1.3%... :r .a. . t . b. i a. ‘1 Q .. 4i. .. 1. . x113, . :5: is: I . A )2. {1: 1:11... I»... 1! I: 5:3 :1 V v.. .1; z: s. z E i‘ufilfii... . .1 "r. nrflnvu IL .8 f‘. ..)U...a$.t: £9,322. at??? 4.5.... 13.3.. .2 . ‘ x . 1., 11.4,! 7 V. I a. .- 5.3.1,: 3:1...) ‘ in; 3...} a... tee: w if: E: y 2211-. RSLITY LIBRARIE llllllllllllllllllllllllHllllllllllllllllllllllllll 3 1293 01037 3307 This is to certify that the dissertation entitled INFLUENCE OF ANTIDO'I‘ES, MFO INHIBITORS, INSECTICIDES, AND CORN HYBRIDS ON ALS-INHIBITING HERBICIDE ACTIVITY presented by Chae-Soon Kwon has been accepted towards fulfillment of the requirements for Ph.D. degreein Cr0p and Soil Sciences Major professor Date/Wilda /8? /973 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State Untverstty PLACE ll RETURN BOX to move this checkout from your rooord. TO AVOID FINES rotum on or baton doto duo. DATE DUE DATE DUE DATE DUE - - L_Jl:]l___l ' { =L__= C—T—l—T MSU loAn Nflmottvo Action/Equal Opponuntty lmtltuton INFLUENCE OF ANTIDOTES, MFO INHIBITORS, INSECTICIDES, AND CORN HYBRIDS ON ALS-INHIBITING HERBICIDE ACTIVITY By CHAE-SOON KWON A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1993 ABSTRACT INFLUENCE or ANTIDOTES, MFO INHIBITORS, INSECTICIDES, AND CORN HYBRIDS 0N SULFONYLUREA HERBICIDE ACTIVITY by CHAE-SOON KWON The effects of antidotes, mixed function oxidase (MFO) inhibitors, insecticides, and corn hybrids on acetolactate synthase (ALS) inhibiting herbicide activity were evaluated in greenhouse and field studies. Cross-resistance of chlorsulfuron [2-chloro-H-[[(4-methoxy-6-l ,3 ,S-triazin-Z-yl) amino] carbonyl] benzenesulfonamide]—resistant kochia (Kochig mm (L.) Schrad.) was evaluated in greenhouse. Normal corn hybrids were sensitive to the interaction of sulfonylurea herbicides with terbufos [S-[[(1,1-dimethylethyl) thio] methyl] 0,0-diethyl phosphorodithioate] . Pioneer 3377 IR and Ciba 4393 RSC corn hybrids showed excellent tolerance to the interaction of AIS-inhibiting herbicides with terbufos regardless of the presence of tank-mixed piperonyl butoxide (PBO) [a-(2-(2- butoxyethoxy) ethoxy)-4,5-methyl enedixoy-Z-propyltoluene] , a mixed function oxidase inhibitor. ICI 8532 IT showed cross-resistance to the interaction of thifensulfuron [3-[[[[(4-methoxy-6-methyl-l ,3,5-triazin-2-yl) amino] carbonyl] amino] sulfonyl]-Z-thiophenecarboxylic acid] and imidazolinone herbicides with terbufos. The antidotes, CGA-154281 [4-(dichloro-acetyl)-3,4—dihydro—3-methyl- 2H-l ,4-benzoxazine] and NA [1,8-naphthalic anhydride], reduced corn injury from metolachlor [2-chloro-H—(2—ethyl—6—methylphenyl)-H-(2—methoxy-l-methylethyl) acetamide] , nicosulfuron [2-[[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] ~15], H—dimethyl-3-pyridinecarboxamide] and primisulfuron [2— [[[[[4,6-bis(difluoromethoxy)-2-pyrimidinyl] amino] carbonyl] amino] sulfonyl] benzoic acid] with terbufos treatments, respectively. The combination treatment of primisulfuron and terbufos did not affect herbicidal activity to wood species. PBO enhanced nicosulfuron activity on barnyardgrass (Echjnmhlga ems-galli (L.) Beauv.), velvetlcaf (Abutflgn 1111921113511 Medicus), and common lambsquarters (Chengpgdjum album L.), thifensulfuron activity on velvetlcaf and common lambsquarters. Also, butylate hydroxyanisole (BHA) [2, [3]-tert-butyl-4- hydroxyanisole] and PEG enhanced nicosulfuron and primisulfuron activities on common lambsquarters and green foxtail m viridis (L.) Beauv.). The enhancement of herbicide activity by 28 % UAN (urea ammonium nitrate) was herbicide, adjuvant, and weed specific. Efficacy of the nonionic adjuvants was herbicide and weed specific. The combination of thifensulfuron with PEG caused injury to Elgin ’87 soybean (Glycine mm; (L.) Merr.), but the W20-STS soybean was tolerant to this combination treatment. Combination of imazethapyr with PBO or BHA had no effect on the growth of Elgin ’87 soybean. The chlorsulfuron resistant-kochia biotype was resistant to six herbicides: Triflusulfuron, 2-[[[[[4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-l ,3 ,5 -triazin-2- yl] amino] carbanyll-amino] sulfonyl]-B-methylbenzoic acid, thifensulfuron, MON 12037, [methyl 3-chloro-5—(4,6-dimethoxypyrinudin-Z-ylcarbamoylsulfamoyl)- l- methylpyrazole—4~carboxylate] , imazamethabenz [(i)-2-[4,5-dihydro—4-methyl]-4— (l-methylethyl)-5-oxo-lH—imidazol-Z-yl]-4(and 5)-methylbenzoic acid(3:2)] , chlorsulfuron, and nicosulfuron. But, the resistant kochia biotype showed sensitivity similar to the susceptible biotype to three herbicides: metsulfuron [2- [[[[(4~methoxy-6-methyl—l ,3,5-triazin-2—yl) amino] carbonyl] amino] sulfonyl] benzoic acid], imazethapyr [2- [4,5 -dihydro—4-methyl-4—(l-methylethyl)-5 -oxo- lfl- imidazol-Z-yl] -5-ethyl-3 -pyridinecarboxylicacid] , andimazaquin [2- [4,5-dihydro-4- methyl-4—(l -methylethyl-5-oxo—lH—imidazol-Z-yl]-3-quinolinecarboxylic acid]. Addition of the PBO at 2 kg/ha to primisulfuron and thifensulfuron increased injury and reduced plant height of the chlorsulfuron resistant kochia biotype. iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Donald Penner, my major advisor, for his guidance, and counsel throughout this project. I would also like to thank Drs. James J. Kells, Bernard H. Zandstra, and Matthew J. Zabik for serving as members of my guidance committee, for their assistance with the research, and for their suggestions in the preparation of this manuscript. A special thanks is extended to Frank C. Roggenbuck for his valuable assistance with all aspects of my research and for his friendship during my study. I also express gratitude to Andrew Chomas for their assistance in field studies. I want to acknowledge the Korean Government for their financial support. Also, appreciation is offered to all weed science graduate students, other fellow graduate students, faculty and staff for their friendship and assistance. Finally, I thank my wife, Ogran Song, and my daughter, Haeji, and my parents whose support and patience have made this goal attainable. TABLE OF CONTENTS Page LIST OF TABLES ................................... x INTRODUCTION ................................... 1 CHAPTER 1. LITERATURE REVIEW Introduction ........................................ 4 Mode of action of sulfonylurea and imidazolinone herbicides 5 Metabolism study .................................. 9 Mechanisms for herbicide resistance ..................... 12 Herbicide resistance crops ........................... 18 Herbicide resistance weeds ........................... 21 Interaction of ALS inhibiting herbicides with organophosphate insecticidefl6 Metabolism of terbufos ............................. 30 Interaction of antioxidants with herbicides ................. 31 Action of antidotes ................................ 33 LITERATURE CITED ................................ 38 CHAPTER 2. THE INTERACTION OF INSECTICIDES WITH HERBICIDE ACTIVITY Abstract ......................................... 51 Introduction ....................................... 5 3 Materials and methods ................................ 56 General greenhouse procedure .......................... 56 Corn hybrid response ............................... 56 Weed response ................................... 57 vi Data presentation and statistical analysis .................... 58 Results and discussion ................................ 59 Interaction of preemergence herbicides with insecticides .......... 59 Interaction of sulfonylurea herbicides with insecticides ........... 59 Antidote effect on the interaction of herbicides with insecticides ..... 6O Interaction effect of primisulfuron and terbufos on the herbicidal activity on weed species ............................ 61 LITERATURE CITED 63 CHAPTER 3. THE EFFECT OF MIXED FUNCTION OXIDASE INHIBITORS ON SULFONYLUREA HERBICIDE ACTIVITY. Abstract ....................................... 72 Introduction ..................................... 74 Materials and methods .............................. 76 Plant materials ................................. 76 MPG inhibitor ................................. 76 Adjuvants studies ................................ 77 Statistical analysis ............................... 78 Results and discussion .............................. 79 Effects of MFO inhibitors on sulfonylurea herbicide activity ..... 79 Effects of PEG rates on sulfonylurea herbicide activity ......... 81 Effects of adjuvants, PBO rates, and 28 % UAN on the phytotoxicity of sulfonylurea herbicides ........ - .................... 81 N icosulfuron ................................. 81 Primisulfuron ................................ 83 Thifensulfuron ............................... 85 LITERATURE CITED .............................. 87 CHAPTER 4. THE EFFECT OF MIXED FUNCTION OXIDASE vii INHIBITORS ON CROP SAFETY TO ALS INHIBITING HERBICIDES Abstract ....................................... 106 Introduction ..................................... 109 Materials and methods .............................. 112 General greenhouse procedure ........................ 112 Corn hybrids .................................. 112 Soybean varieties ................................ 113 Chemical treatments .............................. 113 Field study ............. ‘ ....................... 114 Results and discussion .............................. 116 Field experiment ................................ 119 First year, 1992 ............................... 119 Second year, 1993 ............................. 120 Soybean study .................................. 121 LITERATURE CITED .............................. 123 CHAPTER 5. RESPONSE OF A CHLORSULFURON-RESISTANT BIOTYPE OF W m TO ALS INHIBITOR HERBICIDES AND PIPERONYL BUTOXIDE. Abstract ....................................... 140 Introduction ..................................... 142 Materials and methods .............................. 144 Plant materials ................................. 144 Chemical treatments .............................. 144 Data analysis .................................. 145 Results and discussion .............................. 146 Cross—resistance study ............................. 146 The effect of P80 with sulfonylurea herbicides on the growth of chlorsulfuron- viii LIST OF TABLES CHAPTER 2. The Interaction of Insecticides with Herbicide Activity. Table l. The combination effect of acetanilide herbicides with insecticides on the shoot height of two corn hybrids in the greenhouse 3 WAT. . . 65 Table 2. The combination effect of acetanilide herbicides with insecticides on the visual injury of two corn hybrids in the greenhouse 3 WAT. . . . . 66 Table 3 . The interaction of corn hybrids, terbufos insecticide, and several POST herbicides on corn shoot height in the greenhouse 2 WAT. ..... 67 Table 4. The interaction of terbufos insecticide with several POST herbicides on visual injury to the metolachlor sensitive Northrup King 9283 hybrid in the greenhouse 2 WAT. ............................. 68 Table 5 . The effect of NA on the interaction between herbicides and terbufos on corn plant height in the greenhouse 2 WAT. .............. 69 Table 6. The effect of NA on the interaction of sulfonylurea herbicides with terbufos on corn injury in the greenhouse 2 WAT. .......... 70 Table 7. The interaction effect on the primisulfuron herbicidal activity to three weed species in the greenhouse at 2 WAT. ............... 71 CHAPTER 3. The Effect of Mixed Function Oxidase Inhibitors on Sulfonylurea Herbicides Activity. Table l. The effect of PEG on primisulfuron and nicosulfuron activity to barnyardgrass in the greenhouse 2 WAT. ................ 89 Table 2. The effect of PEG and BHA on primisulfuron and nicosulfuron activity to common lambsquarters in the greenhouse 2 WAT. ........ 90 Table 3 . The effect of PEG and BHA on primisulfuron and nicosulfuron activity to green foxtail in the green house 2 WAT. .............. 91 X Table 4. The effect of PBO on the herbicidal activity of thifensulfuron on the plant height of the two grass weed species in the greenhouse 2 WAT. . . 92 Table 5 . The effect of PBO on the herbicidal activity of thifensulfuron on the growth of velvetlcaf and common lambsquarters in the greenhouse 2 WAT. ................. 93 Table 6. The effect of various PBO rates on nicosulfuron and primisulfuron activity on common lambsquarters in the greenhouse 2 WAT. . . . 94 Table 7. The effect of various PBO rates on nicosulfuron and primisulfuron activity on green foxtail in the greenhouse 2 WAT. .......... 95 Table 8. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of common lambsquarters in the greenhouse 2 WAT. 96 Table 9. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of velvetlcaf in the greenhouse 2 WAT. ........ 97 Table 10. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of barnyardgrass in the greenhouse 2 WAT. . . . . 98 Table 11. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of giant foxtail in the greenhouse 2 WAT. ...... 99 Table 12. The effect of various PBO rates and adjuvants on primisulfuron activity on the growth of common lambsquarters in the greenhouse 2 WAT. 100 Table 13 . The effect of various PBO rates and adjuvants on primisulfuron activity on the growth of velvetleaf in the greenhouse 2 WAT. ....... 101 Table 14. The effect of various PBO rates and adjuvants on primisulfuron activity on the plant height of barnyardgrass in the greenhouse 2 WAT. . . 102 Table 15. The effect of various PBO rates and adjuvants on primisulfuron activity on the growth of giant foxtail in the greenhouse 2 WAT. ...... 103 Table 16. The effect of various PBO rates and adjuvants on thifensulfuron activity on the growth of common lambsquarters in the greenhouse 2 WAT. 104 Table 17. The effect of various PBO rates and adjuvants on thifensulfuron activity on the growth of velvetlcaf in the greenhouse 2 WAT. ....... 105 CHAPTER 4. The Effect of Mixed Function Oxidase Inhibitors on Crop xi Safety to ALS Inhibiting Herbicides. Table 1. The effect of PBO on the response of two corn hybrids to nicosulfuron and primisulfuron in the greenhouse 2 WAT. ............. 125 Table 2. The effect of MFO inhibitors on the response of imazethapyr resistance Pioneer 3377 IR corn hybrids to sulfonylurea herbicides in the greenhouse 2WAT ................. 126 Table 3. The effect of PBO and terbufos on the response of imazethapyr resistant and sensitive corn hybrids to nicosulfuron and primisulfuron herbicides in the greenhouse 2 WAT. ...... . ..................... 127 Table 4. The effect of PBO and terbufos on the responses of imazethapyr resistant and sensitive corn hybrids to nicosulfuron and primisulfuron herbicides in the greenhouse 2 WAT. ........................... 128 Table 5. The effect of PBO and MON 13900 on plant height of imazethapyr resistant and sensitive corn hybrids treated with MON 12000 in the greenhouse 2 WAT. ............................. 129 Table 6. The effects of PBO, terbufos, and the antidote R-29148 on plant height of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides at 2 WAT in the field study in 1992. ...... 130 Table 7. The effects of PBO, terbufos, and the antidote R—29l48 on visual injury of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides at 2 WAT in the field study in 1992. ...... 131 Table 8. The effects of PBO, terbufos, and the antidote R-29148 on plant height of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides at 6 WAT in the field study in 1992. ...... 132 Table 9. The effects of PBO, terbufos, and the antidote R-29l48 on visual injury of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides at 6 WAT in the field study in 1992. ...... 133 Table 10. The effect of PBO, terbufos on plant height of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides 2 WAT in the field study in 1993. ............................. 134 Table 11. The effect of PBO, terbufos on visual injury of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides 2 WAT in the field study in 1993. ......................... 135 Table 12. The effect of PBO, terbufos on plant height of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides 4 WAT in the field study in 1993. ............................. 136 Table 13. The effect of PBO, terbufos on visual injury of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides 4 WAT in the field study in 1993. ......................... 137 Table 14. The effect of PBO on thifensulfuron tolerance by two soybean varieties in the greenhouse at 2 WAT. ....................... 138 Table 15. The effects of PBO and BHA on imazethapyr tolerance of Elgin ’87 in the greenhouse at 2 WAT .......................... 139 CHAPTER 5. Response of a Chlorsulfuron-Resistant Biotype of Mg m to ALS Inhibitor Herbicides and Piperonyl Butoxide. Table 1. The responses of chlorsulfuron-resistant and sensitive biotypes of kochia to the ALS inhibiting herbicides in the greenhouse study. ...... 151 Table 2. The responses of chlorsulfuron-resistant and sensitive biotypes of kochia to the interaction of PBO and sulfonylurea herbicides in the greenhouse 2 WAT ....................................... 152 xiii INTRODUCTION The sulfonylurea and imidazolinone herbicides have high specific activity and have been in commercial use since 1982. Sulfonylurea herbicides reduced the field application rates of herbicide to less than 50 g a.i.lha. Sulfonylurea and imidazolinone herbicides are used to control a broad spectrum of weed species in a variety of crops including corn (Zea mays L.), soybeans (Glycine mm: (L.) Merr.), and small grains. The selective action of these herbicides between crop and weed plants can be attributed to metabolism of the herbicides to inactive products in the various crop species. The mode of action of these compounds has been well established as the inhibition of acetolactate synthase (ALS), one of the enzymes important for the synthesis of branched amino acids. Sulfonylurea herbicides are initially metabolized by a cytochrome P450—dependent monooxygenase system located in the microsomal fraction. This enzyme system supports the hydroxylation of primisulfuron [2-[[[[4,6-bis(difluoromethoxy)-2- pyrimidinyl] amino] carbonyl] amino] sulfonyl] benzoic acid] and nicosulfuron [2- [[[[(4,6-dimethoxy—2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] 4}], N- dimethyl-3-pyridinecarboxamide] at the phenyl ring and at the pyrimidine ring. The recent introduction of the ALS inhibiting-herbicides have stimulated research on the interactions of herbicides with insecticides. Combinations of the 2 organophosphate insecticides, especially terbufos, and primisulfuron or nicosulfuron were shown to interact synergistically resulting in corn foliar and root injury, plant stand reduction, and corn grain yield losses. The synergistic interaction was explained on the basis that the insecticide reduced metabolism and increased absorption of herbicides. Several researchers have tried to find methods to protect crops from the interaction of ALS-inhibiting herbicides and corn insecticides. They found that antidotes or tank-mixing 2,4-D with sulfonylurea herbicides reduced corn injury. Mixed function oxidase (MFO) inhibitors, or antioxidants, are possible herbicide synergist. Piperonyl butoxide [oz-(2-(2-butoxyethoxy) ethoxy)-4,5—methyl enedioxy-2-propyltoluene] increased the activity of EPTC [S-ethyl dipropyl carbamothioate], atrazine [6-chloro—N—ethyl-N’(l-methylethyl)-l ,3,5—triazine-2,4— diamine], bentazon [3-(1-methylethyl)-(IH)-2, 1 ,3-benzothiadiazin—4(3H)-one 2,2- dioxide] and oxadiazon [3-(2,4—dichloro-5-(1-methylethoxy) phenyl)-5-(1,1- dimethylethyl)-1,3 ,4-oxadiazol-2-(3tD-one] to corn. MFO inhibitors inhibit mixed- function oxidase systems which are possibly involved in the metabolism of ALS- inhibiting herbicides in corn. The objectives of this study were 1) to identify interactions between com herbicides and corn insecticides, 2) to evaluate the effect of MFO inhibitors on the activity of several sulfonylurea herbicides, 3) to identify effective adjuvants for the sulfonylurea herbicides, 4) to determine the effect of the MFO inhibitors on corn 3 safety, 5) to evaluate the cross-resistance of a chlorsulfuron resistant kochia (Kochia W (L.) Schrad.) biotype to various ALS-inhibiting herbicides, and 6) to evaluate potential synergistic effects of PBO with sulfonylurea herbicides to a chlorsulfuron-resistant kochia biotype. CHAPTER 1 LITERATURE REVIEW INTRODUCTION The sulfonylurea herbicides are highly active and have been in commercial use since 1982. Among various classes of herbicides used today, sulfonylureas rank at the top in their specific activity. Sulfonylurea herbicides were discovered by Levitt in 1976 (1,2). Sulfonylurea herbicides reduced the field application rates from 0.5 kg a.i.lha or even greater to less than 50 g a.i.lha. Structure of a typical sulfonylurea is characterized by presence of a sulfonylurea ”bridge" connecting two rings. The second class of acetolactate synthase (ALS)-inhibiting herbicides are the imidazolinones. This class of chemiStry is characterized by an imidazolinone ring bonded to an aromatic ring at the 2 position. Sulfonylurea and imidazolinone herbicides are used to. effectively control a broad spectrum of weed species. in a variety of crops including corn (Ea mays L.), soybeans (Glycinc max (L.) Merr.), and small grains. The selective action of these herbicides between crop and weed plants can be attributed to metabolism of the herbicides to inactive products in the various crop species. 4 5 Modes of Action of Sulfonylurea and Imidazolinone Herbicides For the first time in the history of commercial herbicides, the mode of action of the new herbicide was known before the herbicides were widely commercialized. The new classes of herbicide chemistry, sulfonylureas and imidazolinones, were commercialized by separate companies. Two lines of investigation came together to prove that acetolactate synthase (ALS) is the site of action of sulfonylurea herbicides. Experiments by LaRossa and Schloss (67) showed an inhibiting effect of sulfonylureas on growth of bacteria, concurrently, herbicide-resistant mutants of tobacco (Nicctiana tabacum L.) were selected in culture by Chaleff and Ray (19). Other researchers performed biochemical studies showing ALS inhibition by sulfonylureas in susceptible plants but not in selected resistant plants (18). Ultimate proof for this site of action came from studies on regenerated herbicide resistant tobacco, in which breeding experiments showed cosegregation of the herbicide resistance trait and herbicide insensitive enzyme (18,19). Anderson and Shaner (4,108) performed key experiments that lead to discovery of the mode of action of imidazolinones. Anderson and Hibberd (4) found a decline in levels of valine, leucine, and isoleucine in corn cell cultures treated with imazapyr [(i)-2-[4,5- dihydro-4-methyl—4—(l-methylethyl)-5-oxo-lH—imidazol—Z-yll-B-pyridinecarboxylic acid]. Supplementation of these amino acids reversed the growth inhibiting effects of imazapyr. Similar experiments were performed by Shaner (108) using corn root tips. Recently, it has been discovered that ALS is the target of several structurally diverse herbicide compounds such as sulfonylureas, imidazolinones, and triazolpyrimidine sulfonanilides (63,67,76,94,108,113). ALS plays an important role in controlling carbon flow to the amino acids by feedback regulation (73). The mode of action of these compounds has been well established as the inhibition of acetolactate synthase (ALS), one of the enzymes important for the synthesis of branched amino acids. ALS (also known as acetohydroxy acid synthase, AHAS) is the first enzyme in the biosynthesis of Val, Leu, and Ile. This enzyme catalyzes the condensation of an acetoaldehyde moiety derived from pyruvate either with another molecular pyruvate to form 2-acetolactate or with 2-ketobutyrate to form 2—aceto-2-hydroxybutyrate. With microorganisms the enzyme and the inhibitory mode of action have been well studied. In EachccicLia chi and Salmnella tychjmmjnm, three isozymes (ALS I, II, and 111), each of them encoded by a particular gene, differing in substrate preference and feedback regulation have been identified, purified and well characterized (32,104). Most of the enzymological and kinetic studies on inhibition of ALS by herbicides have been carried out with the ALS II from 5. mm, which resembles the plant enzyme with regard to its sensitivity to herbicides, but is different in subunit composition and feedback regulation (94,105). Duner et a1. (30) reported that only partial agreement between plant ALS and bacterial ALS II is evident, while both the time-dependent 7 inhibition and the slow disassociation of the enzyme-inhibitor complex are in accordance, there are decisive differences with regard to reversal of inhibition, i.e. recovery of enzyme activity. LaRossa and Smulski (66) using enzymological analysis found that ALS I activity derived from S. typhjmmjnm and E. ccli species was resistant to the herbicide. Their report demonstrated that the ALS 1 was insensitive to sulfometuron methyl [2-[[[[(4,6-dimethyl-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] benzoic acid]. In contrast, activity of 5. 9mm ALS II and E. chi ALS III was inhibited by sulfometuron methyl (66). An interpretation is that sulfometuron methyl inhibits S nyhimnnmm ALS II but not ALS I (67). If this interpretation is correct, growth inhibition is only manifested when ALS I activity is blocked or absent. ALS I may simply represent an isozyme resistant to the herbicide. Apparently, there are different physical and catalytic properties of plant and bacterial ALS, emphasizing the need for further studies on the plant enzyme (30). ALS is subject to feedback inhibition by the end products Val and Lou in both micro-organisms and in plants (39,74,118). Obviously, the branched-chain amino acid pools eventually decrease after addition of ALS inhibitor herbicides to plants (4). Each herbicide also rapidly increases the intracellular a-ketobutyrate concentration causing metabolic imbalances. LaRossa et al. (68) proposed that these a-ketobutylate-mediated imbalances contribute to the potency of herbicides interacting with ALS. Dumer et a1. (29) tried experiments to Show whether active 8 fractions of barley (chlcum mlgarc L.) ALS represented true isozymes or multiple polymeric forms of a basic ALS subunit. Their data showed that the two enzymically active forms Were not isozymes but were different oligomeric species or aggregates of the basic subunit of ALS. These different ALS species exhibit little difference in feedback inhibition by valine, leucine, and isoleucine or inhibition by the sulfonylurea herbicide chlorsulfuron [2-chloro-N—[[(4—methoxy-6- methyl-1,3,5-triazin-2-yl) amino] carbonyl] benzenesulfonamide] . The lack of cross-resistance in some of the variants has been used to support the hypothesis that there are two separable binding domains for sulfonylureas and imidazolinones on the ALS molecule (103). Differences in sensitivity to nicosulfuron [2-[[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl]-N,N—dimethyl-3-pyridinecarboxamide] in two corn hybrids resistant to imazethapyr [2-[4,5—dihydro—4-methy1-4-( 1 -methylethyl)-5- oxo-1H—imidazol—Z-yl]-5-ethyl—3-py1idinecarboxylic acid] apparently resulted from the differential sensitivity of the ALS target enzyme. These results suggest that the sites of action of two different classes of herbicides, the imidazolinones and the sulfonylureas, may be overlapping but not identical (109). The sulfonylurea herbicide, chlorsulfuron, and the imidazolinone herbicide, imazaquin [2- [4,5 - dihydro-4-methyl-4-(l-methylethyl)-5-oxo-lH-imidazol—Z—yll-3—quinolinecarboxylic acid], were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified ALS from barley with respect to pyruvate (30). Schloss et al. (105) 9 proposed that the herbicide-specific site of ALS is an evolutionary vestige of the quinonc binding site of pyruvate oxidase. Consistent with this proposal, the ubiquinone homologues Q0 and Q1 (potent inhibitors of ALS), and Q0 (an imidazolinone herbicide), and a sulphonanilide herbicide, each compete with a radiolabelled sulfonylurea herbicide for a common binding site on ALS. ALS from several of these variants has also been found to be altered in feedback sensitivity to Val, Lou, and He (38). The sulfonylurea herbicides have been found to act as slow-binding inhibitors of the enzyme (67). Sulfonylurea herbicides have been shown to reduce pollen viability and influence other developmental processes that may account for decreased seed production by otherwise apparently healthy dyer’s woad (Isatis tinctcria L.) plants treated with these materials. Pre-anthesis stage treatment with 3 g a.i.lha metsulfuron [2-[[[[(4—methoxy-6-methyl-l ,3,5-triazin-2-yl) amino] carbonyl] amino] sulfonyl] benzoic acid] was enough to prevent dyer’s woad fruit formation and seed production (5). Metabolism Study Selectivity of ALS-inhibitor herbicides in cereal crops, corn, and soybean has been correlated with the ability of these plants to rapidly convert sulfonylureas to herbicidally inactive products. In contrast, susceptible weeds metabolize these herbicides much more slowly (13). Sulfonylurea tolerance of some weed species 10 has been suggested to result from rapid herbicide inactivation (13). Most tolerant plant species initially metabolize sulfonylurea herbicides by introduction of hydroxyl groups, frequently followed by carbohydrate conjugation (13). There have been several reports implicating cytochrome P—450 monooxygenase in oxidative herbicide metabolism in plants. Cytochrome P-450 is a ubiquitous family of hemoproteins, also referred to as mixed function oxidase; which catalyze a number of N AD(P)H-dependent monooxygenase reactions i.e. , the incorporation of a single atom of oxygen from molecular 02 into organic substances. P-450 are the terminal oxidases of a large number of biotransformations. The enzymic reactions include metabolism of steroids, fatty acids, , prostaglandins, leukotrienes, biogenic amines, pheromones, drugs, plant metabolites, and numerous other substances, including mutagens (82). The metabolic pathways in which these microsomal P—450 proteins participate are primarily involved in the biosynthesis and degradation of cellular components or the detoxification of xenobiotics. Some of the chemical reactions catalyzed by plant P-450 include aryl and alkyl hydroxylation and O— and N-dealkylation. Cytochrome P-450—mediated reactions are vital to the detoxification and selective phytoactivity of many herbicides (123). There is evidence that cytochrome P-450 reactions are responsible for the detoxification of certain herbicides in wheat (Ifincnm acctimm L.) (42,91,108), corn (67,74,118), and other crop species (94) and that these reactions can enable 11 certain crop species to tolerate a herbicide. The soil bacterium Sn-cptgmyccs mimics ATCC 11796 is capable of metabolizing several sulfonylureas. This metabolism is mediated by two inducible cytochrOme P-450 monooxygenases (96) . Each cytochrome P-450 appears to obtain reducing equivalents from N AD(P)H through a reductase and ferredoxin (85); the ferredoxins being the ultimate electron donors for the cytochrome P-450. In plants, the induction of cytochrome P-450—dependent metabolism of xenobiotics has been reported (42,74,91,108,118), but there is no evidence that these reactions are catalyzed by enzymes exhibiting broad and overlapping substrate specificity. To the contrary, the approximately 30 cytochrome P—450 monooxygenase reactions already described in plants showed very narrow substrate specificity (39). Zimmerlin (124) has recently characterized a xenobiotic-inducible cytochrome P-450 from wheat microsomes that catalyzes the aryl hydroxylation of diclofop [(;t-_)-2—[4—(2,4—dichlorophenoxy) phenoxy]propanoic acid], a herbicide selective for wheat crop. Also, Zimmerlin (125) reported that wheat (cv Etoile de Choisy) microsomes catalyzed the cytochrome P-450-dependent oxidation of the herbicide diclofop to three hydroxy-diclofop isomers. Hydroxylation was predominant at carbon 4, with migration of chlorine to carbon 5 (67%) and carbon 3 (25 96). Fonne-Pfister et al. (34) found from microsomal corn seedlings that primisulfuron [2-[[[[[4,6-bis(difluoromethoxy)-2-pyrimidinyl] amino] carbonyl] 12 amino] sulfonyl] benzoic acid] is initially metabolized by a cytochrome P450- dependent monooxygenase system located in the microsomal fraction. This enzyme system supported the in yitm hydroxylation of primisulfuron at the phenyl ring and at the pyrimidine ring, respectively. Corbin et al. (23) suggested that primisulfuron metabolism in excised shoots and microsomal preparations of corn was mediated by cytochrome P450 and that enhanced metabolism induced by an naphthalic anhydride (NA) seed-treatment can overcome the synergistic interaction imposed by terbufos LS-[(l,1-dimethylethyl) thio] methyl Q, Q-diethyl phosphorodithioate] on the metabolism of primisulfuron. Microsomes isolated from 3-day-old etiolated corn (Pioneer 3343 IR) shoots convert nicosulfuron to a single polar metabolite. This metabolite is hypothesized to be the 5- hydroxypyrimidinyl derivative of nicosulfuron found in intact corn plants (37). Formation of the nicosulfuron metabolite by the microsomes is NADPH dependent (9). Barrett (9) suggested that the nicosulfuron metabolism in the microsomal preparations is due to the activity of cytochrome P—450 mixed function oxidase. Mechanisms For Herbicide Resistance Among weeds, triazine resistance was first reported in 1970 (98). Subsequently, resistance to several other herbicides in addition to the triazines have occurred in weeds (8,40). A large number of herbicide-resistant mutants have been isolated and characterized from a wide range of organisms such as bacteria, l3 yeast, and lower and higher plants (65). There are four hypotheses to explain the herbicide resistant mechanisms. 1. Compartmentation of the herbicide preventing this herbicide from reaching the target site. Fuerst et al. (37) showed that paraquat [1, l’-dimethyl- 4,4’—bipyridinium ion] resistance in conyza (Gcnyza bcnancnsis (L.) Cronq.) was related to compartmentation of the paraquat in the resistant biotype. They fed ["C] paraquat through the petiole, and found that [“C] paraquat distributed uniformly in the leaves of the susceptible biotype, but localized in the proximity of vascular tissue, and regions of the lower petiole, in the resistant biotype. From the autoradiograms, paraquat was compartmentalized at the cellular level and was excluded from the active site in the chloroplasts. They suggested that sprayed- paraquat would penetrate the cuticle of leaf but then become rapidly sequestered in leaf mesophyll tissue before reaching the active site in the chloroplast. Resistance to aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CI-ID) herbicides in some populations of annual ryegrass (Lolm ngidnm Gaudin) from Australia and wild oat (Aycna fatna L.) from Canada is correlated with the ability of plants to sequester herbicide at the sub-cellular level (60). The various metabolites of most herbicides include conjugates of either the parent chemical or a phase I metabolite of parent chemical. Conjugation causes physical and chemical compartmentation of herbicides and may be of major importance for the sequestration of phytotoxic chemicals out of the cytoplasm (7). l4 2. Enhanced metabolism of the herbicide. Burnet et al. (15) reported that the resistant rigid ryegrass detoxified simazine [6-chloro-I_\I,N’-diethyl-l ,3,5- triazine-2,4-diamine] , chlortoluron and metribuzin [4-amino-6-(1,1- dimethylethyl)-3-(methylthio)-l ,2,4-t1iazin-5(4I:D-one] faster than a control susceptible. The observation that resistance is reduced if metabolism is suppressed indicates that a major mechanism of resistance and cross—resistance to these herbicides is enhanced metabolism (15). Harms et al. (46) reported that the fate of l‘C-labeled primisulfuron in corn seedling tissues of inbred 4C0 and the hybrid, 4C0 x 4N5 indicated rapid metabolism with a half-er of approximately 3 h, but over 24 h in herbicide-sensitive inbred 4N5. This suggested that the observed primisulfuron tolerance of corn was probably due to a specific metabolic capacity rather than to herbicide-insensitive form of ALS (17) . The differential tolerance of bahiagrass (Paanalnm nctanim Fluegge) and centipedegrass (Emcmmhlqa cnhjncojdcs (Munro) Hack.) to sulfometuron appears to be based on more rapid metabolism of the herbicide in centipedegrass than in bahiagrass (6). Herbicide selection pressure can lead to the mini-evolution of weed biotypes with enhanced MFO capacity to degrade herbicides, and therefore, resistant to herbicides such as chlortoluron, chlorsulfuron or diclofop-methyl (88). 3. Change the characteristics of cell membranes. The mechanism of paraquat resistance in wall barley (chxdcnm glancnm Steud.) is related to an alteration in the membrane transport properties of paraquat in resistant plants (47). 15 Diclofop rapidly depolarized the cell membrane potential in peeled coleoptile sections, with no difference between the R and S biotypes of wild oat. However, when diclofop was removed from the treatment solution, the electrogenic potential remained collapsed in the susceptible cells, but recovered in the resistant cells. In whole plant experiments, acidification of the external medium continued in the presence of diclofop in the R biotype, but was slowly arrested in the S biotype. So, Hall et al. (45) suggested that the difference in the effect of diclofop on transmembrane proton flux in the R and S biotypes may be involved in conferring resistance to the R biotype. 4. Alterations in the target site of the herbicide. Thus far, weeds with alterations in the target site of the herbicide account for the majority of the herbicide-resistant weed problem, especially for sulfonylurea herbicide-resistant weeds. WeStwood and Weller (121) supported a hypothesis that glyphosate [N- (phosphonomethyl) glycine] tolerance in field bindweed (Gcnyolynlua mamas L.) may be due in part to a greater level of activity in the shikimate pathway. This may lead to an increased ability of the tolerant biotype to respond to glyphosate challenge as reflected by EPSP synthase induction patterns. Peniuk et al. (86) reported that the two wild mustard (Sinapic arycnsis L.) biotypes showed similar patterns of adsorption, translocation and exudation following application of ring l“C-labelled 2,4—D [(2,4-dichlorophenoxy) acetic acid] and dicamba [3,6—dichloro- 2—methoxybenzoic acid] to plants grown in a hydroponic system. Furthermore, l6 reverse-phase HPLC indicated that there was no difference in the pattern or extent of metabolism of 2,4-D and dicamba between the resistant and susceptible biotypes. They suggest that sensitivity differences between the two biotypes of wild mustard to 2,4~D and dicamba may be the result of differential sensitivity at the target site(s) of action for auxinic herbicides. I-Iirshberg and McIntosh (57) found that the photosynthetic electron transport in triazine-resistant weeds was 1000-fold less sensitive to symmetrical triazines compared to wild—type chloroplasts. Specific knowledge about the alterations in kinetics of ALS resistant to herbicides is very limited. Rathinasabapathi et al. (92,93) reported that ALS from sulfonylurea—resistant and imidazolinone-resistant variants of Danna inncztia Mill. exhibited different degrees of cross-resistance to sulfonylureas and imidazolinones. The lack of cross-resistance in some of the variants has been used to support the hypothesis that there are two separate binding domains for sulfonylureas and imidazolinones on the ALS molecule (102). A number of ALS mutants have been described which are selectively resistant to sulfonylureas (54) and imidazolinones (55,103). To understand the molecular basis of imidazolinone resistance, Sathasivan et al. (100) isolated the ALS gene from an imazapyr- resistant mutant GH90 of mahidcnsja thaliana (L.) Heynh. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. 17 They found that the mutant ALS gene from GH90 conferred imazapyr resistance in transgenic plants. This is the. first report of the molecular basis of imidazolinone resistance in plants (100). Resistance to both the sulfonylureas and imidazolinones has been shown in haploid Danna imcm’a Mill. lines (102). Since these organisms contain single loci for ALS, multiple resistance may result from independent mutations within the ALS enzyme, each responsible for selective resistance to a specific herbicide class, or from single mutations responsible for cross resistance. One possibility is that each herbicide class interacts with the ALS macromolecule at separate sites. In yeast, at least ten independent sites within highly conserved regions of ALS have been mutated to yield sulfonylurea resistance (70). Hattori et al. (50) demonstrated that multiple-resistance phenotypes can be achieved through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides. ALS from several of these variants also had altered feedback sensitivity to Val, Lou, and Ile (50). Rathinasabapathi et al. (91) suggested that the herbicide resistance mutation somehow altered pyruvate binding to the ALS molecule. This alteration could also have physiological implications, for example, by resulting in less efficient synthesis of branched-chain amino acids in y'm. Nearly all plant mutants resistant to the imidazolinone, sulfonylurea, or triazolopyrimidine herbicides isolated to date have been shown to have numerous 18 similarities: a single semidominant gene conferring resistance, herbicide-resistant ALS enzyme activity, and a direct correlation between the in yiyc, whole plant cross-resistance spectrum, and the in m, ALS activity cross-resistance spectrum. Herbicide Resistance Crops In recent years considerable research in the private and public sectors has been directed toward introducing herbicide “resistance into normally susceptible crop species. Potential benefits of developing herbicide resistant crops include, an increased margin of safety, reduced risk of crop damage from residual herbicides from rotational crops, and introduction of new herbicides for use on normally susceptible crops. The advantage from the use of crop plants with herbicide resistance is not only to increase Crop yield but also to increase quality of that product. ’ Triazine resistant canola (Brassjca napns L.) was the first herbicide resistant crop developed by backcrossing. This procedure used triazine rosistant bird’s rape (Bmaica camncsnja L.) (12). Resistant cultivars were unaffected by atrazine [6- chloro—N-ethyl-N" -( l -methylethyl)-1 ,3 ,5-triazine-2,4-diamine] or cyanazine [2-[[4- chloro—6-(ethylamino)-1,3 ,5-triazine-2-yl] amino] -2-methylpropanenitrile] , and metribuzin [4-amino—6—(1,1-dimethylethyl)-3-(methylthio)-l ,2,4—triazin-5(4H)-one]. However, the crop yield was reduced 20 % to 30% compared with reciprocal susceptible cultivars (11). Researchers at Monsanto have successfully transferred 19 genes conferring a degree of resistance to glyphosate into tomato W W L.) (61), and other crops. Newhouse et al. (83) studied three corn lines resistant to imidazolinone herbicides. For all three lines, resistance was inherited as a single semidominant allele for each of the three independent mutations. All resistant selections have herbicide-resistant forms of ALS. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the ALS activities of the selections (83). Mukaida et al. (79) reported that the ALS enzyme inhibition parallels whole plant injury produced with imazethapyr [2-[4,5-dihydro- 4-(1-methylethyl)—5-oxo-lH-imidazol-2-yl]-5-ethyl-3—pyridinecarboxylic acid] and chlorimuron—ethyl [2-[[[[(4-chloro-6-m’ethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyllbenzoic acid] and suggests that the mechanism for resistance was attributable to differential sensitivity at the target site. Pioneer 3343 IR corn is cross-resistant. to imazethapyr and chlorimuron—ethyl at the whole plant and enzyme levels, while ICI 8532 IT corn is tolerant only to imazethapyr. Also, Pioneer 3343 IR showed resistance to interactions of sulfonylurea herbicides with terbufos (109) with/ without piperonyl butoxide (PBO) [5-[[2-(2-butoxyethoxy) ethoxy] methyl] -6-propyl-1 ,3-benzodioxole] in the field experiment, but ICI 8532 IT did not. Primisulfuron metabolism occurred more rapidly in Pioneer 3343 IR than in the other normal corn hybrids (23). ICI 8532 IT was resistant to imazethapyr regardless of rate, application method, or presence of terbufos at the 20 Illinois locations, but ICI 8532 showed some injury (10,22). A sulfonylurea resistant soybean line (106) and imidazolinone resistant corn have been developed using a Chemical mutagen agent, such as ethyl methane sulfonate (EMS). Resistance in both species was due to an altered ALS binding site. Also, Moseley et al. (78) reported that the resistance of ‘W-20’ soybeans to chlorimuron was due to an altered target site. A chlorsulfuron resistant sugarbeet (Eta yulgans L.) line was selected from cell suspension cultures by Saunders et al. (101). Chlorsulfuron resistant sugarbeet was highly cross-resistant to other sulfonylurea herbicides including. chlorimuron, thifensulfuron [3-[[[[(4»methoxy—6—methyl-1,3,5—triazin-2-y1) amino] carbonyl] amino] sulfonyl]-Z-thiophenecarboxylic acid], and primisulfuron applied at or exceeding field use rates (48). Hart et al. (49) showed that the sulfonylurea resistance in this sugarbeet was inherited in a semi-dominant fashion and that a 2- to 4-fold increase in resistance to primisulfuron and thifensulfuron was observed for homozygous resistant sugarbeet compared to heterozygous resistant sugarbeet. D’Halluin et al. (26) have developed an Agrchactcrjnm mediated transformation procedure for sugarbeet. Sugarbeet was engineered for resistance to sulfonylurea compounds by introducing genes encoding mutant ALS. They confirmed that the transformants expressing a mutant ALS gene were resistant to field levels of sulfonylurea compounds. ' Flax (Linnm naitafissimnm L.) and many other dicotyledonous species are 21 sensitive to sulfonylurea residues in the soil, an interval of up to 4 years may be required before these crops can be sown in sulfonylurea-treated soil. Flax lines were transformed by W mmcfacicns with a gene encoding a resistant ALS enzyme from Anabidcnsja, generating 14 different, independently transformed lines (71). Resistance to chlorsulfuron was stablely inherited in all lines. McSheffrey et al. (72) suggested that the mutation in this particular ALS gene affects the binding of each sulfonylurea herbicide differently, and it is not possible to predict degrees of cross-resistance to other herbicides acting on the same target enzyme. As part of a program to broaden the spectrum of herbicides useful in potato (Scjannm mbcmsnm L.) production, a chimeric gene (min) for bromoxynil [3,5- dibromo—4—hydroxybenzonitrile] resistance has been introduced into potato cultivars. The can gene, which encodes a nitrilase specific for bromoxynil, was derived from chnsjclla czanac, and was introduced into potatoes by Agmhactcrinm nnncfacicna-mediated transformation. The transcription of the bzm gene was directed from a cauliflower (Emsjca clcmcca L. var. botrytis subvar. cultiflora DC.)-mosaic virus 35$ promoter, and resulted in efficient expression in the potato plants (31). Herbicide Resistant Weeds The continued use of a single control agent is often a common feature in cases 22 . of resistance. The first herbicide resistant wood was reported from Belgium around 1950’s. The reports said that dandelion (Imxacum cfficinalc Weber in Wiggers) and ~ buttercup (Ranunculus) showed resistance to 2,4—D. Atrazine/simazine resistant common groundsel (Scnmic Maria L.) was reported in Washington State in 1968 (89). Other triazine-resistant weed species, such as common lambsquarters (thncpcdjnm album L.), pigweed (Amaranthus spp.), witchgrass (Banjcnm canillarc L.), kochia (Emilia sccpmja (L.) Nees), annual bluegrass (Pca annna L.), downy brome (Brcmns tcctcmm L.), barnyardgrass mm M (L.) Beauv.), velvetlcaf (Abntilcn thccphmti Medicus), and giant foxtail (Ma fabcm' Herrm.) have been reported worldwide and in the United States. Radosevich et al. (90) demonstrated that the resistant biotypes of common groundsel were resistant to all s—triazine herbicides due to a mutation in the chloroplast gene that encodes the herbicide binding protein of photosystem II where many photosynthetic inhibitors bind. Rigid ryegrass (Lclinm figidnm Gaudin) in Australia that was sulfonylurea- resistant, was first reported in 1986. This was followed by reports of sulfonylurea resistant prickly lettuce (Lacnlca scmicla L.) from Idaho, kochia, Russian thistle (Salscla ibcrica Sennen & Pau) from Montana, S. Dakota, N. Dakota, Colo., Kansas. Wild mustard (Bmsica kalm: (DC.) L.C. Wheeler) populations resistant to auxin-type herbicides, including 2,4—D, MCPA [(4echloro-2—methylphenoxy) acetic 23 acid], dichlorprop [(_-_|-_)-2-(2,4-dichlorophenoxy) propanoic acid] and dicamba [3 ,6- dichloro-2-methoxybenzoic acid] were identified in western Canada in 1990. Heap et al. (56) reported that plants from the resistant population appeared to be less competitive, had darker green foliage and were shorter than susceptible plants. Bumet et al. (16) reported that rigid ryegrass biotype VLR 69 exhibited resistance to a number of ALS-inhibiting sulfonylurea and imidazolinone herbicides. On the basis that all VLR 69 plants. exhibit resistance to chlorsulfuron but resistance to sulfometuron is restricted only to those that contain an insensitive ALS, it is suggested that more than one mechanism of sulfonylurea resistance is present in the biotype VLR 69. Kochia biotypes that are sulfonylurea resistant have occurred through the continued use of chlorsulfuron in monoculture cereal-growing areas. Saari et al. (99) reported that ALS activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. They also found that no differences were observed in the ALS-specific activities or the rates of [“C] chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. So, they concluded that the mechanism of sulfonylurea resistance in this kochia biotype is due solely to the less sulfonylurea-sensitive ALS enzyme. Guttieri et al. (42) reported that most ALS resistance kochia (chhja accnaria (L.) Schrad.) biotypes had mutation in the codon for the proline residue in Domain 24 A. Also, the nature of the amino acid substitution was highly variable. Four different amino acid substitutions, arginine, threonine, leucine, and alanine were observed in R biotypes. But, some R biotypes did not have an amino acid substitution in Domain A, although in yinc assays of ALS inhibition indicated resistance was due to an altered form of ALS. Therefore, other regions of the ALS gene may be involved in resistance to ALS inhibitors. They suggested that many resistance alleles are present in kochia. The differences between sulfonylureas and imidazolinones with respect to the degree of ALS insensitivity are possibly-due to slightly different binding domains in the common binding site of the protein. Devine et al. (25) reported that the patterns of cross-resistance varied in two chlorsulfuron resistant biotypes of common chickweed (Stcllarja mszfia (L.) Viel) indicating that the alteration in ALS that confers chlorsulfuron resistance does not confer the same level of resistance to other sulfonylurea herbicides. The chlorsulfuron resistant common chickweed showed high levels of cross—resistance among sulfonylurea and triazolopyrimidine herbicides, but low levels of cross- resistance to imidazolinones. Whereas some sulfonylurea herbicides and the triazolopyrimidine herbicide are very effectively excluded from the altered binding niche, other sulfonylureas and the imidazolinone herbicides tested are restricted from the altered binding niche to a much lesser extent. They concluded that the differences in the patterns of cross-resistance observed reflect differences in the 25 binding affinity of the herbicides for the altered ALS (25). Tonks and Westra (117) showed that resistant and susceptible kochia responded similarly to the herbicides other than sulfonylureas. Christoffoleti (21) reported that the resistant biotype of kochia had no physiological disadvantage relative to the susceptible one in terms of biomass and seed production. This suggests that kochia resistant to sulfonylureas may not be less productive than susceptible ones. Thompson (116) found that the resistant kochia biotypes tended to have equal or greater leaf and stem dry weight, shoot height, and stem and shoot diameter than susceptible biotypes 13 weeks after establishment. The resistant and susceptible biotypes, on the average, produced 11,000 and 13,000 seeds per plant. Alcocer- Ruthling et al. (3) studied the seed biology of sulfonylurea-resistant and - susceptible biotypes of prickly lettuce. They found that seed longevity in soil, and fecundity or seed viability were not different between R and S biotypes. Also, they found seed from R biotype plants germinated as fast or faster than seed from S biotypeiplants (3). Triallate [S—(2,3,3-trichloro—2-propenyl) bis(1-methylethyl) carbamothioate] resistant wild oat populations generally had higher seed germination rates than susceptible populations, but no difference in terms of shoot dry weight, shoot number, leaf number or leaf area (84). Powles et al. (87) reported that multiple—resistance in Australian biotypes of rigid ryegrass, which were selected by heavy usage of diclofop-methyl, extends to 26 chemicals in the cyclohexanedione, sulfonylurea, dinitroaniline, triazine, substituted urea, and triazole classes of herbicides. Rubin et al. (97) found that the sulfonylurea resistant redroot pigweed (Amamnthns [cacflcnns L.) from the forest which was annually treated for four years with a mixture of sulfometuron and simazine exhibited a 10 to 40 fold increase in cross-resistance to different sulfonylureas, depending on the method used. N o cross-resistance was detected so far in the resistant population to imazapyr, another ALS inhibitor. Also, the resistant biotype shows a 2 to 5 fold increase in resistance to triazines. These data indicate that mixing herbicides of different modes of action may not be enough to prevent evolution of herbicide resistance especially when resistance is based on altered herbicide metabolism. Interaction of ALS inhibiting Herbicides with Organophosphate Insecticides Herbicides and insecticides may be required in the same growing season to control weeds and insects in corn. However, an interaction may occur between the pesticides that may be synergistic, antagonistic, or additive (52). In herbicide mixtures, Chemicals can interact in the sclution, at the plant surface, in the soil, within the tissues involved in absorption and translocation, as well as at the cellular site of action (69). The first reports of herbicide interactions with insecticides came from studies with cotton (Gcasypjnm 11112311111111 L.). Hacskaylo et al. (44) observed that severe 27 injury or death occurred to seedling cotton when a monuron [N’-(4-chlorophenyl)— N,N—dimethylurea] or diuron [N’-3 ,4-dichlorophenyl)-N,N-dimethylurea] application was preceded with a systemic insecticide application of phorate [Q,Q- diethyl S—[(ethylthio) methyl] phosphorodithioate] or disulfoton [Q,Q—diethyl S-[2- (ethylthio) ethyl] phosphorodithioate] . Subsequent studies showed phosphate and other classes of insecticides to increase the crop phytotoxicity of diuron to com (80). Nicosulfuron, primisulfuron and imazethapyr have the potential to injure corn under unfavorable environmental conditions. Corn injury consisted primarily of height reduction and some malformation of plants. Further the injury may be accentuated by the addition of the insecticide terbufos. Combination of primisulfuron with terbufos in corn field resulted in foliar and root injury, plant height reductions, and yield losses (14). Corn injury with nicosulfuron, primisulfuron or imazethapyr was influenced by com hybrid, terbufos placement, application time (81). Terbufos applied in soil can be absorbed by the corn seedling and moves systmnically throughout the plant (107). In-furrow insecticide treatments in combination with any of the herbicides caused greater crop injury than band applied insecticides in combination with a herbicide (111). The 15 % granular formulation of terbufos applied in-furrow, used in combination with any of the ALS inhibiting-herbicide treatments caused crop injury (chlorotic leaf spotting near the whorl, leaf crinkling, shortened intemodes, and stunting of plant 28 growth). Use of a controlled release formulation of terbufos in-furrow or in a band decreased visual injury symptoms when compared to 15 % granular formulation (111). Addition of the organophosphate insecticide terbufos to the germination medium prevented the metabolism of primisulfuron (23,111), and nicosulfuron (109,110,111) by shoots excised from unsafened seed, but not by shoots from NA- treated seed (23). HPLC analysis of extracted nicosulfuron and metabolites indicated greater parent herbicide longevity in plants treated with terbufos 156. The rate of nicosulfuron metabolism decreased with increasing terbufos concentration (27,28). Herbicide uptake was not affected by terbufos form; however, all of the absorbed nicosulfuron was metabolized at 24 hr after treatment without an organophosphate insecticide pretreatment whereas 90 % , 60 % , and 35 % was metabolized in the terbufos, terbufos-sulfoxide, and terbufos-sulfone treatments, respectively (28). Terbufos form plays a major role in the level of nicosulfuron metabolism in corn (27,28). Terbufos 20 CR plus herbicide was safer than 15 G plus herbicide at 4 weeks after application and to a greater extent at 8 weeks after application (77,120). Corn injury was not increased with combinations of the sulfonylurea herbicides and carbofuran [2,3—dihydro—2,2-dimethyl-7-benzofuranol methylcarbamate], chlorpyrifos [0,0—diethyl-O-(3 ,5,6-trichloro—2-pyridinyl) phosphorothioate],tefluthrin[(2,3,5,6—tetrafluoro—4-methylphenyl)methyl-(1a,3a)- 29 (Z-(j-J-3-(2-chloro-3 ,3 ,3,-trifluoro-1-propenyl)-2,2-dimethylcyclopropane- carboxylate] or chlorethoxyfos. Combination treatment of fonofos [O-ethyl-S- phenylethylphosphonodithioate] plus CGA-136872 resulted in 20% com injury 1 week after treatment (120). Simulated rainfall increased the corn injury from primisulfuron-terbufos interaction (59). Less injury occurred by delaying the application of nicosulfuron when terbufos was used (64). The difference in injury between times of postemergence application .of nicosulfuron may be related to the more extensive root system beyond the insecticide-treated soil. So, corn plants probably were absorbing less insecticide when the herbicide was applied. Second, degradation of terbufos likely was occurring, resulting in less being available for uptake at the later corn stage. Furthermore, degradation of terbufos in the plants was occurring, resulting in less interacting or inhibiting the nicosulfuron metabolism in the plants. Simpson et al. (110) found that tank-mixing 2,4—D with nicosulfuron decreased corn injury resulting from the nicosulfuron/terbufos interaction in field studies. Reduction of injury was observed when 2,4-D was applied at 0.28 kg ha" with and 1 day after nicosulfuron application. The decrease in nicosulfuron metabolism caused by terbufos was reversed when 2,4-D was applied. The 2,4—D did not affect uptake and translocation of nicosulfuron in the presence or absence of terbufos (110). Corbin et al. (23) suggested that primisulfuron metabolism in excised shoots and microsomal preparations of corn is mediated by cytochrome P- 30 450 and that enhanced metabolism induced by an NA seed-treatment can overcome the synergistic interaction imposed by terbufos on the metabolism of primisulfuron. Metabolism of Terbufos Soil-applied insecticides are widely used in corn at planting for wireworm (Mclannls spp. and £2chch spp.), rootworm (Diabmfica spp.), and nematode (142031191118 spp., Iciclmdcms spp., and ijnincma spp.) control. A widely used soil insecticide in the United States is terbufos, an organophosphate insecticide. Terbufos is known to be effective against several soil insect pests that attack corn. Most of the terbufos was lost during the first month after application with corresponding increases in recoveries of terbufos-sulfoxide and terbufos-sulfone. Chapman et al. (20) found that terbufos residue persisted in organic soils because of a rapid conversion of terbufos to the less degradable terbufos sulfoxide. For this reason, terbufos has not been recommended as a soil insecticide 011 organic soil. But its use may be limited because it may be less persistent after a second consecutive year of applications on the same land because of enhanced microbial degradation (20). Szeto et al. (115) also reported that terbufos was oxidized to its sulfoxide and sulfone in soil. Felsot et al. (33) reported that terbufos sulfoxide was the principle oxidative metabolite which formed rapidly from terbufos and was followed by a slower oxidation to terbufos sulfone. Their findings are in general agreement with those reported by Chapman et al. (20). They found that terbufos 31 translocated from soil into broccoli W clcracca L.). The plant residues consisted mostly of terbufos sulfoxide, terbufos oxon sulfoxide, and terbufos sulfone accounting for 90% or more of the total residues in broccoli, but the parent compound accounted for only 5 % of the total. This suggests that terbufos and its sulfoxide and sulfone were translocated from soil into plants and they were further oxidized in the plants to terbufos oxon sulfone. Also they reported that the concentration of total resides was highest in young plants collected at the first thinning, i.e. 22 days after seeding. It decreased steadily as the plants matured. Sellers et al. (107) studied residues of terbufos in Iowa corn and soil. They reported that residues in field corn forage ranged from a high of 0.43 ppm 40 days posttreatment with 4.48 kg ha'1 furrow application to nondetectable residues 60 days posttreatment with 1.12 kg ha" band application. It is apparent that terbufos or its sulfoxide and sulfone were readily taken up by plants grown in treated soil, but residues in the plant tissues, including all toxic oxidative metabolites, degraded rapidly. Interaction of Antioxidants with Herbicides Piperonyl butoxide (PBO) a known insecticide synergist enhanced the herbicidal activity of atrazine and terbutryn [N—(l , l-dimethylethyl)-N’-ethyl-6- (methylthio)—1,3,5-triazine—2,4~diamine] applied postemergence to corn seedlings (119). PBO also reversed the chloroplastic triazine resistance in a resistant biotype 32 of ryegrass, both at the whole plant and isolated chloroplasts levels (119). PBO applied in combination with either atrazine or terbutryn increased the foliar uptake of both herbicides in corn leaves, and lead to a light-dependent damage to membrane integrity. Rapid changes in the glutathionc (GSH) levels were also observed following treatment with PBO alone and PBO with atrazine. PBO treated corn plants contained more terbutryn and its partially-dealkylated metabolites. Etiolated corn seedlings have shown that PBO binds to cytochrome P-450. In the microsomal fraction containing N ADPH, terbutryn conversion to polar metabolites was inhibited by PBO by more than 50%. These results indicate that PBO inhibits terbutryn degradation mediated by cytochrome P-450. Varsano (119) suggest that PBO may act as a herbicide-synergist by several mechanisms simultaneously: increasing herbicide penetration, inducing membrane damage and inhibiting the metabolism of herbicides such as terbutryn. Bentazon [3-(1-methylethyl)-(1_I;l)-2,l,3-benzothiadiazin-4(3I-_I_)-one 2,2- dioxide], tetcyclacis [5-(4-chlorophenyl)-3 ,4,5 ,9 , lO-pentaazatetrracyclo [5.4.102",0‘"‘]—dodeca—3,9-dine] and PBO inhibited the metabolism of both primisulfuron and nicosulfuron in corn shoot tissue. But, tridiphane [2-(3,5— dichlorophenyl—Z—(2,2,2,-trichloroethyl) oxirane] had no effect on metabolism (95). Two hydroxylation reactions in primisulfuron treated corn plants were inhibited in yitrc by tetcyclacis (34). The catalytic reaction required N ADPH and oxygen and was inhibited by the cytochrome P-450 monooxygenase inhibitors, PBO (23) 33 and tetcyclacis(23,125) and carbon monoxide (125). Aminobenzotriazole, tetcyclasis and PBO inhibited nicosulfuron metabolism by com microsomes (9). Metabolism of simazine, chlortoluron, and metribuzin herbicides was inhibited by the mixed-function oxidase (MFO) inhibitor, l-aminobenzotriazole (ABT). ABT in combination with each of the herbicides significantly reduced plant dry weight compared with either the ABT or the herbicide alone (15). Action of Antidotes Hoffrnann (58) was the first researcher to report that a second chemical could selectively protect a whole plant from herbicide injury. He termed these chemicals ‘herbicide antidotes’. The use of chemical antidotes or protectants or safeners has been widely studied. There are significant reasons for using herbicide antidotes including use of higher herbicide rates and therefore more effective weed control, use of herbicides under conditions where crop damage is liable to occur, such as susceptible varieties, carryover of herbicide residues from previous crop, or use of interacting insecticide. Antidotes also provide useful insights into herbicidal action and metabolism. 1 Chemical, biochemical, and competitive or physiological antagonisms of the activity of herbicides by the antidotes are potential mechanisms of protective action. Chemical antagonism occurs when an antidote reacts chemically or physically with herbicide to prevent herbicide absorption by crop. Biochemical 34 antagonism occurs when a protectant reduces herbicide uptake and/ or translocation or stimulates metabolism of herbicides. Competitive or physiological antagonisms occur when an antidote competes with a given herbicide for the same site of action in the cells of the protected plant. Fuerst (35) proposed that two hypotheses for antidote mode of action seem plausible. Antidotes induce fast herbicide metabolism or protect the biOchemical site of action of the herbicide. The other plausible hypothesis is that antidotes protect the biochemical site of herbicide action. Compounds with similar structures to thiocarbamate herbicides are often effective safeners (51,112). For example, dichlormid [2,2-dichloro—N,N-di—Z—propenylacetamide] is structurally very similar to EPTC [S—ethyl dipropyl carbamothioate] and CDAA [2-chloro— N,N-di-2-propnylacetamide] . safeners that protect corn from sulfonylurea herbicides induce both oxidation and glucosylation. Wheat safeners such as CGA-184967 and HOE-70542 induce oxidative metabolism of aryloxyphenoxypropionic acid herbicides. Several investigators have suggested that safeners may be inducing expression of genes that normally are induced by pathogens for the purpose of detoxifying phytotoxins (36). BAS 145138 and naphthalic anhydride. (NA) treatments increased the metabolism of nicosulfuron and primisulfuron in corn shoots but did not increase in the roots (59,95). Also, metabolism was greatly enhanced by microsomes isolated from shoots of NA-treated corn seed (23). 35 Hatzios (53) reported that NA applied as a seed dressing offered good protection to the sensitive cum hybrids against injury caused by the PPI-applied thifensulfuron, but not against the highest rate of 96 g/ha. NA provided limited or no protection to any corn hybrid against injury caused by early postemergence- applied thifensulfuron. NA enhanced significantly the de-esterification of thifensulfuron-methyl causing a 1.5 - 2.0 fold increase in the formation of the parent acid, thifensulfuron. In addition to glutathione S-transferase (GST), MFO, and UDP-glucosyltransferase enzymes, hydrolytic enzymes are enhanced by herbicide safeners which confer tolerance to grass crops. NA seed treatment (0.5 % w/w) induced bentazon 6-hydroxylase activity 2.7 to 11.3 fold but did not induce Cinnamic acid 4-hydroxylase activity (43). Seed treatment with the safener NA or treatment ‘of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAI-I) activity, but decreased cinnamic acid hydroxylation of the wheat microsomes (125). This would suggest that in plants exposed to xenobiotics, some isoenzyme activities may be selectively stimulated, whereas Others are unchanged or decreased. They concluded that cytochrome P—450 induction may be a general mode of action of those safeners that protect crops against herbicides undergoing oxidative metabolism (125). Sweetser (114) has shown that the rate of metabolism of the sulfonylurea herbicides, chlorsulfuron and metsulfuron methyl, was significantly increased in excised leaves from wheat and corn following treatment with crop 36 safeners such as NA, and 'cyometrinil [(Z)-a-[(cyanomethoxy) imino] benzeneacetonitrile] (114). Treatment of corn seed with this safener CGA 154281 [4-(dichloro-acetyl)-3 ,4-dihydro-3-methyl-2H-l ,4-benzoxazine] , dramatically increased specific activities 'of cytochrome P-450 dependent primisulfuron hydroxylation. The total cytochrome P-450 content was also significantly increased in microsomes from safener-treated seedlings as compared to untreated controls (34). They suggested that the safener induces the biosynthesis of distinct cytochrome P-450 isozymes. Zimmerlin et al. (124) reported that the content of cytochrome P-450 was enhanced in etiolated wheat shoot microsomes after treatment with either NA or phenobarbital (PB). A much greater stimulation of enzyme activity occurred when PB and NA were combined. For example, the oxidation of 2,4—D, which is too low to be detected in microsomes from untreated seedling, become measurable. Benoxacor is a dichloroacetamide-safener used to protect corn against injury from metolachlor. Irzyk et al. (50) reported that com (Pioneer 3906) treated at planting with 1 11M benoxacor contained elevated levels of total glutathione S- transferase (GST) activity (24,41). Miller (75) studied that the effects of benoxacor on protein synthesis and GST induction in corn cell suspension cultures. Treatment of cultures with 10 uM benoxacor for 24 hr resulted in a 2.5 fold increase in total GST activity using metolachlor [2-chloro-N—(2-ethy1—6- methylphenyl)-l§l—(2-methoxy-l-methylethyl)acetamide] as the substrate. l“C- 37 Benoxacor metabolism studies, using TLC analysis, reveal that benoxacor is rapidly metabolized to at least seven metabolites more polar that the parent molecule. ’ Gronwald et al. (41) isolated the glutathione S-transferase (GST) isozymes from sorghum (DK 41Y) seedlings treated with the safener CGA-133205. Comparison of chromatogram with one for nonsafened sorghum showed that CGA- l33205-treatment increased the GST activity of two peaks. MON 13900 [3-(dichloroaCetyl)-2,2-dimethyl-5-(2-furanyl) oxazolidine] is a new herbicide safener that can reduce corn injury from several classes of herbicides, especially effective for minimizing the deleterious effects of sulfonylurea herbicides on corn growth and development (122). 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Alternative chemical control of sulfonylurea resistant and susceptible kochia Ma maria). Abstr. Weed Sci. Soc. Amer. 32:17. 118. Umbarger, HE. 1975. Regulation of amino acid biosynthesis in microorganisms. In HRV Armstein, ed, Synthesis of amino acids and proteins. MTP International Review of Science. Butterworths, London, 1-56. 119. Varsano, R., H.D. Rabinowitch, M. Schonfeld, and B. Rubin. 1992. Mode 50 of action of piperonyl butoxide as a synergist of atrazine and terbutryn in corn. Abstr. Weed Sci. Soc. Amer. 32:89. 120. Wells, R.B., C.H. Slack, W.W. Witt, M.D. Cole, and L.D. Boldt. 1992. Effects of CGA-136872 and DPX-V9360 applied to corn treated with soil insecticides. Abstr. Weed Sci. Soc. Amer. 32:99. 121. Westwood J .H. and SC. Weller. 1993. Shikimate pathway activity in glyphosate tolerant and susceptible biotypes of field bindweed (Ccnyclerlrr: arycnai: L.). Abstr. Weed Sci. Soc. Amer. 33:118. 122. White, R.H., B.H. Bussler, E.L. Williams, and W.T. Molin. 1992. MON 13900: A new herbicide safener for corn. Abstr. Weed Sci. Soc. Amer. 32:62. 123. White, R. H. 1992. The cytochrome P—450 system and its involvement in herbicide metabolism. Abstr. Weed Sci. Soc. Amer. 32:113. 124. Zimmerlin A. , and F. Durst. 1992. Aryl hydroxylation of the herbicide diclofop by a wheat cytochrome P-450 monooxygenase. Plant Physiol. 100: 874-881 . ' 125. Zimmerlin A., J-P. Salaun, F. Durst, and C. Mioskowski. 1992. Cytochrome P-450-dependent hydroxylation of lauric acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes. Plant Physiol. 100:868-873. CHAPTER 2 The Interaction of Insecticides with Herbicide Activity ABSTRACT Combination of certain herbicides with insecticides can negatively affect crop growth. Greenhouse studies were conducted to evaluate interaction effects of herbicides and insecticides with/without antidotes, on corn and weed species. Northrup King 9283 hybrid corn showed greater sensitivity to acetanilide herbicides than Cargill 7567. Both hybrids were sensitive to the interaction of chlorimuron, nicosulfuron and primisulfuron with terbufos. Cargill 7567 hybrid corn was more tolerant to the interactions of sulfonylurea herbicides with terbufos than Northrup King 9283. Imazaquin at 70 g/ha reduced corn height of Northrup King 9283 hybrid, but there was no interaction with terbufos. The. antidotes, CGA-154281 and NA, reduced corn injury from metolachlor, and nicosulfuron and primisulfuron with terbufos treatments, respectively. Thus, these antidotes stimulate P-450 mixed function oxidase activity. Nicosulfuron and primisulfuron treatments combined with metolachlor showed less corn injury than metolachlor alone, but these herbicides increased corn injury combined with terbufos treatment. 51 52 . The combination of primisulfuron and terbufos did not enhance herbicidal activity compared with primisulfuron alone to barnyardgrass, giant foxtail and velvetlcaf control. _ Nomenclature: Acetochlor, 2-chloro—N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl) acetamide;alachlor,2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide; chlorimuron, 2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] benzoic acid; imazaquin, 2-[4,5-dihydro—4-methyl—4-(1-methylethyl)-5- oxo-lH—imidazol-2-yl]-3-quinolinecarboxylic acid; metolachlor, 2-chloro-N-(2- ethyl-6-methylphenyl)-N—(2-methoxy-l-methylethyl) acetamide; nicosulfuron, 2,- [[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl]-13,151- dimethyl-3-pyridinecarboxamide ;primisulfuron,2-[[[[[4,6-bis(difluoromethoxy)-2- pyrimidinyl] amino] carbonyl] amino] sulfonyl] benzoic acid; brace; terbufos, S- [[(l , l-dimethylethyl)thio] methyl] Q,Q-diethyl phosphorodithioate; CGA-154281, 4-(dichloro-acetyl)-3 ,4-dihydro-3 -methyl-2H-l ,4-benzoxazine; NA, 1 ,8-naphthalic anhydride; barnyardgrass, Echinmblca curs-gall (L.). Beauv.; common lambsquarters, Gbcncnmlinm album L.; giant foxtail, ma falmri Herrm.; velvetlcaf, Abnfilcn Mhraati Medic.; Corn, an may: ‘Northrup King 9283’, ‘Cargill 7567’; Addition] index words: Interaction, herbicide activity INTRODUCTION To produce corn (an may: L.), farmers need to control weeds, insects, and fungi which may reduce crop yield and quality. Thus, farmers may apply herbicides and insecticides to the same crop, during the same growing season. Interactions between pesticides may occur. The recent introductions of the sulfonylurea and imidazolinone herbicides have stimulated research on the interaction of herbicides with insecticides. Nash (8) reported a synergistic interaction on oat (Aycna :atjya L.), corn, and cotton (Gcssypjnm bimnnrm L.) yields with monuron [N’-(4-chlorophenyl)- N,N-dimethylurea] or diuron [N’-(3,4-dichlorophenyl)-N_,N—dimethylurea] plus phorate [Q,Q-diethyl S- [(ethylthio)methyl] phosphorodithioate] or disulfoton [Q,Q- diethyl S-[2-(ethylthio)ethyl] phosphorodithioate], herbicide-insecticide combinations. He stated that three possibilities of pesticide interaction may be visualized: at the site of absorption where one pesticide affects the absorption of the others; within plants in which one pesticide affects the primary and another pesticide affects a secondary pathway; or both jointly affecting a Single pathway. Rootworms (mabrctica spp.) are a serious corn pest in the corn belt of the United States. Terbufos [S-[[(l,1-dimethylethyl) thio] methyl] cc-diethyl phosphorodithioate] is the organophosphate insecticide widely used for rootworm 53 54 control. The number of potential interactions between combinations of sulfonylurea herbicides and organophosphate insecticides are very high. In 1990, nicosulfuron [(2-[[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] -N,N—dimethyl-3-pyridinecarboxamide] and primisulfuron [2-[[[[[4,6-bis (difluoromethoxy)-2-pyrimidinyl]amino] carbonyl] amino] sulfonyl] benzoic acid] became commercially available for the control of annual and perennial grasses, and annual and perennial broadleafs, respectively, in corn. In the field experiment, combinations of the organophosphate insecticides disulfoton, fonofos [Q-ethyl S- phenyl ethylphosphonodithioate] , isazophos [Q-(S-chloro- l -[methylethyl]- l H l ,2,4- triazol-3-yl) (LG-diethyl phosphorotioate] , or terbufos and primisulfuron were shown to interact synergistically resulting in foliar and root injury, plant height reduction, and corn grain yield losses (2,4). Morton et al. (7) observed that the application of terbufos increased corn injury from nicosulfuron to ‘Pioneer 3751’ and ‘Jubilee’ hybrids. Porpiglia et al. (9) suggests that the synergistic interaction occurs because the insecticide may reduce metabolism and increase the uptake of primisulfuron in plants. Many researchers have reported that injury from nicosulfuron was greater when terbufos had been applied in-furrow at planting (15). Less injury occurred by delaying the application of nicosulfuron when terbufos was used (5). Kapusta et al. (5) suggested that the difference in injury between times of POST application of nicosulfuron may be due to the more extensive root system, and less absorption, 55 and more degradation of terbufos at the later corn stage. Several researchers have tried to find some methods to protect crops from the interaction of herbicides and insecticides. Simpson et al. (13), found that the tank- mixing 2,4—D [(2,4-dichlorophenoxy) acetic acid] with nicosulfuron decreased corn injury resulting from the nicosulfuron/terbufos interaction in the field studies. The decrease in nicosulfuron metabolism caused by terbufos was reversed when 2,4-D was applied. Corbin et al. (3), suggested that metabolism of primisulfuron of corn is mediated by cytochrome P450 and that enhanced metabolism induced by an NA (1 ,8-naphthalic anhydride) seed-treatment can overcome the synergistic interaction imposed by terbufos on the metabolism of primisulfuron. The objectives of this study were: a) to identify interactions between corn insecticides and the acetanilide herbicides or sulfonylurea herbicides, b) to determine whether the antidotes, CGA-154281 in CGA-180937, and NA seed- treatrnent, provided protection against the interaction of herbicides with the insecticide, terbufos, c) to determine whether sensitivity of corn to acetanilide herbicides was related to the other interactions, and d) to determine the interaction effect of the combination of primisulfuron with terbufos on herbicidal activity to several weed species. MATERIALS AND LIETHODS General greenhouse procedure Weed seeds were planted in 945-ml plastic pots, which contained an air-dried Spinks sandy loam (mixed, mesic Psammentic Hapludalfs) soil consisting of 71 .3 % sand, 19.4% silt, and 9.4% clay with a pH of 6.2 or BACCTO soil. After emergence, the plants were thinned to one plant per pot for velvetlcaf (Abntilcn thccpbrzaari Medic.) and common lambsquarters (thncmxljrrm album L.) and two plants per pot for barnyardgrass (Eclnnocblca cm:-gaLi (L.). Beauv.) and giant foxtail Ma falmi Herrm.). Three corn seeds of selected hybrids were planted in 945-ml pots, containing air-dried Spinks sandy loam soil. The plants were grown at 24 C _-_t-_ 2 C with supplemental lighting from high pressure sodium lights to provide a midday light intensity of 1200 rtE m’zs" for both supplemental and natural light. The day length was 18 h. Soil insecticides, terbufos and brace, were incorporated into top 1.5 cm of soil. All herbicide treatments were applied with a flat-fan 8002B nozzle in a spray volume of 280 L/ha at 240 kPa using a chain link-belt compressed air sprayer. After PRE herbicide application, the pots were irrigated to activate herbicides. Corn hybrid response 56 57 Two corn hybrids, which were previously identified as being tolerant (Cargill 7567) or sensitive (Northrup King 9283) to acetanilide herbicides were used in this study. Three corn seeds were planted 1.5 cm deep and covered with the soil which was treated with or without terbufos at 2.9 kg/ha. Metolachlor, alachlor and acetochlor at 1 and 3 times of field rates were applied preemergence with a chain link-belt compressed air sprayer. Antidote, CGA 154281, was used as a pro-mixed with metolachlor. Water was added to the soil surface for incorporation activation of the herbicide. Three factor (corn hybrid, insecticide, and herbicide) factorial, completely randomization design with four replications was used in this study. The POST herbicides, nicosulfuron and primisulfuron, were applied when com plants were in the three- to four-leaf stage. All postemergence treatments included 0.25% (v/v) of X-77‘. Weed response This study was conducted under the previously reported greenhouse conditions. To determine whether the interactions of primisulfuron with terbufos affected herbicidal activity, three weed species were planted in 945 -ml pot and terbufos was ‘ X-77 N onionic surfactant is a mixture of alkylarylpolyoxyethylene glycols, free fatty acids, and isopropanol marketed by Valent U.S.A. Corp., 1333 N. California Blvd., Walnut Creek, CA 94596. 58 applied preplant incorporated (PPI) at 2.9 kg/ha. Primisulfuron was applied at three rates of field rate when the weed species were at the three to five leaf stage or 5 to 8 cm of shoot height. After 14 days, plant height, visual injury, and fresh weight were taken. Three weed species, giant foxtail, barnyardgrass, and velvetlcaf, were used in this study. Data Presentation and Statistical Analysis Shoot height, shoot fresh weight, and injury ratings were taken 3 weeks or 2 weeks, after PRE, and POST treatments, respectively. Plant injury rating was on a scale of 0 (no effect) to 100 (completely dead). The mean of two or three plants in each pot was considered one observation. The data presented are the means of two experiments. The data was analyzw for variance, and means were separated with LSD values at the 5 % level of significance. RESULTS AND DISCUSSION Interaction of preemergence herbicides with insecticides. There is a significant difference of plant height and visual injury of corn between corn hybrids and herbicide treatments (Tables 1 and 2). Cargill 7567 and Northrup King 9283 hybrids showed greater shoot reduction with increasing preemergence herbicides rates. Northrup King 9283 hybrid showed greater sensitivity to acetanilide herbicides than Cargill 7567 hybrid in shoot height reduction and visual injury. No interaction of preemergence herbicides with terbufos or brace was evident except that acetochlor at 6.7 kg/ha + terbufos combination enhanced recovery from high rate of acetochlor in Northrup King 9283 hybrid. The application of terbufos increased the Northrup King 9283 height from 13.7 to 21.8 cm at the acetochlor 6.7 kg/ha treatment. The Northrup King 9283 hybrid showed some visual injury even at the 2.2 kg/ha rate of acetochlor alone, or with insecticide treatment. Rowe et al. (12) reported that ‘Cargill 7567’ was more tolerant than ‘N orthrup King 9283’ to metolachlor. These results conformed their report and also extended herbicide spectrum to alachlor and acetochlor. Interaction of Sulfonylurea herbicides with insecticides. 59 60 The application of sulfonylurea herbicides and imazaquin reduced the corn shoot heights of Cargill 7567 and Northrup King 9283 hybrids (Table 3). Terbufos combination with postemergence herbicides, chlorimuron, primisulfuron, and nicosulfuron further reduced the shoot height of both corn hybrids. Northrup King 9283 showed more sensitivity than Cargill 7567 to the interaction of terbufos and sulfonylurea herbicides. There was no interaction effect of imazaquin with terbufos. Morton et al. (7) reported that ‘Pioneer 3751’ field corn had shown more tolerance than ‘Jubilee’ sweet corn to the interaction of DPX-V9360 with terbufos. Also, the application of terbufos increased injury from DPX-V9360 to both of these hybrids (7). Northrup 9283 hybrid which was sensitive to acetanilide herbicides, showed more shoot reduction due to the interaction of sulfonylurea herbicides with terbufos than Cargill 7567. Antidote effect on the interaction of herbicides with insecticides. The application of a high rate of metolachlor induced considerable corn injury, but addition of terbufos reduced the corn injUry from 69% to 28 % (Table 4). The antidote CGA-154281 (premix with metolachlor) reduced the corn injury from metolachlor treatment. Chlorimuron, nicosulfuron and. primisulfuron reduced corn injury from high rate of metolachlor, but increased corn injury was observed from these herbicides if terbufos was also applied. Antidote CGA- 154281 reduced the corn injury from high rate of metolachlor even if applied with 61 chlorimuron, nicosulfuron, primisulfuron and imazaquin with or without terbufos. The application of terbufos reduced the crop injury from the high rate of metolachlor treatment. The combination of metolachlor with primisulfuron and nicosulfuron reduced crop injury compare to metolachlor alone (Table 4). Rowe et al. (11) also reported CGA-154281 decreased the corn injury from high rate of metolachlor. They concluded that it was due to an enhanced rate of metabolism of metolachlor in the sensitive corn hybrids. The seed treatment of NA reduced corn injury from the combination of terbufos with primisulfuron and nicosulfUron, but the antidote effect of NA was not enough to eliminate all injury (Table 5). The interaction of primisulfuron with terbufos caused greater injury to both corn hybrids than the nicosulfuron terbufos interaction. Seed treatment with NA decreased the visual injury to corn from the interaction of primisulfuron and nicosulfuron with terbufos (Table 6). Rehab et al. (10) reported that NA increased the metabolism of nicosulfuron and primisulfuron in corn shoots up to two-fold. Also, Corbin et al. (3) found that the shoots from N A-treated corn seed did not Show the decreased metabolism of primisulfuron from adding the terbufos treatment. Interaction effect of primisulfuron and terbufos on the herbicidal activity on weed species. With increasing primisulfuron rate, the plant height and fresh weight of 62 barnyardgrass, giant foxtail, and velvetlcaf decreased (Table 7). The application of terbufos did not effect the herbicidal activity of primisulfuron. Arle (1) had found that the interaction of trifluralin with phorate or disulfoton had no effect on the herbicidal activity of trifluralin in annual grasses. The synergistic effect of terbufos was evident with sulfonylurea herbicides, but not with acetanilide herbicides. The sulfonylurea and acetanilide herbicides should be metabolized by a different enzyme system. The enzyme system which metabolizes acetanilide herbicides may not sensitive to terbufos. The degree of sensitivity to the interactions of sulfonylurea herbicides and terbufos was different by hybrid. Terbufos inhibited the metabolism of sulfonylurea herbicides in corn plants. Corn injury might depend on the amount of inhibition of herbicide metabolism. Antidotes, CGA-154281 and NA, stimulated p450 mixed function oxidase activity to increase the metabolism in corn plants. Thus, treatment of antidotes reduced the corn injury from the interaction of sulfonylurea herbicides with terbufos. LITERATURE CITED 1. Arle, H. F. 1968. Trifluralin-systemic insecticide interactions on seedling cotton. Weed Sci. 16:480-482. 2. Bicdiger, D. L., P. A. Baumann, D. N. Weaver, J. M. Chandler, and M. G. Merkle. 1992. Interactions between primisulfuron and selected soil-applied insecticides in corn (an may:). Weed Technol. 6:807-812. 3. Corbin, F. T., D. E. Moreland, and B. Siminszky. 1993. Metabolism of primisulfuron in terbufos and/or naphthalic anhydride-treated corn. Abstr. Weed Sci. Soc. Amer. 33:70. 4. Holshouser, D. L., J. M. Chandler, and H. R. Smith. 1991. The influence of terbufos on the response of five corn (Ea may:) hybrids to CGA-136872. Weed Technol. 5:165-168. 5. Kapusta, G. and R. F. Krausz. 1992. Interaction of terbufos and nicosulfuron on corn (an may:). Weed Technol. 6:999-1003. 6. Lueschen, W. E., R. G. Harvey, J. J. Kells, and T. R. Hoverstad. 1990. Effects of time and rate of application of DPX-V9360 and rate of terbufos on injury to field corn. Res. Rep. North Cent. Weed Sci. 47:241-242. 7. Morton, C. A., R. G. Harvey, J. J. Kells, W. E. Lueschen, and V. A. Fritz. 1991 . Effect of DPX-V9360 and terbufos on field and sweet corn (an may:) under three environments. Weed Technol. 5: 130-136. 8. Nash, R. G. 1968. Synergistic phytotoxicities of herbicide-insecticide combinations in soil. Weed Sci. 16:74-77. 63 64 . 9. Porpiglia, P. J., E. K. Rawls, G. R. Gillespie, and J. W. Peek. 1990. A method to evaluate the differential response of corn (an may:) to sulfonylureas. Abstr. Weed Sci. Soc. Amer. 30:86. 10. Rehab, 1. F., J. D. Burton, E. P. Manes, D. W. Marks, and D. A. Robinson. 1993. Effect of safeners on nicosulfuron and primisulfuron metabolism in corn. Abstr. Weed Sci. Soc. Amer. 33:70. 11. Rowe, L., J. J. Kells, and D. Penner. 1991. Efficacy and mode of action of CGA- 154281, a protectant for corn (an may:) from metolachlor injury. Weed Sci. 39:78-82. 12. Rowe, L., E. Rossman, and D. Penner. 1990. Differential response of corn hybrids and inbreeds to metolachlor. Weed Sci. 38:563-566. 13. Simpson, D. M., K. E. Diehl, and E. W. Stoller. 1993. Mechanism for 2,4-D safening of nicosulfuron/terbufos interaction in corn. Abstr. Weed Sci. Soc. Amer. 33:112. 65 Table 1. The combination effect of acetanilide herbicides with insecticides on the Shoot height of two corn hybrids in the greenhouse 3 WAT. Insecticides Hybrids Herbicides. Rate Control Terbufos" Brace” (kg/ha) -- ------ (cm/plant) ----------- Cargill 7567 Control 0 48.4 48.0 46.2 Metolachlor 2.2 45.9 45.1 47.0 6.7 36.7 39.4 40.1 Alachlor 2.2 47.1 47.6 48.0 6.7 41.5 42.2 44.8 Acetochlor 2.2 p 41.3 41.3 41.7 6.7 34.1 33.8 33.4 NK 9283 Control 0 52.9 52.9 51.0 Metolachlor 2.2 42.2 46.3 46.4 6.7 28.5 34.0 34.7 Alachlor 2.2 I 46.3 48.1 47.6 6.7 35.7 36.0 33.2 Acetochlor 2.2 33.0 31.1 33.7 6.7 13.7 21.8 6.7 LSD at 0.05 7.3 ' Terbufos 11.2 g/100 m row " Brace 1.5 kg/ha 66 Table 2. The combination effect of acetanilide herbicides with insecticides on the visual injury of two corn hybrids in the greenhouse 3 WAT. Insecticides Hybrids Herbicides Rate Control Terbufos‘ Brace” (kg/ ha) """"""" ( % injury) ----------- Cargill 7567 Control 0 0 o o Metolachlor 2.2 3 1 0 6 7 15 26 14 Alachlor 2.2 0 o 0 6.7 Acetochlor 2.2 3 5 6 6.7 29 27 24 NK 9283 Control 0 0 o 0 Metolachlor 2.2 9 7 5 6.7 36 34 33 Alachlor 2.2 ‘ 3 2 2 6.7 21 16 36 Acetochlor 2.2 33 39 41 6.7 84 67 35 LSD at 0.05 15 ' Terbufos 11.2 g/100 m row ” Brace 1.5 kg/ha 67 Table 3. The interaction of corn hybrids, terbufos insecticide, and several POST herbicides on corn shoot height in the greenhouse 2 WAT. Cargill 7567 Northrup King 9283 Herbicide Rate Control Terbufos‘ Control Terbufos (g/ha) (cm/plant) Control - 39.8 39.7 37.7 35.0 Chlorimuron 12 26.4 22.6 32.3 14.0 Nicosulfuron 70 34.7 24.3 33.3 20.3 Primisulfuron 70 31.5 23.6 29. 1 13. 8 Imazaquin 70 24.2 25.2 14.5 13.8 LSD at 0.05 ~ 2.5 ‘ Terbufos 11.2 g/100 m row 68 Table 4. The interaction of terbufos insecticide with several POST herbicides on vkual injury to the metolachlor sensitive Northrup King 9283 hybrid in the greenhouse 2 WAT. Control Metolachlor- CGA-180937” Terbufosc Herbicide Rate - + - + - + (g/ha) : (96 injury) Control - 0 0 69 28 8 6 Chlorimuron 12 31 85 65 75 20 60 Nicosulfuron 70 6 44 43 70 10 55 Primisulfuron 70 1 1 56 54 66 45 50 Imazaquin 70 65 58 70 70 5 3 50 LSD at 0.05 ' 4 ‘ Metolachlor 6.7 kg/ha applied as PRE and supplied water to 12 % moisture level. " CGA-180937 (metolachlor + CGA 154281) 6.7 kg/ha applied as PRE and supplied water to 12 % moisture level. ° Terbufos 11.2 g/100 m row. 69 Table 5. The effect of NA on the interaction between herbicides and terbufos on corn plant height in the greenhouse 2 WAT. Control NA' Hybrid Herbicide Rate Control Terbufosb Control ‘ Terbufos (g/ha) --------- (cm/plant) ----------- Cargill 7567 Control - 59.4 54.3 59.9 53.6 Nicosulfuron 52.5 59.9 ' 46.9 57.0 53.2 Primisulfuron 52.5 51.6 28.2 55.6 41.3 NK 9283 Control - 63.7 56.5 60.3 57.9 Nicosulfuron 52.5 64.8 35.8 61.9 42.3 Primisulfuron 52.5 50.4 28.5 61.9 42.3 LSD at 0.05 4.2 ‘NA seed dressing 1%(w/w) l"I’erbufos 11.2 g/100 m row. 70 Table 6. The effect of NA on the interaction of sulfonylurea herbicides with terbufos on corn injury in the greenhouse 2 WAT. Control NA‘ Hybrid Herbicide Rate Control Terbufos” Control Terbufos (8 Ina) (96 injury)- ------------ Cargill 7567 Control - 0 l 0 2 Nicosulfuron 52.5 0 14 0 3 Primisulfuron 52.5 6 59 o 27 NK 9283 Control 0 2 0 Nicosulfuron 52.5 0 38 1 Primisulfuron 52.5 8 68 0 28 LSD at 0.05 6 'NA seed dressing 1%(w/w) ‘Terbufos 11.2 g/ 100 m row. accesses ”Emma. . fine .56 “<98? menacing 628m. «5 «a m.« «.« 2 2 m.« cm 8... a can as E Q «.o we 92 - - assess + he as 3 ed 3 a: - - 3a confiscate 3 «m _ «.2 we 3 «.2 «.m 3:. Seneca. + 1 5 «m we co «m “.2 as 93 «.3 confiscate 7 . on 2 «.2 3. a. «.2 a: com mecca. + a.« 2 «d «.« a. e.o« «.2. as m.« ceasefire 3 I he «.m c c.«.. e: So was access on o «.9 mm o _.«a «.2 So - .850 9533 3: 92353 €233. 3: access cease sagas 3:3 be? SE 3 58m as; . a .53 S can 3...; a can .3 can 5 USE cam .8828; .525. . 2.2mm buxom : { .33 « a 8:352» o... a. £8... 82. 8.5 a $52. 35.5.. aegiaea 2.. 8 aces sauces... 2: .2. 35. CHAPTER 3 The Effect of Mixed Function Oxidase Inhibitors on Sulfonylurea Herbicide Activity ABSTRACT Greenhouse studies were conducted to evaluate the effect of mixed function oxidase (MFO) inhibitors or antioxidants on the efficacy of sulfonylurea herbicides, and to identify effective adjuvants for the sulfonylurea herbicides with 28 % UAN (Urea Ammonium Nitrate) or piperOnyl butoxide or both. Tank-mixed PBO enhanced nicosulfuron and thifensulfuron activity on barnyard grass, velvetlcaf and common lambsquarters, respectively. Also, BHA and PBO enhanced nicosulfuron and primisulfuron activities on common lambsquarters and green foxtail. The optimal rates of PBO ranged from 1 to 6 kg/ha dependent on weed species and herbicide. All three factors, PBO, nonionic adjuvants, and 28 % UAN , enhanced activity of nicosulfuron on common lambsquarters, velvetlcaf, barnyardgrass and giant foxtail, primisulfuron on giant foxtail and velvetlcaf, and thifensulfuron on common lambsquarters and velvetlcaf. But 28 % UAN did not increase activity of primisulfuron on barnyardgrass, or common lambsquarters. Effective adjuvants with nicosulfuron were K-3000 on common lambsquarters, 72 73 and SYLGARD 309 on velvetlcaf, K-2000, K-3000 and SCOIL on barnyardgrass. Effective adjuvants with primisulfuron were K-2000, SCOIL, and SYLGARD 309 on giant foxtail, X-77, K-2000, K-3000, SCOIL, and SYLGARD 309 on velvetlcaf, K-3000 and SYLGARD 309 on common lambsquarters. Effective adjuvants for thifensulfuron were SCOIL on common lambsquarters, and SCOIL and SYLGARD 309 on velvetlcaf. Nomenclature: nicosulfuron, 2-[[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl]-N,N-dimethyl-3-pyridinecarboxamide; primisulfuron, 2-[[[[[4,6- bis(difluoromethoxy)-2-pyrimidinyl] amino] carbonyl] amino] sulfonyl] benzoic acid; thifensulfuron, 3-[[[[(4-methoxy-6-methyl-l ,3 ,5-triazin-2-yl)amino] carbonyl] amino] sulfonyl] -2-thiophenecarboxylic acid; PBO, oz-(2-(2-butoxyethoxy) ethoxy)- 4,5-methyl enedioxy—2-propyltoluene; barnyardgrass, Echinccblca curs-2am (L.) Beauv.; common lambsquarters, Glrcncpcriium album L.; giant foxtail, m fabcri Herrm.; green foxtail, Scmria y'njrli: (L.) Beauv.; velvetlcaf, Abutilcn W Medicus. Additional index words: 28% UAN, CHEMPRO, K-2000, K-3000, SCOIL, SYLGARD 309, X—77. INTRODUCTION Higher plants may metabolize xenobiotics that they absorb through their roots and leaves. The monooxygenases are enzymes involved in oxidative transformations forming primary metabolites. Mixed function oxidase (MFO) inhibitors, or antioxidants, are compounds used to prevent oxidative reactions. For these reasons, MFO inhibitors have been widely used to protect foods and other products from discoloration and spoilage. O’Brien introduced piperonyl butoxide (PBO) and sesamex (a component of sesame oil) as insecticide synergists (6). PBO, the most effective and widely used insecticide synergist, enhances activity of many organophosphate, carbamate, and pyrethroid insecticides (6). Insecticide synergists are potentially important pest management compounds because they may increase insecticidal activity against resistant insects (1), enhance cost effectiveness, and natural enemy survival (7), and decrease environmental impact by using lower rate of toxic insecticides. PBO significantly enhances the toxicity of certain insecticides because it inhibits microsomal detoxification enzymes (3). Komives et al. (5) and Rubin et al. (9) reported that PBO also acted as a herbicide synergist. PBO increased the phytotoxicity of EPTC [S - ethyl dipropyl carbamothioate] , atrazine [6-chloro-N-ethyl—N: (1-methylethyl)-l,3 ,5-triazine-2,4- 74 75 diamine], bentazon [3-(1-methylethyl)-(1H)-2,1,3-benzothiadiazin-4(3I;l)-one 2,2- dioxidc], and oxadiazon [3-(2,4—dichloro-5-(l-methy1ethoxy)pheny1)-5-(1 ,1- dimethylethyl)-l ,3,4-oxadiazol -2-(3H)-one] to corn (an may: L.)(5 ,9) and of atrazine and bentazon to soybean (Glycinc max (L.) Merr.) (9). They proposed that antioxidants inhibited mixed-function oxidase (MFO) systems which were possibly involved in the metabolism of these herbicides in corn or soybean. Also, Hatzios (4) reported a combination effect of PBO and metolachlor [2-chloro-N-(2- ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] on the growth of sorghum (Scrghrrm bicclcr (L.) Moench ’Funk G522DR’] seedlings and called this a synergistic effect. Varsano et al. (10) observed that PBO reversed the chloroplastic triazine resistance in a resistant biotype of ryegrass Mum n'gidum GAUD.), both at the whole plant and isolated chloroplasts levels. Rehab et al. (8) reported that bentazon, tetcyclacis [5-(4-chlorophenyl)- 3,4,5,9,10-pentaazatetracyclo [5.4.102",03"‘]-dodeca-3,9—diene], and PBO inhibited the metabolism of both primisulfuron and nicosulfuron in corn shoot tissue, but not tridiphane [2-(3,5-dichlorophenyl-2-(2,2,2-trichloroethyl) oxirane]. The objectives of this study were a) to evaluate the effect of antioxidants on the activity of several sulfonylurea herbicides on several weed species, b) to determine the optimal rate of PBO, and c) to identify effective adjuvants for the sulfonylurea herbicides applied with or without PBO. MATERIALS AND METHODS Plant Materials Five weed species, barnyardgrass, green foxtail, giant foxtail, common lambsquarters, and velvetlcaf, were used in this study. Weed seeds were planted in 945-ml pots, which contained BACCTO media. After emergence, the plants were thinned to two plants per pot for barnyardgrass, green foxtail, and giant foxtail, and one plant per pot for common lambsquarters and velvetlcaf. The POST herbicides, nicosulfuron, primisulfuron, and thifensulfuron, were applied when the weed species were at the three to five leaf stage or 5 to 8 cm shoot height. All postemergence treatments were applied with a flat-fan 8002E nozzle in a spray volume Of 280 L/ha at 240 kPa using a chain link-belt compressed air sprayer. All postemergence treatments included 0.25 % (v/v) of X-77‘ MFO Inhibitor Two MFO inhibitors, PBO, and butylated hydroxyanisole [2, [3]-tert-butyl-4- hydroxy-anisole : BHA], were evaluated for effect on the herbicidal activities of the sulfonylurea herbicides, nicosulfuron, primisulfuron, and thifensulfuron. lX-77 nonionic surfactant is a mixture of alkylarylpolyoxyethylene glycols, free fatty acids, and isopropanol marketed by Valent U.S.A. Crop., 1333 N. California Blvd., Walnut Creek, CA 94596. 76 77 INCI'I'E2 (insecticide synergist) was used as the PBO source and contained 92 % technical PBO. PBO and BHA at 4 kg/ha were applied with nicosulfuron and primisulfuron to common lambsquarters and green foxtail. PBO at 2 kg/ha was applied with thifensulfuron. To determine the optimal rate of PBO, rates of 1, 2, 4, and. 6 kg/ha were applied in combination with nicosulfuron and primisulfuron. Adjuvant studies Four weed species, common lambsquarters, velvetlcaf, giant foxtail, and barnyardgrass, were used to identify the most effective adjuvants for the sulfonylurea herbicides, nicosulfuron, primisulfuron and thifensulfuron, with PBO and 28 % UAN (containing urea and ammonium nitrate). Five adjuvants were tested with nicosulfuron. X-77 was added as a standard adjuvant for primisulfuron and thifensulfuron. The concentration of adjuvants was 0.25%, 1.25%, 1%, 1%, 1%, and 0.5% (v/v) for X-77, CHEMPRO’, K-2000‘, K—3000’, SCOIL‘, and SYLGARD 3097, respectively. Four rates, 0, 0.1, 0.5, 2INCITE sold by Loveland Industries Inc. Greeley, Colorado 80632. 3CHEMPRO: Chemorse LTD., Des Moines, Iowa 50322. ‘K-2000: Central Soya. Fort Wayne, IN 46801. ’K-3000: Central Soya. Fort Wayne, IN 46801. “SCOIL: Methylated seed oil from AGSCO Inc; , Grand Forks, ND. 7SYLGARD 309: Dow Corning Corp, Midland, MI 48686. 78 and 1.0 kg/Ila of PBO were applied with nicosulfuron, and three rates, 0, 0.5, and 1.0 kg/ha, of PBO were applied with primisulfuron and thifensulfuron. 4% (v/v) of 28 % UAN was used. The treatments were evaluated 2 weeks after treatments. Plant height and visual injury were recorded. Plant injury rating was on a scale of 0 (no injury) to 100 (completely dead). The mean of two plants in each pot was considered one observation for corn hybrids, barnyardgrass, green foxtail, and giant foxtail. Statistical Analysis All experiments were conducted separately on each species. A three-factor completely randomized design was used in PBO rates and adjuvant experiments. Each treatment was replicated four times and the data presented are the means of two experiments. The data were analyzed by AN OVA and means separated with LSD values at the 5 % level of significance. RESULTS AND DISCUSSION Effects of MFO inhibitors on sulfonylurea herbicide activity. Nicosulfuron was more effective for control of barnyardgrass than primisulfuron (Fable 1). PBO tank-mixed with nicosulfuron increased the herbicidal activity to barnyardgrass, but not with primisulfuron. Nicosulfuron tank-mixed with PBO decreased plant height and fresh weight, and increased visual injury of barnyardgrass more than nicosulfuron alone. Tank-mixture of PBO and BHA with nicosulfuron and primisulfuron increased the herbicidal activities of both herbicides on common lambsquarters (Table 2). PBO and BHA tank-mixed with primisulfuron enhanced control of common lambsquarters. Nicosulfuron at 7 g ai/ha plus PBO was more effective than nicosulfuron 35 g ai/ha alone. The synergistic action of PBO and BHA with nicosulfuron was greater than with primisulfuron for control of common lambsquarters. PBO increased the phytotoxicity of primisulfuron and nicosulfuron on green foxtail, but BHA did not (Table 3). Addition of PBO to primisulfuron increased visual injury and decreased fresh weight, but not plant height of green foxtail. Tank-mixed PBO increased nicosulfuron phytotoxicity on green foxtail as measured by the effects on plant height, fresh weight, and visual injury. 79 80 N icosulfuron at 7 g ai/ha applied with PBO showed the same effective green foxtail control as nicosulfuron at 35 g ai/ha alone. N icosulfuron provided greater control of green foxtail than primisulfuron. PBO tank-mixed with thifensulfuron increased visual injury and decreased plant height of velvetlcaf and common lambsquarters (Table 4). The application of 1.1 g ai/ha of thifensulfuron with PBO decreased plant height from 17.1 to 12.5 cm, and increased visual injury from 24 to 52% compared to thifensulfuron alone. The lowest rate, 0.6 g ai/ha of thifensulfuron, tank—mixed with PBO increased the herbicidal activity to common lambsquarters up to the 2.2 g ai/ha rate of thifensulfuron alone. PBO applied with various rates of thifensulfuron to barnyardgrass and giant foxtail did not significantly increase thifensulfuron activity, except for thifensulfuron at 4.5 g ai/ha plus PBO applied for giant foxtail control (Table 5). Tank-mixing of PBO with thifensulfuron was more effective on broadleaf weed species than grass weed species in increasing weed control (Tables 4 and 5). Mixed function oxidases, PBO and BHA, inhibit oxidative reactions in plants. PBO may inhibit the metabolism of sulfonylurea herbicides (8). Due to the inhibition of sulfonylurea herbicide metabolism in the weed species, tank-mixed PBO may be useful in enhancing activity of nicosulfuron, primisulfuron, and thifensulfuron. The synergistic effect of PBO was greater on broad leaf than grass weed species. 81 Effects of PBO rates on sulfonylurea herbicide activity. Two factors, herbicides and PBO rates, affected plant height and visual injury of common lambsquarters in the greenhouse (Table 6). The synergist action of PBO was evident with both herbicides on the growth of common lambsquarters. PBO alone had no effect on the growth of common lambsquarters or green foxtail (Tables 6 and 7). As PBO rates increased from 1 to 6 kg ai/ha, nicosulfuron activity on common lambsquarters and green foxtail increased (Tables 6 and 7). An increase in primisulfuron activity with increasing rates of PBO was not as evident, although PBO significantly increased primisulfuron activity to both common lambsquarters and green foxtail. Effects of nonionic adjuvants, PBO rates and 28% UAN on the phytotoxicity of sulfonylurea herbicides. Nicosulfuron: All three factors, PBO, nonionic adjuvants, and 28% UAN, increased nicosulfuron activity on common lambsquarters in the greenhouse study. Application of 35 g ai/ha of nicosulfuron alone had no effect on the growth of common lambsquarters (Table 8). PBO tank-mixed with nicosulfuron reduced plant height and increased visual injury. Addition of 28% UAN to nicosulfuron enhanwd common lambsquarters control. All adjuvants treatments increased nicosulfuron activity. Among adjuvants, K-3000 appeared most effective on common lambsquarters. Tank-mixing 1 kg/ha of PBO with nicosulfuron enhanced 82 the visual injury from 76 to 95%, and from 69 to 80%, with SCOIL and SYLGARD 309, respectively. The addition of 28 % UAN to CHEMPRO, or K- 2000 did not affect phytotoxicity. The addition of 28 % UAN to K-2000 plus PBO increased the visual injury up to 0.5 kg/ha rate of PBO. All three factors, nonionic adjuvants, PBO, and 28% UAN, increased nicosulfuron activity on velvetlcaf. Tank-mixed PBO increased nicosulfuron activity to velvetlcaf in the absence of an adjuvant or with 28 % UAN, CHEMPRO i 28% UAN, and SCOIL (Table 9). Addition of 28% UAN alone enhanced velvetlcaf control, and in combined with CHEMPRO, K-2000 and SCOIL. Combination of PBO and 28 % UAN increased nicosulfuron activity in the absence of an adjuvant, or with CHEMPRO, K-2000, and SCOIL. All adjuvant treatments increased nicosulfuron activity to velvetlcaf. Addition of 28 % UAN with SCOIL and K-2000 increased velvetlcaf control at all PBO rates. SYLGARD 309 adjuvant was more effective than the other nonionic adjuvants for velvetlcaf control with nicosulfuron. All treatments of SYLGARD 309 adjuvant showed above 88% of visual injury and below 39% of plant height. From the results, SYLGARD 309 plus 28 % UAN appeared to be the most effective adjuvant for velvetlcaf control with nicosulfuron (Table 9). All three factors, PBO, nonionic adjuvants, and 28% UAN, increased nicosulfuron activity on barnyardgrass. Nicosulfuron at 2.8 g ai/ha alone or with 28% UAN did not affect the growth of barnyardgrass (I‘ able 10). Tank-mixing 83 of l kg/ha of PBO with nicosulfuron increased barnyardgrass control without nonionic adjuvant. The 28 % UAN alone did not increase nicosulfuron activity to barnyardgrass. All nonionic adjuvants increased barnyardgrass control with nicosulfuron. K-2000 appeared to be the most effect of the nonionic adjuvants and SYLGARD 309 the least effective with nicosulfuron for barnyardgrass control (Table 10). Also, combination of 28% UAN and SYLGARD 309 with nicosulfuron increased barnyardgrass control about 20 to 25 %. From the results, K-2000, K-3000 and SCOIL adjuvants were considered good adjuvants to control barnyardgrass with nicosulfuron. With SYLGARD 309, the additions of 28 % UAN and PBO were strongly recommended. All three factors, PBO, nonionic adjuvants, and 28% UAN, enhanced giant foxtail control by nicosulfuron 6 g ai/ha. Nicosulfuron alone or plus 28 % UAN did not affect the growth of giant foxtail (Table 11). Tank-mixed PBO with nicosulfuron increased activity to giant foxtail. In the absence of any nonionic adjuvant, the addition of 1 kg/ha PBO i 28% UAN increased visual injury to giant foxtail by 48 %. All adjuvants increased giant foxtail control with nicosulfuron. With all of the weed species, it appeared that as wwd control increased with the addition of nonionic adjuvant and 28 % UAN, the effect of PBO became less evident. Primisulfuron: 34 Two factors, PBO and nonionic adjuvants, gave a significant increase to primisulfuron activity on plant height of common lambsquarters, but, the addition of 28% UAN did not (Table 12). From the visual injury data, it appeared that only nonionic adjuvants were effective in increasing primisulfuron activity. Application of 21 g ai/ha of primisulfuron alone reduced the growth of common lambsquarters, and tank-mixed PBO provided greater reduction of plant height. Addition of any of the adjuvants to primisulfuron decreased plant height of common lambsquarters. All three factors, PBO, nonionic adjuvants and 28 % UAN, increased primisulfuron activity on velvetlcaf. The 7 g ai/ha rate of primisulfuron alone did not affect the growth of velvetlcaf, but addition of PBO and/or 28 % UAN increased visual injury up to 89% and reduced plant height up to 42% of control without any adjuvant (Table 13). In the absence of 28% UAN and p130, the adjuvant K-3000 and SYLGARD 309 provided exceptionally good enhancement of primisulfuron activity on velvetlcaf. -With 28 % UAN and l kg/ha PBO, all nonionic adjuvant provided excellent enhancement of primisulfuron activity. Two factors, PBO and nonionic adjuvants, increased primisulfuron activity on barnyardgrass, but addition of 28 % UAN did not change primisulfuron activity. Primisulfuron at 35 g ai/ha was not an effective treatment for barnyardgrass control with any adjuvant (Table 14). Only K-2000 enhanced primisulfuron activity. If 1 kg/ha PBO was also applied than SCOIL + 28 % UAN and 85 SYLGARD 309 also enhanced primisulfuron activity. All three factors, PBO, nonionic adjuvants, and 28 % UAN, increased primisulfuron activity on giant foxtail (Table 15). In the absence of 28 % UAN and PBO the nonionic adjuvant, CHEMPRO, K-zooo, K-3000, and SCOIL were most effective in increasing primisulfuron activity on giant foxtail. If both 28 % UAN and 1 kg/ha PBO were present all the nonionic adjuvants were equally good. Thifensulfuron: All three factors, PBO, nonionic adjuvants, and 28% UAN, increased thifensulfuron activity on common lambsquarters in the greenhouse study (Table 16). Treatment of 0.6 g ai/ha of thifensulfuron alone had no effect on the growth of common lambsquarters. Tank-mixed PBO increased thifensulfuron activity, but 28 % UAN did not in the absence of nonionic adjuvants. All adjuvants increased thifensulfuron activity on common lambsquarters. Addition of 28 % UAN with SYLGARD 309 significantly enhanced thifensulfuron activity on visual injury of common lambsquarters. Fielding et al. (2) found that addition of 28 % UAN significantly increased velvetlcaf control, but, it did not enhance common lambsquarters control. The effect of 28 % UAN may be dependent on weed species, adjuvant, and herbicides. All three factors, PBO, nonionic adjuvants, and 28% UAN, increased thifensulfuron activity on velvetlcaf. Treatment of 1.5 g ai/ha of thifensulfuron 86 alone did not effect on the growth of velvetleaf (Table 17). Tank-mixed PBO and/or 28 % UAN increased thifensulfuron activities on velvetleaf. Addition of 28% UAN plus PBO to thifensulfuron provided 81% visual injury without any adjuvant. Tank-mixed PBO and 28 % UAN enhanced velvetleaf control with K- 2000, SCOIL adjuvants, but if both were added, there was no PBO effect. SYLGARD 309 adjuvant efficacy was greatly increased by the addition of 28% UAN . The results of this study are consistent with the hypothesis that at sublethal rates of the herbicide, the activity of these herbicide is a function of the level of the free active herbicide at the site of action. This level is a function of the rate and amount of herbicide absorption and the rate of herbicide metabolism. Since the sulfonylurea herbicides appear to be metabOlized at least to a limited degree in the wood species studies, blocking this metabolism with PBO and increasing herbicide absorption with effective adjuvant or 28 % UAN can raise the herbicide activity to its maximum potential. Similarly if the absorption rate is enhanced or accelerated, metabolism is overwhelmed and maximum potential herbicide activity is observed. This hypothesis explains why a certain maximum level of herbicide activity is observed and why in the presence of effective adjuvants, no effect of PBO is observed. LITERATURE CITED 1. Attia, F. I., G. J. Shanahan, and E. Shipp. 1980. Synergism studies with organophosphorus resistant strains of the Indian meal moth. J. Econ. Entomol. 73:184-185. 2. Fielding, R. J ., and E. W. Stoller. 1990. Effects of additives on the efficacy, uptake, and translocation of the methyl ester of thifensulfuron. Weed Sci. 38: 172-178. 3. Georghiou, G. P. 1980. Implications of the development of resistance to pesticides: basic principles and considerations of counter measures, pp. 116- 129. Vol. 2. In E. G. B. Gooding [ed], Pest and pesticide management in the Caribbean. Proc. of seminar and workshop Barabados, 3-7 Nov. 1980. 316 pp. 4. Hatzios, K. K. 1983. Effects of CGA-43089 on response of sorghum (Scrghum bicclcr') to metolachlor combined with ozone or antioxidants. Weed Sci. 31:280-284. 5. Komives, T. and F. Dutka. 1980. On the mode of action of EPTC and its antidotes on corn. Cereal Res. Commun. 8:627-633. 6. O’Brien, R. D. 1967. InseCticide action and metabolism. Academic Press, NY, Chapter 10, Pyrethroids. 7. Rajakulendran, S. V., and F. W. Plapp, Jr. 1982. Synergism of five synthetic pyrethroids by chlordimeforrn against the tobacco bud worm (Lepidoptera : Noctuidae) and a predator, Gbryacna camca (Neuroptera : Chrysopidae). J. Econ. Entomol. 75: 1089-1092. 8. Rehab, 1. F., J. D. Burton, E. P. Maness, D. W. Monks, and D. A. Robinson. 1993 . Effect of safeners on nicosulfuron and primisulfuron metabolism in corn. Abstr. Weed Sci. Soc. Amer. 33:70. 87 88 9. Rubin, B., J. R. Leavitt, D. Penner, and A. W. Saettler. 1980. Interaction of antioxidants with ozone and herbicide stress. Bull. Environ. Contam. Toxicol. 25:623-629. 10. Varsano, R., H. D. Rabinowitch, M. Schonfeld, and B. Rubin. 1992. Mode of action of piperonyl butoxide as a synergist of atrazine and terbutryn in corn. Abstr. Weed Sci. Soc. Amer. 32:89. 89 Table l. The effect of PBO on primisulfuron and nicosulfuron activity to barnyardgrass in the greenhouse 2 WAT. Treatment Rate Plant height Visual injury Fresh weight (g ai/ha) (cm/plant) (% injury) (g/plant) Control - 55.4 0 21.0 PBO 4000 51.1 0 21.3 Primisulfuron 28 36.4 22 1 1.4 + PBO 28 + 4000 34.8 23 10.3 Nicosulfuron 4 23. 1 61 4.5 + PBO 4 + 4000 17.5 86 1.2 LSD at 0.05 3.2 7 2.1 90 Table 2. The effect of PBO and BHA on primisulfuron and nicosulfuron activity to common lambsquarters in the greenhouse 2 WAT. Treatment Rate Plant height Visual injury Fresh weight (g ai/ha) (cm/plant) (% injury) (g/plant) Control - 21.5 0 7.1 PBO 4000 20.4 0 6.6 BHA 4000 20.2 0 6.5 Primisulfuron 35 8.9 69 1 .3 + PBO 35 + 4000 4.7 86 0.5 + BHA 35 + 4000 6.6 86 0.4 Nicosulfuron 7 17.6 4 6.4 + PBO 7 + 4000 7.5 72 1.1 Nicosulfuron 35 1 1. 1 40 3 .9 + PBO 35 + 4000 5.5 92 0.4 + BHA 35 + 4000 5.9 89 0.3 LSD at 0.05 2.0 8 0.9 IIt—tI *' 91 Table 3. The effect of PBO and BHA on primisulfuron and nicosulfuron activity to green foxtail in the greenhouse 2 WAT. Treatment Rate Plant height Visual injury Fresh weight (g ai/ha) (cm/plant) . (% injury) (g/plant) Control - 38.1 0 12.6 PBO 4000 35.1 0 10.6 BHA 4000 37.5 0 12.6 Primisulfuron 35 14.5 53 l .9 + PBO 35 + 4000 12.6 72 0.7 + BHA 35 + 4000 14.7 48 2.2 Nicosulfuron 7 15.5 51 1.6 + PBO 7 + 4000 8.9 91 0.3 N icosulfuron 35 9.5 86 0.3 + PBO 35 + 4000 1.4 98 0.2 + BHA 35 + 4000 9.2 85 0.4 LSD at 0.05 2.8 5 0.9 92 Table 4. The effect of PBO on the herbicidal activity of thifensulfuron on the growth of velvetlcaf and common lambsquarters in the greenhouse 2 WAT. Velvetleaf Common lambsquarters Treatment Rate Plant ht Injury Plant ht Injury (g ai/ha) (cm/plant) (%) (cm/plant) (96) Control - 27.5 0 28.5 0 PBO 2000 27.6 0 28.5 0 Thifensulfuron 0.6 21.1 17 13.5 51 + PBO 0.6 + 2000 18.3 24 7.0 74 Thifensulfuron 1. 1 l7. 1 24 6.6 69 + PBO 1.1 + 2000 12.5 52 6.8 84 Thifensulfuron 2.2 10.4 ‘ 66 6.7 78 + PBO 2.2 + 2000 10.1 76 6.7 91 LSD at 0.05 2.9 8 2.0 8 93 Table 5. The effect of PBO on the herbicidal activity of thifensulfuron on the plant height of barnyardgrass and giant foxtail in the greenhouse 2 WAT. Treatment Rate Bamyardgrass Giant foxtail (g ai/ha) (cm/plant) (cm/plant) Control - 64.3 68.4 PBO 2000 65.1 67.7 Thifensulfuron 1. 1 64.7 68.8 + PBO 1.1+ 2000 64.1 61.1 Thifensulfuron 2.2 63 .9 67. 1 + PBO 2.2 + 2000 63.7 63.1 Thifensulfuron 4.5 63.8 67.3 + PBO 4.5 + 2000 63.0 53.1 LSD at 0.05 NS 8.6 94 Table 6. The effect of various PBO rates on nicosulfuron and primisulfuron activity on common lambsquarters in the greenhouse 2 WAT. Treatment Rate Plant height Fresh weight Visual injury (g ai/ha) (cm/plant) (g/plant) (% injury) Control - 13.9 3.8 0 PBO 1000 13.3 4.4 0 PBO 2000 13.5 4.6 0 PBO 4000 13.0 4.1 0 PBO 6000 13.1 4.0 0 Nicosulfuron 35 8.7 2.9 49 + PBO 35 + 1000 6.9 1.9 54 + PBO 35 + 2000 5.7 1.1 73 + PBO 35 + 4000 5.4 0.9 76 + PBO 35 + 6000 5.4 0.7 81 Primisulfuron 21 6.4 l .4 57 + PBO 21 + 1000 5.6 0.8 73 + PBO 21 + 2000 5.5 0.7 81 + PBO 21 + 4000 5.2 0.7 80 + PBO 21 + 6000 5.6 1.5 68 LSD at 0.05 1.7 0.9 9 95 Table 7. The effect of various PBO rates on nicosulfuron and primisulfuron activity on green foxtail in the greenhouse 2 WAT. _T-reatment Rate Plant height Fresh weight Visual injury (3 ai/ha) (cm/plant) (g/plant) (% injury) Control - 32.6 7.7 0 - PBO 1000 31.7 8.6 0 PBO 2000 32.9 9.0 0 PBO 4000 32.1 7.6 0 PBO 6000 29.6 6.9 0 N icosulfuron 6 22.2 3. 1 51 + PBO 6 + 1000 20.6 1.6 55 + PBO 6 + 2000 20.8 1.5 59 + PBO 6 + 4000 19.7 1.2 76 + PBO 6 + 6000 19.8 1.1 84 Primisulfuron 21 21.7 3.3 44 + PBO 21 + 6000 19.7 1.9 57 + PBO 21 + 4000 19.6 1.9 53 + PBO 21 + 2000 19.4 1.7 56 + PBO 21 + 1000 19.2 1.6 60 LSD at 0.05 3.3 1.1 7 96 Table 8. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of common lambsquarters in the greenhouse 2 WAT. _ El ll.l| M. 1.. Nicosulfuron (35 g ai/ha) PBO rate (kg/ha) + Adjuvant Rate 0 0.1 0.5 1.0 0 0.1 0.5 1.0 (96 (v/v» (at of control) (9: injury) None 102 91 75 65 0 5 9 17 + 28% UAN 4 102 79 66 48 0 9 27 46 CHEMPRO 1.25 24 29 26 30 79 73 84 80 + 28% UAN 1.25 + 4 27 27 25 31 82 76 84 81 K-2000 l 26 28 30 29 76 77 76 79 + 28% UAN 1 + 4 27 27 27 30 84 89 85 77 K-3000 l 27 28 26 29 94 89 88 87 + 28% UAN l + 4 25 30 29 28 94 86 85 87 SCOIL l 28 28 29 30 76 83 83 95 + 28% UAN 1 + 4 26 27 29 31 89 91 85 83 SYLGARD 309 0.5 32 28 33 29 69 77 68 80 + 28% UAN 0.5 + 4 29 27 30 30 79 77 79 83 LSD at 0.05 10 97 Table 9. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of velvetlcaf in the greenhouse 2 WAT. — _Elant.llsrsht____VlaIaLlalllnl___ N icosulfuron (35 g ai/ha) PBO rate (kg/ha) + Adjuvant Rate 0 0.1 0.5 1.0 o 0.1 0.5 1.0 (9: (v/v)) (at. of control) (96 injury) None ' 93 84 84 79 3 17 19 21 + 28% UAN 4 88 71 73 69 13 24 37 41 CHEMPRO 1.25 66 59 so 53 44 43 64 69 + 28% UAN 1.25 + 4 56 52 43 46 58 59 79 71 K-2000 1 67 62 61 60 38 46 48 58 + 28% UAN 1 + 4 48 50 49 45 66 56 68 71 K—3000 1 45- 49 54 49 66 68 64 66 + 28% UAN 1 + 4 43 45 46 40 69 72 75 81 SCOIL l 62 57 53 49 49 54 57 66 + 28% UAN 1 + 4 43 41 42 43 81 82 76 81 SYLGARD 309 0.5 38 33 39 38 88 95 88 94 + 28% UAN 0.5 + 4 31 32 36 34 98 96 94 95 LSD at 0.05 98 Table 10. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of barnyardgrass in the greenhouse 2 WAT. — _£lmhelsht_. m Nicosulfuron (2.8 g ai/ha) . PBO rate (kg/ha) + Adjuvant Rate 0 0.1 0.5 1.0 o 0.1 0.5 1.0 (% (v/v)) (% of control) (96 injury) None 100 96 97 74 0 3 8 19 + 28% UAN 4 101 95 89 73 o 2 6 19 CHEMPRO 1.25 49 47 53 49 69 66 64 70 + 28% UAN 1.25 + 4 46 49 50 ‘ 46 74 68 69 73 K-2000 1 46 45 49 53 79 76 74 73 + 28% UAN 1 + 4 47 48 47 48 81 79 76 74 K-3000 1 50 48 48 48 73 73 73 74 + 28% UAN 1 + 4 49 50 46 47 79 77 74 78 SCOIL 1 50 51 49 50 71 70 71 68 + 28% UAN l + 4 48 48 47 50 80 76 74 77 SYLGARD 309 0.5 80 76 70 67 17 21 45 50 + 28% UAN 0.5 + 4 62 55 '53 48 42 46 65 71 LSD at 0.05 — 99 Table 11. The effect of various PBO rates and adjuvants on nicosulfuron activity on the growth of giant foxtail in the greenhouse 2 WAT. El 1.] 11' l" N icosulfuron (6 g ailha) _ PBO rate (kg/ha) + Adjuvant Rate 0 0.1 0.5 1.0 0 0.1 0.5 1.0 (% (V/V» (% of control) (% injury) None 100 90 78 52 0 4 28 49 + 28% UAN 4 94 79 66 53 3 11 34 52 CHEMPRO 1 .25 44 45 43 43 67 70 68 70 + 28% UAN 1.25 + 4 42 42 45 41 73 74 70 73 K-2000 1 38 41 42 42 76 74 79 73 + 28% UAN 1 + 4 39 41 42 40 76 76 75 77 K-3000 l 40 44 46 44 72 72 74 74 + 28% UAN 1 + 4 39 41 42 40 79 76 74 81 SCOIL l 40 42 45 41 72 71 73 76 +28%UAN 1+4 8 41 39 43 75 71 78 78 SYLGARD 309 0.5 43 43 44 ' 41 62 68 65 69 + 28% UAN 0.5 + 4 40 45 43 41 73 69 73 72 LSD at 0.05 6 100 Table 12. The effect of various PBO'rates and adjuvants on primisulfuron activity on the growth of common lambsquarters in the greenhouse 2 WAT. 21].] 11' l" Primisulfuron (21 g ai/ha) PBO rate (kg/ha) + Adjuvant Rate 0 0.5 1.0 0 0.5 1.0 ( % (v/v)) (% of control) (% injury) None - 59 29 29 59 66 70 + 28% UAN 4 61 27 30 59 69 68 X-77 0.5 21 22 31 88 81 73 + 28% UAN 0.5 + 4 24 27 24 79 81 91 CHEMPRO 1.25 23 20 26 80 81 71 + 28% UAN 1.25 + 4 24 20 29 78 78 66 K-2000 1 27 22 32 74 80 66 + 28% UAN 1 + 4 22 21 35 83 80 60 K-3000 l 21 23 23 93 74 84 + 28% UAN 1 + 4 21 28 24 86 66 79 SCOIL 1 19 27 28 84 68 75 + 28% UAN 1 + 4 26 25 26 75 75 78 SYLGARD 309 0.5 24 25 31 74 70 63 + 28% UAN 0.5 + 4 24 25 28 95 66 78 [SD at 0.05 ---- 7 -—--- ---- l9 101 Table 13. The effect of various PBO rates and adjuvants on primisulfuron activity on the growth of velvetlcaf in the greenhouse 2 WAT. El 1.] 11' “n. Primisulfuron (7 g ai/ha) PBO rate (kg/ha) + Adjuvant Rate 0 0.5 1.0 0 0.5 1.0 ( % (v/v)) (% of control) (96 injury) None - 102 76 59 0 14 53 + 28% UAN 4 62 49 42 34 85 89 X-77 0.5 89 73 41 7 17 91 + 28% UAN 0.5 + 4 43 35 43 86 91 92 CHEMPRO 1.25 56 47 49 48 71 72 + 28% UAN 1.25 + 4 43 38 39 89 93 91 K-2000 1 66 51 43 31 66 83 + 28% UAN l + 4 42 39 40 81 89 96 K-3000 l 40 39 42 91 91 95 + 28% UAN 1 + 4 36 33 39 97 99 96 SCOIL 1 48 43 44 65 83 81 + 28% UAN l + 4 38 36 38 98 97 97 SYLGARD 309 0.5 37 44 47 93 86 84 + 28% UAN 0.5 + 4 35 40 43 99 96 94 LSDat0.05 ——-- 10----- -—--12--—-— Table 14. The effect of various PBO rates and adjuvants on primisulfuron activity on the plant 102 height of barnyardgrass in the greenhouse 2 WAT. Primisulfuron (35 g ai/ha) PBO (kg/ha) + Adjuvant Rate 0 0.5 1.0 ( % (v/v)) (% of control) None - 101 101 100 + 28% UAN 4 104 100 100 X-77 0.5 103 99 97 + 28% UAN 0.5 + 4 104 102 101 CHEMPRO 1.25 101 101 95 + 28% UAN 1.25 + 4 102 101 100 K-2000 1 83 87 83 + 28% UAN l + 4 79 92 89 K-3000 l 102 100 100 + 28% UAN 1 + 4 98 102 98 SCOIL 1 95 100 93 + 28% UAN 1 + 4 93 98 90 SYLGARD 309 0.5 101 95 85 + 28% UAN 0.5 + 4 101 100 90 M 8 Table 15. The effect of various PBO rates and adjuvants on primisulfuron activity on the growth 103 of giant foxtail in the greenhouse 2 WAT. Plant height Visual injury Primisulfuron (21 g ai/ha) PBO rate (kg/ha) + Adjuvant Rate 0 0.5 1.0 0 0.5 1.0 ( % (v/v)) (% of control) (% injury) None - 81 71 61 10 40 53 + 28% UAN 4 90 70 56 10 45 56 X-77 0.5 69 58 48 31 56 64 + 28% UAN 0.5 + 4 63 51 45 39 65 71 CHEMPRO 1.25 58 45 45 61 74 71 + 28% UAN 1.25 + 4 56 48 46 55 70 74 K—2000 1 53 47 52 56 67 62 + 28% UAN l + 4 50 38 46 62 82 77 K-3000 l 55 47 51 63 66 61 + 28% UAN 1 + 4 49 42 44 70 73 73 SCOIL 1 50 42 39 66 74 77 + 28% UAN l + 4 48 41 38 77 76 84 SYLGARD 309 0.5 69 57 42 4o 58 74 + 28% UAN 0.5 + 4 57 44 40 64 84 79 LSDat0.05 ----l6---- ----13----- 104 Table 16. The effect of various PBO rates and adjuvants on thifensulfuron activity on the growth of common lambsquarters in the greenhouse 2 WAT. El 1.] If I" Thifensulfuron (0.6 g ai/ha) PBO rate (kg/ha) + Adjuvant Rate 0 0.5 1.0 0 0.5 1.0 ( % (v/v)) (% of control) (% injury) None - 97 37 35 1 60 74 + 28% UAN 4 89 35 . 35 3 63 76 X-77 0.5 40 39 36 51 57 73 + 28% UAN 0.5 + 4 36 36 38 61 76 76 CHEMPRO 1.25 34 34 33 73 78 80 + 28% UAN 1.25 + 4 37 36 33 77 77 78 K-2000 l 39 37 40 72 78 77 + 28% UAN 1 + 4 33 32 36 74 82 74 K-3000 1 34 32 34 75 81 79 + 28% UAN 1 + 4 33 33 34 76 79 76 SCOIL 1 35 31 37 73 83 81 + 28% UAN 1 + 4 33 35 35 77 82 82 SYLGARD 309 0.5 38 39 36 53 68 73 + 28% UAN 0.5 + 4 34 35 35 79 76 77 LSD at 0.05 ---- 9 ----- --—-- 8 ----- 105 Table 17. The effect of various PBO rates and adjuvants on thifensulfuron activity on the growth of velvetlcaf in the greenhouse 2 WAT. _ mum—Jamm— Thifensulfuron (1.5 g ai/ha) PBO rate (kg/ha) + Adjuvant Rate 0 0.5 1.0 0 0.5 1.0 ( % (v/v)) (% of control) (% injury) None - 96 90 72 1 14 36 + 28% UAN 4 61 52 50 55 81 81 X-77 0.5 93 77 71 3 26 ' 41 + 28% UAN 0.5 + 4 57 51 49 61 81 88 CHEMPRO 1.25 61 66 56 48 42 56 + 28% UAN 1.25 + 4 46 51 48 81 81 89 K-2000 1 74 66 56 36 45 58 ' + 28% UAN 1 + 4 44 47 46 88 89 87 K-3000 1 55 51 50 66 74 76 + 28% UAN 1 + 4 47 47 46 84 90 88 SCOIL 1 63 58 50 52 53 74 + 28% UAN 1 + 4 48 43 48 92 89 93 SYLGARD 309 0.5 52 69 57 67 38 58 + 28% UAN 0.5 + 4 44 47 45 87 91 92 LSD at 0.05 ---- 8 ----- ---- ll ----- Chapter 4 The Effect of Mixed Function Oxidase Inhibitors on Crop Safety to ALS Inhibiting Herbicides ABSTRACT Greenhouse studies were conducted to determine the response of six corn hybrids and two soybean varieties to acetolactate synthase (ALS) inhibitor herbicides applied with terbufos and/or mixed function oxidase (MFO) inhibitors. Field experiments were also conducted to determine the response of six corn hybrids to the combination treatments, terbufos plus ALS inhibitor herbicides and/or pipemoyl butoxide (PBO) and/or antidote. PBO at 0.33 kg/ha tank-mixed with nicosulfuron and primisulfuron caused injury to the Northrup King 9283 corn hybrid. Great Lakes 584 com showed less sensitivity than Northrup King 9283 to these combination treatments. Pioneer 3377 IR corn hybrid was resistant to the combination of nicosulfuron, primisulfuron plus PBO 2 kg/ha, and also to the combination treatments of imazethapyr herbicide plus PBO or butylated hydroxyanisole (BHA) even though terbufos was previously applied. ICI 85 32 IT, 106 107 ICI 8532 and Pioneer 3377 hybrids showed injury to the combination of nicosulfuron, primisulfuron herbicides and/or terbufos insecticide and/ or PBO 2 kg/ha. ICI 8532 IT corn hybrid showed resistance to the combination treatment of imazethapyr or thifensulfuron with terbufos. In the field study, injury 2 WAT to ICI 8532 IT, ICI 8532, and Pioneer 3377 hybrids from sulfonylurea herbicides plus terbufos was more evident than 6 WAT. Injury to ICI 8532 IT and Pioneer 3377 hybrids by imazethapyr herbicide plus terbufos remained similar to that observed at the early stage (2 WAT). Pioneer 3377 IR and Ciba 4393 RSC hybrids showed cross-resistance to sulfonylurea and imidazolinone herbicides even applied with PBO regardless of the presence of terbufos. ICI 8532 IT was cross-resistant to thifensulfuron and imidazolinone herbicides plus terbufos. All treatments of chlorimuron plus terbufos caused considerable injury to ICI 8532 IT, ICI 8532, Pioneer 3377, and Ciba 4393, but not Pioneer 3377 IR and Ciba 4393 RSC. The combination of thifensulfuron with PBO caused injury to Elgin ’87 soybean, but the W20-STS soybean was tolerant to this combination treatment. Combination of imazethapyr with PBO or BHA did not effect the growth of Elgin ’87 soybean hybrid. Nomenclature: Chlorimuron, 2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] benzoic acid; imazethapyr, 2- [4,5 -dihydro-4~methyl-4- (1-methylethyl)-5-oxo-lH—imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid; 108 nicosulfuron, 2-[[[[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl]-N,N-dimethyl-3-pyridinecarboxamide; primisulfuron, 2-[[[[[4,6- bis(difluromethoxy)-2-pyrimidinyl] amino] carbonyl] amino] sulfonyl] benzoic acid; thifensulfuron, 3-[[[[(4-methoxy-6-methyl-1 ,3,5-triazin-2-yl) amino] carbonyl] amino] sulfonyl]-2-thiophenecarboxylic acid; terbufos, S-[[(1,l-dimethylethyl)thio] methyl] 0,0-diethyl phosphorodithioate; butylated hydroxyanisole (BHA), 2, [3]- tert-butyl-4-hydroxyanisole; piperonyl- butoxide (PBO), 5-[[2-(2-butoxyethoxy) ethoxy] methyl]-6-propyl-l ,3-benzodioxole; corn, an may: L. ‘Great lakes 584’, ‘Northrup King 9283’, ‘Ciba 4393 RSC’, ‘Ciba 4393’, ‘ICI 8532’, ‘ICI 8532 IT’, ‘Pioneer 3377 IR’, ‘Pioneer 3377’; soybean, Glycinc max (L.) Merr., ‘Elgin ‘87 ’, ‘W20-STS’; Additional index words: tolerance, combination, crop safety, cross-resistance, tank-mixture, antidote. INTRODUCTION Commercialization of sulfonylurea and imidazolinone herbicides has accelerated the development of herbicide-resistant crops. Problems associated with some member of these herbicide families include injury to subsequent crops from soil residues and increased crop injury following the use of certain insecticides. Herbicide resistant crops have benefits such as increasing the crop safety margin, reducing crop damage from residual herbicides and widening the choice of herbicides. Resistant crop genotypes have been introduced by selection from naturally existing populations within crop species, selection of resistant mutants within a cultivar at the cell or whole plant level, and insertion of genes for resistance (1,7,10). With genetic engineering techniques now available, if the mechanisms for resistant and the genetic sequence are known, herbicide resistance can be inserted into crops (6). Despite much research on herbicide-resistant crops, few herbicide-resistant crops have been released to date. Metribuzin [4-aminO-6-(1,1-dimethylethyl)-3- (methylthio)-1,2,4-triazin-5-(4ID-one] resistant soybean (Glycjnc max (L.) Merr.) [TracyM] , bipyridylium-resistant forage grass species, atrazine [6-chloro-N-ethyl- N’-( 1-methylethyl)-1 ,3 ,5-triazine-2,4-diamine] resistant canola (Emccica nanu:L.) , 109 110 have been released (5,8,16). Cell culture selection for herbicide resistance has achieved a small portion of success with corn (an may: L.), and sugarbeet (E13 Elgar-i5 L.) (2). Resistance to the sulfonylurea and imidazolinone group of herbicides appeared to be partially dominant. Greater resistance was achieved when both resistant alleles were present. Resistance is due to a site modification in ALS. The imidazolinone resistant corn hybrid, Pioneer 3343 IR, has cross—resistance to the sulfonylurea herbicides which act by inhibiting the same enzyme. Recently, private companies and universities developed and released the ALS inhibiting herbicide-resistant crop cultivars. Bauman et al. (4) reported that imazethapyr [2-[4,5-dihydrO-4—methyl-4-(l-methylethyl)-5-oxo-1II-imidazol-2-yl]-5- ethyl-3-pyridinecarboxylic acid] resistant corn (ICI 8532 IT) showed no injury to imazethapyr regardless of the presence of terbufos [S-[[(1,1-dimethylethyl) thio] methyl] 0,0-diethylphosphorodithioate]. However, they found imazethapyr injury on ICI 8532 was enhanced when terbufos was included in the treatment. Wilcut et al. (17) reported that Pioneer 3343 IR hybrid was not injured by the interaction of either terbufos or carbofuran [2,3-dihydro-2, 2-dimethyl-7-benzofuranyl methylcarbamate] with nicosulfuron [2-[ [[ [[(4,6-dimethoxy-2-pyrimidiny1) amino] carbonyl] amino] sulfonyl] -N,N—dimethyl-3-pyridinecarboxamide] (15 ), imazethapyr, or AC 263,222. Also, Mukaida et al. (9) reported that Pioneer 3343 IR and ICI 8532 IT were unaffected by imazethapyr at 70 g ai/ha. Chlorimuron- 111 ethyl [2-[[[[(4-chloro—6-methoxy-Z-pyrimidinyl) amino] carbonyl] amino] sulfonyl]benzoic acid] at 10 g ai/ha treatment reduced shoot weight of Pioneer 3343, ICI 8532, and ICI 8532 IT. Pioneer 3343 IR was not injured by chlorimuron-ethyl. From the ALS activity study, Mukaida et al. (9) suggested that the mechanism for IR and IT resistance was due to differential sensitivity at the target site. They found Pioneer 3343 IR corn was resistant to both imazethapyr and chlorimuron-ethyl, while ICI 8532 IT was resistant only to imazethapyr. Barrett et al. (3) reported that PBO, tetcyclasis, and aminobenzothiazole inhibited nicosulfuron metabolism by Pioneer 3343 IR microsomes. Based on the observation that nicosulfuron is metabolized by a PBO sensitive MFO, one can hypothesize that PBO treatments to corn plants should increase injury from sulfonylurea herbicides. Sulfonylurea herbicide tolerant weeds could also be tolerant by metabolizing the herbicide in a manner similar to corn. If PBO were tank-mixed with the sulfonylurea herbicide, this should increase the activity to both corn and weeds, but if the ALS enzyme at the corn were less sensitive as in Pioneer 3343 IR it should have less injury. The objectives of this study were; a) to determine the effect of the MFO inhibitors on corn safety from application. of sulfonylurea or imidazolinone herbicides with and without terbufos; b) to determine the effect of MFO inhibitors on soybean tolerance to several ALS inhibitor herbicides . Materials and Methods General greenhouse procedure. Plants were grown from seed in the greenhouse in 946-ml plastic pot containing air-dried Spinks sandy loam (mixed, mesic Psammentic Hapludalfs) soil consisting of 71.3% sand, 19.4% silt, and 9.4% clay with a pH 6.2. Daytime temperatures were 25 i 2 C. Day length was 16 h with 1200 uE./m2/s with both supplemental and natural sunlight. Terbufos treated soil was used to cover seeds to a 1.5 cm depth. Tank-mixed solutions of herbicide and MFO inhibitors were applied postemergence with a flat-fan 8002B nozzle in a spray volume of 280 L/ha at 240 kPa using a chain link-belt compressed air sprayer. Plant height, injury ratings, and fresh weight were evaluated 14 days after treatments. Corn hybrids. The six corn hybrids, Great Lakes 5 84, resistant to high rate of acetanilide herbicides, Northrup King 9283 , sensitive to high rate of acetanilide herbicides (l3), ICI 8532 IT and Pioneer 3377 IR, two imazethapyr resistant corn hybrids, ICI 8532 and Pioneer 3377, two imazethapyr sensitive hybrids, were included in this study. Two corn seeds were planted per 946-m1 plastic pot. Postemergence herbicides and MFO inhibitors were applied when com was at the three- to four- 112 1 l3 leaf stage. In the greenhouse, a completely randomized design was used with four replications per experiment. The data presented are the means of two experiments. Following an analysis of variance, the means were separated using the LSD Test at the 5 % probability level. Soybean varieties. Two soybean varieties were used in this study. W20-STS hybrid was developed and released by Du Pont Company as having enhanced tolerance to sulfonylurea herbicide, thifensulfuron (14). Elgin ’87 variety was used as a standard soybean variety. Two soybean seeds were planted per 946-ml plastic pot. Tank-mixed solutions of herbicides and/or MFO inhibitors were applied postemergence when the soybean reached the three- to four-leaf stage. In the greenhouse, a complete randomized design was used with four replications per experiment. The data presented are the means of two experiments. Following an analysis of variance, the means were separated using the LSD Test at the 5 % probability level. Chemical treatments. Terbufos insecticide was applied as a preplant incorporated (PPI). Corn seeds were covered to a depth of 1.5 cm with the terbufos treated soil (2.9 kg/ha). Nicosulfuron and primisulfuron herbicides were applied at a rate range from 35 114 and 40 g ai/ha, respectively to twice that and MON 12000 and MON 12000 + MON 13900 were applied at 34.7 and 84.1 g ai/ha, respectively. PBO was applied at 4,000 g/ha alone, and 1,000, 500, 333 g/ha rates in combination with the herbicide treatments. In the soybean study, two soybean varieties, Elgin ’87 and W20-STS, were sprayed with thifensulfuron at 8.8, 17.5, and 35 g ai/ha rates and PBO was applied at 2,000 g/ha. Imazethapyr was applied at 70 and 105 g ai/ha to Elgin ’87, and PBO and BHA were applied at 2,000 and 4,000 g ai/ha rates. X-77l surfactant (0.25 % v/v) was added to all spray solutions. Field study. Field studies were conducted in 1992 and 1993 at East Lansing, Michigan on a loam soil with 2.6% organic matter and pH of 7.1. The plots were 3 by 10.5 m, with a 75 cm row spacing. Four corn hybrids (ICI 8532 IT, ICI 8532, and Pioneer 3377 IR, Pioneer 3377) in 1992 and six corn hybrids (added Ciba 4393 RSC and Ciba 4393) were planted with 9,713 seeds per ha population on May 11 1992 and May 10 1993. Terbufos 15 G was applied at 74.4 g/100 m of row, in furrow. Metolachlor (8 BC 2.2 kg/ha) and atrazine (4 F 1.1 kg/ha) herbicides were applied as preemergence treatment to control weed species. Postemergence ‘X-77 nonionic surfactant is a mixture of alkylarylpolyoxyethylene glycols, free fatty acids, and isopropanol marketed by Valent U.S.A. Corp., 1333 N. California Blvd., Walnut Creek, CA 94596. 1 15 treatments were sprayed with a compressed air sprayer with a flat fan 8003 nozzle, 207 kPa, and 206 L/ha. Postemergence herbicides, nicosulfuron, primisulfuron, imazethapyr, chlorimuron were applied atone and/or two times the recommended rates. Preplant incorporation treatment of the herbicides, imazaquin [2-[4,5- dihydro-4-(1-methylethyl)-5-oxo—lH—imidazol—Z-yl]-3-quinolinecarboxylic acid], MON 12000 [methyl 3-chloro—5-(4,6-dimethoxypyrimidin-2-arylcarbamoyl sulfamoyl)-1-methylpyrazde-4—carboxylate] , chlorimuron [2-[[[[(4-chloro-6- methoxy-2-pyrimidinyl)amino] carbonyl] amino] sulfonyl] benzoic acid], CGA- 152005 [1 -(4-methoxy-6-methyl-triazin-Z-yl)-3-[2-(3 , 3 , 3-trifluoropropyl)- phenylsulfonyl]-urea] , and flumetsulam '+ metolachlor, were at recommendated rates or two times these rates. PBO at 2 kg/ha was applied as tank-mixture with nicosulfuron and primisulfuron. To identify the antidote effect on the corn growth, R 29148 [3-(dichloroacetyl)-2,2,5-trimethyloxazolidine] antidote was applied with the sulfonylurea herbicides (1992 only). All postemergence treatments included 0.25% (v/v) X-77 surfactant. At 2 and 6 (1992), and 2 and 4 (1993) weeks after postemergence treatments, plants were evaluated for visual injury and measured plant height (10 plants/row). Field experiments were conducted as a randomized complete block design. Each treatment was repeated three times. Following an analysis of variance, means were separated using the LSD Test at the 5 % probability level. Results and Discussion Applications of 35 g/ha of nicosulfuron, primisulfuron, and 4 kg/ha of PBO alone did not induce injury on the two corn hybrids, Great Lakes 584 and Northrup King 9283 (Table 1). PBO at 1 kg/ha tank-mixed with nicosulfuron and primisulfuron herbicides reduced plant height and fresh weight of both corn hybrids compared to herbicide alone. Addition of PBO at 0.5 kg/ha to nicosulfuron increased injury to Northrup King 9283 corn hybrid, and reduced plant height of Great Lakes 584 hybrid. PBO at 0.5 kg/ha plus primisulfuron caused injury to Great Lakes 584 and Northrup King 9283 hybrids. PBO at 0.33 kg/ha tank-mixed with the two herbicides caused injury to the Northrup King 9283 hybrid. However, addition of 0.33 kg/ha of PBO to primisulfuron did not affect growth of the Great Lakes 584 hybrid. Application of nicosulfuron plus 0.33 kg/ha of PBO decreased plant height of Great Lakes 584, but did not reduce fresh weight (Table 1). From the results, Great Lakes 584 appeared more tolerant to combinations of PBO with sulfonylurea herbicides than Northrup King 9283 hybrid. According to Rowe et al. (13), Great Lakes 584 hybrid also showed more tolerance to high rates of acetanilide herbicides than Northrup King 9283 hybrid. Due to the crop injury, the addition of PBO to sulfonylurea herbicides is not recommended on the normal corn hybrids. The addition of PBO at rates in excess 116 117 of 0.33 kg/ha was sufficient to cause injury to corn from the nicosulfuron and primisulfuron indicating that these herbicides were metabolized by the MEG system in corn. PBO tank-mixed with nicosulfuron, primisulfuron, and imazethapyr did not affect the growth of imazethapyr resistance corn hybrid, Pioneer 3377 IR (Table 2). Also, the addition of BHA to imazethapyr did not cause injury to Pioneer 3377 IR hybrid. Barrett et al. (3) found that PBO inhibited metabolism of nicosulfuron. Despite PBO inhibition of sulfonylurea herbicide metabolism, Pioneer 3377 IR corn was not injured by the combination treatments of sulfonylurea herbicides plus PBO or BHA since the ALS in the Pioneer 3377 IR corn hybrid is known to be less sensitive to this class of herbicide (1 1). Application of terbufos or PBO at 2 kg/ha alone had no effect on the growth of four corn hybrids, but if the two factors were combined, reduced plant height of ICI 85321'1‘ and ICI 8532 hybrids was observed (Tables 3 and 4). The combination of nicosulfuron plus terbufos with or without PBO reduced corn plant height of Pioneer 3377, ICI 8532, and ICI 8532 IT hybrids compared to nicosulfuron alone, but it did not affect the growth of Pioneer 3377 IR hybrid. Primisulfuron alone, or combined with terbufos, reduced plant height and increased visual injury of ICI 8532 IT, ICI 8532, and Pioneer 3377 hybrids, but not of Pioneer 3377 IR hybrid. PBO tank-mixed with primisulfuron resulted in greater visual injury to both sensitive corn hybrids than observed with 118 nicosulfuron, and decreased plant height of all four corn hybrids. The combinations of primisulfuron, terbufos, and PBO induced greater corn injury of three corn hybrids, except Pioneer 3377 IR hybrid. From the results, Pioneer 3377 IR hybrid appeared resistant to the combination of sulfonylureas, terbufos, and PBO (Tables 3 and 4). According to the Mukaida et al. (9), Pioneer 3343 IR corn showed cross-resistance to sulfonylurea and imidazolinone herbicides, but ICI 8532 IT corn was resistant to only imidazolinone herbicides regardless presence of terbufos. These it would appear possible to use PBO in combination with nicosulfuron and prinrisulfuron to increase herbicide activity on weeds without loss of corn safety to Pioneer 3377 IR even if terbufos had been applied for insect control. MON 12000 applied at 34.7 g ai/ha did not affect the plant height of the four corn hybrids, Pioneer 3377, Pioneer 3377 IR, ICI 8532, and ICI 8532 IT (Table 5). However, application of MON 12000 plus antidote MON 13900 reduced plant height of Pioneer 3377 and ICI 8532 corn hybrids, it might be due to the 1.5 times of recommendated rate. PBO at 2 kg/ha did not increase corn injury from MON 12000. The two imazethapyr resistant corn hybrids, Pioneer 3377 IR and ICI 8532 IT, were resistant to a high rate of MON 12000. Since the addition of PBO to MON 12000 did not enhance corn injury to either imazethapyr resistant and sensitive hybrids, PBO may not inhibit the metabolism of MON 12000 in corn plants . 119 Field Experiment. All plots, including control plots, received terbufos 15G at 11.9 kg/ 100 m row, in furrow, metolachlor 8 EC at 2.2 kg ai/ha, atrazine 4F at 1.1 kg ai/ha and X-77 0.25% (v/v), to control insects and weed species. First year, 1992. Postemergence application of nicosulfuron or primisulfuron reduced plant height, and caused injury 2 WAT to ICI 85321T, ICI 8532, and Pioneer 3377 hybrids, due to the prior application of terbufos insecticide (Tables 6 and 7). Addition of PBO at 2 kg/ha, even though tank-mixed antidote R29148 was applied enhanced visual injury to corn except for the Pioneer 3377 IR hybrid. The imazethapyr resistance corn hybrids, Pioneer 3377 IR and ICI 85321T, were not injured by imazethapyr or thifensulfuron herbicides plus terbufos, but the sensitive corn hybrids, Pioneer 3377 and ICI 8532, were injured by that combination treatments (Tables 6 and 7). By 6 WAT, the injury to corn from the primisulfuron and nicosulfuron interaction with terbufos was much less apparent (Tables 8 and 9). However, if PBO had been applied, the interaction was stable apparent. The imazethapyr interaction effect with terbufos was greater at 6 WAT than at 2 WAT. The two imazethapyr-resistant corn hybrids, ICI 8532 IT and Pioneer 3377 IR, showed resistance to the combination of imazethapyr plus terbufos. Only Pioneer 3377 IR hybrid showed resistance to sulfonylureas and imazethapyr herbicides plus terbufos even with added PBO (Tables 6, 7, 8 and 9). ICI 8532 IT corn hybrid was 120 showed resistant to the interaction of thifensulfuron with terbufos regardless of the presence of PBO, but was very sensitive to the combination of nicosulfuron and primisulfuron herbicides plus terbufos. Second year, 1993. Nicosulfuron at 35 or 70 g ai/ha following prior application of terbufos caused corn injury to ICI 8532, Pioneer 3377 and Ciba 4393. The addition of PBO to these combination increased injury to these corn hybrids including the ICI 8532 IT hybrid (Tables 10 and 11). Pioneer 3377 IR and Ciba 4393 RSC showed resistance to the combination of nicosulfuron plus terbufos even with PBO at 2 WAT. Primisulfuron after terbufos with/without PBO caused injury to the ICI 8532 IT, ICI 8532, and Ciba 4393 hybrids. Pioneer 3377 IR, Pioneer 3377 and Ciba 4393 RSC showed no injury from primisulfuron plus terbufos and/or PBO. The Ciba 4393 hybrid was the most sensitive to combination of primisulfuron, terbufos, and PBO. All three imazethapyr sensitive corn hybrids showed sensitivity to the combination of imazethapyr plus terbufos. Chlorimuron postemergence plus terbufos caused injury to ICI 8532 IT, ICI 8532, Pioneer 3377, Ciba 4393, but not Pioneer 3377 IR or Ciba 4393 RSC. MON 12000 applied at 168 g ai/ha prior to terbufos treatment similarly caused injury to ICI 8532 IT, ICI 8532, Pioneer 3377, and Ciba 4393, but not on Pioneer 3377 IR and Ciba 4393 RSC. Flumetsulam/metolachlor, CGA-152005, and imazaquin plus 121 terbufos caused injury to the imazethapyr sensitive corn hybrids. Injury to ICI 8532 IT, ICI 8532, Pioneer 3377, and Ciba 4393 corn hybrids from nicosulfuron and primisulfuron plus terbufos _-|; PBO was less evident at 4 WAT compared to 2 WAT (Tables 12 and 13). But corn injury 4 WAT from imazethapyr was greater or similar to that observed at 2 WAT. From the results, it appear safe to plant Pioneer 3377 IR and Ciba 4393 RSC and apply combination treatments of ALS inhibitor herbicides plus terbufos. Also, PBO tank-mixed with these herbicides did not increase injury to these corn hybrids. The ICI 8532 IT corn hybrid was sensitive to nicosulfuron and primisulfuron with/without PBO, MON 12000, and CGA 152005 plus terbufos, and to chlorimuron ethyl. However, ICI 8532 IT showed resistance to imazethapyr plus terbufos treatment. Soybean Study. Thifensulfuron alone, even at two times the recommended rate, did not affect the growth of the two soybean hybrids, Elgin ’87 and W20-STS (Table 9). Application of tank-mixed PBO at 2 kg/ha with thifensulfuron at 17.5 and 35 g/ha reduced soybean plant height and induced visual injury to Elgin ’87 hybrid, but it did not affect the growth of the W2esrs hybrid. wzesrs hybrid showed excellent tolerance to application of thifensulfuron 35 g/ha with PBO 2 kg/ha compared to Elgin ’87 hybrid (Table 14). Several researchers have reported that PBO inhibited the metabolism of sulfonylurea herbicides in corn (9,12). The 122 inhibition of metabolism of thifensulfuron by PBO would be expected in both soybean hybrids, Elgin ’87 and W20—ST S. Thus, the difference in tolerance to the combination effect of thifensulfuron and PBO may be due to differential sensitivity at the target site of action. W20-STS was shown to have a less sensitive target site (14) . PBO tank-mixed with thifensulfuron applied to W20-STS soybean field could increase weed control without soybean injury. The additions of PBO or BHA to imazethapyr showed no effect on the growth of Elgin ’87 soybean hybrid even at 1.5 fold recommended rate (Table 15). These results suggest that PBO and BHA do not inhibit the metabolism of imazethapyr herbicide in soybean. From the results, the sulfonylurea and imidazolinone herbicides are metabolized by a different MFO, and MFO that metabolize imazethapyr is not sensitive to PBO. But, MFO that metabolize sulfonylurea herbicides is very sensitive to PBO. Certain corn hybrids, Pioneer 3377 IR, Ciba 4393 RSC, are not affected by the combination treatments of PBO or/ and terbufos. Because these corn hybrids changed the sensitivity at the site of action of sulfonylurea herbicides. Literature Cited . Anderson, P. C . , and M. Georgeson. 1989. Herbicide tolerant mutants of corn. Genome 31:994-999. . Anderson, P. C., M. A. Georgeson, K. A., and K. A. Hibberd. 1984. Cell culture of selection of herbicide resistant corn. Abstr. Crop Sci. Soc. p.56. . Barrett, M., and N. Polge. 1993. In-vitro nicosulfuron metabolism by com microsomal preparations. Abstr. Weed Sci. Soc. Amer. p. 86. . Bauman, T. T., M. D. K. Owen, and R. A. Liebl. 1992. Evaluation of Garst 85321T corn for herbicide tolerance. Abstr. Weed Sci. Soc. Amer. p.15. . Faulkner, J. S. 1982. Breeding herbicide tolerant crops cultivars by conventional methods. p.235-256. in H. M. LeBaron and J. Gressel eds. Herbicide resistance in plants. John Wiley & Sons, New York. 401 p. . Fery, R. L. and H. F. Harrison, Jr. 1990. Inheritance and assessment of bentazon tolerance in ’Santaka’ pepper (Gamicum annuum L.). J. Am. Soc. Hort. Sci. 115:854-857. . Gabard, J. M., P. J. Charest, V. N. Iyer, and B. L. Miki. 1989. Cross resistance to short residual sulfonylurea herbicides in transgenic tobacco plants. Plant Physiol. 91 :574-580. . Horsch, R. B., R. T. Fraley, S. G. Rogers, H. J. Klee, J. Fry, M. A. W. Hinchee, and D. S. Shah. 1988. Ambactcliurn-mediated gene transfer to plants; engineering tolerance to glyphosate. Iowa State J. Res. 62:487-502. . Mukaida, H., K. E. Diehl, R. A. Liebl, and L. M. Wax. 1993. Tolerance and acetolactate synthase activity of imidazolinone tolerant corn to imazethapyr and chlorimuron-ethyl. Abstr. Weed Sci. Soc. Amer. p. 64. 123 124 10. Multani, D. S., H. S. Dhaliwal, S. K. Sharma, and K. S. Gill. 1989. Inheritance of isoproturon tolerance in Dnnmj wheat transferred from Initicul mcnmcccum. Plant Breeding 102:166-169. 11. Newhouse K., B. Singh, D. Shaner, and M. Stidham. 1991. Mutations in corn (an may: L.) conferring resistance to imidazolinone herbicides. Theor. Appl. Genet. 83:65-70. 12. Rehab, 1. F., J. D. Burton, E. P. Maness, D. W. Monks, and D. A. RobinSon. 1993. Effect of safeners on nicosulfuron and primisulfuron metabolism in corn. Abstr. Weed Sci. Soc. Amer. 33:70. 13. Rowe, L., E. Rossman, and D. Penner. 1990. Differentialresponse of corn hybrids and inbreds to metolachlor. Weed Sci. 38:563-566. 14. Sebastian, G., M. Fader, J. F. Ulrich, D. R. Fomey, and R. S. Chaleff. 1989. Semidominant soybean mutation for resistance to sulfonylurea herbicides. Crop Sci. 29:1403-1408. 15. Siminszky, B., F. T. Corbin, and B. S. Sheldon. 1993. Nicosulfuron resistance mechanisms and metabolism in terbufos and/ or naphthalic anhydride treated corn. Abstr. Weed Sci. Soc. Amer. p. 112. 16. Souza-Machado, V., S. C. Phatak, and I. L. Nonecke. 1982. Inheritance of tolerance of tomato Wu c:culennrm Mill.) to metribuzin herbicide. Euphytica 31:129-138. 17. Wilcut, J. W., E. F. Eastin, J. S. Richburg, 111., W. K. Vencill, F. R. Walls, and G. Wiley. 1993. Imidazolinone systems for southern weed management in resistant corn. Abstr. Weed Sci. Soc. Amer. p. 5. 125 Table 1. The effect of PBO on the response of two corn hybrids to nicosulfuron and primisulfuron in the greenhouse 2 WAT. Great Lakes 584 Northrup King 9283 Treatment Rate Plant ht Fresh wt Plant ht Fresh wt (g/ ha) (cm/plant) (g/plant) (cm/plant) (cm/plant) Control - 65.7 ' 17.3 70.3 18.3 r PBO 4,000 67.0 16.7 62.5 16.2 Nicosulfuron 35 65.6 17.3 70.6 16.7 + PBO 35 + 1000 48.0 11.3 46.4 8.6 + PBO 35 + 500 60.1 16.8 55.6 13.1 b + PBO 35 + 333 59.0 16.1 60.2 13.9 Primisulfuron 35 63.5 16.7 67.0 16.4 + PBO 35 + 1000 51.8 12.3 48.5 11.4 + PBO 35 + 500 56.8 13.7 56.3 12.5 + PBO 35 + 333 63.4 15.4 62.9 15.3 LSD at 0.05 4.2 2.7 7.0 2.2 126 Table 2. The effect of MFO inhibitors on the response of imazethapyr resistance Pioneer 3377 IR corn hybrids to sulfonylurea herbicides in the greenhouse 2 WAT. Treatment Rate Plant ht Fresh wt (g/ha) (cm/plant) (g/plant) Control - 72.3 31.1 PBO 4000 74.7 33.8 PBO 2000 77.4 36.3 BHA 4000 77.9 35.5 BHA 2000 76.8 35.3 Nicosulfuron 35 74. 1 32.0 + PBO 35 + 2000 74.4 34.1 Primisulfuron 35 72.3 31.6 + PBO 35 + 2000 75.0 36.0 Imazethapyr 70 71.1 31.4 + PBO 70 + 4000 69.3 29.6 + PBO 70 + 2000 68.6 31.1 + BHA 70 + 4000 73.5 32.9 + BHA 70 + 2000 74.4 36.9 LSD at 0.05 N.S. 3.6 127 Table 3. The effect of PBO and terbufos on the responses of imazethapyr resistant and sensitive corn hybrids to nicosulfuron and primisulfuron herbicides in the greenhouse 2 WAT. PR‘ PS" 11‘ IS‘l Terbufos° Treatment Rate - + - + - + - + (g/ ha) A plant ht (cm/plant) Control - 63.3 63.1 62.1 61.6 62.5 61.8 59.8 57.6 PBO 2000 64.2 60.8 61.1 59.1 62.8 58.5 59.0 55.7 Nicosulfuron 35 59.0 57.7 59.9 51.0 62.7 58.1 57.3 51.1 + PBO 35 + 2000 57.3 57.5 38.0 30.6 51.5 30.4 37.9 28.5 Primisulfuron 40 61.2 58.8 54.5 42.2 56.3 50.9 52.9 42.8 + PBO 40 + 2000 58.1 58.8 39.2 31.4 51.7 32.5 43.4 28.6 LSD at 0.05 3.0 3.6 3.6 3.6 ‘ PR : Pioneer 33771R, imazethapyr resistant " PS : Pioneer 3377, imazethapyr sensitive ° IT : ICI 85321T, imazethapyr resistant ‘ IS : ICI 8532, imazethapyr sensitive ‘ Terbufos : Terbufos at 2.9 kg ha'1 applied PPI. 128 Table 4. The effect of PBO and terbufos on responses of imazethapyr resistant and sensitive corn hybrids to nicosulfuron and primisulfuron herbicides in the greenhouse 2 WAT. PR‘ PS" ITc IS‘I Terbufos‘ Treatment Rate - + - + - + - + (3/ ha) (96 injury) Control - 0 0 0 0 0 0 PBO 2000 0 0 0 0 0 0 Nicosulfuron 35 0 2 0 3 ‘ 0 2 l 5 + PBO 35 +2000 1 2 32 56 12 71 34 64 Primisulfuron 40 0 1 1 18 1 7 2 11 + PBO 40+2000 1 l 24 54 4 58 11 62 LSD at 0.05 2 6 5 5 ' PR : PiOneer 3377IR, imazethapyr resistant " PS : Pioneer 3377, imazethapyr sensitive ° IT : ICI 85321T, imazethapyr resistant " IS : ICI 8532, imazethapyr sensitive ° Terbufos : Terbufos at 2.9 kg ha" applied PPI. . 129 Table 5. The effects of PBO and MON 13900 on plant height of imazethapyr resistant and sensitive corn hybrids treated with MON 12000 in the greenhouse 2 WAT. Corn hybrids Treatment Rate PS‘ PR" 18‘ IT“ (g/ha) Plant ht (cm/plant) Control - 73.9 72.8 76.5 75.0 MON 12000 34.7 71.0 72.5 74.2 74.1 + PBO 34.7+2000 70.6 73.4 72.8 73.8 MON 12000 84.1 67.4 71.1 69.8 72.4 MON 13900 + PBO 84.1+2000 67.0 71.5 67.8 72.1 LSD at 0.05 5.7 N.S. 3.5 N .S. ‘ PS: Pioneer 3377, imazethapyr sensitive " PR: Pioneer 3377 IR, imazethapyr resistant ° IS: ICI 8532, imazethapyr sensitive " IT: ICI 8532 IT, imazethapyr resistant 130 Table 6. The effects of PBO, terbufos, and, the antidote R-29148 on plant height of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides at 2 WAT in the field‘ study in 1992. Corn hybrids Treatment Rate IT" 18‘ PR‘ PS0 Plant ht (cm/plant) Control - 63.4 64.3 62.0 64.9 Nicosulfuron 35 50.9 45.1 60.7 41.9 + PBO 35 + 2000 34.6 28.6 58.9 28.6 +PBO+R29148 35 + 2000 + 605 34.2 27.-7 58.1 27.2 Nicosulfuron 70 ' 49.0 39.3 57.9 39.0 I" Primisulfuron 40 50.3 44. 1 55 .6 39.2 + PBO 40 + 2000 36.7 27.0 59.7 28.5 +PBO+R29148 40 + 2000 + 605 32.7 27.4 54.5 27.2 Primisulfuron 80 48.6 41.8 61.4 38.2 Imazethapyr’ 70 58.4 28.7 58.3 28.4 Imazethapyr' 140 54.9 25.9 57.3 26.6 Thifensulfuron 4.4 . 56.8 49.9 57.2 45.2 LSD at 0.05 7.9 7.0 5.4 6.9 ‘ All plots were treated with terbufos 156 74.4 g ml 100 m row, in furrow, metolachlor 8 BC 2.2 kg/ha, Atrazine 4 F 1.1 kg/ha, and X-77 0.25% (v/v) " IT: ICI 85321T, imazethapyr resistant ° IS: ICI 8532, imazethapyr sensitive " PR: Pioneer 33771R, imazethapyr resistant ° PS: Pioneer 3377, imazethapyr sensitive ‘ Tank-mixed 28% UAN .1 _. E r 131 Table 7. The effects of PBO, terbufos, and the antidote R-29148 on visual injury of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides at 2 WAT in the field‘ study in 1992. Corn hybrids Treatment Rate 11* 18° PR‘ PS‘ ( % injury) Control - ‘ 0 0 0 0 Nicosulfuron 35 18 25 2 32 + PBO 35 + 2000 43 53 0 57 +PBO+R29148 35 + 2000 + 605 43 52 . 0 55 Nicosulfuron 70 20 30 2 42 Primisulfuron 40 25 32 2 38 + PBO 40 + 2000 43 55 0 60 +PBO+R29148 40 + 2000 + 605 48 50 0 60 Primisulfuron 80 23 27 0 35 Imazethapyrf 70 3 53 0 55 Imazethapyr' 140 3 63 0 60 Thifensulfilron 4.4 3 13 0 20 LSD at 0.05 11 10 2 13 ‘ All plots were treated with terbufos 15G 74.4 g pr/100 m row, in furrow, metolachlor 8 BC 2.2 kg/ha, and atrazine 4 F 1.1 kg/ha, and X-77 0.25% (v/v). " 1T: ICI 85321T, imazethapyr resistant ° 18: ICI 8532, imazethapyr sensitive " PR: Pioneer 33771R, imazethapyr resistant ‘ PS: Pioneer 3377, imazethapyr sensitive ‘ Tank-mixed 28% UAN 132 Table 8. The effects of PBO, terbufos, and the antidote R—29148 on plant height of imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides at 6 WAT in the field‘ study in 1992. Corn hybrids Treatment Rate TI" 18" PR‘I PS“ Plant ht (cm/plant) Control - 179.1 177.0 186.1 185 .0 Nicosulfuron 35 179.1 169.8 183.7 175.5 + PBO 35 + 2000 144.7 90.9 170.9 113.7 + PBO+R29148 35 + 2000 + 605 140.4 96.5 174.7 115.1 Nicosulfuron 70 167.8 160.9 183.4 150.0 Primisulfuron 40 167.7 151.0 174.2 159.5 + PBO 40 + 2000 138.5 97.7 175.5 125.5 + PBO+R29148 40’ + 2000 + 605 135.3 127.7 179.1 125.9 Primisulfuron 80 162.6 154.8 175.4 _ 155.1 Imazethapyr' 70 173.1 67.6 170.5 116.8 Imazethapyr‘ 140 164.9 33.1 172.7 63.2 Thifensulfuron 4.4 172.7 174.9 179.5 174.4 LSD at 0.05 10.6 23.4 11.9 28.8 ‘ All plots were treated preemergence with Terbufos 156 74.4 g pr/ 100 m row, in furrow, metolachlor 8 BC 2.2 kg/ha, atrazine 4 F 1.1 kg/ha, and X-77 0.25% .(v/v). " IT: ICI 85321T, imazethapyr resistant ° IS: ICI 8532, imazethapyr sensitive ‘ PR: Pioneer 3377IR, imazethapyr resistant ° PS: Pioneer 3377, imazethapyr sensitive ' Tank-mixed 28% UAN 133 Table 9. The effect of PBO, terbufos, and the antidote R-29148 on visual injury of imazethapyr resistant and sensitive corn hybrids treated ALS inhibitor herbicides at 6 WAT in the field' study in 1992. — Corn hybrids Control 0 O 0 0 Nicosulfuron 35 O O O 0 + PBO 35 + 2000 13 47 0 37 + PBO+R29148 35 + 2000 + 605 17 37 O 43 Nicosulfuron 70 O 3 O 7 Primisulfuron 4O 3 3 O 0 + PBO 40 + 2000 20 37 0 33 + PBO+R29148 40 + 2000 + 605 33 40 O 43 Primisulfuron 80 0 O O 3 Imazethapyr" 70 0 73 O 40 Imazethapyr' 140 O 93 O 77 Thifensulfuron 4.4 O O O O LSD at 0.05 10 . 14 NS 15 ' All plots were treated preemergence treatments with Terbufos 156 74.4 g pr/ 100 m row, in furrow, metolachlor 8EC 2.2 kg/ha, atrazine 4P 1.1 kg/ha, and X-77 0.25% (v/v). ' " IT: ICI 85321T, imazethapyr resistant ° IR: ICI 8532, imazethapyr sensitive " PR: Pioneer 3377IR, imazethapyr resistant ° PS: Pioneer 3377, imazethapyr sensitive " Tank-mixed 28% UAN Table 10. The effect of PBO, terbufos on imazethapyr resistant and sensitive com 134 hybrids treated with ALS inhibitor herbicides 2 WAT in a field' study in 1993. Corn hybrids Treatment Rate IT" IS° PR‘I PS° CRf CS‘ (g/ha) Plant m (cm/plant) Control - 73.9 75.3 85.6 82.9 85.2 84.0 POST Nicosulfuron 35 81.8 59.1 86.0 60.3 86.1 74.3 + PBO 35+2000 57.0 47.0 77.5 54.2 81.0 47.6 Nicosulfuron 70 68.5 54.8 77.5 55.2 87.0 55.0 Primisulfuron 40 71.9 77.8 77.8 74.2 78.7 72.1 + PBO 40+2000 61.9 60.7 75.4 72.6 81.8 39.0 Primisulfuron 80 77.9 73.5 76.4 71.2 86.8 70.4 Imazethapyr 70 76.1 33.5 75.2 36.1 78.9 38.7 Imazethapyr 140 81.2 31.8 72.6 31.0 73.9 28.3 Chlorimuron 12 33.6 32.7 80.4 36.6 81.7 31.6 PPI Chlorimuron 14 45.1 55.8 79.8 47.1 80.7 57.3 Chlorimuron 28 40.5 42.7 73.6 27 . 1 77.4 40.9 MON 12000 , 168 56.7 59.8 77.4 38.0 81.3 37.8 Flumetsulam/ 2417 70.0 60.5 72.2 67.7 79.9 44.8 metolachlor CGA-152005 40 61.7 60.9 72.2 57.3 72.0 44.0 Imazaquin 70 79.6 47.5 74.6 26.3 81.8 64.0 LSD at 0.05 17.5 21.7 9.5 19.0 10.0 20.4 WW furrow, metolachlor 8EC 2.2 kg/ha, atrazine 4F 1.1 kg/ha, and X-77 0.25% (v/v). " IT: ICI 8532 IT, imazethapyr resistant ° IS: ICI 8532, imazethapyr sensitive " PR: Pioneer 3377 IR, imazethapyr resistant ° PS: Pioneer 3377, imazethapyr sensitive ' CR: Ciba 4393 RSC, sulfonylurea resistant ‘ CS: Ciba 4393, sulfonylurea sensitive 135 Table 11. The effect of PBO, terbufos on imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides 2 WAT in a field‘ study in 1993. Corn hybrids Treatment Rate IT“ IS° PR‘ PS" CRf CS‘ (g/ha) Visual injury (96) Control ' - 0 0 0 0 0 0 POST Nicosulfuron 35 18 32 0 32 2 23 + PBO 35 +2000 37 57 2 43 5 55 Nicosulfuron 70 18 37 0 33 2 37 Primisulfuron 40 20 20 0 7 3 15 + PBO 40+2000 23 28 2 3 57 Primisulfuron 80 20 22 2 5 2 23 Imazethapyr 70 2 70 2 65 7 65 Imazethapyr 140 5 75 5 75 8 75 Chlorimuron 12 78 78 7 70 5 75 m . . Chlorimuron 14 48 33 0 42 3 40 Chlorimuron 28 48 52 23 47 2 42 MON 12000 168 32 32 3 50 5 47 Flumetsulam/ 2417 17 23 3 23 3 33 metolachlor - CGA-152005 40 27 23 15 33 7 38 Imazaquin 70 2 35 5 57 3 33 LSD at 0.05 20 16 15 24 6 21 mm furrow, metolachlor 8EC 2.2 kg/ha, atrazine 4P 1.1 kg/ha, and X-77 0.25% v/v. " IT: ICI 8532 IT, imazethapyr resistant ° IS: ICI 8532, imazethapyr sensitive " PR: Pioneer 3377 IR, imazethapyr resistant ° PS: Pioneer 3377, imazethapyr sensitive ' CR: Ciba 4393 RSC, sulfonylurea resistant ' CS: Ciba 4393, sulfonylurea sensitive Table 12. The effect of PBO, terbufos on imazethapyr resistant and sensitive com 136 hybrids treated with ALS inhibitor herbicides 4 WAT in a field‘ study in 1993. Corn hybrids Treatment Rate IT" ISc PR‘I PS° CR' CS‘ (g/ha) Plant ht (cm/plant) Control - 142.1 154.8 162.9 164.9 164.7 158.0 POST Nicosulfuron 35 157.2 133.6 161.4 147.0 164.5 155.5 + PBO 35+2000 123.9 83.9 148.1 137.3 160.8 114.0 Nicosulfuron 70 149.3 118.1 149.6 129.6 166.2 140.0 Primisulfuron 40 142.2 156.6 155.7 153.1 161.1 156.2 + PBO 40+2000 135.7 141.3 145.1 155.8 154.9 102.1 Primisulfuron 80 142.5 139.4 154.5 150.7 170.3 159.3 Imazethapyr 70 137.4 50.8 141.2 68.1 148.5 78.5 Imazethapyr 140 153.1 45.7 130.0 40.6 150.8 45.4 Chlorimuron 12 46.8 44.0 148.2 71.0 151.9 73.9 PPI Chlorimuron 14 94.7 136.7 156.7 108.6 160.1 106.7 Chlorimuron 28 74.5 91.0 141.6 52.1 155.9 95.4 MON 12000 168 114.2 106.2 144.0 93.4 145.9 79.6 Flumetsulam/ 2417 126.2 127.7 145.6 138.8 149.4 104.6 metolachlor CGA-152005 40 130.3 137.7 139.6 121.4 149.7 108.7 Imazaquin 70 149.4 105.5 145.9 65.5 154.5 129.6 LSD at 0.05 31.7 40.8 19.0 38.4 18.2 44.1 mm furrow, metolachlor 8EC 2.2 kg/ha, atrazine 4P 1.1 kg/ha, and X-77 0.25% v/v. " IT: ICI 8532 IT, imazethapyr resistant ° IS: ICI 8532, imazethapyr sensitive ‘ PR: Pioneer 3377 IR, imazethapyr resistant ‘ PS: Pioneer 3377, imazethapyr sensitive ' CR: Ciba 4393 RSC, sulfonylurea resistant ' CS: Ciba 4393, sulfonylurea sensitive 137 Table 13. The effect of PBO, terbufos on imazethapyr resistant and sensitive corn hybrids treated with ALS inhibitor herbicides 4 WAT in a field‘ study in 1993. Corn hybrids Treatment Rate IT" 18‘ PR‘I PS° CRf CS‘ (g/ha) Visual injury (96) Control - 0 0 0 0 0 0 POST Nicosulfuron 35 0 20 0 13 0 15 + PBO 35 +2000 23 45 0 17 0 33 Nicosulfuron 70 8 25 0 20 0 22 Primisulfuron 40 15 17 0 7 0 7 + PBO 40+2000 15 17 0 0 33 Primisulfuron 80 8 8 O 3 O 12 Imazethapyr 70 2 82 3 68 0 60 Imazethapyr 140 0 58 7 88 0 82 Chlorimuron 12 78 80 3 58 2 68 PPI Chlorimuron 14 18 23 0 37 0 32 Chlorimuron 28 45 47 7 72 0 48 MON 12000 168 37 35 7 47 0 52 Flumetsulam/ 2417 13 13 2 15 0 20 metolachlor CGA-152005 40 25 20 7 20 3 33 Imazaquin 70 5 35 0 65 2 33 LSD at 0.05 17 29 7 23 3 29 mm furrow, metolachlor 8EC 2.2 kg/ha, atrazine 4F 1.1 kg/ha, and X-77 0.25% v/v. " IT: ICI 8532 IT, imazethapyr resistant ° IS: ICI 8532, imazethapyr sensitive " PR: Pioneer 3377 IR, imazethapyr resistant ° PS: Pioneer 3377, imazethapyr sensitive ' CR: Ciba 4393 RSC, sulfonylurea resistant ‘ CS: Ciba 4393, sulfonylurea sensitive 138 Table 14. The effect of PBO on thifensulfuron tolerance by two soybean varieties in the greenhouse 2 WAT. Elgin ’87 W20-STS Treatment Rate Plant ht Visual injury Plant ht Visual injury (g ail ha) (cm/plant) (96) (cm/plant) (96 ) Control 15 .3 0 13.0 0 PBO 2000 15.4 0 13.0 0 Thifensulfuron 8.8 15.4 0 12.8 _+ PBO 8.8+2000 14.5 0 13.6 Thifensulfuron 17 .5 15.4 0 13. 1 0 + PBO 17.5-+2000 14.1 30 12.7 Thifensulfuron 35 15.8 0 12.9 + PBO 35+2000 11.0 44 11.9 LSD at 0.05 1.0 4 1.1 2 139 Table 15. The effects of PBO and BHA on imazethapyr tolerance of Elgin ’87 in the greenhouse 2 WAT. Treatment Rate Plant ht Fresh wt ( g ai/ha) (cm/plant) (s/plant) Control 11.4 5.0 PBO 4000 11.5 5.7 BHA 4000 11.3 5.6 Imazethapyr 70 11.2 4.7 + PBO 70 + 2000 11.3 4.8 + PBO 70 + 4000 11.9 5.3 + BHA 70 + 2000 11.5 4.6 + BHA 70 + 4000 11.1 5.0 Imazethapyr 105 11.3 5.1 + PBO 105 + 2000 11.8 4.8 + PBO 105 + 4000 11.5 5.0 + BHA 105 + 2000 11.3 5.0 + BHA 105 + 4000 12.0 6.1 LSD at 0.05 N.S. 0.9 Chapter 5 Response of a Chlorsulfuron-Resistant Biotype of Mia mm to ALS Inhibitor Herbicides and Piperonyl Butoxide ABSTRACT Greenhouse studies were conducted to determine kochia resistance to a spectrum of ALS-inhibiting herbicides. The chlorsulfuron resistant biotype was resistant to six herbicides; triflusulfuron, thifensulfuron, MON 12037, imazamethabenz, chlorsulfuron, and nicosulfuron. But, the resistant biotype showed sensitivity similar to the susceptible biotype to three herbicides; metsulfuron, imazethapyr, and imazaquin. The resistant biotype was slightly less sensitive to primisulfuron, chlorimuron and flumetsulam than the sensitive biotype. Addition of a mixed function oxidase inhibitor, PBO at 2 kg/ha, to primisulfuron and thifensulfuron increased visual injury and reduced plant height of chlorsulfuron sensitive kochia biotype. And PBO tank-mixing to primisulfuron enhanced R biotype control at low rate of primisulfuron. Nomenclature: chlorimuron, 2-[[[[(4—chloro—6-methoxy—2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] benzoic acid; chlorsulfuron, 2-chloro-N—[[(4-methoxy- 14o 141 6-methyl-l ,3 ,5-triazin-2-yl) amino] carbonyl] benzenesulfonamide; flumetsulam, N - [2,6-difluorophenyl] -5-methyl( 1 ,2,4) triazolo-[l ,5a] -pyrimidine-2-sulfonamide; imazamethabenz, (i)-2-[4,5-dihydro-4-methyl]-4-(1-methylethyl)-5-oxo—1H- imidazol-2-y1]-4(and 5)-methylbenzoic acid(3 :2); imazaquin, 2-[4,5-dihydro—4~ methyl—441-methylethyl)-5-oxo—1H-imidazol-Z—yl]-3-quinolinecarboxylic acid; imazethapyr, 2-[4,5-dihydro—4-methyl-4-(l-methylethyl)-5-oxo—lH—imidazol-Z—yl]-5- ethyl-3-pyridinecarboxylic acid; metsulfuron, 2-[[[[(4-methoxy—6-methyl-1,3,5- triazin-2—yl) amino] carbonyl] amino] sulfonyl] benzoic acid; MON 12037, methyl 3—chloro-5-(4,6-dimethoxypyrimidin—2-ylcarbamoyl sulfamoyl)—l-methylpyrazole—4~ carboxylate; nicosulfuron, 2-[[[[(4,6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl]-H,H—dimethyl-3-pyridinecarboxamide; primisulfuron, 2-[[[[[4,6- bis (difluoromethoxy)-2—pyrimidinyl] amino] carbonyl] amino] sulfonyl] benzoic acid; Thifensulfuron, 3-[[[[(4-methoxy-6-methyl-l ,3,5-triazin-2—yl) amino] carbonyl] amino] sulfonyl] -2-thiophenecarboxylic acid; triflusulfuron, 2—[[[[[4— (dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2—yl] amino] carbonyl]- amino] sulfonyl] -3 -methylbenzoic acid; piperonyl butoxide (PBO) , a-(2-(2- butoxyethoxy) ethoxy)-4,5-methyl enedioxy—Z-propyltolune; kochia, K h' m (L.) Schrad. Additional index words: Cross resistance, MFO inhibitor, interaction. INTRODUCTION Repeated use of herbicides with the same mode of action on the same site has been implicated in the emergence of resistant weed populations. Resistance to pesticides is a world-wide phenomenon and exists for fungicides, insecticides, and herbicides (5). Since 1970, herbicide resistance has become well known in scientific and agricultural communities (15). Newer types of herbicides with high specific activity are sulfonylureas and imidazolinones. The mode of action of these compounds have been demonstrated to be the inhibition of acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS) (2,12,17). Recently, chlorsulfuron-resistant biotypes of four weed species have appeared in the USA, Canada, and Australia. These include prickly lettuce (1am 32111913 L.), kochia (Emilia maria (L.) Schrad), and Russian thistle (Sflsgla ibefiga Sennen & Pan), in the US, chickweed (Slim 11153113 (L.) Vill), in Canada, and rigid ryegrass (mm LigidJan Gaud.), in Australia (3,4,7,9,10,11, 16). Many reports concluded that the mechanism for sulfonylurea resistance is a less sulfonylurea-sensitive ALS enzyme (3,11,13,16). Devine et al. (3) reported that the altered ALS in chlorsulfuron resistance-biotypes did not confer the same level of resistance to other ALS-inhibiting herbicides. Also, 142 143 Primiani et al. (10) reported that the degree of cross resistance of a chlorsulfuron resistant kochia biotype to ALS-inhibiting herbicides varied. Piperonyl butoxide (PBO), a widely used as a pesticide synergist, has been effective with many organophosphate, carbamate, and pyrethroid insecticides, increasing insecticidal activity against resistant insects (1,8). PBO has shown potential to increase activity of several herbicides, such as EPTC (S-ethyl dipropyl carbamothioate) (6), and bentazon (3-(1-methylethy1)-(ltD-2,l ,3-benzothiadiazin- 4(3I-D-one 2,2-dioxide) (14). The objectives of this study were a) to determine the cross-resistance of chlorsulfuron resistant kochia biotype to various ALS-inhibiting herbicides, and b) to evaluate potential synergistic effects of PBO with sulfonylurea herbicides to the chlorsulfuron—resistant kochia biotype. MATERIALS AND METHODS Plant Materials. Seeds of kochia resistant and sensitive to chlorsulfuron were obtained from du Pont de Nemours & Co., Inc. Seeds were planted in 27 by 53 cm plastic boxes which contained BACCTO soil mix, and germinated in the greenhouse. The plants were grown at 24 C j; 2 C with supplemental lighting from high pressure sodium lights to provide a midday light intensity of 1200 p E m'zs" for both supplemental and natural light. The day length was 18 h. After emergence, the 2-cm seedlings were transplanted one per pot to 945-ml plastic pots containing BACCTO soil. Uniform plants 4 to 5 cm tall were selected for postemergence herbicide treatments. Chemical Treatments. To determine the cross-resistance to various ALS inhibiting-herbicides, nicosulfuron (70 g/ha), primisulfuron (80 g/ha), thifensulfuron (8.8 g/ha), chlorimuron (24 g/ha), metsulfuron (8.4 g/ha), chlorsulfuron (28 g/ha), imazethapyr (135 g/ha), imazamethabenz (1008 g/ha), imazaquin (280 g/ha), MON 12307 (100 g/ha), flumetsulam (100 g/ha), and triflusulfuron (35 g/ha) herbicides were applied to both kochia biotypes. All treatments were applied as 144 145 postemergence, and included X-77l (0.25 % v/v). All herbicide treatments were applied with a flat-fan 80025 nozzle in a spray volume of 280 L/ha at 240 kPa using a chain link-belt compressed air sprayer. To evaluate PBO effect on ALS inhibiting herbicide on the growth of chlorsulfuron-resistant and —sensitive kochia biotypes, thifensulfuron (1.1 and 4.4 g/ha) and primisulfuron (20 and 40 g/ha) herbicides were applied to both kochia biotypes with 2 kg/ha PBO. Data Analysis. Plant height and visual injury ratings were evaluated 14 days after postemergence treatments. Data presented are the means of two experiments with four replication in each. Experiments were conducted as a completely randomize design. Two (herbicide, biotype) and three (treatment, PBO, biotype) factorial design were used in cross-resistance and PBO effects experiments. Means were separated by LSD Test at the 5 % level using the MSTAT program. lX-77 nonionic surfactant is a mixture of alkylarylpolyoxyethylene glycols, free fatty acids, and isopropanols marketed by Valent U.S.A'. Corp., 1333 N. California Blvd., Walnut Creek, CA 94596. RESULTS AND DISCUSSIONS Cross-resistance study. All two factors, kochia biotypes and herbicides, affected the response on plant height and visual injury. All ALS inhibiting-herbicides applied at two times the usual use rates reduced plant height, and caused more than 50% injury to the chlorsulfuron sensitive kochia biotype (Table 1). Response of this sensitive kochia biotype to the various ALS-inhibiting-herbicides, ranked from the highest to the lowest level of sensitivity, imazethapyr > chlorsulfuron > metsulfuron _>_ flumetsulam Z nicosulfuron, primisulfuron 2 imazaquin 2 MON 12037 _>_ thifensulfuron _>_ chlorimuron 2 triflusulfuron Z imazamethabenz. Application of imazethapyr and metsulfuron at two times field use rates controlled chlorsulfuron resistant kochia 99 % , and 89 % ,respectively. The chlorsulfuron resistant biotype was very resistant to thifensulfuron, MON 12037, and triflusulfuron herbicides applied at two times the usual use rates causing less than 10 % injury. The magnitude of resistance of the chlorsulfuron-resistant kochia biotype to ALS-inhibiting herbicides ranked from the highest to the lowest levels, triflusulfuron, thifensulfuron Z MON 12037 > imazamethabenz _>_ chlorsulfuron 2_ nicosulfuron 2 chlorimuron _>_ primisulfuron > imazaquin, flumetsulam 2 metsulfuron _>_ imazethapyr (Table 1). . 146 147 Primiani et al. (4) found that the 50% growth reduction (GRSO) value of chlorsulfuron and metsulfuron to the chlorsulfuron resistant kochia was 30 and 8 times higher than that of susceptible kochia biotype, respectively. Reed et al. (13) reported that 2 and 46 g/ha of metsulfuron were needed to obtain 90% control of susceptible and resistant kochia, respectively. The results cannot be compared to each other directly, due to the difference in methods. But, other researchers reported that the chlorsulfuron resistant kochia was more resistant than the susceptible biotype to metsulfuron, whereas this study showed the same responses, 90% and 89% injury, for both biotypes to metsulfuron. Either the metsulfuron rate was too high or the mutation was different. The kochia response to chlorsulfuron in this study was similar to that reported by others (10,13,16). Friesen et al. (4) ranked the levels of chlorsulfuron resistance of kochia as following ; thifensulfuron > > chlorsulfuron > imazethapyr > metsulfuron. The two most effective herbicides, imazethapyr and metsulfuron, showed no differential herbicide activity to the both biotypes in this study. The mutation in one particular ALS gene affects the binding of each ALS inhibiting herbicide differently and it is not possible to predict degrees of cross-resistance to other herbicides acting on the same target enzyme. According to Sivakumaran et al. (18), the magnitudes of ALS resistance and cross-resistance were highly variable among kochia populations. They explained that the differences might be due to the type of mutation in the gene encoding ALS. 148 PBO effect on the activity of two sulfonylurea herbicides to the kochia biotypes. All three factors, kochia biotypes, herbicide treatment and PBO, affected differential responses on the growth of kochia biotypes. Application of PBO at 2 kg/ha alone had no effect on the growth of either biotypes of kochia (Table 2). The herbicide treatments, 1.1 and 4.4 g. ai/ha of thifensulfuron, and 20 and 40 g ai ha of primisulfuron, reduced plant height of both chlorsulfuron resistant and sensitive biotypes. PBO tank-mixed to primisulfuron at 20 g/ha enhanced visual injury of R biotype from 44 (no PBO) to 71% (with PBO). The chlorsulfuron resistant biotype appeared more sensitive to primisulfuron than the thifensulfuron. Addition of PBO increased visual injury indicating that both herbicides are metabolized by a mixed function oxidase sensitive to PBO (Table 2). The addition of PBO to sulfonylurea herbicides may provide farmers with a tool to increase activity of these herbicides against kochia. LITERATURE CITED . Attia, F. I., G. J. Shanahan, and E. Shipp. 1980. Synergism studies with organophosphorous resistant strains of the Indian meal moth. J. Econ. Entomol. 73:184-185. . Chaleff, R. S., and C. J. Mauvais. 1984. Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants. Science 224: 1443- 1445. F . Devine, M. D., M. A. S. Marles and L. M. Hall. 1991. Inhibition of acetolactate synthase in susceptible and resistant biotypes of Maria madia. Pestic. Sci. 31:273-280. . Friesen, L. F., I. N. Morrison, A. Rashid, and M. D. Devine. 1993. Response of a chlorsulfuron-resistant biotype of m maria to sulfonylurea and alternative herbicides. Weed Sci. 41 : 100-106. . Georghiou, G. P. 1986. The magnitude of the resistance problem. p.14—43 in Pesticide resistance: strategies and tactics for management. Natl. Acad. Press. Washington, DC. . Komives, T., and F. Dutka. 1980. On the mode of action of EPTC and its antidotes on corn. Cereal Res. Commun. 8:627-633. . Mallory-Smith, C. A., D. C. Thill, and M. J. Dial. 1990. Identification of sulfonylurea herbicide-resistant prickly lettuce (Laying serrigla). Weed Technol. 4:163-168. . O’Brien, R. D. 1967. Insecticides action and metabolism. Academic Press, New York, NY, Chapter 10, Pyrethroids. . Powles, S. B., and P. D. Howat. 1990. Herbicide-resistant weeds in Australia. Weed Technol. 4:178-185. 149 10. ll. 12. 13. 14. 15. l6. 17. 18. 150 Primiani, M. M. J. C. Cotterman, and L. L. Saari. 1990. Resistance of kochia Mia ma) to sulfonylurea and imidazolinone herbicides. Weed Technol. 4: 169-172. Rathinasabapathi, B. and J. King. 1991. Herbicide resistance in mm m. Plant Physiol. 96: 255-261. Ray, T. B. 1984. Site of action of chlorsulfuron inhibition of valine and isoleucine biosynthesis in plants. Plant Physiol. 75:827-831. Reed, W. T., J. L. Saladini, J. C. Cotterman, M. M. Primiani, L. L. Saari. 1989. Resistance in weeds to sulfonylurea herbicides. Brighton Crop Protection Conf. weeds :295-300. Rubin, B., J. R. Leavitt, D. Penner, and A. W. Saettler. 1980. Interaction of antioxidants with ozone and herbicide stress. Bull. Environ. Contam. Toxicol. 25:623-629. Ryan, G. F. 1970. Resistance of common groundsel to simazine and atrazine. Weed Sci. 18:614-616. Saari, L. L., J. C. Cotterman, and M. M. Primiani. 1990. Mechanism of sulfonylurea herbicide resistance in the broadleaf weed, Kmhia scaparia. Plant Physiol. 93:55-61. Shaner, D. L., P. C. Anderson, and M. A. Stidham. 1984. Imidazolinones potent inhibitors of acetohydroxyacid synthase. Plant Physiol. 76:545-546. Sivakumaran, K., D. Mulugeta, P. K. Fay, and W. E. Dyer. 1993. Differential herbicide response among sulfonylurea-resistant Emilia Ma L. accessions. Weed Sci. 41:159-165. 151 Table 1. The responses of chlorsulfuron-resistant and sensitive biotypes of kochia to the ALS inhibiting-herbicides in the greenhouse study 2 WAT. Plant height Visible injury Treatment Rate S biotype R biotype S biotype R biotype (g ail ha) ----- (cm/plant) -------------- (%) ------- Control 22.8 21.3 0 0 Nicosulfuron 70 6.3 9.9 84 46 Primisulfuron 80 5.9 6.9 84 66 Thifensulfuron 8.8 8.9 18.3 66 3 Chlorimuron 23 9.3 10.3 62 51 Metsulfuron 8.4 5.6 5.6 90 89 Chlorsulfuron 28 4.9 12.9 96 38 Imazethapyr 135 5 .0 4.6 100 99 Imazamethabenz 1008 10.3 13.2 50 27 Imazaquin 280 7. 1 5.9 74 73 MON 12037 100 7.8 17.1 71 9 Flumetsulam 100 6.4 6.2 89 74 Triflusulfuron 35 9.3 21 .4 56 3 LSD at 0.05 2.9 18 152 Table 2. The responses of chlorsulfuron-resistant and sensitive biotypes of kochia to the interaction of PBO and sulfonylurea herbicides in the greenhouse 2 WAT. Plant height Visual injury Mmem Rate 8 biotype R biotype S biotype R biotype (g ai/ha) --- (cm/plant) --- ------ (%) ------ Control 26.4 24.9 PBO 2000 26.3 24.4 Thifensulfuron 1.1 17.8 21.3 21 3 " + PBO 1.1-+2000 14.9 21.3 45 6 Thifensulfuron 4.4 ' 13.5 19.3 43 10 " + PBO 4.4+2000 11.0 16.3 58 19 Primisulfuron 20 1 1.6 1 1.9 52 44 " + PBO 20+2000 7.9 7.8 79 71 Primisulfuron 40 11.0 9. 8 58 4 59 " + PBO 40+2000 8.8 6.3 76 77 LSD at 0.05 4.4 21 153 SUMMARY AND CONCLUSION The effects of antidotes, mixed function oxidases (MFO) inhibitors, insecticides, and corn hybrids on the activity of acetolactate synthase (ALS) inhibiting herbicides were evaluated in greenhouse and field studies. Chlorsulfuron-resistant kochia was evaluated in greenhouse for cross-resistance. Northrup King 9283 and Cargill 7567 hybrids were sensitive to the interaction of chlorimuron, nicosulfuron and primisulfuron with terbufos. Cargill 7567 hybrid corn was more tolerant to acetanilide herbicides and to the interactions of sulfonylurea herbicides with terbufos than Northrup King 9283 . But, there was no interaction of imazaquin with terbufos to corn hybrids. The antidotes, CGA- 154281 and NA reduced corn injury from metolachlor, nicosulfuron and primisulfuron applied with or after terbufos treatment, respectively. NA stimulates MFO activity and can partially overcome the sulfonylurea herbicide- terbufos interaction. The combination of primisulfuron and terbufos did not enhance herbicidal activity to weed species. Tank-mixed piperonyl butoxide (PBO) enhanced nicosulfuron and thifensulfuron activity on barnyardgrass, velvetlcaf and common lambsquarters, respectively. Also, butylated hydroxyanisole (BHA) and PBO enhanced nicosulfuron and primisulfuron activity on common lambsquarters and green 154 foxtail. All three factors, PBO, nonionic adjuvants and 28 % UAN, enhanced activity of nicosulfuron on common lambsquarters, velvetlcaf, barnyardgrass, and primisulfuron on giant foxtail and velvetlcaf, and thifensulfuron on common lambsquarters and velvetlcaf. But, addition of 28 % UAN to primisulfuron did not enhance activity to common lambsquarters and barnyardgrass. Effective adjuvants with nicosulfuron were K-3000 on common lambsquarters, and SYLGARD 309 on velvetlcaf, K-2000, K-3000 and SCOIL on barnyardgrass. Effective adjuvant with primisulfuron were K—2000, SCOIL, and SYLGARD 309 on giant foxtail, X- 77, K-2000, K-3000, SCOIL, and SYLGARD 309 on velvetlcaf, K-3000 and SYLGARD 309 on common lambsquarters. Effective adjuvants for thifensulfuron were SCOIL on common lambsquarters, and SCOIL and SYLGARD 309 on velvetlcaf. PBO at 0.33 kg/ha tank-mixed with nicosulfuron and primisulfuron caused injury to the Northrup King 9283 corn hybrid. Pioneer 3377 IR corn hybrid was tolerant to the combination of nicosulfuron, primisulfuron plus PBO 2 kg/ha, and also to the combination treatments of imazethapyr or thifensulfuron with terbufos. In the field study, Pioneer 3377 IR and Ciba 4393 RSC hybrids showed cross- resistance to sulfonylurea and imidazolinone herbicides even treatments with PBO regardless of the presence of terbufos. ICI 8532 IT was cross-resistant to thifensulfuron and imidazolinone herbicides plus terbufos. 155 The combination of thifensulfuron with PBO caused injury to Elgin ’87 soybean, but the W20-STS soybean was tolerant to this combination treatment. Combination of imazethapyr with PBO or BHA had no effect on the growth of Elgin ’87 soybean variety. The chlorsulfuron resistant-kochia biotype was resistant to six herbicides: triflusulfuron, thifensulfuron, MON 12037, imazamethabenz, chlorsulfuron, and nicosulfuron. But, the resistant kochia biotype showed sensitivity similar to the susceptible biotype to three herbicides: metsulfuron, imazethapyr, and imazaquin. Addition of PBO at 2 kg/ha to primisulfuron and thifensulfuron increased injury and reduced plant height of chlorsulfuron resistant kochia biotype. From the my research, I would like to recommend several methods to protect corn from the interaction of ALS-inhibiting herbicides with corn insecticides, and to enhance activity of these herbicides. First, selection of an appropriate corn hybrid, resistant to the interaction of ALS-inhibiting herbicides with terbufos can protect corn from interaction effects. The degree of corn injury from the interaction of ALS-inhibiting herbicides with corn insecticides showed greatly variability with hybrid. Use of Pioneer 3377 IR and Ciba 4393 RSC hybrids appear to allow use of sulfonylurea herbicides POST with terbufos, the use of imazethapyr, and the use of PBO. Also, the W20-STS soybean appear to allow use of thifensulfuron plus PBO to increase weed control. Second, I would recommend the addition of PBO and/or 28% UAN to 156 sulfonylurea herbicides to enhance wwd control. Tank-mixing PBO and/or 28 % UAN to certain sulfonylurea herbicides increased the activity on several weed species, especially broad-leaf weed species. Third, herbicide activity can be increased by the selection of an appropriate adjuvant. Efficacy of the nonionic adjuvants was herbicide and weed specific. Fourth, a weed biotype resistant to one inhibitor of ALS may be effectively controlled by other inhibitors of ALS. APPENDIX : Analyst of Variance Table 157 Chapter 2. Table 1. _ K Sum of Mean F Value Source DF Squares Square Value 1 Rep. 7 1033 148 2.7 2 Hybrid 1 2642 2642 48.5 4 Insect. 2 122 61 1.1 6 H x l 2 136 68 1.2 8 Herb. 6 22547 3757 68.9 10 H x H 6 4472 745 13.7 12 I x H 12 704 59 1.1 14 11:1le 12 468 39 .7 -15 Error 287 15646 55 Total 335 47771 c.v.: 18.58% 158 Table 2. K Sum of Mean P Value Source DP Squares Square Value 1 Rep 7 969 138 0 6 2 Hybrid 1 28527 28527 118.7 4 Insect 2 161 80 0 3 6 H x l 2 740 370 1.5 8 Herb. 6 101176 16863 70.2 10 H x H 6 25341 4223 17.6 12 I x H 12 2281 190 0.8 14 HxIxH 12 1535 128 0.5 -15 Error 287 68981 240 1 1 Total 335 47771 c.v.: 90.69 159 K Sum of Mean P Value Source DF Squares Square Value 1 Rep. 7 969 138 0.6 2 Hybrid 1 28527 28527 1 18.7 4 Insect. 2 161 80 0.3 6 H x l 2 740 370 1.5 8 Herb. 6 101176 16863 70.2 10 H x H 6 25341 4223 17.6 12 I x H 12 2281 190 0.8 14 HxIxH 12 1535 128 0.5 -15 Error 287 68981 240 Total 335 47771 Table 5. K Sum of Mean F Value Source DF Squares Square Value — 1 Rep 7 325 46 l 5 2 Hybrid 1 453 453 14.5 4 Antidote 1 5472 5472 175 .6 6 HA 1 366 366 1 1.7 8 Insect. 1 17537 17537 562.9 10 HI 1 339 339 10.9 12 AI 1 3545 3545 113.8 14 HA] 1 313 313 10.0 16 Herbi 2 19252 9626 309.0 18 an 2 ' 378 189 6.1 20 AH 2 3561 1781 57 .2 22 HAH 2 180 90 2.9 24 [H 2 14049 7024 225 .5 26 HIH 2 354 177 5 .7 28 AIH 2 1859 929 29.8 30 HAIH 2 232 1 16 3 7 ~31 Error 161 5016 31 Total 191 73230 c.v.: 52.15 5 161 Chapter 3. Table 6. Visual injury. K Sum of Mean F Value Source DF Squares Square Value 2 Herbicide 2 2605 1303 16.3 4 PBO 4 74868 18717 233.6 6 Herb x PBO 8 60251 7531 94.0 -7 Error 105 8413 80 c.v.: 19.39% Table 7. Visual injury. K Sum of Mean F Value Source DF Square Square Value -- . - ,-,_,A____ . - , A 7777777 — —nA~A—- A — “—4— m 2 Herbicide 2 2383 1 191 24.5 4 PBO 4 56672 14168 291.3 6 Herb. x PBO 8 45775 5722 117.7 -7 Error 105 5106 49 c.v.: 17.58% 162 Table 8. Visual injury. K ’ Sum of Mean P Value Source DF Squares Square Value — 2 P30 3 2148 716 7.0 4 Adjuvant - 5 249166 49833 485 6 PA 15 11324 755 7.3 8 Nitrogen 1 2395 2395 23.3 10 PN 3 299 100 1.0 12 AN 5 2143 429 4.2 14 PAN 15 4908 327 3.2 -15 Error 336 34551 103 o 1‘ .3 8 38 Table 9. Visual injury K Sum of Mean F Value Source DF Squares Square Value _ 2 PBO 3 6955 2318 12.3 4 Adjuvant 5 178043 35609 189.6 6 PA 15 7457 497 2.7 8 Nitrogen 1 18634 18634 99.2 10 PN 3 793 264 1.4 12 AN 5 4443 889 4.7 14 PAN 15 3343 223 1 .2 -15 Error 336 63103 188 c.v. : 22.30% 163 Table 10. Visual injury. K Sum of Mean F Value Source DP Squares Square Value — 2 PBO 3 952 317 7.7 4 Adjuvant 5 244766 48953 1183.3 6 PA 15 6496 433 10.5 8 Nitrogen 1 6017 6017 145.4 10 PN 3 171 57 1 4 12 AN 5 1W5 2013 48 7 14 PAN 15 1217 81 2.0 -15 Error 336 13900 41 c.v. : 11.15% Table 11. Visual injury K Sum of Mean P Value Source DF Squares Square Value — 2 PBO 3 5833 1945 31.7 4 Adjuvant 5 138244 27649 451 .2 6 PA 15 19663 1311 21.4 8 Nitrogen 1 1258 1258 20.5 10 PN 3 81 27 0.4 12 AN 5 313 63 1 0 14 PAN 15 662 44 0 7 -15 Error 336 20591 61 c.v. : 12.13% K Sum of Mean P Value Source DF Squares Square Value _ 2 PBO 2 565 282 0.8 4 Adjuvant 6 17004 2834 7.7 6 PA 12 18652 1554 4.3 8 Nitrogen 1 107 107 0.3 10 PN 2 833 416 1.1 12 AN 6 4215 703 1 .9 l4 PAN 12 7451 621 1.7 ~15 Error 294 107638 366 164 c.v. : 25.77% Table 13. Visual injury. K Sum of Mean P Value Source DF Squares . Square Value — 2 PBO 2 24810 12405 79.7 4 Adjuvant 6 86033 14339 92.2 6 PA 12 29462 2455 15.8 8 Nitrogen 1 62022 62022 398.7 10 PN 2 7484 3742 24.1 12 AN 6 225 10 3752 24. 1 l4 PAN 12 16242 1353 8.7 -15 Error 294 45741 156 . c.v. : 16.32% Table 14. Plant height. K Sum of Mean P Value Source DF Squares Square Value — 2 PBO 2 1807 904 12.9 4 Adjuvant 6 11022 1837 26.3 6 PA 12 3772 314 4.5 8 Nitrogen 1 85 85 1.2 10 PN 2 114 57 0.8 12 AN 6 316 53 0.8 14 PAN 12 445 37 0.5 -15 Error 294 20574 70 c.v. : 8.67% Table 15. Visual injury. K Sum of Mean P Value Source DP Squares Square Value _ 2 P80 2 1 1884 5942 24.0 4 Adjuvant 6 24645 4108 16.6 6 PA 12 4518 376 1 .5 8 Nitrogen 1 1189 1189 4.8 10 PN 2 48 24 0. 1 12 AN 6 1 133 189 0.8 14 PAN 12 860 72 0.3 —15 Error 294 72818 248 c.v. : 30.02% 165 ”HE—a m n. fit 166 Table 16. Visual injury. K Sum of Mean P Value Source DP Squares Square Value _ 2 P80 2 17668 8834 132 4 Adjuvant 6 39722 6620 99.3 6 PA 12 34674 2889 43.3 8 Nitrogen 1 1277 1277 19. 1 10 PN 2 648 I 324 4.9 12 AN 6 2248 375 5.6 14 PAN 12 1216 101 1.5 if -15 Error 294 19609 67 K Sum of Mean F Value Source DF Squares Square Value — 2 P80 2 1 1318 5659 45 .3 4 Adjuvant 6 48795 8132 65 . 1 6 PA 12 10274 856 6.9 8 Nitrogen 1 123050 123050 985. 1 10 PN 2 2622 A 131 1 10.5 12 ' AN 6 13161 2194 17.6 14 PAN 12 3254 271 2.2 -15 Error 294 36725 125 c.v.: 17.19% 167 Chapter 5. Table 1. Visual injury. K Sum of Mean P Value Source DP Squares Square Value — 2 Biotype 1 . 37156 37156 106.0 4 Herbicide 12 151911 12659 36.1 6 BH 12 ' 31185 2599 7.4 -7 Error 182 63974 351 K Sum of Mean P Value Source DP Squares Square Value — 2 Biotype 1 8023 8023 18.3 4 Herbicide 4 103750 25938 59.0 6 BH 4 9300 2325 5 3 8 PBO 1 7659 7659 17.4 10 BP 1 328 328 0.8 12 HP 4 3003 751 1.7 14 BHP 4 695 174 0.4 -15 Error 140 61538 440 c.v.: 58.33% "7111111111111114111111“