitor (such as a diphenylether, a N-phenylphthalimide, an aryl triazinone, or a pyrimidinedione), and a long-chain fatty acid inhibitor (such as a chloroacetamide, an oxyacetamide, or a pyrazole).
This application claims priority to U.S. Provisional Application Ser. No. 63/621,371, filed Jan. 16, 2024, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63621371 | Jan 2024 | US |
