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' 1 ngL'111E-AIV11ifi‘fi 1. g-1v3“:511%.;3:2£_11'1IL2:111§111+ 1 -. - .«I. “21.7.;31119: 1I4L‘L41Lw1znf 1,- ' liki-‘LfiJ '.,ILI1'-1.11'111!¢ I) ”II ;1II|hS'1:..:'KIIII .1131.“ ‘—~ . ’5‘ v' 'F’n 11 ., ’ "*9 T155 ' WV 19' 3219 :?'«’1~5‘2",'I!‘ . 191;. a gym; 5% X . .—».~on.’o',’.‘ ‘ ‘ .. ‘;~':?T‘, ~'".$.—"J.ll"..)- ~~‘ - .0 _‘ a . - .4 . . ,~ V Z, {X . t 1 ~.' : 1'"? q, “L Li, .21.} .1141: aux it y THESIS This is to certify that the dissertation entitled Efficacy of CGAr43089 E1-(Cyanomethoximino)-Benzacetonitrile) As A Herbicide Antidote for Sorghum (Sorghum Vulgare Pers.) presented by Gary Lynn Leek has been accepted towards fulfillment of the requirements for Ph.D. degreein Crop & Soil Sciences Major professor Date November 5, 1981 MS U is an Affirmative Action/Equal Opportunity Institution 0-1277! MSU LIBRARIES m RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. EFFICACY 0F GSA-43089 [a-(CYANOMETHOXIMINO)-BENZACETONITRILE] AS A HERBICIDE ANTIDOTE FOR SORGHUM (SORGHUM VULGARE PERS.) By Gary Lynn Leek A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Science 198] ABSTRACT EFFICACY OF CGA-43089 [a-(CYANOMETHOXIMINO)-BENZACETONITRILE] AS A HERBICIDE ANTIDOTE FOR SORGHUM (SORGHUM VULGARE PERS.) By Gary Lynn Leek Seed treatment with CGA-43089 [a-(cyanomethoximino)-benzaceto— nitrile] provided protection to sorghum (Sorghum vulgare Pers.) against the herbicides metolachlor [2-chloro-N:(2-ethyl-6-methyl- phenyl)-flf(2-methoxy-l-methylethyl)acetamide], alachlor [Z-chloro- 2',6'-diethyl-N:(methoxymethyl)acetanilide], diethatyl [N:(chloro- acetyl)-N;(2,6-diethylphenyl)glycine], ethofumesate [(f)-2-ethoxy-2,3- dihydro-3,3-dimethyl-5-benzofuranyl methanesulfonate], butylate + R-25788 [(Sfethyl diisobutylthiocarbamate) + (N,N;diallyl-2,2-dichloro- acetamide)], and atrazine + metolachlor [2-chloro-4-ethylamino-6- isopropylamino-§:triazine + Z-chloro-N;(2-ethyl-6-methylphenyl)-Nr(2- methoxy-l-methylethyl)acetamide] under greenhouse conditions. CGA-43089 did not protect sorghum from the phytotoxic effects of diphenamid [Ngfl:dimethyl~2,2-diphenylacetamide], EPTC [Srethyl dipropylthiocarbamate], EPTC + R-25788 [§:ethyl dipropylthiocarbamate + N,N:diallyl-2,2-dichloroacetamide], pronamide [3,5-dichloro(fl:l,l- dimethyl-Z-propynyl)benzamide], metribuzin [4-amino-6-tert:butyl-3- (methylthio)-g§:triazin-5(4fl)-one or buthidazole {3-[5-(l,l-dimethyl- ethyl)-l,3,4-thiadiazol-2-yl]-4-hydroxy—l-methyl-2-imidazolidinone}. Under field conditions, CGA-43089 protected sorghum against high rates of metolachlor, alachlor, diethatyl, and atrazine + metolachlor (4.5. 6.7, 6.7, and 3.4 + 4.2 kg/ha respectively), and low rates of ethofumesate and butylate + R-25788 (l.7 and 3.4 kg/ha respectively). Both forage and grain yields were significantly increased when sorghum was protected from herbicide damage by CGA-43089 compared with those plants not receiving the antidote. The protective action of CGA-43089 on sorghum against metolachlor did not require light. The protective action of the antidote was not evident when sorghum was grown under flooded conditions. Untreated sorghum plants were not protected from metolachlor injury by the lateral displacement of GOA-43089 from treated seedlings growing in close proximity, unless the two types of seed were placed immediately adjacent to each other. Storage of antidote-treated sorghum seed for ll months or more reduced germination. GOA-43089 does not protect sorghum from metolachlor injury via reduced metolachlor absorption or retention. After germinating 24 h in lO'5M 14C-metolachlor, GSA-43089 treated sorghum seeds 14C-metolachlor than untreated seeds. CGA-43089 absorbed 36% more treated seeds retained 78% of the radioactivity detected as parent metolachlor and 22% of the radioactivity as a polar metabolite, while untreated seeds converted 50% of the parent herbicide to a polar metabolite. The protective action of GSA-43089 does not involve alterations in translocation of metolachlor to the site of action. Sorghum seedlings grown from seed treated with CGA-43089 translocated MC-metolachlor similarly to untreated seedlings. The protective effects of CGA-43089 do not appear to involve increased rates of metolachlor metabolism. GSA-43089 treated sorghum seedlings exposed to 14C-metolachlor 24 h or 5 days following germination absorbed more 14C-metolachlor and metabolized the 14C-metolachlor at the same rate or less rapidly than unprotected seedlings. These results suggest that the protective effects of GOA-43089 are not due to reduced herbicide absorption, modified herbicide translocation, or increased rates of metolachlor metabolism, but may involve other factors, such as interfering with the mechanism of herbicide action. ACKNOWLEDGMENTS The author wishes to express sincere appreciation to his major professor, Dr. Donald Penner, for his patience, guidance, technical expertise, and deep friendship that endured throughout the entire project. Special thanks are also expressed to Dr. William Meggitt, particularly for his assistance in supplying the mechanical equipment and technical help necessary to complete the field work associated with this project. The author would also like to express thanks to Dr. Alan Putnam, Dr. John Kaufmann, and Dr. Matthew Zabik for their help in planning this project and manuscript review. Thanks also to Ciba-Geigy and Dr. Homer LeBaron for supplying the untreated and treated sorghum seed, radiolabeled metolachlor, and financial assis- tance involved in this project. Special thanks are also expressed to fellow graduate students that helped make difficult days and nights infinitely more bearable, particularly my very close friends Dale Aaberg, Rich Voorman, Martin Mahoney, and Lynn Oakes. A final and special thanks is expressed to Claudia Haas for her deep friendship, caring, and help in the final preparation of this dissertation. ii TABLE OF CONTENTS Page LIST OF TABLES.... ....... . ............. . ....... . ................. iv LIST OF FIGURES ......................... . ..... . ......... ......... v INTRODUCTION..................................................... 1 CHAPTER 1. EVALUATION OF CGA-43089 AS A POTENTIAL ANTIDOTE AGAINST SELECTED HERBICIDES FOR SORGHUM (SORGHUM VULGARE PERS.)......... ..................... l2 Abstract ...... ....... ................................ 12 Introduction ............. . ....... . ............. ...... 13 Materials and Methods.. ............. ................. 14 Results and Discussion............................... 16 Literature Cited..................................... 25 CHAPTER 2. FACTORS INFLUENCING EFFICACY OF CGA-43089 ............ 26 Abstract....... ..................................... . 26 Introduction........ ..................... . ....... .... 26 Materials and Methods...... ......... . ..... . ..... ..... 27 Results and Discussion ......... ... ....... . ......... .. 29 Literature Cited.. ....... . ....... . ....... . ........... 37 CHAPTER 3. INFLUENCE OF CGA-43089 ON METOLACHLOR ABSORPTION, TRANSLOCATION, AND METABOLISM IN SORGHUM (SORGHUM VULGARE PERS.)....................................... 38 Abstract.... ........................................ . 38 Introduction........ ........... ..... ......... . ....... 39 Materials and Methods................................ 39 Results and Discussion............................... 43 Literature Cited..................................... 65 APPENDIX A. Structures of l,8-naphthalic anhydride, R-25788, and CGA-43089........................................ 66 LIST OF TABLES CHAPTER l. Page l. Antidote potential of GOA-43089 - Greenhouse data.. ..... . 20 2. Antidote potential of CGA-43089 - Field data for 1979 herbicides applied preplant incorporated ....... ..... 21 3. Antidote potential of CGA- 43089 - Field data for 1979 herbicides applied preemergence. .... ............... 22 4. Antidote potential of CGA-43089 - Field data for 1980 herbicides applied preplant incorporated..... ..... .. 23 5. Antidote potential of CGA-43089 - Field data for 1980 herbicides applied preemergence............. ........ 24 CHAPTER 2. 1. Effect of light on efficacy of CGA-43089 in protecting sorghum against metolachlor injury ............ 32 CHAPTER 3. 1. Radioactivity detected in twenty sorghum seedlings due to absorption, metabolism, and combined absorption and metabolism .......... ..... ................. 48 iv CHAPTER 1. CHAPTER 1. LIST OF FIGURES 2. Top photo: Sorghum growing in half-strength Hoagland's No. l nutrient solution ..... . ............................ Bottom photo: Sorghum growing in half-strength Hoagland's No. 1 nutrient solution supplemented with 1x1o-4M metolachlor ......... . ..... ... ....... . ..... ....... Top photo: Container on the left holds three treated sorghum seeds, each surrounded by four untreated sorghum seeds planted 0.5 cm away. Soil was not sprayed with herbicide. Container on the right holds sorghum planted in similar fashion. Soil sprayed with Page . 34 . 34 1x10-5M metolachlor ........ ... ....... . ....... ....... ..... . 36 Bottom photo: Container on the left holds three treated sorghum seeds. each surrounded by four untreated sorghum seeds planted adjacent. Soil was not sprayed with herbicide. Container on the right holds sorghum planted in similar fashion. Soil was sprayed with 1x10'5M metolachlor .................. . ...... 3. Radioscans of thin-layer chromatograms of extracts from sorghum seed germinated in I C-metolachlor solution for 24 h. Scan of unprotected seeds on top, scan of CGA-43089 treated seeds on bottom ........................ Translocation of 14C-metolachlor in sorghum seedlings. Unprotected plants are on the upper tier, GOA-43089 treated plants on the lower tier ................. ........ Radioscans of thin-layer chromatograms of extracts from sorghum seedlings. Seedlings were treated with 14C-metolachlor 24 h after initiation of germination, and extracts taken 12 h later. Scan of unprotected seedlings on top, scan of GSA-43089 treated seedlings on bottom .................................. .............. . 36 . 5O . 52 . 54 Page 4. Radioscans of thin-layer chromatograms of extracts from sorghum seedlings. Seedlings were treated with 14C—metolachlor 24 h after initiation of germination, and extracts taken 24 h later. Scan of unprotected seedlings on top, scan of CGA-43089 treated seedlings on bottom ............................... 56 5. Radioscans of thin-layer chromatograms of extracts from sorghum seedlings. Seedlings were treated with 14C-metolachlor 24 h after initiation of germination, and extracts taken 3 days later. Scan of unprotected seedlings on top, scan of GOA-43089 treated seedlings on bottom ............................... 58 6. Effect of CGA-43089 on 14C—metolachlor metabolism in sorghum seedlings ...................................... 59 7. Radioscans of thin-layer chromatograms of extracts from sorghum seedlings. Seedlings were treated with 4C-metolachlor 5 days after initiation of germination and at the time of shoot emergence, and extracts were taken 24 h later. Scan from unprotected seedlings on top, scan from CGA-43089 protected seedlings on bottom. ................................................... 61 8. Radioscans of thin—layer chromatograms of extracts from sorghum seedlings. Seedlings were treated with 14C-metolachlor 5 days after initiation of germination and at the time of shoot emergence, and extracts were taken 3 days later. Scan from unprotected seedlings on top, scan from CGA-43089 protected seedlings on bottom .................................................... 63 9. Effect of GOA-43089 on 14C-metolachlor metabolism in sorghum seedlings ......................................... 64 APPENDIX A. Structures of 1 ,8-naphthalic anhydride, R- 25788, and CGA- 43089 ................................................. 66 vi Introduction The use of chemicals to protect crops from herbicide injury is a relatively new approach to selective weed control. These chemicals, termed "protectants", “safeners”, or "antidotes", selectively protect crop plants from herbicide injury without protecting weeds. Antidotes can be used to widen the selectivity or margin of safety of a herbicide so that higher herbicide doses can be applied, more potent herbicides can be used, longer periods of weed control can be obtained, and greater reliability under varying environmental conditions is possible. Antidotes may also permit the use of normally less selective herbicides, more economical herbicides. or more environmentally desirable herbicides. A new plant protectant, [a-(cyanomethoximino)-benzacetonitri1e], known as CGA-43089 and trademarked Concep has recently been registered for use in sorghum against metolachlor injury. Existing herbicides used for sorghum have not provided sufficiently broad spectrum weed control. Broad spectrum herbicides, such as metolachlor, injured sorghum at rates required for effective weed control. The objectives of this research were: a) to evaluate CGA-43089 as a potential antidote against selected herbicides for sorghum under greenhouse and field conditions, b) to determine the influence of light, flooding, leaching, and storage on the protective action of CGA-43089, and c) to determine if the protection provided to sorghum 2 by CGA-43089 was due to reduced herbicide absorption, modified trans- location of the herbicide to the target site, or altered herbicide metabolism. REVIEW OF LITERATURE Introduction A herbicide antidote is a compound that selectively protects crop plants from herbicide injury without protecting weeds. The site of antidote action can be external or internal. External protection could involve purely physical barriers to herbicide uptake, as in the case with activated carbon, or could involve competition with the herbicide for site of entry. Internal protection would involve more complex biochemical interaction, such as competition for a binding site of toxic action, or increased detoxication of the herbicide. External Crop Protection - Activateg_Carbon Activated carbon (activated charcoal) was one of the first protectants used, with varying degrees of success (1). Since it adsorbs most organic herbicides it can provide a physical barrier to herbicide uptake by the plant, thus reducing the risk of damage to the crop caused by herbicide treatment. The use of activated carbon as a protectant has its limitations. Most obvious is that it can only be used with soil applied herbicides. Another drawback is that its range of action is severely localized. Seed coating with activated carbon generally does not protect the emerging shoot from herbicide injury since the seedlings rapidly grow out of the protected zone (8) (12). Placing the activated carbon in rows or as spot treatments is often uneconomical and also results in weed protection in the immediate vicinity of the crop (15) (27). 4 Furthermore, relatively large amounts of charcoal are required to obtain the desired protection (14) (23). Internal Protection The first observation that led ultimately to the concept of herbicide antidotes was made by Hoffmann in 1947 (28). He observed that tomato plants treated with both 2,4,6-T and 2,4-D did not show phenoxy-related injury symptoms. Later, in 1962, he described the use of chemical seed treatments that protected wheat from barban injury (16). These compounds were not developed for commercial use, but in 1969 Hoffmann reported the discovery of what ultimately became the first commercially developed herbicide protectant, 1,8-naphthalic anhydride (17) (Appendix A). Despite a number of reports on various chemicals having protectant action (28), only two other compounds have been released for commercial use as herbicide protectants. These are N,N7dially1-2,2-dichloro- acetamide, known as R-25788 (Appendix A), and a newer compound [a-(cyanomethoximino)-benzacetonitri1e], known as CGA-43089 and trademarked Concep (Appendix A). 1,8-Naphthalic Anhydride 1,8-Naphtha1ic anhydride was introduced commercially in 1972 as a seed treatment that protected corn against EPTC (§;ethyl dipropyl- thiocarbamate) injury, and to a lesser extent butylate (Srethyl diisobutylthiocarbamate) damage (28). Naphthalic anhydride has subsequently been shown to protect a number of crops against a variety of herbicides. Naphthalic anhydride has been reported to protect corn from various acetanilides (21), nearly all the thiocarbamates (3), and 5 limited protection from buthidazole {3-[5-(1,1-dimethylethy1)-l,3,4- thiadiazol-Z-yl]-4-hydroxy-l-methyl-2-imidazolidinone} injury (13). Naphthalic anhydride has also been reported to protect sorghum from alachlor [2-chloro-2',6'-diethyl-N7(methoxymethyl)acetanilide] injury (29); sorghum, corn, and cotton from damage by the pyrrolidine urea herbicide 5328 [cis 2,5-dimethyl-l-pyrrolidine carboxanilide] (18); and corn (2) and oats (7) from foliar applications of barban [4-chloro-2- butynyl mfchlorocarbonilate]. Mechanism of Action of Naphthalic Anhydride Little is known of the mode of action of naphthalic anhydride. However, it almost certainly does not act by preventing herbicide uptake into the plant (11) (18) (25). Data concerning the effect of naphthalic anhydride on herbicide metabolism are fragmentary. There are conflicting reports on whether naphthalic anhydride alters EPTC metabolism in corn tissue (11) (25). Wilkinson and Smith (30) have demonstrated that 10‘7M naphthalic anhydride in combination with 10‘5M EPTC reversed EPTC-induced inhibition of acetate into lipids. They suggest that naphthalic anhydride may work by reversing the inhibition of fatty acid synthesis caused by thiocarbamate herbicides. Lay and Casida (19) were unable to demonstrate naphthalic anhydride induced stimulation of GSH levels or GSH-S-transferase activity in corn roots. Since this conflicts with results they obtained for R-25788, they concluded that naphthalic anhydride had a different mode of action from R-25788 (l9). R—25788 R-25788 was introduced commercially in 1973 as a protectant against EPTC and butylate injury in corn. R-25788 differs from naphthalic anhydride in that l) R-25788 is equally effective in pre- venting EPTC injury to corn when applied as a soil spray as well as a seed treatment, 2) no weed species examined to date are protected (5), 3) R-25788 is more effective than naphthalic anhydride in protecting corn from injury by acetanilide herbicides and high rates of EPTC and butylate (3). Mechanism of Action of R-25788 R-25788 could protect corn from EPTC injury by l) inhibiting uptake and distribution of EPTC within the plant, 2) reversing EPTC induced inhibition of lipid synthesis, 3) enhancing EPTC detoxication, 4) any combination of the above. Chang gt_al, (6) examined the effects of R-25788 on the uptake and distribution of EPTC by corn seedlings, and found that R-25788 did not inhibit uptake of [14C]-EPTC or affect the distribution of [14C]- EPTC when measured over l-7 days. In fact, greater rates of uptake of [14C]-EPTC were reported upon treatment with R-25788. Wilkinson and Smith (30) have demonstrated reversal of EPTC- induced inhibition of lipid synthesis in isolated spinach chloroplasts by R-25788. They suggest that R-25788 may act by reducing EPTC injury at a site of lipid synthesis. Lay and Casida (19) have suggested that the mode of action of the protectant involves an increase in the rate of detoxication of the herbicide, via conjugation to glutathione. Specifically, they have 7 suggested that R-25788 acts by increasing the levels of GSH and GSH-S-transferase activity, and demonstrated these increases in corn following pretreatment with the protectant. They propose that plants sensitive to thiocarbamate herbicides are ones which lack initially higher GSH levels as well as the mechanism for synthesis of high GSH-S-transferase levels (20). The mechanisms by which R-25788 could increase GSH levels and GSH-S-transferase activity have not been elucidated. Leavitt and Penner (22) have suggested that R-25788 protects corn primarily by stimulating sulfoxidation of EPTC, and as a result enhances its subsequent detoxication via conjugation to GSH. This hypothesis is supported by the work of Casida and co-workers (4) who have shown that corn treated with EPTC sulfoxide is not damaged, and that the EPTC sulfoxide is rapidly detoxified. This observation indicates that the levels of GSH or GSH-S-transferase activity cannot be the limiting factor, as even high levels of the sulfoxide can be detoxified. Thus, if EPTC is administered to corn, any sulfoxide formed can be detoxified, but sulfoxidation may not be fast enough to prevent EPTC from manifesting its toxicity. It follows then, that an R-25788 stimulated increase in levels of GSH or GSH-S-transferase activity in corn would not be critical in protecting corn. GOA-43089 A new plant protectant, [a-(cyanomethoximino)-benzacetonitri1e], known as CGA-43089 and trademarked Concep has recently been registered for use as a seed treatment in sorghum against metolachlor herbicide injury. Existing herbicides used for sorghum have not provided 8 sufficiently broad spectrum weed control. Broad spectrum herbicides, such as metolachlor, injured sorghum at rates required for effective weed control. Seed treatment with CGA-43089 protected sorghum from metolachlor injury whether applied as a seed treatment or soil spray (9). However, since CGA-43089 is not specific for sorghum, and provides protection to a limited number of weed species (26), it is applied as a seed coating. Presently, nothing is published explaining the mechanism of action of GOA-43089 in sorghum. The site of uptake of CGA-43089 has been shown to be the shoot zone (26). No protection to metolachlor is obtained when CGA-43089 is applied to the roots (26). Since metolachlor is also taken up in the shoot zone (10), the site of action of GSA-43089 may also be in the coleoptile of young seedlings. MON-4606 In 1980, a safening agent [S-thiazolecarboxylic acid, benzyl ester, 2-chloro-4-(trifluoromethyl)] known as MON-4606 and trademarked Screen was discovered that will allow the use of alachlor on sorghum with a commercial level of selectivity (24). This safening agent can be applied as a seed treatment or in-furrow granule. Currently, there is no published data concerning the mode of action of MON-4606 in sorghum. 10. 11. LITERATURE CITED Arle, H.F., 0.A. Leonard, and V.C. Harris. 1948. Inactivation of 2,4-D on sweet potato slips with activated carbon. Science, New York, 197:247-248. Blair, A.M. 1978. Interactions between barban and protectants on maize, oats, and barley. Weed Res. 18:77-81. Blair, A.M., C. Parker, and L. Kasasian. 1976. Herbicide Protectants and Antidotes - A Review. PANS Vol. 22, No. 1, pp. 65-74. Casida, J.E., R.A. Gray, and H. Tilles. 1974. Thiocarbamate sulfoxides: Potent, selective, and biodegradable herbi- cides. Science 184:573-574. Chang, F.Y., J.D. Bandeen, and G.R. Stephenson. 1972. A selective antidote for prevention of EPTC injury in corn. Can. J. Plant Sci. 52:707-714. Chang, F.Y., G.R. Stephenson, and J.D. Bandeen. 1974. Effects of N,N7dia11yl-2,2-dichloroacetamide on ethyl N,N7di-ny propylthiocarbamate uptake and metabolism by corn seedlings. J. Agr. Food Chem. 22:245-248. Chang, F.Y., G.R. Stephenson, G.W. Anderson, and J.D. Bandan. 1974. Control of wild oats in oats with barban plus antidote. Weed Sci. 22:546-548. Croxford, D.E., D.M. Elkins, and G. Kapusta. 1975. Crop pro- tectants and herbicides for orchardgrass-alfalfa establishment. Weed Sci. 23:414-418. Ellis, J.F., J.W. Peek, J. Boehle, Jr., and G. Mfiller. 1980. Effectiveness of a new safener for protecting sorghum (Sorghum bicolor) from metolachlor injury. Weed Sci. 28:1-5. Gerber, H.R., G. Mfiller, and L. Ebner. 1974. CGA-24705, a new grasskiller herbicide. Proc. Brit. Weed Control Conf. 12:787-794. Guneyli, E. 1971. Factors affecting the action of 1,8-naphthalic anhydride in corn treated with S-ethyl dipropylthio- carbamate (EPTC). Dissertation Abstracts International (B)32:l957-1958. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 10 Gupta, 0.P. and N.K. Niranwal. 1976. Increasing herbicide selectivity in maize and cowpeas by seed treatment with activated carbon and NA. PANS 22:86-89. Hatzios, K.K. and D. Penner. 1980. Potential antidote against buthidazole injury to corn (Zea Mays). Weed Sci. 28:273-276. Helweg-Anderson, A. 1968. The inactivation of simazine and linuron in soil by charcoal. Weed Res. 8:58-60. Henne, R.C. and R.T. Guest. 1974. Activated carbon as a method of reducing metribuzin phytotoxicity to seeded tomatoes. Proceed. of the Northeastern Weed Sci. Soc. 28:242-248. Hoffmann, 0.L. 1969. Chemical antidotes for EPTC on corn. Weed Sci. Soc. of Amer. Abstracts 12, pp. 17-26. Hoffmann, 0.L. 1978. In "Chemistry and Action of Herbicide Antidotes" (Pallos, F.M. and J.E. Casida, Eds.), p. 1. Academic Press, New York. Holm, R.E. and 5.5. Szabo. 1974. Increased metabolism of a pyrrolidine urea herbicide in corn by a herbicide antidote. Weed Res. 14:119-122. Lay, M.M. and J.E. Casida. 1976. Dichloroacetamide antidotes enhance thiocarbamate sulfoxide detoxification by elevating corn root glutathione content and glutathione- S-transferase activity. Pest. Biochem. and Physiol. 6:442-456. Lay, M.M., 0.P. Hubbell, and J.E. Casida. 1975. Dichloro- acetamide antidotes for thiocarbamate herbicides: mode of action. Science 189:287-289. Leavitt, J.R.C., and D. Penner. 1978. Potential antidotes against acetanilide herbicide injury to corn (Zea_Mays). Weed Res. 18:281-286. Leavitt, J.R.C. and D. Penner. 1979. Ig_vitro conjugation of glutathione and other thiols with acetanilide herbicides and EPTC sulfoxide and the action of the herbicide antidote R-25788. J. Agr. Food Chem. 27:533-538. Long, C.E. and R.F. Scranton. 1969. The action of charcoal on the herbicidal activity of several herbicides. Proceed. North Central Weed Control Conf. 24:55-56. MON-4606 safening agent for Lasso herbicide on grain sorghum (milo) 1980. Available from: Monsanto Agricultural Products Company, St. Louis, MO. 25. 26. 27. 28. 29. 30. 11 Murphy, J.J. 1972. Effect of 1,8-Naphthalic anhydride on the uptake of S-ethyl N,N-dipropylthiolcarbamate (EPTC) by Zga_mays. Chem. BibTI Interactions 5:284-288. Nyfeller, A., H.R. Gerber, J.R. Hensley, 1980. Laboratory studies on the behavior of the herbicide safener CGA-43089. Weed Sci. 28:6-10. Olson, P.D. 1971. The use of activated carbon to establish crops with various herbicides. Comm. Washington State Weed confo 9 pp. 59-60. Pallos, F.M. and J.E. Casida, editors. 1978. "Chemistry and action of herbicide antidotes". Academic Press, New York, l71pp. Spotanski, R.F., and 0.C. Burnside. 1973. Reducing herbicide injury to sorghum with crop protectants. Weed Sci. 21:531-536. Wilkinson, R.E. and A.E. Smith. 1975. Reversal of EPTC-induced fatty acid synthesis inhibition. Weed Sci. 23:90-92. CHAPTER 1 EVALUATION OF CGA-43089 AS A POTENTIAL ANTIDOTE AGAINST SELECTED HERBICIDES FOR SORGHUM (SORGHUM VULGARE PERS.) Abstract Seed treatment with CGA-43089 [a-(cyanomethoximino)-benzaceto- nitrile] provided protection to sorghum (Sorghum vulgare Pers.) against the herbicides metolachlor [2-chloro-N;(2-ethy1-6-methy1phenyl)-N- (2-methoxy-l-methylethyl)acetamide], alachlor [2-chloro-2',6'-diethy1- N-(methoxymethyl)acetanilide]. diethatyl [N-(chloroacetyl-N-(2,6- diethylphenyl)glycine], ethofumesate [(I)-2-ethoxy-2,3-dihydro-3,3- dimethyl-5-benzofuranyl methanesulfonate], butylate + R-25788 [(S-ethyl diisobutylthiocarbamate) + (N,N-dial1y1-2,2-dichloro- acetamide)], and atrazine + metolachlor [2-ch1oro-4-ethylamino-6- isopropylamino-g-triazine + 2-chloro-N-(2-ethyl-6-methylphenyl)-N- (2-methoxy-1-methylethyl)acetamide] under greenhouse conditions. CGA-43089 did not protect sorghum from the phytotoxic effects of diphenamid [N,N;dimethyl-2,2-diphenylacetamide], EPTC [§;ethyl dipropylthiocarbamate], EPTC + R-25788 [S-ethyl dipropylthiocarbamate + N,N-diallyl-2,2-dichloroacetamide], pronamide [3,5-dichloro-(N-l,l- dimethyl-Z-propynyl)benzamide], metribuzin [4-amino-6-tgrt-butyl-3- (methylthio)a§-triazin-5(4fl)-one, or buthidazole {3-[5-(l,l-dimethyl- ethyl)-l,3,4-thiadiazol-2-yl]-4-hydroxy-l-methyl-2-imidazolidinone}. Under field conditions, CGA-43089 protected sorghum against high rates of metolachlor, alachlor, diethatyl, and atrazine + metolachlor (4.5, 6.7, 6.7, and 3.4 + 4.2 kg/ha respectively), and low rates of 12 l3 ethofumesate and butylate + R-25788 (1.7 and 3.4 kg/ha respectively). Both forage and grain yields were significantly increased when sorghum was protected from herbicide damage by CGA-43089 compared with those plants not receiving the antidote. Introduction Numerous compounds have been examined for antidotal activity since the discovery by Hoffmann (2) that 4'-chloro-2-hydroxy-imino- acetanilide selectively protects wheat (Triticum aestivum L.) from injury caused by subsequent foliar applications of barban. Two compounds that have been evaluated as antidotes to protect sorghum against herbicide injury are NA (1,8-naptha1ic anhydride) and R-25788 (N3N-diallyl-2,2-dichloroacetamide). Jordan and Jolliffe (3) and Rains and Fletchall (4) found that NA provided substantial protection to sorghum from high rates of alachlor. Spotanski and Burnside (5) confirmed in a number of field experiments that alachlor could be used for the selective control of annual grasses in sorghum provided the seeds were coated with NA. R-25788 as a tank mix did not protect sorghum from herbicide damage; however, seeds treated with a wettable powder formulation of R-25788 provided limited protection against both alachlor (5) and EPTC (1). Sorghum seed treatment with CGA—43089 has been introduced as a protective measure against metolachlor injury. In addition to sorghum, rice, wheat, and proso millet have shown some increased tolerance to metolachlor following CGA-43089 seed treatment, but not to the extent obtained with sorghum. Some weeds, exemplified by Brachiaria plantagingg_[(Link) Hitchc.] and Eleusine indica have also exhibited 14 increased tolerance to metolachlor following treatment of the seeds with the antidote. Materials and Methods Greenhouse Study Plants were grown in greenhouse soil (1:1:1 soil, sand, peat) in 946 ml waxed cups. Formulated emulsifiable concentrates of the herbicides were sprayed on the surface of soil contained in 26 by 20 by 6 cm aluminum foil trays using a link belt sprayer at 2.1 kg/cm2 pressure in 376 L/ha spray volume. The herbicide treated soil in these aluminum foil trays was placed in a rotary mixer and incorporated for l min. This herbicide incorporated soil was placed on top of untreated soil to a depth of 5.0 cm. The antidote-treated seed was provided by Ciba-Geigy and contained 1.25 9 active ingredient of CGA-43089/kg seed prepared by spraying a concentrated formulation (2.09 F) of the antidote on seed rotating in a roller mill apparatus. The untreated seed was also.provided by Ciba-Geigy and was similar in every respect to the treated seed except that it had not been treated with CGA-43089. Six antidote-treated or untreated Funk's G499 sorghum seeds were planted 25 cm deep into the soil of each cup. After planting, the cups were placed in a greenhouse with supplementary high pressure sodium lighting (240 uE m‘zs’I) to give a 16 h day. Temperature ranged from 20 C at night to 33 C during the day. All plants were fertilized daily with a 150 ppm concentration of a commercial fertilizer testing 20:20:20 for NPK in a volume of water equal to that necessary to keep the plants fully turgid. 15 Thirty days after planting, the sorghum plants were photographed, harvested, and fresh weights determined. The data are expressed as g fresh weight per cup, and are the means of two experiments with four replications per experiment. A completely randomized design was used. Field Study In 1979, CGA-43089 treated and untreated sorghum seeds were planted the fourth week in June in 76 cm rows 6 m long at a rate of 2.3 kg/ha. Soil texture was a sandy clay loam with an organic matter content of 2.5%. The herbicides metolachlor, alachlor, diethatyl, ethofumesate, and butylate + R-25788 were applied both preplant Herbicides were incor- Plants incorporated and preemergence on the surface. porated twice to a depth of 5 cm using a spring tooth harrow. were harvested 14 weeks after planting. Plant fresh weights and seed head dry weights were the parameters measured. In 1980, CGA-43089 treated and untreated sorghum seeds were Inlanted June 11 in 76 cm rows 7.6 m long at a rate of 2.3 kg/ha. The scril was a clay loam with an organic matter content of 2.8%. The her1>icides metolachlor, alachlor, diethatyl, ethofumesate, butylate + R-25788, and atrazine + metolachlor were applied both preplant incx>rporated and preemergence. Herbicides were incorporated twice to a depth of 5 cm with a spring tooth harrow. Sorghum plants were harvested 18 weeks after planting. The parameters measured were plant fresh weights and seed head dry weights. In all the treatments, herbicides were applied with a tractor mounted sprayer which traveled 6.4 km/h and delivered 234 L/ha at a Pressure of 2.1 kg/cmz. During both years of field tests, the study 16 was laid out in a split-split plot design to examine no antidote versus antidote treatment and preplant incorporated versus preemergence treatments. Each treatment was replicated three times. Results and Discussion 'In greenhouse tests CGA-43089 provided significant protection to sorghum from the phytotoxic effects of the herbicides metolachlor, alachlor, diethatyl, butylate + R-25788, and ethofumesate as measured by fresh weight (Table l). CGA-43089 did not protect sorghum from injury caused by diphenamid, EPTC, pronamide, metribuzin, or buthidazole (Table 1). Propachlor did not injure sorghum even at relatively high use rates, and the addition of the antidote thus did not provide additional protection (Table 1). In field studies CGA-43089 protected sorghum against high rates of metolachlor, alachlor, diethatyl, and atrazine + metolachlor, and low rates of ethofumesate and butylate + R-25788 (Tables 2-5). Sorghum receiving herbicide + CGA-43089 showed significantly less visible injury (data not presented) and had greater fresh weights (Table 2-5) and yield (Tables 2-5) than sorghum which did not receive the antidote. In all studies, the antidote alone did not significantly alter either the fresh weight or grain production of the sorghum (Tables 2-5). In the 1979 field study, when the herbicides were applied preplant incorporated, use of the antidote resulted in significantly greater fresh weight production of sorghum exposed to the intermediate and high rates of the acetanilide herbicides metolachlor, alachlor, and diethatyl (Table 2). Sorghum was not damaged severely enough at 17 the low rates of these acetanilide herbicides that use of the antidote resulted in a significant increase in fresh weight production (Table 2). Against the non-acetanilide herbicides, CGA-43089 offered significant protection only against the low rate of butylate + R-25788 (Table 2). In terms of seed head yield, CGA-43089 offered significant protection to sorghum from all the acetanilide herbicides at all the rates tested (Table 2). Against the non-acetanilide herbicides, use of the antidote resulted in significantly higher seed head yield only against the low rate of butylate + R-25788 (Table 2). In the 1979 preemergence study, use of the antidote resulted in significantly greater sorghum fresh weight production and seed head yield against all of the acetanilide herbicides at all the rates tested, except for the high rate of diethatyl (Table 3). Concerning the non-acetanilide herbicides, use of CGA-43089 resulted in a significant increase in sorghum fresh weight production and seed head yield against the low rate of butylate + R-25788 (Table 3). Use of CGA-43089 also resulted in a significant increase in seed head yield against the low rate of ethofumesate (Table 3). In the 1980 preplant incorporated study, use of CGA-43089 resulted in significantly greater sorghum fresh weight production against the high rates of metolachlor and diethatyl and all rates of alachlor (Table 4). Against the other three herbicides tested, use of CGA-43089 resulted in significant increases in fresh weight against the high rate of ethofumesate and atrazine + metolachlor (Table 4). In terms of seed head yield, the antidote offered significant protection to all the acetanilide herbicides at all the rates tested except the low rates of metolachlor and diethatyl (Table 4). Use of CGA-43089 18 also resulted in significant increases in seed head yield against the high rates of ethofumesate and atrazine + metolachlor (Table 4). In the 1980 preemergence study, use of CGA-43089 resulted in significant increases in fresh weight production and seed head yield against all rates of all herbicides tested (Table 5). During both years' field studies, the antidote provided greater protection when the herbicides were applied preemergence rather than preplant incorporated (Table 2-5). This is probably related to the heavy rains which occurred shortly after spraying both years, causing the incorporated herbicides to leach through the soil. Since those plants in the preplant incorporated trial were not exposed to high enough levels of herbicide to cause severe injury, the protecting effect of the antidote was not as evident. CGA-43089 offered significant protection to sorghum from three (:1asses of herbicides, namely the acetanilides metolachlor, alachlor, arud diethatyl, the thiocarbamate herbicide butylate + R-25788 and the herbicide ethofumesate. However, there were differences in the rwalative efficacy of CGA-43089 to reduce phytotoxic damage dependent upon the class of herbicide. CGA-43089 was most effective in pro- tracting sorghum from injury against the three acetanilide herbicides Inertolachlor, alachlor, and diethatyl, and less effective against birtyJate + R-25788 and ethofumesate. The herbicides metolachlor, a1 achlor, diethatyl, butylate + R-25788, and ethofumesate all inflict fSirnilar injury symptoms in sorghum. The characteristic symptoms often 1°hc21ude abnormal leaf emergence from the coleoptile, leaves that don't IJrIrwal] normally, or leaves that emerge twisted and stunted. Since the 1:.“wee classes of herbicides all cause similar injury symptoms at 19 approximately the same developmental stage in sorghum seedlings, it may be that the antidote acts at a similar site of action, and differences in the relative efficacy of CGA-43089 protection may be due to specific differences in the nature and structure of the herbicides in each class. 20 TABLE 1. Antidote potential of CGA-43089 - Greenhouse dataa. Fresh weight Herbicide Rate -antidote +antidote (kg/ha)_fii (gm/pot) Control 39.2a 39.8a Metolachlor 2.8 7.6e 28.3abc Alachlor 5.6 1.2e 25.0bc Diethatyl 5.6 0.9e 33.2ab Butylate + R-25788 5.6 1.1e 19.4cd Ethofumesate 2.8 1.3e 33.2ab Diphenamid 4.5 9.4de 10.2de EPTC 2.8 0 e 1.4e Pronamide 2.8 29.9abc 22.9bc Propachlor 6.7 39.5a 33.9ab Metribuzin 2.8 0 e 0 e Buthidazole 1.7 0 e 0 e aMeans followed by the same letter are not significantly different at the 5% level according to Duncan's multiple range test. 21 .ummu mace; mpawp_:e m.cmo::n op mcwveouum —m>mp em mgu pm pcmemwmwu >~ucmuwww=mwm yo: mew Loupmp mamm one an uwzoppow mcmmzm 5-;amm chem w-cm.m em._ 5.8 wmmm~-m m-u__w ¥-;Nmm a-ne.m “-5m.m ¢.m + agapsuzm m-u~mm “-mmae ;-ua.m ;-um.w s.m uuoo__ m-emmm u-a~.m_ e-am.__ A., mammae=eogum m-uoom ¥-mep e-am._. n-mw.e 5.8 m-emma x-:-m a-ao.P_ n-5u.m m.a u.~__ L-Lomm c-am.op ;-ue.m N.N _sam;paaa m-m_mo xmm e-am.oF no._ “.8 0-6Nom x-epm_ e-ae.__ n-m~.¢ m.¢ cummp_ 5-8008 u-am.mF ;-n_.m N.N Lo_=uap< c-6FNm chomp e-am.F_ n-mo.¢ m.a magmas 5-;LNN unao.m_ n-ma.m s.m unmemp e-m~N¢ u-a~.mp _-ea.a N.N Lo_;uapoumz moom_ aaocmp nau.se am.m_ _ocacoo apoewoca+ muoewpca- wooeao=a+ wooewwma- Am;\mxv A30; E _.m\5mv Azog e F.m\mxv mung muwupngm: a_mws new; ummm unmwmz smug; .umpacoacoocw p=a_amga ewe—age mmuauwacm; mam, toe mama o_aea - mmome-m_ em wcp pm pcogmeewu appcmuwewcmwm no: men meump mama msu ma umzoPPoe mcmmzm ;-emmm 5mm e-ee.¢ Pc._ N.m mwamm-m comaw cmmmm na~.o_ mem.m e.m + mumpspzm emcee so wem.m Po.o ¢.m eoepm 5-8mmm L-g~.m m-ao.e N._ mummasacoeom emmmm em P-uo.m wa.o “.0 a-uapm emu u-m~.o_ _N._ m.a uao_op emmom o-a_.o_ aco.m N.~ _xoa;umwo c-0mmo ;N_ e-am.m Pm._ N.o nammN_ nae neo.mp _N._ m.e a__o_ ;m_F ak.e_ L-mN.N ~.N Lo_eomP< m-u~mm LN m-am.~ PN.o m.¢ unmoo_ go“ nam.m_ _s._ ¢.m nmpmmp emo_ nam.mp 5N.N N.N Lo_;uapoaaz awmmp epomp naa.m_ nae.m_ _oap=ou wpouwpca+ apoeapca- mpoe_p=m+ muouwpea- Am;\me A30; 2 F.o\Emv A30; E P.o\mxv mpmm mcwowncm: a_mws new; eamm osmwmz smock mocmmameamaa emm_aaa maewownam; meme Lac mama u_aea - mmome-mp em on“ no “cosmewwu xppcmowewcmwm no: men empum— mEmm we“ En wwzoppom mcmwzm e_mws uam; ummm pcmwmz Emmgd 6-0mmoe Lemmmm u-am.m_ EEE.N N.¢ + e.m Leagua_oumz u-ao¢_c m-uompm c-am.a_ L-uo.ep m.N + N.~ + m=_~aep< a-a~mme m-umo_m c-6m.a_ L-na.¢_ m.e mmcmm-m c-aoo_o a-ammmm E-am.mp E-am.op N.N + apa_>u=m o-eNNNE E-Lmope u-mm.mp ¥-_m.o_ e.m u-amo_~ m-omamm E-mm.op P-C_.m_ N._ mpamme=cogom e-ammpe E-cmmme e-am.wp E-m¢.m_ E.m u-mom¢~ E-aamme c-a~.mF L-aN.m_ m.¢ u-ae-~ 8-3mmmo m-mc.ae E-am.m_ ~.N _xoa;umwo nawoam wema_ am.o~ xe.m “.8 nammow Pgm_mN a~.om xeo.~ m.¢ nawmmm mmmm amo.m_ E-mm.P_ N.N Lo_;ua_< nam__w Emaamm ao.o~ A-Em.mp m.¢ u-ammea m-e_m_m e-am.ap L-eo.m_ e.m u-ewmaa m-aemmo m-a¢.a_ m-ao.a_ N.N Lopeum_0pmz a-am¢_a u-mNo¢N E-am.m_ m-am.mF Fozpcou mpouwocm+ mpouwoca- apouwp=a+ .lmpoewpca- Am;\mxv A30; E N.m_\mv Azoc E o.~\mxv mama wuwuwngm: umpacoacouce Scapamca umwpaaa mmequaLmE ommp Lac mama upmwa - mmome-mp em as“ pm “cosmeewu EFHEmuwwwcmvm we: mew mepmp wEmm on» Ea um3o__o$ mcmmzm 24 u-ampoa cameo nmo.mp m-mm.~ ~.¢ + ¢.m Lopeua_oamz aawmea L-e_~mP nmm.mp c-6m.o m.~ + N.N + mcw~acu< muemmm coo_ 6-6m.__ mo._ m.¢ mmcmm-m u_N¢m C-EQMEP na~.mp c-6m.~ N.~ + mumpaoam o-am_eo cmam no..w_ aco.m e.m u-aoe¢~ uaomm amo.m_ c-ua.w A., apammaaeocpm nasmau c-ee_mF aam.o~ m-mo.m “.8 o-ammmk c-como~ nam.om c-u~.m m.¢ neomaa summon amo.o~ L-ue.m N.N Espaepawo namo_m LEON aa.- men.F N.o neommm cmmm nao.om m-mm.~ m.a amoem c-ummm_ aam.a_ m-em.¢ N.N Lofigua_< nammwc c-amum_ nam.m_ c-u_.a m.¢ u-aommk c-emmap nam.m. c-um.o ¢.m nameem L-emom_ nam.m_ c-um.m N.N Lo_;oapopmz u-amamo u-e-mo onm.m_ na_.wp _ocpcou mooe_p=a+ mpoeeoca- apoevp=a+ mpoe_p=m- Aa5\mxv A30; E ~.m_\Emv Azoc E o.m\mxv ovum moquan: u_aws cam; eaam “coca: gamed mucmmeEmmsa ummpanm mwuwuwngw; owm— wpmv upmwd n mmom¢ump em me“ go mmocmgmmmwo pzoowmvcmwm mpoowocw mzoe cwgmwz Arvmxm_smummp Em me» am pomsmmmwo xpucoowmwcmwm mo: mLo emppmp mEom mew Ea omzoFFom mcomZo memoowp ommmm mmNF omm oop ommm.op E Nu omomm mommFN once oom oo_ omo_.m ; om ommm momowm wmmm poem moom oop ommn.m : m_ ompomte ememmm_ aemmee to“, emm ow aea0m.m e we gmmmop mopmnm oom once ram omPN.o ; om ommm memo“, mwomm owe“ momm rmo mmwm.~ 5 NF ompomgucn mowowocm: mpwpooopmz mowowogmz mmPFooopmz ommm ompompma oowcma ucmgom toe 2mg LoPom Lom Zoo ucmcom E eopom & ompomep mo & 2mg cowuoospxm Emwpooopmz oco EmFFoooumz cowemgoma< cowHQMomn< cowuocomo< omconou o.emuop E we so .5 om .; ~_ mommoowooe tom ompoocmxm oco .cowpocwEgmm memo ; om gopgoopomeioo Ewe: ompomgp mcmz mmEFFommm Eogmgom .EmwponoumE oco coppogomoo om:_nEoo one .Emvfiooome .cowwosomoo op moo mmcwpommm Eocmcom zucmzp =_ ompomumo xuw>wpooowoom ._ m4m c: o“ H win- I: a. c) jf .mn- "HW- l-L‘ 1.4 1 TTTT 0.71 0.12 0.47 Rf values FIGURE 7. FIGURE 8. 62 Radioscan of thin-layer chromatogram of extracts from 14C- sorghum seedlings. Seedlings were treated with metolachlor 5 days after initiation of germination and at the time of shoot emergence, and extracts were taken 3 days later. Scan for unprotected seedlings on the top, scan for CGA-43089 treated seedlings on bottom. The polar metabolite had an Rf vaer of 0.12 and the parent metolachlor had an Rf of 0.71. The developing system was Hexane:Chloroform:Ethanol (70:20:10). CPM x 10,000 63 10 CPM x 10,000 he; __... A IA 41...: I. hum. Rf values 10 #4 5 1 Rf values ‘ FIGURE 8. 64 .coaumEHEumw mo ummco umuwm mzmc m>fiw vmfiaaom uoHSomHoum210 .mmczooom 83:90» E EmzonEoE ..oEoEosPoElo: co mmom¢l