Methods for detecting acetohydroxyacid synthase inhibitors

Abstract

The invention provides a method for determining whether a compound inhibits acetohydroxyacid synthase. The invention further provides a method for determining whether a plant is resistant to an acetohydroxyacid synthase inhibiting compound.

I claim: 
     
       1. An in vivo method for determining whether a compound inhibits acetohydroxyacid synthase which comprises: (a) treating a first acetohydroxyacid synthase inhibitor-susceptible plant or plant part with an effective amount of the compound and an effective amount of a ketol-acid reductoisomerase inhibitor;   (b) treating a second acetohydroxyacid synthase inhibitor-susceptible plant or plant part from the same population of the same species with the effective amount of the ketol-acid reductiosomerase inhibitor alone; and   (c) measuring the amounts of acetolactate and acetohydroxybutyrate present in the treated plants or plant parts to determine if the amount of acetolactate and acetohydroxybutyrate present in (a) is less than the amount of acetolactate and acetohydroxybutyrate present in (b).   
     
     
       2. The method according to claim 1 wherein the ketol-acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl)glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodium N-hydroxy-N-alkyloxamate and sodium N-hydroxy-N-aralkyloxamate. 
     
     
       3. The method according to claim 2 wherein the alkyl component of the N-hydroxy-N-alkyloxamate is selected from C 1  -C 6  alkyl and C 3  -C 7  cycloalkyl. 
     
     
       4. The method according to claim 3 wherein the alkyl component is isopropyl. 
     
     
       5. The method according to claim 2 wherein the aralkyl component is benzyl. 
     
     
       6. The method according to claim 1 wherein the acetohydroxyacid synthase inhibitor susceptible plant is selected from the group consisting of a monocotyledonous plant and a dicotyledonous plant. 
     
     
       7. The method according to claim 1 wherein the plant parts are young rapidly growing tissue. 
     
     
       8. The method according to claim 1 wherein the amount of acetolactate and acetohydroxybutyrate present in the treated plant or plant part is determined by: (a) extracting the acetolactate and acetohydroxybutyrate present in the treated plant or plant part into water;   (b) treating the water extract with a sulfuric acid solution;   (c) treating the acidified water extract solution from (b) with a 0.5% creatine solution and a 5% α-naphthol in sodium hydroxide solution; and   (d) measuring and comparing the color of the product of (c) to a known standard.   
     
     
       9. The method according to claim 1 wherein the plant or plant part is treated with a 1 μM to 1,000 μM solution of the compound and a 1 μM to 1,000 μM solution of the ketol-acid reductoisomerase inhibitor. 
     
     
       10. An in vivo method for determining whether a population of a plant species is resistant to an acetohydroxyacid synthase inhibitor which comprises: (a) treating a first plant or a part of the plant with an effective amount of the acetohydroxyacid synthase inhibitor and an effective amount of a ketol-acid reductoisomerase inhibitor;   (b) treating a second plant or plant part from the same population of the same species with the effective amount of the ketol-acid reductoisomerase inhibitor alone;   (c) measuring the amounts of acetolactate and acetohydroxybutyrate present in the treated plants or plant parts to determine if the amount of acetolactate and acetohydroxybutyrate present in (a) is at least about 15% of the amount of acetolactate and acetohydroxybutyrate present in (b).   
     
     
       11. The method according to claim 10 wherein the acetohydroxyacid synthase inhibitor is selected from the group consisting of an imidazolinone, a sulfonylurea, a sulfonamide and a pyrimidyloxybenzoate. 
     
     
       12. The method according to claim 11 wherein the imidazolinone is selected from the group consisting of 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid;   2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylic acid;   isopropylammonium 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate;   methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate; and   2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid.   
     
     
       13. The method according to claim 10 wherein the ketol-acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl)glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodium N-hydroxy-N-alkyloxamate and sodium N-hydroxy-N-aralkyloxamate. 
     
     
       14. The method according to claim 13 wherein the alkyl component of the N-hydroxy-N-alkyloxamate is selected from C 1  -C 6  alkyl and C 3  -C 7  cycloalkyl. 
     
     
       15. The method according to claim 14 wherein the alkyl component is isopropyl. 
     
     
       16. The method according to claim 13 wherein the aralkyl component is benzyl. 
     
     
       17. The method according to claim 10 wherein the plant parts are young rapidly growing tissue. 
     
     
       18. The method according to claim 10 wherein the amount of acetolactate and acetohydroxybutyrate present in the treated plant or plant part is determined by (a) extracting the acetolactate and acetohydroxybutyrate present in the plant or plant part into water;   (b) treating the water extract with a sulfuric acid solution;   (c) treating the acidified water extract solution from (b) with a 0.5% creatine solution and a 5% α-naphthol in sodium hydroxide solution; and   (d) measuring and comparing the color of the product of (c) to a known standard.   
     
     
       19. The method according to claim 10 wherein the plant or plant part is treated with a 1 μM to 1,000 μM solution of the acetohydroxyacid synthase inhibitor and a 1 μM to 1,000 μM solution of the ketol-acid reductoisomerase inhibitor. .Iadd. 
     
     
       20.  A method for determining whether a material to be tested is capable of inhibiting acetolactate synthesis in a given plant tissue sample containing living cells which comprises the steps of: (a) combining in an aqueous medium the plant tissue sample, an effective amount of the material, and an effective amount of an inhibitor of keto acid reductoisomerase, so that acetolactate will accumulate in the mixture unless the material inhibits acetolactate synthesis; and   (b) detecting accumulation of acetolactate..Iaddend..Iadd.21. The method according to claim 20 wherein the keto acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl)glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodium N-hydroxy-N-alkyloxamate and sodium N-hydroxy-N-aralkyloxamate..Iaddend..Iadd.22. The method according to claim 21 wherein the alkyl component of the N-hydroxy-N-alkyoxamate is selected from C 1  -C 6  alkyl and C 3  -C 7     
     
     
        cycloalkyl..Iaddend..Iadd.23.  The method according to claim 22 wherein the alkyl component is isopropyl..Iaddend..Iadd.24. The method according to claim 21 wherein the aralkyl component is benzyl..Iaddend..Iadd.25. The method according to claim 20 wherein the given plant tissue sample is selected from the group consisting of a monocotyledonous plant and a dicotyledonous plant..Iaddend..Iadd.26. The method according to claim 20 wherein the given plant tissue sample is young rapidly growing 
     
     
        tissue..Iaddend..Iadd.27.  The method according to claim 20 wherein step (b), detecting accumulation of acetolactate, comprises the steps of: (c) extracting the acetolactate present in the given plant tissue sample into water;   (d) treating the water extract with a sulfuric acid solution;   (e) treating the acidified water extract solution from (d) with a 0.5% creatine solution and a 5% α-naphthol in sodium hydroxide solution; and   (f) measuring and comparing the color of the product of (e) to a known standard..Iaddend..Iadd.28. The method according to claim 20 wherein the given plant tissue sample is treated with a 1 μM to 1,000 μM solution of the material and a 1 μM to 1,000 μM solution of the keto acid reductoisomerase inhibitor..Iaddend..Iadd.29. A method of claim 20 wherein step (b), detecting accumulation of acetolactate, comprises the steps of:   (c) allowing time for acetolactate to accumulate;   (d) rupturing the cells;   (e) acidifying said mixture to convert any accumulated acetolactate to acetoin, and   (f) colorimetrically detecting the presence of acetoin in the mixture..Iaddend..Iadd.30. The method of claim 29 wherein step (f) comprises the step of adding an effective amount of a compound containing the guanidino group, 1-naphthol, and base to the mixture..Iaddend..Iadd.31. The method of claim 30 wherein the compound   
     
     
        containing the guanidino group is creatine..Iaddend..Iadd.32.  A method for determining whether a given plant is resistant to a herbicide known to have inhibition of acetolactate synthase as its mode of action, which comprises: (a) combining in an aqueous medium a fresh sample of tissue from said plant, an effective amount of said herbicide, and an effective amount of a keto acid reductoisomerase inhibitor; and   (b) detecting the accumulation of acetolactate..Iaddend..Iadd.33. The method according to claim 32 wherein the herbicide is an acetohydroxyacid synthase inhibitor selected from the group consisting of an imidazolinone, a sulfonyl-urea, a sulfonamide and a pyrimidyloxybenzoate..Iaddend..Iadd.34. The method according to claim 33 wherein the imidazolinone is selected from the group consisting of   5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid;   2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylic acid;   isopropylammonium 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate;   methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate; and   2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid..Iaddend..Iadd.35. The method according to claim 32 wherein the keto acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl)glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodium N-hydroxy-N-alkyloxamate and sodium N-hydroxy-N-aralkyloxamate..Iaddend..Iadd.36. The method according to claim 35 wherein the alkyl component of the N-hydroxy-N-alkyloxamate is selected from C 1  -C 6  alkyl and C 3  -C 7  cycloalkyl..Iaddend..Iadd.37. The method according to claim 35 wherein the alkyl component is isopropyl..Iaddend..Iadd.38. The method according to claim 35 wherein the aralkyl component is benzyl..Iaddend..Iadd.39. The method according to claim 32 wherein the given plant tissue sample is   
     
     
        young rapidly growing tissue..Iaddend..Iadd.40.  The method according to claim 32 wherein step (b), detecting the accumulation of acetolactate, comprises the steps of: (c) extracting the acetolactate present in the given plant tissue sample into water;   (d) treating the water extract with a sulfuric acid solution;   (e) treating the acidified water extract solution from (d) with a 0.5% creatine solution and a 5% α-naphthol in sodium hydroxide solution; and   (f) measuring and comparing the color of the product of (e) to a known standard..Iaddend..Iadd.41. The method according to claim 32 wherein the given plant tissue sample is treated with a 1 μM to 1,000 μM solution of the acetohydroxyacid synthase inhibitor and 1 μM to 1,000 μM solution of the keto acid reductoisomerase inhibitor..Iaddend..Iadd.42. The method of claim 32 wherein said herbicide is known to have inhibition of acetolactate synthase as its mode of action..Iaddend..Iadd.43. The method of claim 32 wherein the herbicide is selected from the group consisting of flumetsulam, imazaquin, chlorsulfuron, sulfometuron-methyl, imazapyr, and imazethapyr..Iaddend..Iadd.44. The method of claim 32 wherein step (b), detecting the accumulation of acetolactate, comprises the steps of:   (c) rupturing the cells of said plant tissue sample;   (d) acidifying said mixture, formed by the combining step (a), to convert any accumulated acetolactate to acetoin; and   (e) adding effective amounts of a compound containing the guanidino group, 1-naphthol, and base to the mixture, so that the color of the resulting mixture indicates whether acetolactate synthesis was inhibited..Iaddend..Iadd.45. The method of claim 44 wherein the compound containing the guanidino group is creatine..Iaddend.