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I _ . . r . . . . .1, v I: ‘ ; -r: I .. 1;... qt :9... .r 4:..,,w..h_.......r L37 -.h¢...\l.......u...... .. 8 n.6,. ... ..s.._ . .r:u’l.. L .L. m.» ......c v -c v . . ..... o . . ., , ~. . l x “ . . . . > _ ‘ ' ’l Ir ‘I‘ g 53 0 V 753 umlil‘ffll/filfillllnWWI “3&2... Unlvonlty This is to certify that the '"Ssertation entitle: Corn (Zea mazs L.) Tolerance to Chloroacetanilide Herbicides presented by Loston Rowe has been accepted towards fulfillment of the requirements for Ph.D. Crop and Soil Sciences degree in Major professor Date 7/10/89 on v 'f ' {‘11 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before die due. DATE DUE DATE DUE DATE DUE ! I __—'l fil—fifll MSU Is An Affirmative ActiorVEquel Opportunity lnetltmion —.—___.—_ a.._. _ f CORN (ZEA Mfllfi L.) TOLERANCE TO CHLOROACETANILIDE HERBICIDES BY Loston Rowe 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 1989 (90422 4330 ABSTRACT CORN (ZEA MAXS L.) TOLERANCE TO CHLOROACETANILIDE HERBICIDES by Loston Rowe Greenhouse, laboratory, and field studies were con- ducted to evaluate factors influencing corn tolerance to chloroacetanilde herbicides. Studies were conducted to determine the effectiveness of GSA-154281 in protecting corn from metolachlor injury and to determine the mechanism for the protective action. Applications of alachlor and metolachlor to ten Great Lakes corn hybrids at four application rates showed some of the hybrids were more tolerant of alachlor and others were more tolerant of metolachlor. There was a linear response of increasing injury with increasing application rate. In . a soil moisture study, more injury was evident as the soil moisture content increased. Evaluation of 200 commercial corn hybrids and 29 corn inbreds revealed a high degree of variability in metola- chlor tolerance. The distribution of tolerance resembled a normal distribution curve, with most of the hybrids and inbreds having a midlevel of tolerance. Laboratory studies with metolachlor tolerant and sensitive hybrids indicated that the variability of tolerance was due to differences in absorption, metabolism, as well as differences at the site of action of metolachlor. Greenhouse and field studies showed that CGA-154281 was very effective in protecting corn seedlings from meto- lachlor injury. This protection was evident even with sensitive hybrids at high soil moisture levels and high herbicide application rates. Studies with 14C-metolachlor indicated that the protectant CGA-154281 did not reduce metolachlor absorption or alter the pathway of metolachlor metabolism. However, the protectant did appear to enhance the metabolism of metolachlor to a non-phytotoxic gluta- thione conjugate. Nomenclature: Corn, (Zea mm L.): alachlor, 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)- acetamide; metolachlor, 2-chloro-n-(2-ethyl-6—methylphe- nyl)-H-(2-methoxy-1-methylethyl)acetamide; CGA-154281, 4- (dichloro-acetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine. W index m. Corn tolerance, distribution of tolerance, soil moisture content, protectant, glutathione conjugate. ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Donald Penner for his guidance, and counsel throughout this project. I also offer appreciation to Drs. James J. Kells, Elmer Rossman, Alan Putnam, Bruce Branham, and Bernard H. Zandstra 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 re- search and for his sincere friendship during my study. I want to acknowledge the Ciba Geigy Corporation for their financial support. Also, appreciation is offered to Dr. James Jay for his kindness and assistance. Finally, appreciation is extended and my wife, Rita, whose support and patience has made this goal attainable. iv TABLE OF CONTENTS Page LIST OF TABLES I C O O O O O O O O O O O O O O O C O O O O O O O O O O O O O O O O ..... Vii LIST OF FIGURES . O O O O O O O C O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 ix INTRODUflION.’.OOOOOCOOOOOOOOOOOOOOOOO0.0.0.0....O... 1 U CHAPTER 1. A REVIEW OF LITERATURE................... Chloroacetanilides....................... 3 Mode of Action....................... 3 Metabolism........................... 4 Soil Activity........................ 6 Factors Affecting Crop Injury............ 9 Soil Moisture........................ 9 Genetic Variability.................. 11 Other Factors........................ 12 Chemical Protectants..................... 14 Literature Cited......................... 21 CHAPTER 2. FACTORS AFFECTING CHLOROACETANILIDE INJURY TO CORN (ZEA MAXS L.)............. 31 Abstract................................. 31 Introduction............................. 33 Materials and Methods.................... 34 Computer Survey...................... 34 General Greenhouse Procedure......... 35 Influence of Hybrid, Herbicide, and Rate........................... 36 Influence of Soil Moisture Content... 36 Results and Discussion................... 37 Computer Survey...................... 37 Influence of Hybrid, Herbicide, and Rate........................... 38 Influence of Soil Moisture Content... 39 Literature Cited......................... 48 CHAPTER 3. RESPONSE OF CORN HYBRIDS AND INBREDS To “TomcnmR. O O O O O O O O O O O O O O O O I O O O O O O O O O 49 Abstract. I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 49 V Introduction............................. 51 Materials and Methods.................... 52 Inbred Study......................... 52 Hybrid Study and Selection........... 53 Metolachlor Absorption and Metabolism......................... 54 Results and Discussion................... 55 Inbred Study......................... 55 Hybrid Study and Selection........... 56 Metolachlor Absorption and Metabolism......................... 56 Literature Cited.................. ..... .. 72 CHAPTER 4. EFFICACY OF CGA-154281 AS A PROTECTANT FOR CORN FROM METOLACHLOR INJURY......... 73 Abstract................................. 73 Introduction............................. 75 Materials and Methods.................... 76 General Greenhouse Procedure......... 76 Hybrid and Rate Response............. 77 Moisture Response.................... 78 Weed Response........................ 78 Field Studies........................ 78 Results and Discussion................... 80 Hyrbid and Rate Response............. 80 Moisture Response.................... 81 Weed Response........................ 81 Field Studies........................ 82 Literature Cited......................... 95 CHAPTER 5. INFLUENCE OF THE PROTECTANT CGA-154281 ON THE ABSORPTION AND METABOLISM OF “Tommeeoooooeoooeooooeeoeoooooooeooo 96 Abstract................................. 96 Introduction............................. 97 Materials and Methods.................... 98 Results and Discussion................... 99 Literature Cited......................... 108 SUMRYAND CONCLIJSIONS...OOOOOOOOOOOOOOOOOOOOO0...... 109 LIST OF TABLES CHAPTER 2 1. Comparison of corn grain yield for hybrids receiving alachlor versus metolachlor in public sector trail from 1984-1986........... ...... 2. The effects of alachlor and metolachlor at four application rates on 10 Great Lakes hybrids........ 3. The effects of soil moisture on the response of Andersons 103 hybrid to alachlor and metolachlor... CHAPTER 3 1. Response of 29 corn inbred lines to metolachlor applied at 4.5 kg/ha.OOOOOOOOOOOOOOOOOOO0..00...... 2. Response of 200 hybrids to metolachlor applied at 4.5 kg/haOO ..... OOOOOOOOOOOOOOOOOOOOOOOO00.0.00...O 3. Response of 24 hybrids to metolachlor applied at 6.7 kg/haOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 4. Absorption and metabolism of 14C-metolachlor by tolerant and sensitive corn hybrids...... ....... 5. Internal concentration of 14C-metolachlor in tolerant and sensitive corn hybrids................ CHAPTER 4 1. Response of four hybrids to metolachlor at 8 application rates with and without CGA-154281 ...... 2. Response of Pioneer 3744 to metolachlor at four soil moisture regimes with and without CGA-154281.. 3. Response of eight weed species to metolachlor at two application rates with and without CGA—154281.. vii Page 40 41 43 58 59 64 65 65 83 85 86 4. Field response of six hybrids to metolachlor with and without CGA-154281 in 1987..................... 87 5. Field response of four hybrids to metolachlor with and without CGA-154281 in 1988..................... 88 CHAPTERS 1. Effect of CGA-154281 on the absorption of 14C- metolachlor by four corn hybrids................... 102 2. Effect of CGA-154281 on the metabolism of 14C- metolachlor by four corn hybrids................... 103 viii LIST OF FIGURES CHAPTER 1 Page 1. Corn injury symptom................................ 19 2. Chemical structures of alachlor and metolachlor.... 20 CHAPTER 2 1. Comparisons of corn yield between alachlor and metolachlor in 1984-1986..................... ...... 44 2. Effects of alachlor and metolachlor at four application rates on shoot height of 10 Great ukes corn hYbridSOOOOOOOOOOOOOO0.0.0.0....00...... 45 CHAPTER 3 1. Picture of four selected corn hybrids, Cargill 7567, Northrup King 9283, Great Lakes 584, Pioneer 3744.0...OOOOOOOOOOOOIOOOOOOOOOO0.0.0....O. 66 2. Flow diagram for absorption and metabolism study... 67 3. Distribution of injury for 29 corn inbred lines treated with 4.5 kg/ha of metolachlor.............. 69 4. Distribution of injury for 200 corn hybrids treated with 4.5 kg/ha of metolachlor....... ....... 7o 5. Output from AMBIS Radioactivity Scanner............ 71 CHAPTER 4 1. Chemical structure of CGA-154281................... 89 2. Response of 4 corn hybrids to metolachlor and CGA- 180937 at 8 application rates................ ...... 90 3. Response of Pioneer 3744 to metolachlor and CGA- 180937 at four soil moisture contents.............. 92 ix 4. Field response of four corn hybrids to metolachlor and CGA-180937 . . . ........ . ................. . ....... 93 CHAPTER 5 1. Flow diagram for absorption and metabolism study with C-metOIaChlor. O I O O O O O O O O O O O O O O O O O O O O O O O O O O O O 104 2. Mass balance for 14C applied............ ........... 106 3. Output from AMBIS Radioactivity Scanner... ......... 107 INTRODUCTION The chloroacetanilides are a class of herbicides that are commonly used in corn (Zea may: L.) and soybean (911; cine max (L.) Merr.) production. They control many annual grasses and certain small seeded broadleaf weeds and are generally selectively safe on corn. However, under certain conditions injury symptoms can occur. The factors that affect the extent of corn injury exhibited are not clearly understood. Alachlor (2-chloro-H-(2,6-diethy1phenyl)-u-(methoxyme- thyl)acetamide) and metolachlor (2-chloro-u-(2-ethyl-6- methylphenyl)-n-(2-methoxy-1-methylethyl)acetamide) are the primary chloroacetanilide herbicides used in corn. These compounds have very similar chemical structures and control essentially the same weeds. However, differences in corn tolerance to the two herbicides have been reported. The accuracy and basis of these reported differences have been disputed. Soil conditions, specifically soil moisture content, may also influence the observed corn injury. Genetic va- riability among corn hybrids and inbred lines has been linked to the differences in corn susceptibility to chlo- roacetanilides. Inherited traits like herbicide tolerance could result in observed differences in injury. However, the range of variability in chloroacetanilide tolerance among commercial hybrids is not well defined. If the factors that cause variability in corn tole- rance to the chloroacetanilides can be identified, then management practices and products such as chemical protec- tants can be developed to prevent the problem. Protectants are already used extensively in sorghum (Sorghum mm L.) production for protection from alachlor and metola- chlor. A new protectant, CGA-154281 (4-(dichloroacetyl)- 3,4-dihydro-3-methyl-2H-1,4—benzoxazine), is currently being evaluated for the protection of corn from metolachlor injury. The objectives of this research were to: 1) identify the factors associated with chloroacetanilide injury to corn, including herbicidal differences, soil moisture con- ditions, and genetic variability; 2) determine the range of chloroacetanilide tolerance among inbreds and hybrids; 3) determine the basis for the observed difference in tolerance between tolerant and sensitive hybrids; 4) evaluate the effacacy of CGA-154281 in protecting corn from metolachlor injury: and 5) determine the mechanism of the protection associated with CGA-154281. Chapter 1 REVIEW OF LITERATURE CHLOROACETANILIDES m 9.: 89:12:: Chloroacetanilides are preemergence herbicides which control many grass and several broadleaf weed species. Chloroacetanilides inhibit the early development of suscep- tible weed species. The treated seeds usually germinate, but the seedlings do not emerge from the soil. These compounds are generally selective and safe for use on corn. However, under certain conditions, chloroacetanilides may cause stunted or abnormal growth of corn seedlings (Figure 1). Most of the research on the modes of action of chlo- roacetanilides indicates that these herbicides inhibit growth (21,28,56), inhibit protein synthesis (24,87), alter lipid synthesis (25,104), or interact with plant hormones (75,107). Deal and Hess (21) concluded that the growth inhibi- tion of plants caused by chloroacetanilides results from an inhibition of cell division and cell elongation. The de- gree of growth inhibition is mostly a function of con- centration and duration of the treatment. They found that significant inhibition of etiolated oat (Avepa sativa L.) coleoptiles was caused by slightly lower concentrations of alachlor than metolachlor. Chloroacetanilides are absorbed by both shoot and root. Grass species are generally more suseceptible when the herbicide is absorbed by the emerging shoot, especially when absorbed near the coleoptilar node (34,58,59,76,82,95). Translocation of these herbicides can occur both in the xylem and phloem. However, data indicating primarily xylem transport were obtained on emerged plants, which would have a much more active transpiration stream than unemerged plants (17,34). Metabolism Several studies have been conducted to determine the fate of chloroacetanilides in higher plants. Most re- searchers agree with Breaux et al. (14), that the basis of chloroacetanilide selectivity is related to the plant's success in metabolizing these compounds (23). The metabolism of chloroacetanilides herbicides in higher plants is not fully understood. However, most re- searchers have concluded that glutathione plays a major role in the inactivation of these compounds (11,12,13,14,34, 63,64,67) . When a chloroacetanilide herbicide enters a tolerant plant seedling, the glutathione conjugates to it, producing a non-phytotoxic metabolite, which is harmless to the plant. Glutathione conjugates chloroacetanilides nonen- zymatically in vitro (38,67) and enzymatically in vitro with. glutathione 'S-transferases isolated from etiolated corn (72) and sorghum (38). Isozymes of glutathione s- transferase isolated from etiolated corn seedlings varied in their reactivity with the chloroacetanilides (72). The chloroacetanilides have been reported to be alkylating agents (70). The conjugation of these herbicides with glutathione could be considered an alkylation reaction. Alachlor and metolachlor have very similar structures (Figure 2) and serve the same applications in corn produc- tion. While some researchers have determined that there is no significant difference in the phytotoxicity of alachlor and metolachlor to corn (9,47,102) , others maintain that there are differences, and that the differences are due to differences in glutathione conjugation (19,30,80). Harvey et al. (47) stated that preliminary greenhouse studies indicated that metolachlor had greater potential for injury to corn than alachlor. However, field studies over a 12-year period indicated corn yields following meto- lachlor treatments were at levels not significantly diffe- rent from those following alachlor treatments. Thus, there was no evidence that either herbicide caused more injury to corn than the other. In growth chamber studies, Boldt and Barrett (9) found that alachlor applications generally caused more injury and yield loss to Pioneer 3780 corn than metolachlor, while the response of Pioneer 3320 to the two herbicides was not consistently different. A research team at Monsanto Chemical Company found that both alachlor and metolachlor were absorbed by the seedlings at the same rate. However, plants converted the alachlor into harmless by-products twice as easily. The researchers concluded that, because of the difference in their chemical structures, glutathione conjugation occurred more readily with alachlor than with metolachlor, and therefore, alachlor was less phytotoxic to corn seedlings (19,30,80). Attempts to determine differences in metabolism and phytotoxicity between alachlor and metolachlor show many discrepancies, especially between times, concentrations and hybrids used. Studies usually compare the two herbi- cides at equal rates. However, metolachlor is labeled for and usually used at lower rates than alachlor. This factor also complicates the comparison of the two herbicides for chemical effects. 521152121131: The primary factors affecting soil inactivation of chloroacetanilide herbicides are adsorption to soil compo- nents and microbial degradation. The herbicide degradation rate by soil microbes decreased and adsorption to the soil components increased. with increasing' organic :matter' and clay content (51). Ninety percent of all chloroacetanilide loss in soil is due to microbial degradation (51). Because chloroacetanilides are degraded quickly by microbes, their soil persistence is relatively short. Beestman (8) found half-life values of 4.0 and 7.3 days for alachlor in a silt and a silty clay soil, respectively. The half-life of metolachlor has been estimated at 30 to 50 days in the northern areas and 15 to 25 days in the southern areas of the United States (51) . Studies have shown that degra-‘ dation of alachlor and metolachlor was greater at 50 and 80% than at 20% field capacity at 20 C. Degradation rates of alachlor and metolachlor at 50% field capacity were greater at higher temperatures (113). These results indicate that metolachlor persists longer in the soil than alachlor. Therefore, metolachlor has the potential to provide a longer period of weed control. The persistence of the herbicides and their injury seems to be amplified under cool, wet conditions. The rate of herbicide required to achieve a certain level of weed control on a particular soil has often been related to the capacity of the soil to sorb the herbicide. It has been shown that alachlor moves more readily through the soil than metola- chlor. Although metolachlor is more soluble in water than alachlor, less movement occurs in the soil because metola- chlor is adsorbed more tightLy to the soil particles (20,51,105). Weber and Peter (105) found that adsorption of the herbicides was not related to molecular size (weight or volume), or molecular surface area. However, differences in adsorption were apparently due to slight, molecular structural differences in the two herbicides. In a study by Banks and Robinson (5), less than 10% of the original alachlor remained in the soil 10 days after treatment, compared to 26% of the original metolachlor. They con- cluded that straw and decaying organic matter provided for a greater retention of the metolachlor than of alachlor. Peter and Weber (86) found that alachlor and metola- chlor adsorption was postively correlated with soil organic matter content, clay content, and surface area as measured by ethylene glycol monoethyl ether (EGME) or benzyl ethyl ether (BBB) and inversely correlated with herbicidal acti- vity. Alachlor was adsorbed in slightly greater amounts by soil than metolachlor. Metolachlor had slightly greater bioactivity than alachlor on grass weeds, but the herbi- cides had similar activity on broadleaf weeds. Slightly greater amounts of metolachlor than alachlor were leached through the soil and slightly greater amounts of alachlor were retained in the upper soil zones. A study of adsorption and mobility of the chloroace- tanilides by Jordan (57) indicated that adsorption of ala- chlor and metolachlor did not differ in 10 different soils. He also reported that the mobility of the chloroacetani- lides was inversely related to their adsorptivity. Although previous studies of adsorption and mobility are conflicting, their results indicate a difference in corn tolerance to alachlor and metolachlor could be due to differences in their location and availability in the soil. FACTORS AFFECTING CROP INJURY 3.9.11 1421512113 Soil water content influences the phytoxicity of seve- ral herbicides (15,43,44,59,61,62,71,99,100,103,106). Rainfall or irrigation is accepted as being necessary for the activation of preemergence herbicides, such as chlo- roacetanilides. Surface applied water moves the herbicide into the soil thus preventing its loss from the soil sur- face via phototransformation and volatization. This move- ment of the herbicide into the soil by rainfall also brings it into contact with the germinating weed seedlings. Along with herbicidal activity, herbicidal injury to crops, which is associated with increased rainfall, could be due to movement of the herbicide into the soil. Although rainfall usually has been associated with herbicide effectiveness and injury, perhaps soil moisture should be the primary consideration with rainfall of secon- dary importance as it affects soil moisture content. 10 Walker (103) stated that herbicidal response depends, in part, on the soil water content, which influences herbicide concentrations in the soil solution, the rates of mass flow, diffusion, and plant root extension. He further concluded that herbicidal phytotoxicity generally increases as soil water content increases. However, it may not be closely correlated with the amount or concentration of herbicide in the available soil solution (103). Green and Obien (37) concluded that the principle ef- fect of soil water content on herbicide phytotoxicity pro- bably is associated with herbicide transport, which is more sensitive to changes in water content than is the concen- tration of herbicide in the soil solution (55). Stickler et al. (99) showed that the effectiveness of atrazine (6-chloro-N-ethyl-n-(l-methylethyl)-1,3,5-tria- zine-2,4-diamine) and EPTC (S-ethyl dipropylcarbamothioate) was increased when soil moisture was raised from 25% to 31%, but no further increase was obtained at 37% moisture. Response to chloramben (3-amino-2,5-dichlorobenzoic acid) increased linearly and response to trifluralin (2,6-dini- tro-n,N-dipropyl-4-(trifluoromethyl)benzenamine) decreased linearly with increased moisture. They also concluded that three possible functions of surface-applied moisture were to: 1) move the herbicide into the soil and thus reduce loss of the herbicide from the soil surface, 2) move the herbicide into the soil for contact with germinating 11 weeds or emerging weed seedlings, and 3) create suffi- ciently moist conditions in the soil for absorption of the herbicide by the seedlings. There is some data that indicates certain herbicides have less activity with increasing soil water content. Grover (40) found that bioactivity of picloram (4-amino- 3,5,6-trichloro-2-pyridinecarboxylic acid) decreased as the soil moisture content increased. He stated that this was due to the effect of varying soil moisture levels on the concentration of the picloram in the soil-water phase. Therefore increasing soil moisture had a dilution effect on the herbicide concentration. Genetic W Differential tolerance to the same herbicides has been reported in several crops (2,3,4,10,36,45,53,78,89,93,96). Narsaiah and Harvey (74) found differential alachlor tole- rances among both corn inbreds and hybrids. Alachlor se- verely injured inbreds W117, W182E, and A65, but inbreds W153R and W59M were not affected, even at 10.0 kg/ha. Penner et al. (84,92) evaluated the sensitivity of 108 inbred lines and several hybrids and found the tolerance followed a normal distribution curve. Francis and Hamill (31) found significant differences in corn shoot weight in a study with three alachlor rates and 21 inbred lines. They also stated that hybrids appeared to exhibit a smaller 12 range of response to high rates of alachlor than inbred lines. They concluded that variation in inbred and hybrid tolerance to alachlor would suggest that screening is necessary before assessing the suitability of alachlor for foundation and seed production fields. Studies conducted by Renner et al. (90,94) showed that corn hybrids showed differential responses to imazaquin (2— (4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol- 2-yl)-3-quinolinecarboxylic acid) for all parameters that were measured. Great Lakes 422 and 5922, Cargill 921, and Pioneer 3901 were significantly more tolerant to imazaquin than the others tested. They concluded that the tolerance shown did not appear related to corn maturity groups. Wright and Rieck (111) found that Pioneer 3030 and Coker 71 were tolerant to butylate (fi-ethyl cyclohexylethylcarba- mothioate) , whereas Pioneer 511A and PAG644 were observed as being sensitive. Laboratory studies showed that the tolerant hybrids absorbed less 14C-butylate and metabolized more to 14C02 than the sensitive ones. Merriam Susceptibility of a plant to a herbicide differs with variation in environmental factors. High temperatures and high humidities generally increase susceptibility (15,41, 60,101). However, Penner (85) found that alachlor was injurious to navy beans (Engggglgg yglgazig L.) at a high 13 application rate at 20 and 25 C but not at 30 C. The ala- chlor injury to the navy beans was characterized by plant growth reduction and growth inhibition of the leaf apex. Muzik and Mauldin (73) found that 2,4-D ((2,4-dichlorophe- noxy)acetic acid) absorption and translocation in both leaves and roots was less under low temperatures. There- fore, sensitivity of wheat (Tritiggm aeggiygm L.) to 2,4-D was greater at 26 C than at 10 C and 5 C, at all stages of growth. Burt and Akinsorotan (15) noted that EPTC and butylate reduced corn growth more at 30 C than at 20 C. High tempe- ratures before coleoptile emergence was more critical than high temperatures after coleoptile emergence. Penner (83) also reported that the phytotoxicity of linuron (ti-(3,4- dichlorophenyl)-N-methylurea) to corn and soybean seedlings increased with increasing temperatures from 20 C to 30 C. He concluded that there was a relationship between in- creased herbicide transport to the shoot and higher tempe- ratures, therefore causing increased toxicity. However, Thompson et al. (101) noted that cold, wet conditions pro- duced more severe inj ury than warmer temperatures. They concluded that lower temperatures caused a decrease in the detoxication rate of atrazine in corn. Therefore, the accumulation of absorbed atrazine was responsible for the injury to corn under cooler temperatures. Other resear- chers agree that an increase in phytotoxicity can occur at 14 high temperatures because of increased herbicide uptake. On the other hand, an increase in phytotoxicity at lower temperatures may be attributed to reduced detoxication of the herbicide (16,60,73). Several reports indicate that soil pH may play a role in the amount of herbicide injury which occurs (46,108). Harris and Warren (46) showed that soil pH altered the adsorption and desorption properties of several herbicides, thereby, altering the amount available for plant uptake. Soil texture is also known to influence the activity of soil applied herbicides. Generally, more injury was observed when the herbicides were applied to coarser tex- tured soils (39,42,77,81,106). Soils with higher organic matter contents showed less herbicide activity than did soils of lower organic matter (39). CHEMICAL PROTECTANTS The use of chemical antidotes or protectants has been widely studied. Because of the extensive commercial use of protectants for protection of sorghum (Sorghum giggle: L.) from chloroacetanilide injury, these studies have pri- marily dealt with chloroacetanilide protectants for sorghum (18,22,27,68,91,97). However, recent reports indicate that chemical protectants may be useful for other crops and other herbicides (6,7,49,52,65,69,79,109,110). 15 When used at higher rates, alachlor and metolachlor are often injurious to corn seedlings. Thus, the use of crop protectants or antidotes to minimize corn injury be- comes important (69) . Leavitt and Penner (66) reported that of the six potential protectants they evaluated di- chlormid (3,5, diallyl-2-2—dichloroacetamide) provided the most protection to corn from alachlor, metolachlor and other chloroacetanilide herbicides. Spotanski and Burnside (97) said that 1,8 naphthalic anhydride was the most effec- tive protectant they tested in reducing alachlor injury to sorghum. They concluded that the seed treatment was more effective than tank mixes. Rains and Fletchall (88) found that dichlormid was more effective than CDAA (2-chloro- N,N-di-z-propenylacetamide) in preventing yield reductions to corn from alachlor or metolachlor in the greenhouse. The mechanism of the protective action of chloroaceta- nilide protectants is not fully understood. Herbicide protectants do indeed selectively reduce the biological activity of herbicides, which would otherwise result in crop injury. This reduction in crop injury must be the result of the protectant reducing or eliminating the her- bicide from its site of action, or by an induction of an alternate pathway that will compensate for or override the effects of the herbicide. The biochemical antagonism can be a result of one or more factors, including reduced herbicide uptake, reduced herbicide translocation, enhanced 16 herbicide ‘metabolism, herbicide compartmentalization, or the induction of alternate plant metabolic pathways. There are conflicting reports in the literature as to which actually occurs. Hatzios (50) proposed that biochemical, competitive, and physiological antagonisms of the activity of herbicides by the protectants are potential mechanisms of protective action. He said that biochemical antagonism occurs when a protectant prevents the penetration and/or translocation of a given herbicide into the protected plant, or when the protectant enhances the metabolic detoxication of the her- bicide in the protected plant. Competitive or physiologi- cal antagonisms occur when a protectant competes with a given herbicide for the same site of action in the cells of the protected plant. Chemical antagonism involves a chemi- cal reaction of the protectant with the herbicide to form an inactive herbicide-protectant complex. Fuerst (34) proposed that two hypotheses for protec- tant mode of action seem plausible. Protectants induce rapid herbicide metabolism or they protect the biochemical site of action of the herbicide. Protectants may induce rapid herbicide metabolism by increasing levels of glutathione and/or glutathione trans- ferase which enhances the conversion of the herbicide to inactive metabolites. While looking at the effect of pro- tectants on glutathione content and glutathione fi-trans- 17 ferase (GST) activity in sorghum, Gronwald et al. (38) found a significant increase in GST activity when metola- chlor was used as a substrate. The degree of protection from metolachlor injury conferred by a particular antidote was strongly correlated with its ability to enhance GST activity. This theory of protectant interaction with glu- tathione fi-transferase enzymes to enhance chloroacetanilide metabolism has been reported from other studies as well (1,29,32,33,35,54,65,112). Ebert (26) reported that cyometrinil (((cyano-methoxy) imino)benzeneacetonitrile) prevented the loss of cuticular integrity and therefore greatly reduced the amount of meto- lachlor taken up by sorghum seedlings. The other plausible hypothesis is that protectants protect the biochemical site of herbicide action. Com- pounds with similar structures to thiocarbamate herbicides are often effective protectants (50,58,98) . For example, dichlormid is structurally very similar to EPTC. There is evidence that there is an intermediate step in the metabolism of chloroacetanilide herbicides. As with the thiocarbamate herbicides, this intermediate step in- volves the oxidation of the herbicide to a sulfoxide. The oxidation occurs either by a mixed function oxidase or a peroxidase. The sulfoxide could then be conjugated enzyma- tically or nonenzymatically to the glutathione conjugate that has been widely observed (34). Recent studies 18 showed that CGA-43089 fails to counteract metolachlor injury to sorghum grown in nutrient-solution culture or under conditions of excessive soil moisture (58). These reports indicate that the presence of molecular oxygen might be related to the protective effect offered by some herbicide protectants. The theory of an intermediate sulfoxidation step in the metabolism would explain the importance of molecular oxygen (48). 19 Figure 1. Corn injury symptom. EH3 CHaCHa ? 0 H / \ .3" ‘44.... CHECHa alachlor O “\P ' l CHECH3 metolachlor Figure 2. Chemical structures of alachlor and metolachlor. 10. 11. LITERATURE CITED Adams, C.A., E. Blee, and J.E. Casida. 1983. Dichloroacetamide herbicide antidotes enhance sulfate metabolism in corn roots. Pestic. Biochem. Physiol. 19:350-360. Anderson, R.N. 1964. 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Influence of soil moisture on the safening effect of CGA-43089 in grain sorghum. Weed Sci. 29:281-287. Knake, E.L., A.P. Appleby, and W.R. Furtick. 1963. Soil incorporation and site of uptake of preemerge herbicides. Weeds. 11:228-232. Kozlowski, T.T., S. Saski, and J.H. Torrie. 1967. Influence of temperature of phytotoxicity of triazine herbicides to pine seedlings. Amer. J. Bot. 54: 790-796. Kratky, B.A. and G.F. Warren. 1973. Water-soil-plant interactions with terbacil. Weed Sci. 21:451-454. Lambert, S.M. 1966. The influence of soil moisture content on herbicidal response. Weeds. 14:117-121. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 26 Lamoureux, G.L. and D.G. Rusness. 1987. EPTC meta- bolism in corn, cotton, and soybean: Identification of a novel metabolite derived from the metabolism of a glutathione conjugate. J. Agric. Food Chem. 35:1-7. Lamoureux, G.L., L.E. Stafford, and F.S. Tanaka. 1971. Metabolism of 2-chloro-n-isopropylactanilide in the leaves of corn, sorghum, sugarcane, and barley. J. Agri. Food Chem. 19:346-350. Lay, M. and A.M. Niland. 1985. Biochemical response of inbred and hybrid corn to R-25788 and its distri- bution with EPTC in corn seedlings. Pestic. Biochem. Physio. 23:131-140. Leavitt, J.R.C. and D. Penner. 1978. Protection of corn from acetanilide herbicidal injury with the anti- dote R-25788. Weed Sci. 26:653-659. Leavitt, J.R.C. and D. Penner. 1979. In vitro con- jugation of glutathione and other thiols with acetani- lide herbicides and EPTC sulfoxide and the action of the herbicide antidote R-25788. J. Agri. Food Chem. 27:533-536. Leif, J.W., O.C. Burnside, and A.R. Martin. 1987. Efficacy of CGA-92194 and flurazole in protecting grain sorghum from herbicide injury. Weed Sci. 35:547-553. Martin, A.R. and O.C. Burnside. 1982. Protecting corn from herbicide injury with R-25788. Weed Sci. 30:269- 272. McFarland, J.E. and F.D. Hess. 1985. Alkylation differences in the chloroaceanilide herbicides. Abstr. Weed Sci. Soc. Amer. p. 81. Moyer, J.R. 1987. Effect of soil moisture on the efficacy and selectivity of soil-applied herbicide. Weed Sci. 3:19-34. Mozer, T.J., D.C. Tiemeier, and E.G. Jaworski. 1983. Purification and characterization of corn glutathione S-transferase. Biochem. 22:1068-1072. Muzik, T.J. and W.G. Mauldin. 1963. Influence of environment on the response of plants to herbicides. Weeds 11:142-145. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 27 Narsaiah, D.B. and R.G. Harvey. 1977. Differential responses of corn inbreds and hybrids to alachlor. Crop Sci. 17:657-659. Narsaiah, D.B. and R.G. Harvey. 1977. Alachlor and gibberellic acid interaction on corn tissues. Weed SCi. 25:197-199. Narsaiah, D.B. and R.G. Harvey. 1976. Alachlor place- ment in the soil as related to phytotoxicity to maize seedlings. Weed Res. 17:163-168. Neuberger, B. and M.D.K. Owen. 1985. 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Lipid metabolism in grain sorghum treated with ala- chlor plus flurazole. Weed Sci. 33:25-28. Weber, J.B. and C.J. Peter. 1982. Adsorption, bio- activity, and evaluation of soil test for alachlor, acetochlor, and metolachlor. Weed Sci. 30:14-20. Wiese, A.F. and D.T. Smith. 1970. Herbicidal acti- vity as affected by soil incorporation and rainfall. Weed Sci. 18:515-517. Wilkinson, R.E. 1982. Alachlor influence on sorghum growth and gibberellin precursor synthesis. Pestic. Biochem. Physiol. 17:177-184. Wilkinson, R.E., E.L. Ramseur, R.R. Duncan, and L.M. Shuman. 1988. Metolachlor sensitivity and low pH tolerance interactions in sorghum. Agron. Abstr. p. 101. Winkle, M.E., J.R.C. Leavitt, and O.C. Burnside. 1978. Control of weedy sorghum in corn with acetanilide herbicides. Proc. North Central Weed Contr. Conf. 33:46. 110. 111. 112. 113. 30 Winkle, M.E., J.R.C. Leavitt, and O.C. Burnside. 1980. Acetanilide-antidote combinations for weed control in corn and sorghum. Weed Sci. 28:699-704. Wright, T.H. and C.E. Rieck. 1974. Factors affecting butylate injury to corn. Weed Sci. 22:83-85. Zama, P. and R.R. Hatzios. 1986. Effects of CGA-92194 on the chemical reactivity of metolachlor with glutathione and metabolism of metolachlor in grain sorghum. Weed Sci. 34:834-841. Zimdahl, R.L. and S.R. Clark. 1982. Degradation of three acetanilide herbicides in soil. Weed Sci. 30:545-548. Chapter 2 FACTORS AFFECTING CHLOROACETANILIDE INJURY TO CORN ABSTRACT A computer survey was conducted to evaluate public research reports in which alachlor and metolachlor were compared in the same trial. A summary of the data indi- cated that there was no significant difference in corn yield between alachlor and metolachlor treatments when used at labelled application rates. Greenhouse studies were conducted to determine the effects of herbicide, herbicide rate, genetic variability and soil moisture content on the tolerance of corn seedlings to two chloroacetanilide herbi- cides. Alachlor and metolachlor were applied preemergence at 2.2, 3.4, 4.5, and 6.7 kg/ha to ten Great Lakes corn hybrids. As was expected, there was a linear response of increasing' herbicide injury with increasing application rate. When comparison was made between the two herbicides, metolachlor appeared to be less injurious at the low rate and more injurious at the high rate. There was a signifi- cant degree of variability in injury among the ten hybrids tested. This variability was more evident at higher herbi- cide application rates. Some of the hybrids appeared to be 31 32 more tolerant of alachlor, while others were more tolerant of metolachlor. Six soil moisture levels ranging from 8% to 22% soil moisture were evaluated for their effect on alachlor and metolachlor injury to corn seedlings. In- creased herbicide injury occurred as the soil moisture level increased for both herbicides. The injury ranged from no injury at the lowest soil moisture level to about 70% at the highest soil moisture level. Nomenclature: Corn, Zea mg L.; alachlor, 2-chloro-N-(2,6-diethylphe- nyl)-N_- (methoxymethyl)acetamide: metolachlor, 2-chloro-fl- (2-ethyl-6-methylphenyl)-n-(2-methoxy-1-methyl-ethyl)aceta- mide. Miguel iggex Me. moisture level, genetic variability, tolerance. INTRODUCTION Chloroacetanilide herbicides are commonly used in corn production to control a wide range of weed problems. The two most commonly used chloroacetanilides in corn are ala- chlor and metolachlor. These two compounds comprise a major segment of the United States corn herbicide market. Alachlor and metolachlor are generally safe for use on corn, however, under certain conditions injury symptoms do occur. These symptoms range from a twisting and curling of the leaves early in development to a more severe stunting and malformation of the plant, which may result in de- creased corn yield. The factors influencing the degree of chloroacetani- lide injury to corn are not very well documented. Research and marketing claims by Monsanto Company imply that struc- tural differences between alachlor and metolachlor afford a significantly reduced amount of corn phytotoxicity from alachlor than from metolachlor (1,2,3,8) . However, inde- pendent research has found that there is really no signifi- cant difference in corn injury between the two compounds (5,9) . Inherited differences between inbreds and hybrids has been shown to provide differential tolerance to chloroace- tanilide herbicides (7). Francis and Hamill (4) found 33 34 significant differences in tolerance of 21 inbred lines when treated with alachlor. Narsaiah and Harvey (6) found that alachlor severely injured inbreds W117, W182E, and A65, but inbreds W153R and W59M were not affected at 10.0 kg/ha. Soil moisture content is an important factor in the amount of herbicide injury which occurs. With most soil applied herbicides there is the response of increasing crop injury and weed control effectiveness with increasing soil moisture. waever, Grover (5) found that the bioactivity of picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxy1ic acid) decreased as the soil moisture content increased. The objective of these studies was to evaluate the factors which influence the degree of corn injury from chloroacetanilide herbicides. The factors evaluated in- cluded herbicide, herbicide application rate, genetic va- riability, and soil moisture conditions. MATERIALS AND METHODS Wm With the aid of Ball Research Services, East Lansing, MI., a computer survey was conducted to evaluate the public research reports in which alachlor and metolachlor were used in head to head comparisons. The data included corn grain yield for hybrids which received treatments of 35 alachlor and metolachlor in 1984, 1985, or 1986. Compari- sons included those that occurred at one location, done by one researcher, with tank mixed additional herbicide ap- plied at the same rates. Alachlor and metolachlor were applied at the same rate or at the recommended differential application rate considered as an appropriate comparison by the researcher (i.e. alachlor 2.8 kg/ha, metolachlor 2.2 kg/ha). General greenhguse RIQEQQQIE Corn seed were planted in 946-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. The herbicides were applied preemergence with a chain-link belt, compressed air sprayer, which delivered a volume of 280 L/ha at 240 kPa. A known amount of water was then added to the soil surface for incorporation of the herbicide. The pots were then placed in the greenhouse which maintained 16 hr days at 25 +/- 2 C. The plants were grown with supplemental lighting from high-pressure sodium lamps. The light intensity was 500 uE.m"'2.s'1 with only supplemental lights to 1200 uE.m' 2 '1 with both supplemental and natural sunlight. The .s greenhouse was maintained at 40 to 75% relative humidity. Plant height and injury ratings were evaluated 10 days after planting. Plant height as percent of the control was 36 calculated. Each hybrid was compared to its own control, thus correcting for differences in shoot height among the hybrids. Plant injury rating was on a scale of 0 (no effect) to 100 (complete kill). The mean of three plants in each pot was considered one observation. Each treatment was replicated four times and the data are the means of two experiments. Following analysis of variance, means were separated at the 5% level of significance according to Duncan’s multiple range test. Influenceefhmmlhsrhiciderandrafe Ten Great Lakes hybrids were evaluated under the con- ditions described above. This experiment tested the effects of alachlor and metolachlor on the ten hybrids at four application rates. The ten Great Lakes hybrids used were 579, 313, 381, 422, 437, 466, 498, 516, 547, and. 599. Alachlor and metolachlor were applied preemergence at rates of 2.2, 3.4, 4.5, and 6.7 kg/ha. After the herbicide application, 125 ml of water was applied to the soil sur- face for herbicide incorporation and activation. This gave a moisture content of 12% for each pot. Equal amounts of water were added to each pot thereafter. After 10 days, plant height and visual injury ratings were taken. Influenceefseilmgisiiireecnfenf Anderson 103 corn seed was evaluated as previously 37 described in the general greenhouse procedure. Herbicide applications of alachlor and metolachlor at 4.5 kg/ha were used. Applications of surface applied water were made to obtain six moisture levels of 8, 10, 12, 14, 18, and 22 % soil moisture. Preliminary studies verified that adequate corn seedling growth could be obtained at these levels of soil moisture. The moisture levels were maintained daily by weighing and adding the appropriate amounts of water as needed. After 10 days plant height and injury ratings were taken as previously described. RESULTS AND DISCUSSION Wanner The computer data base contained a total of 158 compa- risons of alachlor and metolachlor. These comparison were made with 31 different corn hybrids. The response of the hybrids showed that some were more tolerant to metolachlor while others are more tolerant to alachlor (Table 1). For example, in twelve comparisons with Pioneer 3906, the ala- chlor treatments averaged 625 kg/ha (10.8 bu/A) more yield than the metolachlor treatments. However, in six compari- sons with Pioneer 3603, the metolachlor treatments averaged 387 kg/ha (6.2 bu/A) more than alachlor treatments. Of the 31 hybrids included in the trials, those receiving the 38 alachlor treatments yielded more with 17 of the hybrids, while the corn receiving metolachlor treaments yielded more with 15 hybrids (Figure 1) . The mean difference in corn yield between alachlor and metolachlor treatments for all 158 comprisons was only 25 kg/ha in favor of alachlor. This difference is negligible and insignificant. mummmm In this experiment both parameters used for measuring injury showed a significant interaction between herbicide, hybrid, and application rate (Figure 2). The interaction of hybrid, herbicide, and rate indi- cates that at the low herbicide application rate of 2.2 kg/ha there was generally no significant difference in in- jury between hybrids or herbicides. With seven of the 10 hybrids, the application rate of 2.2 kg/ha metolachlor was . less injurious than alachlor, although this difference was rarely significant (Table 2). As the herbicide appplication rate increased, the differences among the hybrids and between the herbicides also increased. At the 4.5 kg/ha appplication rate, Great Lakes hybrid 313 was more tolerant to metolachlor, whereas, hybrid 516 was more tolerant to alachlor. At the highest application rate of 6.7 kg/ha, metola- chlor was generally more injurious than alachlor for all 39 hybrids. Based on visual observations over a period of time (data not included), corn seedlings treated with meto- lachlor at the highest appplication rates were unable to overcome the injury as well as those seedlings treated with alachlor. Influence 9.: 5.9.11 meietere 92053.01; In this experiment there was an interaction between herbicide and soil moisture level. The interaction indi- cated a linear response of increasing injury with increa- sing moisture for both alachlor and metolachlor (Figure 3). However, at 12% soil moisture, which was about field capa- city, there was no significant difference between alachlor and metolachlor treatments. There was significantly grea- ter injury from metolachlor compared to alachlor at the higher moisture levels. At the 22% soil moisture level the alachlor treated seedlings were 40% of the control height compared to 25% for the metolachlor treated plants (Table 3). From these studies it was conluded that the corn hybrid, the herbicide, the herbicide application rate, and the soil moisture content at the time of plant emergence all play a role in the degree of chloroacetanilide injury which occurred. 4O Teple 1. Comparisona of corn grain yield for hybrids receiving alachlor versus metolachlor in public sector trails from 1984-1986. Corn Number of Sum of yielg hybrid Comparisons difference (bu/A) Bojac 2 -3 Carhart 793 2 -1 Coker 22 2 +2 Dekalb T-12-30 7 +10 Dekalb 1100 2 0 Dekalb 636 l -3 Funk’s 4733 2 +28 Punk's 4740 3 -15 Nebraska 611 3 -16 Olds 95 2. +9 Pioneer 3147 2 +4 Pioneer 3352 2 +7 Pioneer 3377 2 -7 Pioneer 3378 5 -18 Pioneer 3413 1 +17 Pioneer 3475 1 +21 Pioneer 3535 2 -22 Pioneer 3603 6 -37 Pioneer 3732 13 +45 Pioneer 3747 48 -10 Pioneer 3780 1 -5 Pioneer 3901 2 -18 Pioneer 3906 12 -130 Pioneer 3732 25 +59 Pioneer 3347 1 +6 Pioneer 3382 1 +7 Sokota 270 2 -2 Stuaffer 57751 1 -5 Sunbelt 1876 l -25 Terra 3203 l -10 Wilson 1700 2 +10 Unknown 4 -73 Totals 158 -69 aMean difference between herbicide treatments (-69/158) was -0.44 bu/A. bNegative numbers indicate the alachlor treatment had a yield advantage, positive numbers indicate the metolachlor treatment had a yield advantage. 41 Table 2. The effects of alachlor and metolachlor at four application rates on ten Great Lakes hybrids. Great Lakes Shoot Hybrid Herbicide Rate height Injury (kg/ha) (% of untreated) (%) 579 Alachlor 2.2 94.5 28 3.4 75.6 36 4.5 62.0 39 6.7 50.0 55 Metolachlor 2.2 96.6 11 3.4 95.8 11 4.5 56.8 51 6.7 39.4 69 313 Alachlor 2.2 85.8 34 3.4 53.4 59 4.5 44.6 64 6.7 24.8 71 Metolachlor 2.2 94.6 13 3.4 77.6 34 4.5 68.1 48 6.7 25.7 81 381 Alachlor 2.2 93.9 8 3.4 79.9 24 4.5 68.6 17 6.7 55.8 27 Metolachlor 2.2 99.2 4 3.4 73.6 21 4.5 56.1 25 6.7 35.8 56 422 Alachlor 2.2 78.0 34 3.4 67.7 44 4.5 49.8 41 6.7 36.7 63 Metolachor 2.2 88.6 16 3.4 85.1 31 4.5 52.3 59 6.7 34.6 68 437 Alachlor 2.2 91.5 29 3.4 68.8 38 4.5 53.6 53 6.7 36.4 54 Metolachlor 2.2 83.6 28 3.4 80.5 33 4.5 59.2 52 6.7 35.9 67 leele g. Continued. 42 Great Lakes Shoot Injury Hybrid Herbicide Rate height rating (kg/ha) (% of untreated) (%) 466 Alachlor 2.2 83.3 19 3.4 85.9 17 4.5 57.2 23 6.7 44.4 37 Metolachlor 2.2 91.2 9 3.4 69.5 26 4.5 57.3 38 6.7 30.4 57 498 Alachlor 2.2 83.2 17 3.4 88.0 24 4.5 53.4 45 6.7 38.9 40 Metolachlor 2.2 97.1 21 3.4 81.6 30 4.5 59.9 42 6.7 35.4 65 516 Alachlor 2.2 84.8 14 3.4 70.8 16 4.5 56.9 36 6.7 30.0 37 Metolachlor 2.2 71.5 21 3.4 56.9 36 4.5 44.0 52 6.7 27.9 70 547 Alachlor 2.2 82.4 18 3.4 55.9 35 4.5 52.9 26 6.7 41.8 35 Metolachlor 2.2 108.2 10 3.4 68.4 22 4.5 50.4 37 6.7 32.2 58 599 Alachlor 2.2 87.6 21 3.4 71.4 17 4.5 44.0 20 6.7 34.3 29 Metolachlor 2.2 75.9 47 3.4 64.3 42 4.5 34.5 67 6.7 35.5 73 LSD (0.05) 12.0 12 Table 1- 43 Effect of soil moisture on response of Andersons 103 corn hybrid to alachlor and metolachlor. Shoot Injury height rating Soil moisture Alachlor Metolachlor Alachlor Metolachlor (%) 8 10 12 14 18 22 LSD (0.05) ...... (%)------ --(% of untreated)-- 95.5 98.3 8 11 80.5 75.7 13 10 74.8 73.9 15 16 56.7 58.4 34 35 51.8 28.2 49 59 40.2 25.2 69 7O 44 m II...I.III.....IIII'll-II: lllll Udbhmflbr ILILIIILIIIIIIHIIIII IIIFIILIII I: 2%,; III. q‘qI IE“ no; «0 E0981 [All Comparisons Hybrid Comparisons (155) (31) Comparisons of corn yield between alachlor and metolachlor treatments in 1984-1986. Figurel. 45 Eiggxe 2. Effects of alachlor and metolachlor at four application rates on shoot heights of 10 Great Lakes corn hybrids (LSD (0.05) = 12.0). \\\\\\\\\\\§§\\\\\\\\\\\\\\\\\\\\\\\\\\_\‘ ___—_..-- A--m_- *-._.——w .\\\\\\\\.\)§\\\\\\\\H\\\\\\\\\\\\\\\\\\S |I‘I““““““II EMU 23 Alachlor .\\\\\\\\\\\H5§§\\\\\\\\\\\\\\\\ \\ rI‘I‘I““““““I‘I‘I \\\_\\\\\\\\\\\)\\\\\\\\\\\\\\\}\ \\\\3 \\\\}\\\\\\\V 3.4 kg/ho 579 313 381 422 437 466 498 516 547 599 "V'I'vv'I'VUVIVTVV'VV'Y‘VVT‘I‘V‘U'"V'IVVUIIWT'IVVVVIV'V' 89888€8m eve-e- mmgmgmmwmmmwmmmwwmmmmmm nmuuuremuuunmmuunnnmuu \\_\_\_\\\\\\\§W\\\\\\\\\\\\\\\\\\\\\\V I|I!‘I‘I“‘\“I\““‘\“|‘I‘I 2.2 kg/ho \\\\\\\\\\\\\\5\\\\\\\\ \\\\\\\\\\ \ \\\\\ \\\\\\\\\\\\‘ II“‘I‘|““‘I‘|“ ““‘I III‘|“““I“‘I““|‘I“‘I‘I‘ I““\“\“““I“I“ “|‘II‘I 66 498 5 579 313 381 mil'T11‘1 O O '- O F ' Yt‘I'Ur‘I'Y—V'IVY“"V'V'V'V'l""[""""VIVV‘V O O O O O O (O ID V 0305 n (paw-914m :0 z) NBPH 100145 0 GOOD ION- ¢ 37 ‘- 2 N 4 GOOD Ne- 46 ~\.\1\1\1\\.\.\.\. 93333 0': OJ U .C \\ U'1 x P\ CV ‘0 \\_\\\.\_\}.\_..\\\.\.\}.\)\\_\‘ N P m I") n P Wm o: [x m 7".'I""""“er"tfl1"""V'Vl','U"""""'U"""" sesasessesseo O O O O! O O h ‘0 O In 0 0 Ne- ? $3 (pazomun 10 z) W5!9H zoous 8 O '— O 47 120. E lulrflmdmu' {3110? Hummer 310% ° 5 e! 901 "E E 350‘: “570:: soc-g 30.: § - ‘20-: ”’10-: o‘f-,-.h--.----.--rr 5 10 15 20 25 Soil Moisture Content (7:) Figure 3. Effects of soil moisture content on the response Andersons 103 hybrid corn to alachlor and metola- chlor applied at 4.5 kg/ha (LSD (0.05) = 10.4). LITERATURE CITED Breaux, E.J. 1985. Chloroacetanilide herbicide se- lectivity: the initial fate of acetochlor in tolerant and susceptible plants. Proc. North Central Weed Contr. Conf. 40:123. Christy, A.L., A.S. Wideman, J.S. Mc.Laren. 1985. Canopy photosynthesis as a possible indicator of herbi- cide stress in corn. Proc. North Central Weed Contr. Conf. 40:106. Farm Chemicals. 1986. Meister Publishing Co. Farm Chemicals Intl. 2:82-83.. Francis, T.R. and A.S. Hamill. 1980. Inheritance of maize seedling tolerance to alachlor. Can. J. Plant Sci. 60:1045-1047. Harvey, R.G., R.E. Doersch, and J.W. Albright. 1985. Twelve year comparison of alachlor and metolachlor. Proc. North Central Weed Contr. Conf. 40:51. Narsaiah, D.B. and R.G. Harvey. 1977. Differential responses of corn inbreds and hybrids to alachlor. Crop Sci. 17:657-659. Niccum, C.E. 1970. Variations in inbred and varietal tolarance of corn to butylate, alachlor, and propachlor. Proc. North Central Weed Contr. Conf. 25:33-35. O’Connell, K.M., E.J. Breaux, and R.T. Fraley. 1988. Different rates of metabolism of two chloroacetanilide herbicides in Pioneer 3320 corn. Plant Phys. 86: 359- 363. Viger, P.R. and C.V. Eberlein. 1986. Corn tolerance to acetanilide herbicides. Proc. North Central Weed Contr. Conf. 41:7. 48 Chapter 3 RESPONSE OF CORN HYBRIDS AND INBREDS TO METOLACHLOR ABSTRACT Greenhouse studies were conducted to determine the response of 200 corn hybrids and 29 inbreds to metolachlor applied at 4.5 kg/ha. Both hybrids and inbreds varied in their response to the herbicide. The distribution of injury resembled a normal distribution curve with most of the hybrids having a midlevel of tolerance. waever, some of the hybrids were very tolerant, while others were quite sensitive. Laboratory studies were conducted to evaluate absorption and metabolism of 14C-metolachlor for a subset of tolerant and sensitive hybrids. These studies showed that their was no difference in the pathway of metabolism for metolachlor in the tolerant and sensitive hybrids. The studies revealed that the basis fer observed variability in metolachlor tolerance among hybrids was due to diffe- rences in rates of absorption and metabolism of meto- lachlor, and differences at the site of action of metola- chlor. The tolerant Great Lakes 584 hybrid absorbed sig- nificantly less 14C-metolachlor than did the sensitive Pioneer 3744, while the tolerant Cargill 7567 metabolized 49 50 l4C-metolachlor significantly faster than the other hy- brids. The internal concentrations of available 14C-meto- lachlor were the same for the tolerant Cargill 7567 and the sensitive Northrup King 9283, indicating differences at the site of action of metolachlor for these two hybrids. No- menclature: Corn, Zee neye L.: metolachlor, 2-chloro-fi-(2- ethyl-6-methylphenyl)-n-(2-methoxy-1-methylethyl)acetamide. Additienel inflex m5. Tolerance, normal distribution curve, midlevel tolerance, internal concentration. INTRODUCTION Metolachlor is a herbicide that is commonly used in corn production without significant injury to corn for the control of many grasses and several broadleaf weeds. How- ever, under certain circumstances injury symptoms have been reported. The factors contributing to this increased crop injury from metolachlor have not been fully studied. In addition to several enviromental factors, genetic dif- ferences among hybrids is believed to play a role in the amount of visual injury observed (2,6,7). Several other crop species are known to exhibit dif- ferential tolerance among cultivars to specific herbicides. The most documented of these is the response of soybean (glyeine max (L.) Merr.) cultivars to metribuzin (4-amino- 6-(1,1-dimethylethyl)-3-(methylthio)1,2,4-triazin-5(4H)- one) (4). However, through extensive research and selec- tive breeding, soybean tolerance to metribuzin can be iden- tified and utilized at will. Differential tolerance of corn cultivars to several other herbicides has been report- ed. These include atrazine (6-chloro-n-ethyl-EL-(1-methyl- ethyl)-1,3,5-triazine-2,4-diamine), trifluralin (2,6-dini- tro-n,fl-dipropyl-4-(trifluoromethyl)benzenamine) , EPTC (S- ethyl dipropylcarbamothioate), and imazaquin (2-(4,5-dihy- 51 52 dro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl)-3- quino-linecarboxylic acid) (1,8,9,10). The objectives of these studies were to determine the distribution and degree of metolachlor injury among corn hybrids by testing a representative number of hybrids, and also to select and evaluate tolerant and sensitive hybrids to determine the physiological basis for the observed dif- ferences in tolerance. MATERIALS AND METHODS Inbred SEES! Twenty-nine public corn inbreds were obtained from the corn breeding program of Michigan State University, East Lansing, Michigan. The corn seed were planted in 50 by 30 cm pans with 8 inbreds in rows per pan. The soil was a Spinks sandy loam (mixed, mesic Psammentic Hapludalfs) consisting of 71.3% sand, 19.4% silt, and 9.4% clay with a pH of 6.2. The seeds were planted 4.0 cm deep and 4.5 kg/ha of metolachlor was then applied preemergence with a chain-link belt compressed air sprayer, which delivered 280 L/ha at 240 kPa. A total of 1200 ml of water was added per pot to the soil surface for incorporation and activation of the herbicide. This amount gave approximately 12% moisture content (w/w) to the previously air-dried soil. Greenhouse 53 conditions were 16 h days at 24 C +/-2. The plants were grown with supplemental lighting from high-pressure sodium lamps. The light intensity was 500 uE.m'2's-1 with only 2.s_1 supplemental lights to 1200 uE'm- with both supplemen- tal and natural sunlight. The greenhouse was maintained at 40 to 70 % relative humidity. After 10 days, plant height was measured and visual injury was evaluated. The mean of four plants was considered one obsevation. Data is expres- sed as percent of control for that particular inbred. The experiment included four replications and was repeated. mid 53.1.1511 and eeleetien Corn hybrids were evaluated in the greenhouse by ob- taining 200 corn hybrids from 17 major seed companies across the Midwest. Twenty-four tolerant and sensitive hybrids were selected for further study based on the re- sults from the 200 hybrids. The 24 hybrids were treated with 6.7 kg/ha of metolachlor for further evaluation and from these two tolerant and two sensitive hybrids were selected for 14C -metolachlor absorption and metabolism studies. Cargill 7567 and Great Lakes 584 were tolerant, while Pioneer 3744 and Northrup King 9283 were sensitive to metolachlor (Figure 1). 54 3321321212: sesemeien end nefebeliem 14C-Metolachlor (specific activity 7.26 uCi/umole, uniformly ring labelled) was obtained from the CIBA-Geigy Corporation. The corn seed from the four hybrids mentioned above were placed on germination blotters and covered with paper towels. They were then placed in a dark growth cham- ber at 25 C. After 3 days the etiolated seedlings were removed from the growth chamber and the herbicide treatment was applied (Figure 2). A 2 ul drop, which contained 67.3 ug of metolachlor was applied just above the coleoptilar node of the corn seedling. Of the metolachlor applied only 2% was 1‘tC-metolachlor. The seedlings were placed back into the growth chamber for an 8-h absorption period. Based on preliminary studies this period of time allowed for maximum measurability of absorption and metabolism activity. After the 8-h absorption period the seedlings were rinsed with 3 ml of 100% methanol. Preliminary work verified that all surface radioactivity could be removed with 3 ml of 100% methanol. The seedlings were then placed in dry ice at -30 C until extraction. Two plants for each treatment were weighed, combined, and extracted with a Virtis grinder in 50 ml of 90% methanol for 5 min. The extract was filtered with Whatman No. 1 paper. The residue was oxidized with a Harvey Biological Oxidizer and counted with a Tri-Carb Liquid Scintillation Spectrometer. The volume of the extract was reduced under vacuum at 35 C and 55 1 ml of 90% methanol was added. A 100 ul aliquot of the concentrated extract was radioassayed with the spectrometer and a 50 ul aliquot was spotted on a Silica Gel GF TLC plate. The plate was then eluted with butanol:acetic acid: water (12:3:5) and radioactivity distribution was deter- mined with an AMBIS Radioactivity Scanner. The results for absorption are presented as the percent 14C absorbed. The results for metabolism are presented as the percent of 14C absorbed that was converted to metabolite as determined by the scanner. Rf values were calculated for each area of radioactivity. The results represent the means of ten replications. By calculation based on absorption, metabo- lism, and weight of the corn seedling, internal concentra- tion of metolachlor for each hybrid was determined. RESULTS AND DISCUSSION Wendi The results of this study showed a wide range in response of 29 corn inbreds to metolachlor applied at 4.5 kg/ha. The amount of injury ranged from a low of 16.3% for a tolerant inbred to 80.0% for a sensitive one. The shoot height of corn inbred LH91 was 82.4% of the height of the control compared to 1.1185 which was only 23.6% of its control height (Table 1). The majority of the hybrids had 56 a midlevel of tolerance with their injury ranging from 35 to 55% of their controls (Figure 3). mmmw This study also indicated a high degree of variability in metolachlor injury among the 200 commercial hybrids tested (Table 2). The range of distribution resembled that of a normal distribution curve with some of the hybrids being very tolerant, while others were quite sensitive (Figure 4) . As in the inbred study the majority of the hybrids had a midlevel of tolerance. The subset of 24 hybrids included the hybrids on the ends of the injury response spectrum. Further evaluation of these revealed distinct differences in injury when meto- lachlor was applied at a higher rate (Table 3) . These differences led to the selection of two tolerant and two sensitive hybrids which exhibited dramatic visual dif- ferences in metolachlor injury (Figure 1). Great Lakes 584 and Cargill 7567 were metolachlor tolerant, while Northrup King 9283 and Pioneer 3744 were metolachlor sensitive. Metoleehlereeserpeienendmetabelisn The four selected hybrids were evaluated to identify differences in the absorption and metabolism of 14C-metola- chlor. From the qualitative analysis of the TLC plates we concluded that there was no difference in the path of meta- bolism for tolerant and sensitive hybrids. The radioacti- 57 vity in all hybrids was divided into two components (Figure 5). One was the parent compound metolachlor, which had a Rf value of about 0.82. The other component was a more polar metabolite which had a Rf value of 0.49. Based on previous reports (3,5,11) this metabolite is probably the inactive or non-phytotoxic conjugate of glutathione and metolachlor. The laboratory study indicated significant differences in the absorption and rate of metabolism of 14C-metolachlor for the four hybrids tested (Table 4). Great Lakes 584 absorbed only 23.0% of the l4C-metolachlor, which was sig- nificantly less than the other three hybrids. Cargill 7567 metabolized greater amounts of metolachlor than the three other hybrids, while Great Lakes 584 metabolized more than the two sensitive ones. The calculations of the internal concentrations of metolachlor remaining as parent compound revealed, as expected, a significantly higher amount in the sensitive Pioneer 3744 and a lower amount in the tolerant Great Lakes 584 (Table 5). However, the tolerant Cargill 7567 and the sensitive Northrup King 9283 contained the same internal concentrations of available parent metola- chlor. Based on the highly significant difference in vi- sual injury symptoms exhibited by these two hybrids, there may be differences in the sensitivity at the site of action for metolachlor. Perhaps there is a difference in the number of sites or the nature of site of action. 58 Eagle 1. Response of 29 corn inbred lines to metolachlor applied at 4.5 kg/ha. Shoot Injury Inbred Line height rating (% of untreated) (%) MBS838 82.4 16 LH91 71.5 30 LH108 70.0 43 LH82 69.7 40 LH146 63.3 46 FR31 62.5 43 LH109 61.5 48 M871 60.6 46 M88847 55.4 44 A632 55.1 49 LH119 53.5 45 LH132 51.6 49 LH74 48.9 51 FR19 46.0 61 LH59 45.4 60 FR1141 44.7 60 DF14 43.9 61 LH136 43.8 55 LH38 42.9 59 596 41.5 56 M38501 41.3 61 LBS? 40.0 61 FR23 39.6 58 LH145 36.1 60 LH54 34.6 70 M876 32.7 63 OF9 28.9 65 LHSl 26.4 80 LH85 23.6 73 LSD (0.05) 14.9 12 59 Table 2. Response of 200 hybrids to metolachlor at 4.5 kg/ha. Shoot Injury Hybrid height rating (% of untreated) (%) Asgrow 2545 88.1 18 Cargill 130411 86.7 43 Great Lakes 584 81.5 33 Northrup King 9470 79.2 32 Great Lakes 516 78.2 40 Northrup King 9251 76.0 32 Andersons 85 75.6 33 Terra 1125 75.6 48 Renk 68 75.4 23 Great Lakes 381 74.4 40 Asgrow 2330 74.0 35 Renk 76 73.9 35 Great Lakes 365 73.5 50 Terra 975 71.8 37 Callahan 19097x 71.5 37 Great Lakes 547 71.3 32 Callahan 19101X 71.3 42 Golden Harvest 2343 70.5 47 Callahan 19102x 70.3 43 Pioneer 3352 70.1 37 Crows 181 69.4 32 Cargill 7567 69.0 42 Asgrow 6882 69.0 35 Northrup King 9385 68.9 47 Great Lakes 487 68.9 42 Golden Harvest 2572 68.6 43 Great Lakes 482 68.2 48 Great Lakes 85553 68.1 33 Pioneer 3540 68.0 37 Dekalb 547 67.9 58 Great Lakes 437 67.6 48 Pioneer 3704 67.5 45 Asgrow RX788 67.1 43 Great Lakes 420 67.0 55 Dekalb 572 66.6 42 Great Lakes 5922 65.7 38 Dekalb 484 65.6 33 Great Lakes 498 65.2 38 Terra 3203 65.2 47 Andersons 107 65.1 35 Andersons 110 64.8 35 Callahan 19925X 64.5 30 Renk 73 64.4 33 Renk 1060 64.2 52 60 Table 2. Continued. Shoot Injury Hybrid height rating (% of untreated) (%) Northrup King 9540 63.8 43 Stauffer 7751 63.8 43 Cargill HT115 63.7 45 Asgrow XP4506 63.4 50 Golden Harvest 2492 62.9 32 Great Lakes 599 62.6 45 Great Lakes 466 62.4 38 Crows 199 62.4 32 King 1184 62.3 40 Glenn-Garno 1005 62.0 42 Northrup King 9161 61.9 50 Payco 800 61.8 40 Golden Harvest EX536 61.5 50 Voris 2491 61.4 40 Glenn-Garno 1012 61.1 50 Glenn-Garno 988 61.0 38 Stauffer 4474 61.0 55 Stauffer 4590 60.9 37 Dekalb 524 ; 60.7 50 Andersons 95 7 60.3 50 Asgrow RX498 60.1 48 Terra 1040 59.9 67 King 596 59.7 52 Renk 21 59.7 60 Crows 212 59.4 37 Voris 2515 59.2 40 Dekalb 464 59.1 58 Great Lakes 86647 59.1 45 Stauffer 4454WX 58.7 43 Asgrow 180 58.7 47 Cargill 7877 58.6 43 Andersons 103 58.6 52 Stauffer 5750 58.6 50 Pioneer 3902 58.5 55 Cargill HT110 58.3 52 Great Lakes 86601 58.3 50 Callahan 19908x 58.0 43 King 237 58.0 48 Great Lakes 87680 58.0 58 Pioneer 3790 57.8 53 Great Lakes 87671 57.8 57 Great Lakes 579 57.6 51 Callahan 766 57.2 42 Glenn-Garno 900x 57.1 42 Stauffer 4402 57.1 48 Table 2. Continued. 61 Shoot Injury Hybrid height rating (% of untreated) (%) Stauffer 2184 56.8 38 Cargill 893 56.6 48 Northrup King 9527 56.6 42 Pioneer 3475 56.5 50 Callahan 747 56.4 50 Renk 64 56.2 53 Glenn-Garno 1003 56.2 57 Cargill 809 55.6 58 Terra 32 55.4 52 Glenn-Garno 8885 55.1 57 Stauffer 5722WX 55.1 48 Northrup King 9060 55.0 40 Terra 162E 54.9 53 Terra 3100 54.9 48 Cargill SX239 54.8 60 Callahan 754 54.8 53 Cargill 853 54.7 45 Voris 2465 54.7 47 King 5574 54.5 58 Andersons 93 54.4 40 Glenn-Garno 944 54.3 42 Andersons 99 54.3 58 Cargill HT120 54.2 60 Stauffer 2206 53.8 50 Crows 444 53.5 40 Asgrow RX578 53.5 48 Crows 442 53.1 50 Payco 611 53.1 52 Renk 138 52.6 50 Payco 847 52.5 45 Golden Harvest 2465 51.6 62 Callahan 738 51.5 55 Andersons 100 51.5 55 Payco 786 51.3 47 Dekalb 397 51.3 43 Terra 3102 51.3 52 Voris 2365 51.2 60 Stauffer 6707WX 50.9 33 Asgrow 2230 50.8 53 Cargill HT95 50.8 62 Crows 488 50.6 50 King 4422 50.5 58 Northrup King 9353 50.5 52 Northrup King 4325 50.3 62 Payco 342 50.3 57 62 Table 2. Continued. Shoot Injury Hybrid height rating (% of untreated) (%) Northrup King 9292 49.9 67 Pioneer 3803 49.5 57 Great Lakes 414 48.9 62 Crows 482 48.6 60 Pioneer 3901 48.3 57 Terra 29 48.1 63 King 416 47.9 50 Great Lakes 422 47.8 65 Renk 148 47.6 63 Golden Harvest EX615 47.4 70 Cargill 937 47.1 57 Cargill SX123 47.0 52 Crows 344 46.7 58 Terra 262E 46.7 45 Golden Harvest 2250 46.6 68 Renk 60 46.5 47 Renk 19 46.1 58 Voris 2331 46.1 62 Cargill 6127 45.8 68 Renk 27 45.5 60 Dekalb 435 45.3 63 Andersons 90 45.3 50 Stauffer 5340 45.3 60 Callahan 726 45.2 57 Great Lakes 82351 45.0 53 Stauffer 5650 44.5 62 Pioneer 3744 44.4 57 Golden Harvest 2344 44.3 72 King 647 44.2 63 Payco 686 43.7 50 Pioneer 3949 43.6 78 Cargill 3477 43.6 48 Asgrow RX626 43.6 57 Renk 7A 43.2 58 Payco 872 43.1 58 Cargill 3987 42.5 58 Northrup King 39 42.2 70 Dekalb 461 42.2 48 Cargill HT105 40.8 55 Great Lakes 313 40.4 77 Glenn-Garno 1007 40.0 63 Andersons 105 39.8 63 Crows 210 39.6 60 King 2203 39.5 45 Cargill 5157 39.1 68 63 Table 2. Continued. Shoot Injury Hybrid height rating (% of untreated) (%) Pioneer 3737 38.5 78 King 2204 37.5 70 Stauffer 3306 36.9 57 Northrup King 9283 36.8 72 King 4484 36.5 65 Cargill 859 36.3 67 King 4464 36.0 60 Cargill 2787 35.7 68 Stauffer 3303 35.6 62 Cargill SX310 35.0 66 Voris 2471 34.1 63 Stauffer 2101WX 33.4 65 Great Lakes SXllZ 33.1 65 Terra 3200 32.6 72 Callahan 728 31.3 68 Pioneer 3779 31.0 73 Cargill 819 30.6 60 Glenn-Garno 900 29.6 72 Dekalb 415 28.2 77 Renk 24 26.3 60 Payco 500 24.7 75 LSD (0.05) 16.0 11 Table 1. Response of 24 hybrids to metolachlor applied at 6.7 kg/ha. 64 Shoot Injury Hybrid height rating (% of untreated) (%) Cargill 7567 74.2 30 Crows 181 65.0 40 Great Lakes 516 62.0 39 Great Lakes 584 61.2 42 Crows 212 60.3 41 Cargill 8x239 59.7 41 Terra 3203 58.3 44 Renk 68 54.4 42 Asgrow 2545 54 . 3 44 Great Lakes 547 51.2 51 Golden Harvest 2343 51.1 46 Stauffer 3303 48.6 58 Renk 64 46.9 59 Northrup King 39 43.7 55 Terra 29 43.7 62 Pioneer 3949 43.2 54 Payco 500 42.9 58 Dekalb 415 41.7 61 Andersons 93 37.8 56 Pioneer 3779 37.5 61 Glenn-Garno 1007 33.6 63 Great Lakes 313 31.7 69 Pioneer 3744 30.3 72 Northrup King 9283 27.2 68 LSD (0.05) 9.8 9 65 Table i- Absorption and metabolism of 14C-metolachlor by tolerant and sensitive corn hybrids. Hybrid 14C Absorbed Metabolite (% of applied) (% of absorbed) Cargil 7567 33.2 32.3 Great Lakes 584 23.0 26.6 Northrup King 9283 30.5 21.6 Pioneer 3744 36.6 18.7 LSD (0.05) 6.3 3.9 Table §- Internal concentration of 14C-metolachlor in tolerant and sensitive corn hybrids. Hybrid Concentration (Hg/9) Cargill 7567 1.13 Great Lakes 584 0.74 Northrup King 9283 1.04 Pioneer 3744 1.49 LSD (0.05) 0.20 66 Elgnre 1. Four selected corn hybrids, from left to right, Cargill 7567, Northrup King 9283, Great Lakes 584, Pioneer 3744. 67 figure 2.- Flow diagram for absorption and metabolism study. 68 concocecms_ mm:_o> E 8:25:30 8263830 5:58 335000601 umccoom 335000631 oczanoo Emcee All IIIV 3:03032 to .8 85.8 coesoaem 0.: Axes .8650: £3, 62683 8968952 All IIIV Umouomo< 6:01:08 5:; most .5 m tote. 628.868 0.1me 8:95 . moEEmmm Eco Eolxoolm 69 Ml m 1 g 12. :1 4 'o 10* fl 0 ,\ e ‘ ' x c 84 r. ‘ \ '4- J ,’ \ O 6" I \ , r L. , \ 0 J ' “ I.\ .0 r , ’ E 44 ”. . \‘ v \ 3 4 r s‘ I 2 2:1 ’I k‘ 4 ,’ ‘I~~ o ‘1'r':‘f*f*"'3"f'f""rff"r*"'f'f"r""r‘f‘fi U 10 20 30 ‘0 50 50 70 DO 80 100 Shoot Height (76 of Control) Figure ;. Distribution of injury for 29 corn inbred lines treated with 4.5 kg/ha of metolachlor. 69 Number of Inbred Lines m figure;- I" , \ , \ , t , \ , \ , \ , \ , t \ l ‘ v’ ‘ a I’ ‘ ’1 \ a \ r s ’ ‘ I h. I ‘~ I s I 's‘ I s~ 'U""r""U'f"rv'fir""r""'U‘fV'rTh 10 20 30 40 60 70 90 100 Shoot Height (9: of Control) Distribution of injury for 29 corn inbred lines treated with 4.5 kg/ha of metolachlor. 0| 0 7O .. re N u or a 4. 91-53.1911Engineuufi’lil AA Number of Hybrids A d O a 0 .AAA AIAAA- I- C memes. ’\ I’ i I t I t I \ I \ p \ I \ , I I \ \ ,1 \ e’ ‘ ’ \ I , K ' ‘ . \ , I I \ I \ ’ l .I’ I‘ ‘ 'f'Uv'V'I""T""U '''''''' j'jfi' 10 20 30 4o 50 60 7'0 so 90 Shoot Height (7; of Control) Distribution of injury for 200 corn hybrids treated with 4.5 kg/ha of metolachlor. 71 figure:- Output from AMBIS Radioactivity Scanner. 10. 11. LITERATURE CITED Anderson, R.N. 1964. Differential response of corn inbreds to simazine and atrazine. Weeds. 12:60-61. Francis, T.R. and A.S. Hamill. 1980. Inheritance of maize seedling tolerance to alachlor. Can. J. Plant Sci. 60:1045-1047. Fuerst, E.P. and J.W. Gronwald. 1986. Induction of rapid metabolism of metolachlor in sorghum shoots by CGA-92194 and other antidotes. Weed Sci. 34:354-361. Harcastle, J.S. and W.A. Krueger. 1974. Differences in tolerance of metribuzin by varieties of soybean. Weed Res. 14:181-184. Leavitt, J.R.C. and D. Penner. 1979. In vitro conjugation of glutathione and other thiols with acetanilide herbicides and EPTC sulfoxide and the action of the herbicide antidote R-25788. J. Agric. Food Chem. 27:533-536. Narsaiah, D.B. and R.G. Harvey. 1977. Differential responses of corn inbreds and hybrids to alachlor. Crop Sci. 17:657-659. Niccum, C.E. 1985. Variations in inbred and varietal tolerance of corn to butylate, alachlor, and propa- chlor. Proc. North Central Weed Contr. Conf. 25:33-35. Renner, K.A., W.F. Meggitt and D. Penner. 1988. Response of corn cultivars to imazaquin. Weed Sci. 36:625-628. Roggenbuck, F.C. and D. Penner. 1987. Factors influencing corn tolerance to trifluralin. Weed Sci. 35:89-94. Sagaral, E.G. and C.L. Foy. 1982. Response of seve- ral corn cultivars and weed species to EPTC with and without the antidote R-25788. Weed Sci. 30:64-69. Zama, P. and K.K. Hatzios. 1986. Effects of CGA- 92194 on the chemical reactivity of metolachlor with glutathione and metabolism of metolachlor in grain sorghum. Weed Sci. 34:834-841. 72 Chapter 4 EFFICACY OF CGA-154281 AS A PROTECTANT FOR CORN FROM METOLACHLOR INJURY ABSTRACT Greenhouse and field studies were conducted to deter- mine the influence of herbicide rate, hybrid variability, and soil moisture content on the effectiveness of CGA- 154281 in protecting corn seedlings from metolachlor injury. In greenhouse studies, metolachlor and CGA-180937 (metola- chlor + CGA-154281) were applied preemergence at seven rates ranging from 1.1 kg/ha to 7.8 kg/ha. Four corn hybrids, which were previously identified as being tolerant or sensitive to metolachlor, were used. As expected, high rates of metolachlor caused significant injury to the corn seedlings, especially the sensitive hybrids. However, with CGA-180937, very few injury symtoms were observed, even at the highest herbicide rate and with the most sensitive hybrid. Four watering regimes were used to evaluate pro- tection by CGA-154281 at various soil moisture contents. Corn seedlings treated with CGA-180937 showed no signifi- cant injury, whereas, those treated with metolachlor alone showed 70% injury at the highest moisture level. Metola- chlor injury increased as soil moisture content increased. 73 74 In field studies in 1987 and 1988, metolachlor and CGA- 180937 were applied at rates up to 6.7 kg/ha to hybrids ranging in sensitivity to metolachlor. These studies also indicated that CGA-154281 was effective in protecting corn seedlings under conditions conducive to metolachlor injury. Nomenclature: Corn, Zea naye L.: metolachlor, 2-chloro-N- (2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)aceta- mide: CGA-154281, 4-(dichloro-acetyl)-3,4-dihydro-3-methyl- 2H-1,4-benzoxazine; CGA-180937, metolachlor' + CGA-154281 (30:1). aggibienal ingex,uezgee Protectant, hybrid varia- bility, soil moisture contents. INTRODUCTION Metolachlor is generally safe for use on corn, however, it may injure corn seedlings under certain conditions. Factors which enhance metolachlor injury to corn are high application rates, inherent sensitivity of hybrids or in- bred lines, and high soil moisture content (1,4,5). Chemical antidotes or protectants are known to protect grain sorghum (fieggbum bieele; L.) from metolachlor. These antidotes have also been shown to protect corn seedlings as well (2,3,6). Since a seed treatment with some expense is required and the metolachlor injury to corn is infrequent and limited, these compounds have not been utilized in corn production. The new experimental protectant CGA-154281 (Figure 1) is being evaluated specifically for the protection of corn from metolachlor. Very little is known about this protec- tant, thus the objectives of our research were to evaluate the effectiveness of this new protectant for corn seedlings growing under conditions that are known to be conducive to metolachlor injury. Also, we wanted to determine whether the CGA-154281 provided protection to several weed species. 75 MATERIALS AND METHODS WWW Corn seed of selected hybrids were planted in 946 ml 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. The herbicides were applied preemergence with a chain-link belt, compressed air sprayer which delivered a volume of 280 L/ha at 240 kPa. Varying amounts of water were added to the soil surface for incorporation of the herbicide. The pots were placed in the greenhouse which was maintained at 16 h days at 25 +/- 2 C. The plants were grown with supplemental lighting from high-pressure sodium lamps. The 2.8-1 with only sup- light intensity ranged from 500 uE.m' plemental lights to 1200 uE.m'2.s"1 with both supplemental and natural sunlight. The greenhouse was maintained at 40 to 75% relative humidity. Shoot height and injury ratings were taken after 10 days. Shoot height is expressed as percent of the untreated plant's height. Plant injury rating was on a scale of 0 (no effect) to 100 (completely dead). The mean of three plants in each pot was considered one observation. Each treatment was replicated four times and the data are the means of two experiments. The data was analyzed, and means were separated with LSD values at the 5% level of significance. 76 77 Magnetism Four corn hybrids, which were previously identified as being tolerant or sensitive to metolachlor, were evaluated under the above conditions at application rates ranging from 1.1 to 7.8 kg/ha of metolachlor in the Dual formula- tion or the CGA-180937 formulation (metolachlor + CGA- 154281). The tolerant hybrids were Cargill 7567 and Great lakes 584. The sensitive ones were Pioneer 3744 and Northrup King 9283. Metolachlor was applied preemergence at rates of 1.1, 2.2, 3.4, 4.5, 5.6, 6.7, and 7.8 kg/ha. After the herbicide application, 125 ml of water was ap- plied to the soil surface for incorporation. This gave a moisture content of 12% for each pot, which was equilavent to field capacity for the soil. Equal amounts of water were added to each pot thereafter until the data was col- lected. Seil meiseure resumes Pioneer 3744 corn hybrid was grown as previously des- cribed. Metolachlor was applied alone or in the presence of CGA-154281 at 4.5 kg/ha. Applications of water were made to obtain soil moisture contents of 8, 12, 18, and 22% moisture. Preliminary studies were conducted to verify that adequate corn seedling growth could be obtained at these soil moisture levels. The soil moisture contents were maintained daily by weighing and adding the appro- 78 priate amounts of water as needed. After 10 days plant heights and injury ratings were taken as previously report- ed. Heed respense This study was conducted to determine if CGA-154281 provided protection from metolachlor to certain weed spe- cies. The study was conducted under the previously re- ported greenhouse conditions. Eight weed species were planted in 30 by 50 cm pans and metolachlor or CGA-180937 was' applied preemergence at 1.1 and 2.2 kg/ha. After 14 days injury ratings were taken. The weed species planted were giant foxtail (eeberia fiabezii Herrm.), barnyardgrass (Eehineehlea emezseli (L). Beaum). fall panicum. (Banieum dishetmniflenm Michx.) . johnsongrass (Serehum helepense (L.) Pers.), common ragweed (Ma W L.), common lambsquarters (Qbenenediun album L.) , redroot pig- weed (Anazanebue zebzefilexue L.), and shattercane (Sergbum bieele; L. Moench.). Eieldefeeies Field studies were conducted at two locations in 1987 and 1988 to determine if CGA-154281 was effective in pro- tecting sensitive corn hybrids from high rates of metola- chlor under field conditions. In these experiments, 6.7 kg/ha of metolachlor in the Dual formulation or in the CGA- 79 180937 formulation (metolachlor + CGA-154281) was applied on hybrids which were previously selected in greenhouse studies as being metolachlor tolerant or sensitive. In all experiments the herbicides were applied premergence with a tractor mounted compressed air sprayer which delivered 205 L/ha at 206 kPa. The experiments were arranged as split- plot design with herbicide treatment being the whole plot and hybrids being subplots. Plant height and injury ra- tings were taken after 21 days, and the height data was converted to percent of control. In 1987, both locations were on the campus of Michigan State University in East Lansing. One location was on the Crops farm, which has a Riddles sandy loam (mixed, mesic Typic Hapludalfs, 67.6% sand, 10.4% silt, 21.1% clay) soil with a pH of 6.9, while the second location in 1987 was on the Soils farm. The soil type there is a Capac sandy clay loam (mixed, mesic Aric Ochraqualfs, 61.6% sand, 24.4% silt, 14.1% clay) with a pH of 6.8. Six hybrids marketed by three seed companies were used in 1987. In preliminary studies a relatively tolerant and sensitive hybrid from each company was selected. The tolerant hybrids were Pio- neer 3352, Andersons 85, and Dekalb 584. The sensitive hybrids were Pioneer 3475, Andersons 103, and Dekalb 415. Metolachlor was applied at 2.2 and 6.7 kg/ha while CGA- 180937 was applied at 6.7 kg/ha. In 1988, one location was on campus in East Lansing, 80 and the other location was near Battle Creek, Michigan at the Kellogg Biological Field Station (KBS). The campus soil is a Capac sandy loam (mixed, mesic Aeric Ochraqualfs) consisting of 71.6% sand, 10.4% silt, and 18.1% clay with a pH of 6.5. The KBS soil is a Oshtemo sandy loam (mixed, mesic Typic Hapludalfs) consisting of 71.3% sand, 19.4% silt, and 9.4% clay with a pH of 5.9. Four hybrids which were selected as being tolerant or sensitive were used. The tolerant hybrids were Cargill 7567 and Great Lakes 584, while the sensitive ones were Pioneer 3744 and Northrup King 9283. In 1988, both metolachlor and CGA-180937 were applied at 2.2 and 6.7 kg/ha. Plant height and injury ratings were taken after 21 days, and percent of control was calculated. RESULTS AND DISCUSSION Rate and maria respense In this study the CGA-154281 protected even the sen- sitive corn seedlings at the high metolachlor application rates (Table 1) . The metolachlor alone treatment caused significant injury to Cargill 7567 at 4.5 kg/ha. However, for that same hybrid, the protectant protected the seed- lings even at the highest rate of 7.8 kg/ha. With the other tolerant hybrid, Great Lakes 584, the protectant 81 protected up to 5.5 kg/ha (Figure 2). For the sensitive hybrid, Northrup king 9283, signifi- cant injury occurred with the metolachlor alone treatment at 2.2 kg/ha, while the protectant protected this hybrid up to 6.6 kg/ha. Significant injury was also observed with Pioneer 3744 at 2.2 kg/ha with the metolachlor alone treat- ment, however with the protected treatment no injury occur- red up to 5.5 kg/ha. MeisSureresnenee CGA-154281 significantly protected Pioneer hybrid 3744 corn seedlings form the 4.5 kg/ha metolachlor application even at the highest soil moisture level (Figure 3). At 22% moisture the injury from metolachor was 78.8% compared to the 10% injury for the metolachlor plus protectant treat- ment (Table 2). Feedreeesmse When metolachlor plus protectant was used at the la- belled rate of 2.2 kg/ha, there was no significant protec- tion of any of the eight weed species tested. However, at one-half the labelled use rate or 1.1 kg/ha a significant degree of protection occurred for johnsongrass and shatter- cane (Table 3). 82 Field studies In the field studies in 1987 there was a significant location effect so the study could not be combined over the two locations. In 1988 the study was combined over loca- tion and the data is presented as such. In 1987 on the Crops Farm, there was no significant injury to any of the hybrids even at the high rate of meto- lachlor, as no significant rainfall occurred within 24 days after the preemergence application of the herbicides. Therefore, the protectant potential of CGA-154281 could not be accurately determined. However, that year at the Soils Farm 3.5 cm of rain fell within 5 days after appplication, resulting in significant injury to the sensitive hybrids at the high rate of metolachlor. This injury was not evident in the CGA-180937 treatment, indicating adequate protection with the antidote (Table 4). In 1988 in the combined experiments, protection was again evident at the high rate and on the more sensitive hybrids (Table 5). The metolachlor alone treatment at 6.7 kg/ha resulted in a reduction in plant height to 72.4 and 72.9 percent of control for Northrup King 9283 and Pioneer 3744, respectivery. However, the addition of the protec- tant prevented significant injury (Figure 4) . In all the field studies there was generally no significant injury when metolachlor was applied alone at the labelled use rate of 2.2 kg/ha. 83 Table l. Response of four hybrids to metolachlor at 8 application rates with and without CGA-154281. Metolachlor Shoot rate Hybrid CGA-154281 height (kg/ha) (% of untreated) 1.1 Cargill 7567 - 99.2 + 103.7 NR 9283 - 98.5 + 103.1 CL 584 - 98.5 + 102.7 Pioneer - 96.9 + 101.8 2.2 Cargill 7567 - 96.7 + 102.2 NR 9283 - 82.8 + 97.8 GL 584 - 91.3 + 97.8 Pioneer 3744 - 83.8 + 91.5 3.4 Cargill 7567 - 90.2 + 100.8 NR 9283 - 72.0 + 92.9 GL 584 - 86.5 + 92.2 Pioneer 3744 - 71.1 + 91.9 4.5 Cargill 7567 - 81.1 + 95.5 NR 9283 - 54.8 + 92.3 CL 584 - 70.9 + 95.9 Pioneer 3744 - 61.8 + 90.2 5.6 Cargill 7567 - 75.9 + 98.3 NR 9283 - 53.1 + 93.8 GL 584 - 70.8 + 90.1 Pioneer 3744 - 44.4 + 86.4 6.7 Cargill 7567 - 69.3 + 91.5 NR 9283 - 40.5 84 Table 1. Continued. Metolachlor Shoot rate Hybrid CGA-154281 height (kg/ha) (% of untreated) 86.4 64.6 83.9 28.8 80.7 55.7 88.5 31.5 80.8 47.6 85.8 27.0 76.3 GL 584 Pioneer 3744 7.7 Cargill 7567 NR 9283 GL 584 Pioneer 3744 + r+-r-+r + r+-r-+r + LSD (0.05) 13.4 85 Table 2. Response of Pioneer 3744 to metolachlor at four soil moisture regimes with and without CGA-154281. Soil moisture Shoot Injury content CGA-154281 height rating (%) (% of untreated) (%) 8 - 89.8 15 + 98.9 0 12 - 45.6 45 + 89.1 1 l8 - 34.1 64 + 87.3 11 22 - 19.9 79 + 70.1 10 LSD (0.05) 8.7 8 86 Table ;. Response of eight weed species to metolachlor at two application rates with and without CGA- 154281. Metolachlor Injury Weed rate CGA-154281 rating (kg/ha) (%) Giant foxtail 1.1 - 100 + 98 2.2 - 100 + 100 Barnyardgrass 1.1 - 99 + 98 2.2 - 100 + 100 Fall panicum 1.1 - 100 + 100 2.2 - 100 + 100 Johnsongrass 1.1 - 97 + 78 2.2 - 98 + 93 Common ragweed 1.1 - 72 + 87 2.2 - 98 + 99 Common lambsquarters 1.1 - 92 + 88 2.2 - 99 + 99 Redroot pigweed 1.1 - 98 + 98 2.2 - 100 + 100 Shattercane 1.1 - 97 + 72 2.2 - 98 + 97 LSD (0.05) 10 87 Table 5. Field response of six hybrids to metolachlor with and without CGA-154281 in 1987. Shoot height Metolachlor Crops Soils Hybrid rate CGA-154281 Farm Farm (kg/ha) --(% of untreated)-- Pioneer 3475 2 2 98.7 93.8 6 7 96.5 70.1 103.5 95.7 Pioneer 3352 2 2 101.5 98.2 6 7 99.1 82.0 97.8 96.2 Andersons 103 2.2 93.5 101.7 6.7 95.8 84.2 99.7 101.6 Andersons 85 2 2 102.3 92.3 6 7 92.0 86.1 106.7 99.8 Dekalb 415 2 2 99.4 91.3 6 7 100.3 73.4 105.6 98.1 Dekalb 584 2 2 97.1 100.9 6 7 96.4 89.1 105.4 99.5 LSD (0.05) ns 11.5 88 Table 5- Field response of four hybrids to metolachlor with and without CGA-154281 in 1988. Metolachlor Shoot Hybrid rate GOA-154281 heighta (kg/ha) (% of untreated) Cargill 7567 2.2 - 100.2 + 97.7 6.7 - 96.8 + 102.9 Great Lakes 584 2.2 - 91.9 + 98.4 6.7 - 86.4 + 98.2 Northrup King 9283 2.2 - 96.7 + 102.5 6.7 - 72.4 + 99.7 Pioneer 3744 2.2 - 98.3 + 95.8 6.7 - 72.9 + 94.5 LSD (0.05) 12.5 aData combined over two locations in Michigan. 89 /\l/O\ V\ /\ # ['9 CH3 OZC—CHC12 CGA— 154281 film 1. Chemical structure of CGA-154281. 90 Figure 2. Response of 4 hybrids to metolachlor and CGA- 180937 at 8 application rates (LSD (0.05) = 13.4). 91 Asia; 26m 62656: A285 23. 62636: Nb who Wm Ev WM. «bu Hp m 0 Wu who new It nun NAN J— m o -9 .8 .ON -om S.R., 62.2.. .on nmum 95. 9:562 ”on -9. ".9. .on won -8 .8 .2 AK .00 .om .8 ".8 .69 J\\ woo— o: -.. . o: 0 RN. mww who the fin NW J _ Lo 0 320230: III M .oF human—I38 9.6 now .ON wow :3 48.3 6.5 .8 BE :98 -on .0? uOv .Om -On .8 -8 -os -os .om .8 .O@ .OO .00— -oo— o: y o: (Ionuoo l0 :4) 1451914 locus (IOJlUOO 10 2) NEW 10093 92 110. 100% 53> O .1 *3 905 o 80': 0 : 15 71%: R 60-‘1‘ V .1 E; 504-." "6 40% I: : .. 305 O : .2 we 0) : 10': one GOA-180837 0 e—n Metolachlor r I r fit I 5 fi I I T r I T r I T—r T 5 10 15 20 25 Soil Moisture Content (3) Figure 3- Response of Pioneer 3744 to metolachlor and CGA- 180937 at four soil moisture contents (LSD (0.05) = 8.7). 92 llllu'lllllllllllllllllllllllIIUII Shoot Height (X of control) :2 ES 23 8: 23 £3 £3 23 £3 £3 53 23 { one GOA-180937 : H Uetoloonlor r ‘ r I I U V r U V r t I I 1 U r r S 10 15 20 25 Soil Moisture Content (%) Iiggzg 1. Response of Pioneer 3744 to metolachlor and CGA- 180937 at four soil moisture contents (LSD (0.05) = 8.7). 93 Eiggrg A. Field response of four corn hybrids to metola- chlor and GSA-180937 in 1988 (LSD (0.05) = 12.5). 94 3////////////////////////////////////. « 55555555554 my 7%,///////////////////////////////////é 55555555554 2%???//////////////////////,. 33555552 mm 73//////////////////////////////////// 5555555555: ““ c228 3 5 22.: .85 7//////////////////////////////////// « 555554 «I. 7/////////////////////////////////////////. .m 5555 / .m. w a???//////////////////////é 555552 mm MW. Z////////////////////éé/é/é Wm 2 35555552 ‘1“ 93:8 .o 5 22.: .86 LITERATURE CITED Boldt, L.D. and M. Barrett. 1988. Factors in alachlor and metolachlor injury to corn seedlings. Abstr, Weed Sci. Soc. Amer. 28:84 Hatzios, K.K. 1984. Interactions between selected herbicides and protectants on corn. Weed Sci. 32:51-58. Leavitt, J.R.C. and D. Penner. 1978. Protection of corn from acetanilide herbicide injury with the antidote R-25788. Weed Sci. 26:653-659. Rowe, L. and D. Penner. 1987. Variability in corn tolerance to acetanilide herbicides. Proc. North Central Weed Contr. Conf. 42:32. Viger, P.R. and C.V. Eberlein. 1986. Corn tolerance to acetanilide herbicides. Proc. North Central Weed Contr. Conf. 41:7. Winkle, M.E., J.R.C. Leavitt, and O.C. Burnside. 1980. Acetanilide-antidote combinations for weed con- trol in corn and sorghum. Weed Sci. 28:699-704. 95 Chapter 5 INFLUENCE OF THE PROTECTANT CGA-154281 ON THE ABSORPTION AND METABOLISM OF METOLACHLOR ABSTRACT Laboratory studies were conducted to determine the effect of the protectant GSA-154281 on the absorption and metabolism of metolachlor in two metolachlor sensitive and two metolachlor tolerant corn hybrids. During an 8 h period, the CGA—154281 did not alter the absorption of 14C- metolachlor. Qualitative comparison of the metabolism of metolachlor in the presence or in the absence of the pro- tectant revealed that GSA-154281 did not alter the pathway of metolachlor metabolism. In both instances the metola- chlor was metabolized to a more polar metabolite, believed to be a glutathione conjugate. However, CGA-154281 signi- ficantly enhanced the rate of metabolism of metolachlor in three of the four hybrids tested. It appears from the data that the mechanism by which the antidote enhanced metola- chlor metabolism activity was already at a maximum in the unaffected hybrid, Cargill 7567. Nomenclature: Corn, Zea mags 1...: metolachlor, 2-chloro-N_-(2-ethyl-6-methylphenyl)- N-(2-methoxy-1-methylethyl) acetamide: CGA-154281, 4-(di- chloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine. 96 INTRODUCTION Metolachlor is commonly used for weed control in corn. However, injury symtoms to the corn are known to occur under certain conditions. It has been reported that the new protectant CGA—154281 is effective in alleviating the injury of corn from metolachlor (9). Since this is a new protectant, very little is known about its mechanisms of protective action. Previous hypotheses, which have been proposed for other chemical protectants suggest that these compounds may act in several ways, depending on the protec- tant, the herbicide, and the crop which is protected. One popular hypothesis is that the protectant simply enhances the rate of degradation or metabolism of particular herbi- cide in the crop (1,6). Others believe that the protectant may reduce the absorption of the herbicide, thereby main- taining a sub-toxic dose in the crop plant (2) . Another hypothesis is that the protectant may somehow alter or compete for the site of action for a particular herbicide, and by doing so, causes the herbicide to be inactive or non-phytotoxic in the crop (3,5,7). The objective of this research was to study the ab- sorption and metabolism of metolachlor in the presence or in the absence of CGA-154281 to determine the mechanism of its protective action. 97 MATERIALS AND METHODS This study was conducted to determine the effects of GSA-154281 on the absorption and metabolism of metolachlor in the corn hybrids Cargill 7567, Northrup King 9283, Great Lakes 584, and Pioneer 3744. The corn seed were placed in plastic containers on germination blotters and covered with moist paper towels. The containers were then placed in a dark growth chamber which was maintained at 25 C. After 3 days the etiolated seedlings were removed and the herbicide treatment was applied (Figure 1). A 2 ul drop, which con- tained 67.3 ug of metolachlor in the Dual formulation or in the CGA-180937 formulation (metolachlor + CGA-154281), was applied just above the coleoptilar node of the corn seed- ling. The metolachlor treatment contained 2% l4C--metola- chlor (specific (activity' 7.26 ‘uCi/umole, ‘uniformly' ring labelled). The seedlings were returned to the growth cham- ber for a 8-h period of herbicide absorption. Based on preliminary studies this period of time allowed for maximum measurability of absorption and metabolism activity. After the 8-h absorption period, the seedlings were rinsed with 3 ml of 100% methanol. Preliminary work verified that all surface radioactivity could be removed with 3 ml of metha- nol. The seedlings were then placed in dry ice at -30 C until extraction. Two plants for each treatment were weighed, combined, and extracted with 50 ml of 90% methanol 98 99 for 5 min. The extract was filtered with Whatman No. 1 paper. The residue was oxidized with a Harvey Biological Oxidizer and counted with a TRI-CARB Liquid Scintillation Spectrometer. The extract was dried down completely under vacuum, and 1 ml of 90% methanol was added. A 100 ul aliquot of the concentrated extract was radioassayed and 50 ul was spotted on a Silica Gel GF6O TLC plate. The plate was eluted in butanol:acetic acid:water (12:3:5) and ra- dioassayed with an AMBIS Ratioactivity Scanner. The re- sults are shown as calculated Rf values. The results for absorption were calculated by dividing the amount of 14C- metolachlor absorbed by the amount which was applied to get the percent of metolachlor absorbed. The results for meta- bolism were calulated by dividing the amount of 14C-metola- chlor that was metabolite by the total amount that was metobilite plus parent compound as quantified by the ra- dioactivity scanner. The mass balance of 14C was also calcuated (Figure 2). The results are from two experiments which included five replications each. RESULTS AND DISCUSSION No significant difference was observed in the amount of metolachlor absorbed by the corn seedlings in the pre- sence or absence of CGA-154281 (Table 1). This indicates 100 that the protectant did not protect corn by reducing the amount of herbicide absorbed. Two distinct areas of radioactivity were found on the TLC plate both in the presence and the absence of CGA- 154281. The Rf values for these areas were 0.82 and 0.49 (Figure 3). Previous studies (4,8,10), along with a stan- dard included in this study, suggest that the high Rf value of 0.82 is the parent compound metolachlor. Any metola- chlor at this Rf is still in the active form and is consi- dered to be available for herbicide activity. Previous studies (4,8,10) also indicate that the radioactivity at the lower Rf of 0.49 is the product of radioactive metola- chlor conjugation with glutathione to form the inactive metabolite. These results show that the protectant did not protect corn seedlings by changing the pathway of metola- chlor metabolism. In both cases the metoachlor was con- verted to a non-phytotoxic metabolite via the conjugation with glutathione. Quantitation of the metabolite of metolachlor metabo- lism showed that in the presence of GSA-154281, there was significantly greater amounts of the metabolite present. The increase in the metolachlor metabolism rate occurred in three of the four hybrids tested (Table 2). The metabolism was not enhanced in Cargill 7567. The rate of metolachlor metabolism in this hybrid in the absence of the antidote was already significantly greater than in the other three 101 hybrids. Apparently, in the Cargill 7567 the metolachlor metabolism was already functioning at a maximum level. Several hypothesis are available as to what mechanism is affected and therefore in turn speeds up metabolism. These include the increase of an enzyme which catalyzes the conjugation of metolachlor, or the increase in the gluta- thione concentration in the plant. Both of these possibi- lities deserve further study and review before final asses- ments can be made. 102 Tabla 1. E fect of CGA-154281 on the absorption of C-metolachlor by four corn hybrids. CGA-154281 Hybrid - + ------ (% absorbed)----- Cargill 7567 33.2 ab 28.6 bc Great Lakes 584 23.0 cd 21.2 d Northrup King 9283 30.5 ab 26.5 bcd Pioneer 3744 36.6 a 31.7 ab Means followed by a common letter are not significantly different at the 0.05 level according to Duncan’s multiple range test. 103 Tabla 2. f fect of CGA-154281 on the metabolism of C-metolachlor by four corn hybrids. CGA-154281 Hybrid - + ----- (% metabolite)---- Cargill 7567 32.3 a 30.4 ab Great Lakes 584 25.6 c 32.9 a Northrup King 9283 21.6 d 29.0 abc Pioneer 3744 18.6 d 27.3 bc Means followed by a common letter are not significantly different at the 0.05 level according to Duncan’s multiple range test. 104 Elgar; ;. Flow diagram for absorption and metabolism study with C-metolachlor. 105 cocooczcmn_ 323 E 8:35:30 8:05:30 mecoom géooofiom tmccoom 3_>:ooo_oom ocnanoo Lcmtod AIII IIV 3:09:22 to so 8:5 8:83me ofi $08 .0858: 55 828:3 8908952 All .IIV pontomb< 6:059: 53, must .3 m Eta. 52020me minfim pm__ad< mmczommm Eoo Eolxoplm e14C—Metolochlor Av vg. Rec very Rate = 95.08% 107 — £13313 1. Output from AMBIS Radioactivity Scanner. 10. LITERATURE CITED Adams, C.A., E. Blee, and J.E. Casida. 1983. Dichlo- roacetamide herbicide antidotes enhance sulfate metabolism in corn roots. Pest. Bio. Phys. 19:350- 360. Ebert, E. 1982. The role of waxes in the uptake of metolachlor into sorghum in relation to the protectant CGA-43089. weed Res. 22:305-311. Ezra, G. and G.R. Stephenson. 1985. Mechanism(s) of action of dichloroacetamide antidotes. Abstr. Weed Sci. Soc. Amer. 25:73. Fuerst, E.P. and J.W. Gronwald. 1986. Induction of rapid metabolism of metolachlor in sorghum shoots by CGA-92194 and other antidotes. Weed Sci. 34:354-361. Fuerst, E.P. 1987. Understanding the mode of action of the chloroacetamide and thiocarbamate herbicides. Weed Tech. 1:270-277. . Gronwald, J.W., E.P. Fuerst, C.V. Eberlein, and M.A. Egli. 1987. Effect of herbicide antidotes on glutathione-s-transferase activity of sorghum shoots. Pest. Bio. Phys. 29:66-76. Hatzios, K.K. 1985. Uses and potential mechanisms of action of herbicide safeners. Abstr. Weed Sci. Soc. Amer. 25:72-73. Leavitt, J.R.C. and D. Penner. 1979. In vitro conjugation of glutathione and other thiols with acetanilide herbicides and EPTC sulfoxide and the action of the herbicide antidote R-25788. J. Agric. Food Chem. 27:533-536. Rowe, L. and D. Penner. 1989. Efficacy of CGA-154281 as an antidote for protection against metolachlor. Abstr. Weed Sci. Soc. Amer. 29:5-6. Zama, P. and K.K. Hatzios. 1986. Effects of CGA- 92194 on the chemical reactivity of metoalchlor with glutathione and metabolism of metolachlor in grain sorghum. Weed Sci. 34:834-841. 108 SUMMARY AND CONCLUSIONS A review of research literature indicated that corn tolerance to the chloroacetanilides involves a complicated interaction of many factors associated with the production system. Distinct differences between herbicides, enviro- mental factors, genetic variability, and use of protectants all seem to play roles in determining if and to what extent corn will be injured. Our research was an effort to eval- uate these factors and increase our knowledge in this area of weed science. From our first series of studies we concluded that the corn hybrid planted, the herbicide, the herbicide applica- tion rate, and the soil moisture content at the time of early corn emergence all play a significant role in the amount of chloroacetanilide injury which occurred. Some of the hybrids were relatively tolerant to both alachlor and metolachlor, while others appeared to be more tolerant to one or the other of the two herbicides. There was general- ly a linear response of increasing herbicide injury with increasing herbicide application rate and with increasing soil moisture content._ However, we concluded that under normal conditions of using labelled herbicide application rates and soils at field capacity, there is generally not a 109 110 significant difference in injury between hybrids and herbi- cides since under these conditions no herbicide injury is likely to occur. We found that there was a high degree of variability in chloroacetanilide tolerance among corn inbred lines and commercial corn hybrids available to growers. The variabi- lity in tolerance resembled that of a normal distribution curve with some hybrids having a very high level of tole- rance, while others had a low level of tolerance. However, the vast majority of the hybrids had a midlevel of tole- rance. From the laboratory studies with 1’I‘C-metolachlor, we concluded that the variability of tolerance appeared due to differences in absorption, metabolism, and perhaps dif- ferences at the site of action of metolachlor. From our research with the new protectant, CGA-154281, we found that this compound was very effective in protect- ing corn seedlings from metolachlor injury. This protec- tion occurred even under the conditions that are known to enhance metolachlor injury. In field studies, we showed that metolachlor injury generally did not occur when ap- plied at the labelled use rates. However, CGA-154281 did give added assurance for safe use of metolachlor on corn under the most extreme conditions of high application rate on sensitive hybrids. Finally, we concluded that CGA-15428l did not protect corn from metolachlor injury by reducing the amount of 111 metolachlor absorbed by the seedling. Also, the protectant did not protect corn by altering the pathway of metolachlor metabolism. The protective action of CGA-154281 appeared due to the enhanced metabolism of metolachlor to a gluta- thione conjugate. "‘ainiiiilmmiiES