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Y Michigan State ‘ University Ifllllllllfljflfllllllllllfllflflll\lllMlfllfljl This is to certify that the . thesis entitled FATE AND SELECTIVITY 0F PHENOXY-PHENOXY HERBICIDES IN PLANTS presented by Paul Frederick Boldt has been accepted towards fulfillment of the requirements for Ph.D Horticulture degree in Major professor OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. FATE AND SELECTIVITY OF PHENOXY-PHENOXY HERBICIDES IN PLANTS By Paul Frederick Boldt A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1979 ABSTRACT FATE AND SELECTIVITY OF PHENOXY-PHENOXY HERBICIDES IN PLANTS By Paul Frederick Boldt The objectives of this study were to determine: l) the response of selected annual grass and broadleaf species to HOE 29152 [methyl 2-[4-(4-trifluoromethyl phenoxy)phenoxy] propanoate] and diclofop—methyl [methyl 2-[4-(2,4-dichlorophenoxy) phenoxy] propanoate] and 2) the selectivity mechanisms of foliar applications of diclofop-methyl. In greenhouse and field studies HOE 29l52 was more toxic to annual grass and broadleaf species than diclofop-methyl at equivalent rates. The selectivity of annual grass species to the two herbicides was fairly uniform, with barnyardgrass [Echinochloa crusfigalli (L.) Beauv.] being the most sensitive and longspine sandbur [Cenchrus longjspinus (Hack.) Fern.) the most tolerant. Large crabgrass (Digitaria sanguinalis L.) and proso millet (Panicum miliaceum L.), both moderately tolerant species to diclofop-methyl were sensitive to HOE 29152. Formulations of‘ HOE 29152 containing additional adjuvants were more toxic to annual grasses and crops than the normal farmulation at a given rate. The use of tank-mixed adjuvants with either herbicide caused a greater Paul Frederick Boldt fresh weight reduction in barnyardgrass. Site of uptake studies showed that phenoxy-phenoxy herbicides caused the greatest reduction in fresh weight in barnyardgrass when both the plant and soil were sprayed. Most of the activity was attributed to foliar uptake. Retention, absorption, translocation and volatility of foliarly- applied diclofop-methyl were compared in barnyardgrass - a susceptible grass, proso millet - moderately susceptible grass, longspine sandbur - a tolerant grass, soybean [Glycine max (L.) Merr. 'Hack'] and cucumber (Cucumis sativa L. 'Green Star') - both tolerant broadleaf species. On a ug/plant basis, the order of diclofop- methyl spray retention was cucumber > soybean > proso millet > longspine sandbur = barnyardgrass. On a ug/mg dry weight basis, proso millet retained 3 to l0 times more diclofbp-methyl than all other species. One day after treatment (DAT) absorption of 14C diclofop-methyl was l4 to l8% less in lonQSpine sandbur than the other species, 3 DAT absorption in cucumber was 8 to 14% greater than the other species, and 5 DAT absorption in soybean was 3 to l2% less 14c diclofop-methyl did than the other Species. Translocation of not differ among species with 98% of the applied radioactivity located in the treated leaf. Loss of radioactivity applied to the surface of intact living plants and excised dead plants of cucumber, soybean, and barnyardgrass as well as glass cover slips showed an average loss of ll% of the applied radioactivity. Differences in retention and absorption of diclofop-methyl were not related to selectivity. Paul Frederick Boldt In metabolism studies, more than 95% of the radioactivity washed from the leaf surface from all species was diclofop-methyl. Plant extracts contained diclofop-methyl, diclofop and water- soluble conjugates with higher levels of diclofop-methyl and diclofop being found in barnyardgrass, proso millet and soybean. Higher levels of water soluble conjugates were present in cucumber and longspine sandbur. Acid hydrolysis of the water soluble conjugates yielded high amounts of diclofop in barnyardgrass and proso millet, and high amounts of ring-OH diclofop in longspine sandbur, soybean and cucumber. Alkaline hydrolysis of the non- extracted plant residue yielded diclofop as the major component in all species. ACKNOWLEDGMENTS A special thanks is extended to Dr. Alan "Putt" R. Putnam for his guidance, time and most importantly, friendship during the course of my studies. Appreciation is given to Drs. Stanley K. Ries, William Meggitt, James Flore and Don Penner for serving on my guidance committee. The partial financial support of these studies by American Hoechst Company is acknowledged; and the expert technical assistance of Ms. Cathy Braue, Ms. Sylvia Dooley and Mr. William Chase is appreciated. Finally, but most important of all, thanks is given to Linda, my wife, who gave so much so I and we could have this day. ii TABLE OF CONTENTS 3393 INTRODUCTION . . . . . . . . ................ l CHAPTER 1 - PHENOXY—PHENOXY HERBICIDES - A LITERATURE REVIEW ........................... 3 HISTORY ........................ 3 PHYSICAL AND CHEMICAL PROPERTIES ........... 4 ACTIVITY AND SELECTIVITY ............... 4 FATE IN PLANTS .................... 9 Retention, Uptake and Translocation ....... 9 Metabolism .................... ll Physiological Effects . . ............ l2 FATE IN SOILS ..................... l4 LITERATURE CITED . .................. 15 CHAPTER 2 - RESPONSE OF SELECTED ANNUAL GRASS AND BROADLEAF SPECIES TO FOLIAR APPLICATIONS OF PHENOXY—PHENOXY HERBICIDES ......................... 19 ABSTRACT ....................... 19 INTRODUCTION ..................... 20 MATERIALS AND METHODS ................. 22 Phenoxy-Phenoxy Comparisons ........... 22 Field Studies with HOE 29l52 ........... 23 Adjuvant Studies ................. 24 Site of Uptake .................. 25 iii RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 25 Phenoxy-Phenoxy Comparisons ..... . ..... 25 Field Studies with HOE 29l52 . . . ........ 27 Adjuvant Study . . . ............... 34 Site Of Uptake .................. 34 LITERATURE CITED .......... . ........ 38 CHAPTER 3 - SELECTIVITY MECHANISMS TO FOLIAR APPLICATIONS OF DICLOFOP-METHYL. I. RETENTION, ABSORPTION, TRANSLOCATION AND VOLATILITY ................ 40 ABSTRACT ........................ 40 INTRODUCTION ..................... 42 MATERIALS AND METHODS ................. 43 Cultural Practices ................ 43 Retention Study ................. 44 Absorption Study ................. 44 Translocation Study ............... 45 Volatility Study ................. 45 Statistical Considerations ............ 47 RESULTS AND DISCUSSION ................ 47 Retention Study ................. 47 Absorption Study ................. 48 Translocation Study ............... 5] Volatility Study ................. 51 LITERATURE CITED ................... 55 TV Page CHAPTER 4 - SELECTIVITY MECHANISMS TO FOLIAR APPLICATIONS OF DICLOFOP-METHYL. II.' METABOLISM ............ 57 ABSTRACT ....................... 57 INTRODUCTION ..................... 59 MATERIALS AND METHODS ................. 60 Cultural Practices ................ 50 Chemical Treatment ................ 50 Sample Handling and Extraction .......... 5i Radioactivity Quantification ........... 53 Thin Layer Chromatography ............ 64 Statistical Considerations ............ 54 RESULTS AND DISCUSSION ................ 54 Leaf Nash .................... 54 Plant Extract .................. .57 Acid Hydrolysis ................. 71 Solid Residue .................. 71 LITERATURE CITED ................... 77 BIBLIOGRAPHY ........................ 79 LIST OF TABLES Table CHAPTER 2 Page 1 6R5 values (kg/ha) for foliar-applications giclofOp-methyl or HOE 29152 on selected plant species .................. 26 2 Control of annual grasses with postemergence applications of three formulations of HOE 29152 applied when grasses had two or three leaves and rated 14 days later ....... 28 3 Visual ratings Of longspine sandbur control in asparagus after foliar applications of phenoxy- phenoxy herbicides ................ 29 4 Injury to vegetable crops with postemergence applications of three formulations of HOE 29152 applied to small seedlings and rated 14 days later . . . . ................. 30 5 Interaction of HOE 29152 with several post- emergence broadleaf herbicides on weed control and onion injury ................. 32 6 Control of annual grasses with postemergence applications of non-ionic acetylenic adjuvants tank-mixed with HOE 29152 applied when grasses had two to three leaves and rated 14 days later ...................... 33 7 Influence of adjuvant type and concentration on postemergence activity of phenoxy-phenoxy herbicides on the fresh weight Of barnyardgrass 14 days after treatment ................. 35 8 Site of uptake of foliarly-applied diclOfOp-methyl and HOE 29152 in barnyardgrass and yellow foxtail grown on various soils . . . . . . . . . . . V1 Table CHAPTER 3 Page Retention Of foliarly-applied diclofop—methyl (36 EC) by five species measured immediately after application ............... . .49 Percent absorption of foliarly-a plied 14C diclOfOp-methyl, 1,3 and 5 DAT y five species. .50 Distribution Of foliarly- applied 14C diclofop- methyl in 5 species over time ..... . . .52 Loss of foliarly-applied 14C diclofop-methyl from plant and glass surfaces .......... 53 CHAPTER 4 Rf values for 14C diclofop-methyl and its metabolites in two solvent systems ........ 55 Percentage of total applied radioactivity in the leaf wash, plant extract and plant residue . . . .66 TLC separation of radioactivity in 50% methanol leaf wash on five species, 1, 3 and 5 DAT with C diclofop-methyl ............... 58 TLC separation of radioactivity in 80% methanol extract of five species, l, 3 and 5 DAT with diclofop-methyl ................. 59 TLC separation of radioactivity in the 6 N HCl hydrolyzed water phase of the 80% methanol extract of absorbed C diclofop-methyl ..... 72 Absorbed 14C diclofop-methyl not extracted with 80% methanol in five species, 1, 3 and 5 DAT as determined by combustion ........... 73 TLC separation of radioactivity removed from plant residue by 1 N NaOH hydrolysis in five species, 1, 3 and 5 DAT ................... 75 vii LIST OF FIGURES Figure CHAPTER 1 Page 1 Structures Of phenoxy—phenoxy herbicides released to date ................ 5 2 Proposed metabolism of diclofop-methyl in wheat and wild oat (28) ....... . . . . . 13 CHAPTER 4 1 Flow diagram for extraction, separation and quantification Of 14C diclofop-methyl and its metabolites in five plant species ..... 62 viii INTRODUCTION In the early l940's, the introduction of 2,4-D, for selective postemergence broadleaf weed control in grass crops, signaled the beginning of modern weed science. Since then many herbicides have been introduced for pre- or postemergence control, however, selective postemergence grass herbicides have been rare. Seven Of the ten worst weeds in the world are members of the Poaceae family. As economic and ecological pressures increase, the need for species specific chemical weed control increases. It is evident a need for a selective postemergence grass herbicide exists. In 1973, Hoechst, A. G. introduced HOE 22870 [4-(4'-chloro- phenoxy)phenoxy a-propionic-isobutyl ester] and HOE 23408 [methyl 2-(4-(2,4-dichlorophenoxy)phenoxy)propanoate] phenoxy-phenoxy herbicides for the control Of annual grass weeds in broadleaf crops and certain cereal grains. HOE 23408, now known as diclofop-methyl, was developed and should soon be approved by the Environmental Protection Agency for use on certain crops in the United States. Species tolerant to foliarly-applied diclofop-methyl include wheat (Triticum aestivum L.), quackgrass (Agropyron repens L.), Johnsongrass (Sorghum halpense L.), large crabgrass [Digitaria sanguinalis (L.) Scop.], sandburs (Cenchrus spp.) and all broadleaf plants. Three of these species, quackgrass, large crabgrass and sandbur are serious weed problems in horticultural crops. Selectivity studies have centered on the tolerance within the grass species particularly wheat, a tolerant species, and wild oat (Avena fatua L.), a susceptible species. 1 In 1977, HOE 29l52 [methyl 2-(4-(4-trif1uoromethyl phenoxy) phenoxy)propanoate] was released. Early reports indicated that many Of the species tolerant to diclofop-methyl, including perennial grasses, were susceptible to HOE 29152. This study had two objectives, the first being to determine the response of selected grassy weed Species and broadleaf crOps to foliarly-applied HOE 29152 and compare the efficacy of this compound with diclofop-methyl. The second Objective was to determine the primary mechanism(s) for selectivity Of foliarly-applied diclofop-methyl, on a whole plant basis, in broadleaf and tolerant grass species. CHAPTER 1 PHENOXY-PHENOXY HERBICIDES - A LITERATURE REVIEW The phenoxy-phenoxy compounds, a new class of herbicide chemistry, present a unique opportunity for selective postemergence control of grassy weeds in broadleaf and certain cereal crops. Early compounds in this Class were developed in the laboratories of Hoechst, A. 6., Frankfurt, Germany. HISTORY Two new phenoxy-phenoxy herbicides, HOE 22870 and HOE 23408, were introduced to the agricultural research community in 1973. HOE 22870 was discontinued after one year Of evaluation. A third compound, HOE 29152, was introduced in 1976 and fUrther development was terminated in early 1978. The Weed Science Society Of America assigned the common name diclofop to HOE 23408, with a corresponding chemical name of 2-(4-(2,4-dichlorophenoxy)phenoxy)propanoic acid. The methyl ester formulation has been termed diclofop-methyl. Early literature reports (19,32) have also referred to this herbicide as dichlofop, or dichlorfop. In Canada the herbicide is marketed as HOE-GRASSTM, while in the United States it is known as HOELONTM. PHYSICAL AND CHEMICAL PROPERTIES Chemical structures and names Of released phenoxy-phenoxy herbicides are shown in Figure 1. Physical properties of diclofop- methyl are: colorless, odorless solid, melting point - 39-41 C, boiling point - 175-177 C, volatility - 3 x To-7 mm mercury (20 C), 5 x 10'6 mm mercury (40 C), solubility - 50 g/100 ml xylene, 40 g/100 ml acetone or ethanol, 5 mg/100 ml water (1). Minimal information for HOE 29152 indicates it is soluble in organic solvents, 1.4 ppm solubility in water, vapor pressure Of 0.97 x 10'4 torr (43 C). The conventional formulation for the phenoxy-phenoxy herbicides has been a 36 EC, however, additions of adjuvants have been made to HOE 29152 and diclofop-methyl to make 24 EC formulations. ACTIVITY AND SELECTIVITY The phenoxy-phenoxy herbicides are used for selective grass control in broadleaf and certain cereal crops. All broadleaf plants treated with phenoxy-phenoxy herbicides have been tolerant at a dosage 2X or greater than that needed for acceptable grass control (2,5,17,20,22,24,25). The herbicides can be used preemergence to the weeds, either surface applied or incorporated, or postemergence. Evaluating l3 grassy weeds for response to diclofop-methyl applied preplant incorporated, preemergence or postemergence, Wu and Santelman (37), found the herbicide to be most toxic when incorporated into soil or applied early postemergence to the weeds. DiclOfOp-methyl HOE 22870 CH3 CH3 I l Cl @ 0.0 - CH - COOCHZ - CH - CH3 4-(4'-chlorophenoxy)-phenoxy-a-propionic-isobutyl ester diclofop-methyl (HOE 23408) CH3 I ' Cl methyl 2-(4-(2',4'-dichlorophenoxy)phenoxy)prOpanoate HOE 29152 CH3 I CF3© 0©0 - CH - CO0CH3 methyl 2-(4-(4'-trifluoromethyl phenoxy)phenoxy)propanoate Figure 1. Structures Of phenoxy-phenoxy herbicides released to date. gave greater control of wild oats (Avena fatua L.) and green foxtail (Setaria viridis L.) when applied postemergence (8,13). Since the need for selective postemergence grass herbicides far outweighs the need for more preemergence grass herbicides, the majority Of research conducted has concentrated on the postemergence use of the phenoxy- phenoxy herbicides. In the greenhouse, Anderson (3) evaluated the response Of 27 annual grass species to postemergence applications of HOE 22870 and diclofop-methyl at 1.1 kg/ha. Control ranged from 0-100% indicating that control Of grass weeds within grass crops was possible with these herbicides. Species most sensitive were barnyardgrass [Echinochloa crusegalli (L.) Beauv.], corn (;gg_mays L.), foxtails (Setaria spp.) and witchgrass (Panicum capilare L.), while wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and downy brome (Bromus tectorum L.) were tolerant. The perennial grasses, quackgrass [Agropyron repens (L.) Beauv.] and Johnsongrass [Sorghum halepense (L.) Pers.], were not controlled with these herbicides. Rao and Sweet (22), evaluating ll vegetable crops and 11 annual grass species for their response to postemergence Sprays of diclofop-methyl at 0.5 to 4 kg/ha, found all broadleaf crops and wheat tolerated the highest dosage. Studies by Putnam et al. (20) indicate excellent vegetable crop selectivity. Yields of flax (Linum usitatissimum L.) (10), rape (Brassica rapus L.) (10), and soybeans [Glycine max (L.) Merr.] (2) treated with diclofop-methyl were not influenced by the herbicide itself. Friesen et a1. (13) have shown diclofop-methyl will control wild oat and green foxtail in wheat and barley at 0.84 to 1.69 kg/ha. As the annual grasses advanced in age from 2 to 5 leaf, the effectiveness of the herbicide decreased. However, all treatments provided satisfactory control. Effective control of wild oat and Italian ryegrass (Lolium multiflorum Lam.) in winter was achieved with 0.84 kg/ha of diclofop-methyl applied at the 2 to 3 leaf stage of growth (6). Susceptibility Of annual grasses decreased by as much as 80 fold when treated at the 6 leaf stage as opposed to the 3 leaf stage (37). HOE 29152 controlled the same annual grasses as diclofop-methyl in addition to the perennial grasses, quackgrass and Johnsongrass (25,38). Annual grasses were controlled at 0.25 to 0.50 kg/ha (5). Johnsongrass at 0.86 to 1.68 kg/ha, quackgrass at 1.1 to 3.4 kg/ha. Wheat and barley exhibited the same degree of tolerance to HOE 29152 as they did to diclofop-methyl, while broadleaf plants tolerated l to 4 kg/ha (25). The addition of adjuvants to the spray solution of phenoxy- phenoxy herbicides indicated that diclofop—methyl at 0.56 kg/ha with an adjuvant gave postemergence wild oat control equal to diclofop- methyl alone at 1.12 kg/ha (l9). Adjuvants used were various crop Oils; petroleum Oil; Triton X-363, Triton X-100, Triton X-A; Renex 36; and CD 189. The oils were used at a rate Of 2.8 liters/ha while the surfactants were used at 0.5% v/v. Results of Nalewaja et al. (16) supported these studies. In addition to increased wild oat control, green foxtail and tame oat (Avena fatua L.) control was enhanced. Renex 36 (0.5% v/v) was more effective than Triton X-100 (0.5% v/v) at the 2 leaf stage but less effective at the 4 leaf stage. The adjuvants with diclofop-methyl did not increase phytotoxicity to the crops. Schrieber et al. (26) report that the addition of a wetting agent to postemergence sprays Of diclofop-methyl did not enhance control of weed grasses. Adjuvants added to the formulation of HOE 29152 increased the activity Of the herbicide on annual grasses, but also increased toxicity to seven vegetable crops evaluated (5). Tank-mixed adjuvants have been reported to increase the control of yellow foxtail, green foxtail, proso millet, and large crabgrass (5), as well as quackgrass (38). Combinations of 2,4-D [(2,4 dichlorophenoxy)acetic acid], MCPA [[(4-chloro-gftolyl)oxy] acetic acid] and dicamba (3,6 dichlorO-g: anisic acid) with diclofop-methyl caused a decrease in wild oat control when compared to diclofop-methyl alone (12). The antagonism was not due to spray solution incapability or the solvents in the formulations, but rather the active ingredients of the herbicides. The ester formulations were less antagonistic than the amine form- ulations. Broadleaf weed control by the phenoxy herbicides was not influenced by diclofop. Other interactions with the phenoxy-phenoxy herbicides have been reported. Bentazon [3-isopropyl-lflyz,l,3-benzothiadiazin-4 (NH)-one 2,2-dioxide] and diclofop-methyl tank-mixed at 1.12 kg/ha of each herbicide caused a leaf weight reduction in treated soybeans (36). Wild oat control with desmidipham (ethyl-m hydroxy carbanilate carbanilate) (18) or dinoseb acetate (Z-sggfbutyl-4,6-dinitro- phenol) (34) with diclofop-methyl has been reduced, Combinations Of diclofop-methyl and nitrofen (2,4-dichloropheny1 penitrophenyl ether) or bifenox [methyl 5-(2,4-dichlorOphenoxy)-2 nitrobenzoate] reduced annual grass control in onions compared with diclofop-methyl alone (4). Finally, Nalewaja et al. (17) reported that combinations of HOE 29152 with bentazon or dinoseb reduced annual grass control and increased injury to flax or soybeans. FATE IN PLANTS Retention, Uptake and Translocation. .In studies with green foxtail, wild oat, wheat and barley, green foxtail had greater f 14c diclofop-methyl than the other spray retention and uptake 0 species (32). This increase in dosage may explain the increased susceptibility of green foxtail. Excised shoots Of wheat and wild oat, immersed in a solution containing 14C diclofop-methyl, absorbed 81 and 87% Of the applied radioactivity respectively after 24 h (28). Penetration and toxicity of diclofop-methyl increased as the chemical application was made nearer the leaf base of wild oat (38). 14 Penetration of C diclofop-methyl continued over a 192 h period in green foxtail, wild oat, wheat and barley (32). Gorbach et a1. (14) indicated only 3% of the radioactivity applied to wheat as 14C diclofop-methyl could be rinsed from the foliage 16 DAT. 10 With combinations of 2,4-D and diclofop-methyl, retention and uptake of diclofop-methyl in wild cat was not changed from that of diclofop-methyl alone (31). However, with MCPA and diclofop- methyl combinations in wild oat diclofop-methyl penetration was decreased (21). 14C diclofop-methyl was a function of chemical Root uptake of concentration and the amount Of water absorbed, with wheat having greater absorption than wild oat or green foxtail. Shimabukuro et al. (28) found chemical concentration in the roots of root- treated wild oat and wheat plants was 0.3 and 0.5 ug/g dry weight. Differential root uptake was not considered a mechanism for selectivity. Walter (35) indicated that diclofop-methyl applied to the roots of wild oat was more toxic in nutrient solution than in soil. Site of uptake studies, assessing the relative importance of foliage and root uptake of postemergence applications of diclofop- methyl, have shown that root uptake contributes only a minor portion to total growth reduction (16,23). The greatest effect is achieved when both the soil and foliage are treated together. With soil applications Of diclofop-methyl, Friesen et al. (13) reported shoot uptake in wild oat to be most important while Crowley et al. (11) reported the opposite. The difference in results is attributed to technique differences with Friesen et al. not using a charcoal barrier between soil layers and possibly allowing diclofop-methyl to leach into the root zone and biasing the results of the shoot uptake. Yellow foxtail was more sensitive than barnyardgrass to soil applications of diclofop-methyl with both the roots and shoots being ll equally sensitive Sites of uptake (12). Shoot uptake was greater than root uptake, with barnyardgrass. Translocation of diclofop-methyl is very limited in wild oat or wheat (7,28). Brezeanu et al. (7) found 95% of the absorbed diclofop-methyl remained in the treated zone of both wheat and wild oat. Shimabukuro et a1. (28) found limited symplastic and apoplastic translocation of diclofop-methyl in both species. Metabolism. Metabolism studies with diclofop-methyl have centered on its fate in grassy plants. Gorbach et al. (14) applied 14C diclofop-methyl to tolerant summer wheat. Eighteen days after treatment, 58% of the applied radioactivity could be extracted with CH3C13:H20 (1:1, v/v), 32% could be extracted with 1 N NaOH digestion and 0.6% was unextracted. The major metabolites were diclofop, 2-(4-(2,4-dichlorO-5‘ hydroxy phenoxy)phenoxy)propionic acid and possible glucoside conjugates of the hydroxy metabolite. Todd and Stobbe (30) studied metabolism in vacuum infiltrated leaf segments of green foxtail, and wild oat, susceptible species, and wheat and barley, tolerant species. They concluded selectivity was a function of the level of diclofop present in the plant. Either rapid de- esterification Of diclofop-methyl or slow conjugation of diclofop in a plant would lead to susceptibility of that species. Shimabukuro et al. (28) studied metabolism of diclofop-methyl in leaf segments of wheat and wild oat which were incubated in a solution Of 14 C diclofop- methyl for 24 h. DiClofop-methyl was rapidly hydrolyzed to diclofop in both species. In wheat, diclofop is irreversibly ring hydroxylated 12 which in turn is sugar conjugated (Figure 2). In wild oat, diclofop is conjugated as a neutral glycosyl ester which can hydrolyze to reform diclofop (Figure 2). Thus, a large free acid pool may be main- tained in the susceptible plants. Qureshi and Vanden Born (21) have studied the metabolism of diclofop-methyl when applied with MCPA in wild oat. Hydrolysis of diclofop-methyl to diclofop was slowed and the diclofop formed was rapidly conjugated. They felt these conjugates remained inactive, opposite to Shimabukuro's proposal (28). Todd and Stobbe (31) studied the metabolism of diclofop-methyl when vacuum infiltrated with 2,4-0 in wild oat leaf segments. They found an enhanced rate of de-esterification of diclofop-methyl with a minimal increase in conjugates formed. They hypothesize that the high levels of diclofop cause tissue necrosis, isolating the chemical which negates its ability to move within the wild oat plant. Physiological Effects. In studies by Shimabukuro et al. (27) diclofop-methyl (10 um) was found to be a strong auxin antagonist, inhibiting the IAA-stimulated elongation of wheat and oat coeloptile segments, 13 and 51% respectively. Dicamba partially overcame these effects. Diclofop inhibited oat, but not wheat coeloptile elongation. From these studies it is postulated that both diclofop-methyl and diclofop are herbicidally active, but have different modes of action and act at different sites. 13 @M—@" -OC-COOCH3 (ocflve) @l wheat a wild out (343 0 @‘-@—O-C-COOH . (ocnve) cu whe__g_i w____ild oot ;/© OH H C 0 -§-coow C: o Q-@—O-fi-COOR' l Onocflve) \\:\:\\ Hnocflve) ”Sig CH3 R O-C- COOH H Dnocfive) Figure 2. Proposed metabolism of diclofop-methyl in wheat and wild oat (28). l4 Brezeanu et al. (7) studied ultrastructural modification in wheat and wild oat treated with diclofop-methyl. Ultrastructural ' damage was greater in wild oat than in wheat, with the chloroplasts being the organelles most affected in both species. Chow and La Barge (9) found decreased chlorophyll content and photosynthetic activity in wild oat treated with diclofop-methyl. Photosynthate translocation was also inhibited. FATE IN SOILS A greater portion of a postemergence application of diclofop- methyl may reach the soil rather than a plant surface. Residual activity was found 3 weeks after application (8), but no soil activity was found 8 weeks after treatment in the field (35). Degradation Of diclofop-methyl occurs more rapidly under warm, moist soil conditions (15). Metabolism studies (15,29) indicate that diclofop-methyl rapidly hydrolyzes to diclofop with 85, 68 and 40% hydrolysis 24 h after application on heavy clay, sandy loam and silty clay soils, respectively. Hydrolysis occurred equally among soils with moisture levels at the wilting point or field capacity. The acid is adsorbed by the soil, and can be removed by alkaline digestion. Traces of 4-(2-4-dichlorophenoxy)phenol were also recovered from the treated soils. Diclofop-methyl was found to be relatively immobile in the soil, comparable to the mobility of trifluralin (37) and as indicated previously, both the roots and shoots of annual grasses are able to absorb diclofop-methyl from the soil (8,15). 10. ll. LITERATURE CITED American Hoechst Corp. 1976. HOE 23408, technical information bulletin. American Hoechst Corp., Agric. Chem. Dept. Somerville, NJ. pp. 7. Anderson, R. N. 1976. Control Of volunteer corn and giant foxtail in soybeans. Weed Sci. 24: 253-256. Anderson, R. N. 1976. Response Of monocotyledons to HOE 22870 and HOE 23408. Weed Sci. 24: 266-269. Boldt, P. F. and A. R. Putnam. 1976. Onion and weed response to several newer herbicides. Proc. North Cent. Weed Contr. Conf. 31: 107. Boldt, P. F. and A. R. Putnam. 1977. Annual grass control in vegetable crops with HOE 29152. Proc. North Cent. Weed Contr. Conf. 32: 25. Brewster, B. D., A. P. Appleby and R. L. Spinney. 1977. Control of Italiah ryegrass and wild oats in winter wheat with HOE 23408. Agron. J. 69: 911-913. Brezeanu, A. G., D. G. Davis and R. H. Shimabukuro. 1976. Ultrastructural effects and translocation of methyl-2-(4- (2,4-dichlorophenoxy)phenoxy)propanoate in wheat (Triticum aestivum) and wild oat (Avena fatua). Can. J. Bot. 54:.2038- 204B. Chow, P. N. D. 1978. Selectivity and site of action in relation to field performance to diclofop. Weed Sci. 26: 352-358. Chow, P. N. D. and D. E. La Berge. 1978. Wild oat herbicide studies. 2. Physiological and chemical changes in barley and wild oats treated with diclofop-methyl herbicide in relation to plant tolerance. J. Agric. Food Chem. 26: 1134-1137. Chow, P. N. P. and D. G. Dorrell. 1979. Response Of wild oat (Avena fatua), flax (Linum usitatissimum) and rapeseed (Brassica campestris and 8, nagus) to diclofop-methyl. Weed Sci. 27: 212-215. Crowley, J., J. T. O'Donovan, and G. N. Prendeville. 1978. Phytotoxicity gf soil-applied dichlorfOp-methyl and its effect on uptake Of 4 Ca in wild oats, barley and wheat. Can. J. Plant Sci. 58: 395-399. 15 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 16 Dekkar, J. H., W. F. Meggitt, P. F. Boldt and T. Malefyt. 1978. Soil herbicidal activity from postemergence applications Of HOE 29152 and diclofop. Proc. North Cent. Weed Contr. Conf. 33: 115. Friesen, H. A., P. A. O'Sullivan and W. H. Vanden Born. 1976. HOE 23408, a new selective herbicide for wild oats and green foxtail in wheat and barley. Can. J. Plant Sci. 56: 567-578. Gorbach, S. G., K. Kuenzler and J. Asshauer. 1977. On the metabolism Of HOE 23408 OH in wheat. J. Agric. FOOd Chem. 25: 507-511. Martens, R. 1978. Degradation of the herbicide 14C diclofop- methyl in soil under different conditions. Pestic. Sci. 9: 127-134. Nalewaja, J. 0., K. A. Adamezewski, L. Garcis-Torres, E. Pacholak and S. D. Miller. 1976. Factors affecting HOE 23408 phytotoxicity. Proc. North Cent. Weed Contr. Conf. 31: 132- 134. Nalewaja,.J. D., E. Pacholak, L. C. Liu and S. D. Miller. 1976. BAS 9021 and HOE 29152 for grass weed control. Proc. North Cent. Weed Contr. Conf. 31: 137-140. Olson, W. A. and J. D. Nalewaja. 1976. HOE 23408 combinations with MCPA and desidipham. Proc. North Cent. Weed Contr. Conf. 31: 134-136. O'Sullivan, P. A., H. A. Friesen and W. H. Vanden Born. 1977. Influence of herbicides for broad-leaved weeds and adjuvants with dichlorfOp-methyl on wild oat control. Can. J. Plant Sci. 57: 117-125. Putnam, A. R., A. P. Love and R. P. Rice, Jr. 1974. Control of annual grasses in vegetable crOps with HOE 23408 and HOE 22870. Proc. North Cent. Weed Contr. Conf. 29: 74. Qureshi, F. A. and W. H. Vanden Born. 1979. Interaction of diclofop-methyl and MCPA on wild oats (Avena fatua). Weed Sci. 27: 202-205. Rao, S. A. and R. 0. Sweet. 1977. Weed and crop response to methyl 2-(4-(2,4-dichlorphenoxy)phenoxy)propanoate (HOE 23408). I. Rates, timings and environmental factors. Abstr. Weed Sci. Soc. Amer. pp. 43. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 17 Rao, 5. A. and R. 0. Sweet. 1977. Weed and crop response to methyl 2-(4-(2,4-dichlorOphenoxy)phenoxy)propanoate (HOE 23408). 11. Possible causes for differential responses of grasses. Abstr. Weed Sci. Soc. Am. pp. 43. Richardson, W. G. and C. Parker. 1976. The activity and postemergence selectivity Of some recently developed herbicides: HOE 22870, HOE 23408, flamprop-methyl, metamitron and cyperquat. Technical Report Agricultural. Research Council Weed Research Organization, 39. pp. 50. Richardson, W. G. and C. Parker. 1977. The activity and post- emergence selectivity Of some recently developed herbicides: KUE 2079 A, HOE 29152, RH 2915, triclopyr, and Dowco 290. Technical Report Agricultural. Research Council Weed Research Organization, 42. pp. 53. Schreiber, M. M., G. F. Warren and P. L. Orwick. 1976. Effect of wetting agent, stage of growth and species on differential selectivity Of HOE 23408. Proc. North Cent. Weed Contr. Conf. 31: 134. Shimabukuro, M. A., R. H. Shimabukuro, W. S. Nord and R. A. Hoerauf. 1978. Physiological effects of methyl 2-(4(2,4- dichlorophenoxy)phenoxy)propanoate on oat, wild oat and wheat. Pestic. Biochem. Physiol. 8: 199-207. Shimabukuro, R. H., W. C. Walsh and R. A. Hoerauf. 1979. Metabolism and selectivity of diclofop-methyl in wild oat and wheat. J. Agric. Food Chem.: in press. Smith, A. E. 1977. Degradation of the herbicide dichlorfop methyl in prair e soils. J. Agri. Food Chem. 25: 893-898. Todd, B. G. and E. H. Stobbe. 1976. Selectivity of diclofop- methyl among wheat, barley, wild oat and green foxtail. Proc. North Cent. Weed Contr. Conf. 31: 140. Todd, 8. G. and E. H. Stobbe. 1976. Basis of the antagonistic effect of 2,4-D on diclofop-methyl toxicity in wild oats. Proc. North Cent. Weed Contr. Conf. 31: 40. Todd, B. G. and E. H. Stobbe. 1977. Selectivity Of diclofop- methyl among wheat, barley, wild oat (Avena fatua) and green foxtail (Setaria viridis). Weed Sci. 25: 382-385. 33. 34. 35. 36. 37. 38. 18 Walter, H. and F. Dischof. 1976. Effectiveness Of new post- emergence herbicides against wild oats (Avena fatua L.) in relation to application site. 2. Pflanzenknankheiten 83: 338-351. Walter, H., F. Muller and W. Koch. 1977. Interaction of diclofop-methyl and other post-emergence herbicides. Z. Pflanzenknankheiten, Sanderh. VIII. 389-402. Walter, H. 1977. Investigations on the effectiveness of post-emergence herbicides against wild oat‘(Avena fatua L.). Ph.D. Thesis, University of Hohenheim Library, West Germany. 101 pp. Woldelatios, T. and R. G. Harvey. 1977. Diclofop and bentazon interactions on soybeans and annual weeds. Proc. North Cent. Weed Contr. Conf. 32: 42-43. Wu, C. H. and P. W. Santelman. 1976. Phytotoxicity and soil activity of HOE 23408. Weed Sci. 24: 601-604. Young, F..L. and D. L. Wyse. 1977. Evaluation of HOE 29152 for quackgrass control in soybeans. Proc. North Cent. Weed Contr. Conf. 32: 32-33. CHAPTER 2 RESPONSE OF SELECTED ANNUAL GRASS AND BROADLEAF SPECIES TO FOLIAR APPLICATIONS OF PHENOXY-PHENOXY HERBICIDES ABSTRACT Greenhouse and field studies were initiated to determine the response of selected annual grass and broadleaf species to diclofop- methyl [methyl 2-[4-(2,4-dich1orophenoxy)phenoxy]propanoate] and/or HOE 29152 [methyl 2-[4-(4-trif1uoromethyl phenoxy)phenoxy]propanoate]. At equivalent rates HOE 29152 was more toxic to annual grass and broadleaf species than diclofop-methyl. The sensitivity of annual grass species to the two herbicides was fairly uniform, with barnyardgrass [Echinochloa crus:galli (L.) Beauv.] being the most sensitive and longspine sandbur [Cenchrus longjspinus (Hack.) Fern.] the most tolerant. Large crabgrass (Digitaria sanguinalis L.) and proso millet (Panicum miliaceum L.), both moderately tolerant species to diclofop-methyl were sensitive to HOE 29152. Form- ulations of HOE 29152 containing additional adjuvants were more toxic to annual grasses and crOps than the normal formulation at a given rate. The use of tank-mixed adjuvants with either herbicide caused a greater fresh weight reduction Of barnyardgrass. Site of uptake studies showed phenoxy-phenoxy herbicides caused the greatest reduction in fresh weight Of barnyardgrass when both the plant and soil were sprayed. Most Of the activity could be attributed to foliar uptake. 19 20 INTRODUCTION Several postemergence herbicides are available for broadleaf weed control in vegetable crops. However, there is a lack Of effective postemergence herbicides for wide Spectrum annual grass control. In 1973, Hoechst, A. G. released diclofop-methyl for evaluation. Field and greenhouse studies established that species sensitive to diclofop-methyl included barnyardgrass, wild oat (Avena fatua L.), green foxtail (Setaria viridis L.), witchgrass (Panicum capillare L.); species moderately sensitive included yellow foxtail [Setaria lutescens (Weigel) Hubb.] and proso millet while tolerant species included wheat, (Triticum aestivum L.), barley, (Hordeum vulgare L.), quackgrass [Agropyron repens (L.) Beauv.], and all broadleaf plants (1,2,8,15,18). Factors, such as the use of adjuvants or interactions with other herbicides, may influence the postemergence activity of diclofop-methyl. O'Sullivan et a1. (12) and Nalewaja et al. (10) reported wild oat control with diclofop-methyl at 0.56 kg/ha with selected adjuvants equal to diclOfOp-methyl at 1.12 kg/ha without additives. However, Schreiber et a1. (17) did not enhance diclofop- methyl activity with wetting agents. Since the weed ecosystem is composed of a variety of broadleaf and grassy weeds, it may be advantageous to tank mix broadleaf and grass postemergence herbicides for once over treatment. However, reduced wild oat control has been reported when diclofop-methyl was tank mixed with 2,4-0 [(2,4-dichlorophenoxy)acetic acid], MCPA [(4-chlorO-g-tolyl)oxy) acetic acid], dinoseb acetate [(2-sgg-butyl-4,6-dinitrophenol)acetate] 21 acetate, or desmidipham (ethyl-m-hydroxycarbanilate carbanilate) (11,12,20). Combinations of diclOfOp-methyl and nitrofen (2,4— dichlorophenyl p-nitrophenyl ether) or bifenox [methyl 5-(2,4- dichlorophenoxy)-2 nitrobenzoate] reduced annual grass control in onions compared with diclofop-methyl alone. The role of soil vs. foliar uptake Of postemergence applications has been investigated. In wild oats (5,10) or barnyardgrass (14), foliar uptake caused a much greater fresh weight reduction than soil uptake. However, the combined foliar and soil treatment caused the greatest fresh weight reduction. Placement experiments with germin- ating seeds, having diclofop-methyl treated soil either above or below the seed, show the greatest toxicity occurs when the shoot is exposed to the chemical (5). Using 1050 values established for root growth, Crowley et a1. (6) found that root applications of diclofop-methyl were more toxic than Shoot applications to sub- sequent root and shoot growth. In 1976 another phenoxy-phenoxy herbicide, HOE 29152, was introduced. Preliminary studies have shown that many species tolerant to diclofop-methyl are susceptible to this herbicide (4,22). The Objectives of this study were to determine the: 1) relative sensitivity of selected annual grass species to diclofop-methyl and HOE 29152; 2) influence of selected chemical, soil and plant factors on the postemergence activity and selectivity Of HOE 29152: and 3) Site Of uptake for foliar applications Of diclofop-methyl and HOE 29152 to annual grasses grown on several soil types. 22 MATERIALS AND METHODS ' Phenoxy-Phenoxy COmparisons. Greenhouse experiments were initiated to evaluate the relative toxicity and selectivity Of HOE 29152 as compared to diclofop-methyl using GR50 values which "express the dosage (kg/ha) of herbicide needed to reduce the fresh weight of the treated species by 50%. Species response evaluated for both chemicals were barnyardgrass, yellow foxtail, proso millet, green foxtail, large crabgrass and longspine sandbur. Cucumber (Cucumis sativa L. 'Green Star') and soybean [Glycine max (L.) Merr. 'Hark'] response was determined for diclofop-methyl only. Seeds were planted in 20 cm styrofoam pots filled with a sand:peat:soil (1:1:1, v/v/v) potting media and placed in a greenhouse with temperatures of 25 C - day, 20 C - night. Natural light was supplemented with metal halide lighting, with a 16 h photoperiod at an average intensity of 842 DE m'zs'I. Relative humidity ranged from 30 to 60%. All plants were watered overhead and by subirrigation on alternate days except after chemical application, when only sub- irrigation was employed. On a weekly basis, 200 ml of a water soluble fertilizer solution (22.5% N - 22.5% P205 - 22.5% K20, 2 g/L) was applied to each pot. After emergence five uniform and equally spaced plants were selected in each pot. At the time of treatment, the plants had three leaves and were 10 to 14 days Old. They were sprayed on a moving belt Sprayer delivering 234 L/ha at a pressure of 2.1 kg/cmz. Single applications were made to the annual grasses while multiple applications of 20 kg/ha per application were made to the broadleaf 23 plants. Commercial formulations (36 EC) of both diclofop-methyl and HOE 29152 were used with rates of applications ranging from 0.01 to 5 kg/ha for the annual grasses and 20 to 140 kg/ha for cucumber and soybean. After spraying, the plants were returned to the greenhouse. Ten days after treatment (DAT) the plants were harvested and dry and fresh weights determined. The data were converted to percent of untreated controls and GR50 values calculated. The experiment was arranged in a randomized complete block design with five replications and was repeated twice. Field Studies with HOE 29152. Experiments were conducted at three locations with soil types of: Russo fine sand (O.M. - 0.8%, pH - 6.2), Hillsdale sandy loam (O.M. - 2.2%, pH - 6.5) and a Houghton muck (O.M. - 80%, pH - 6.8). Annual grass species evaluated were green and yellow foxtail, large and smooth crabgrass (Digitaria spp.), proso millet, barnyardgrass, stinkgrass [Erogratis cilianensis (All.) Lutati], longspine sandbur; vegetable crops evaluated were asparagus (Asparagus afficinalis L. 'Martha Washington'), carrots (Daucus carota L. var. sativa 'Spartan Delite'), cucumbers (Cucumis sativa L. 'Carolina'), cabbage (Brassica Oleracea L. var. capitata 'Harris Resistant Danish'), lettuce (Lataca sativa L. 'Ithaca'), onions (Allium cepa L. 'Spartan Banner'), snap beans (Phaesolus vulgaris L. var. humilis Alef. 'Spartan Arrow') and tomatoes (Lycopersicon esculentum Mill. 'Heinz 1350'). Not all annual grass or vegetable species were treated and evaluated at all locations. Three form- ulations of HOE 29152 were tested at 0.13 to 2.24 kg/ha. HOE 29152-1 (36 EC) contained solvents and emulsifiers, HOE 29152-2 (24 EC) and HOE 29152-3 (24 EC) had Genapol X80 (Hoechst) and Renex 36 (ICI), 24 polyoxyethylene alkyl ether surfactants, respectively. In one study Blend C and S-104-ES-75, non-ionic acetylenic surfactants (Air Products and Chemicals), were used as tank-mixed adjuvants with HOE 29152-1 (36 EC) at 0.5% v/v. In another study, HOE 29152-1 (36 EC), 0.56 or 1.12 kg/ha, was applied singly and in combination with acifluorfen [5-[2—chloro-4(trifluoromethyl)phenoxy]-2- nitrobenzoic acid], 0.56 or 1.12 kg/ha; nitrofen, 1.12 or 2.24 kg/ha; bromoxynil (3,5-dibromO-4-hydroxy benzonitrile), 0.34 or 0.68 kg/ha, or methazole [2-(3,4-dichlorophenyl)-4-methyl-l,2,4- oxadiazolidine-3,5-dione], 0.84 or 1.68 kg/ha, to onions, common purslane (Portulara Oleracea L.) and large crabgrass grown on organic soil. All experiments were designed as a randomized complete block with three or four blocks. Adjuvant Studies. Greenhouse studies were initiated to evaluate adjuvant types and rates on activity of phenoxy-phenoxy herbicides. Adjuvants used were Blend C, S-104-ES-75, Renex 36 and Genapol X80 at 0.125, 0.25 and 0.5% v/v. These were tank mixed with diclofop- methyl or HOE 29152 and applied at 0.10 or 0.05 kg/ha, respectively, to barnyardgrass. Growing conditions and chemical application were as previously described. Plants were harvested at 10 DAT, fresh weights recorded, and data expressed as percent of the corresponding chemical treatment without adjuvant. The experiment was a 2 x 2 x 3 x 4 factorial in a completely randomized block design with four blocks, and was repeated. All data were subjected to an analysis of variance with mean separation using Fishers protected LSD at the 5% probability level. 25 Site of Uptake. Greenhouse studies were conducted to evaluate the role of foliar, soil and foliar plus soil uptake Of postemergence applications of HOE 29152 and diclofop-methyl. Seeds Of yellow foxtail and barnyardgrass were planted in three soil types: Houghton muck, Miami sandy loam (O.M. - 1.2%, pH 7.2) and the sand:peat:soil potting mix. Ten days after planting, the plants were divided into three groups: one having the plants covered with glass test tubes, one having the soil covered with 1 cm of vermiculite and the remaining group having the plants and soil uncovered. The plants were sprayed with diclofop-methyl at 0.3 and 0.6 kg/ha on barnyardgrass and yellow foxtail respectively. HOE 29152 was applied at 0.15 and 0.3 kg/ha on barnyardgrass and yellow foxtail, respectively. Plants were placed in the greenhouse and harvested 10 days after treatment. The experiment was a 2 x 2 x 3 x 3 factorial in a completely randomized block design with four replications, and was repeated. Data were subjected to analysis Of variance and mean separation was by Fishers protected LSD at the 5% level. RESULTS AND DISCUSSION Phenoxy-Phenoxy Comparisons. Comparison of GR50 values for diclofop-methyl and HOE 29152 Showed the latter herbicide to be 1.5 to 2 times more toxic than diclofop-methyl to all annual grass species tested (Table 1). 0f the grasses, longspine sandbur was most tolerant of both compounds and barnyardgrass most sensitive. Proso millet and large crabgrass, moderately susceptible to diclofop- methyl, were very sensitive to HOE 29152. Excluding those two 26 Table 1. GR50 values (kg/ha) for foliar-applications Of diclofop-methyl or HOE 29152 on selected plant species. GR5oa Species Diclofop-methyl HOE 29152 kg/ha Barnyardgrass 0.10 0.05 Green foxtail 0.12 0.08 Yellow foxtail 0.30 0.16 Proso millet 0.70 0.06 Large crabgrass 0.70 0.06 Longspine sandbur 1.50 0.70 Cucumber 83.00 NTb Soybean 112.00 NTb a 50% reduction in fresh weight of harvested plants expressed as a % of untreated plants. b NT = not tested. 27 species, HOE 29152 was approximately twice as toxic as diclofop- methyl to annual grasses. In the additional study with diclofop-methyl, cucumber and soybean were 82 and 110 times more tolerant, respectively, than longspine sandbur, the most tolerant annual grass tested. The values for cucumber and soybean may reflect not only diclofop- methyl toxicity but also surfactant and solvent injury. Todd and Stobbe (18) found E050 values for barley and wheat, grass species tolerant to diclofop-methyl, to be similar to the GRSO values found in these studies for cucumber and soybean. Field Studies with HOE 29152. Annual grass response to post- emergence Sprays Of HOE 29152 under field conditions closely followed the trend of greenhouse determined GR50 values (Tables 2,3). Order of sensitivity from greatest to least, to HOE 29152 was: barnyardgrass = proso millet > smooth crabgrass > green foxtail > large crabgrass = stinkgrass > yellow foxtail >> longspine sandbur. All annual grasses except yellow foxtail and longspine sandbur were acceptably controlled with 0.25 kg/ha of HOE 29152-1 (36 EC), and the former species was controlled at 0.25 kg/ha with formulations containing additional additives (Table 2). Longspine sandbur was tested with only the 36 EC formulation, with 1.50 kg/ha needed for acceptable control (Table 4). CrOps treated with three formulations of HOE 29152 showed no economic injury at 0.25 kg/ha Of any formulation. Applications Of 1.0 kg/ha of HOE 29152-1 (36 EC) was safe to all crops but 1.0 kg/ha of HOE 29152-2,3 (24 EC) caused significant foliar injury to 28 .mm xmcmm umvum u mummpmm mo: .omx Foamcmw omuum u Nummpmm mo: .cowumP35com vcmucmum u Plumpmm we: a .Focucou mumFano u op .Pocucou mPQMHamoum u m.~ .Pogucoo o: u o m o.o_ o.oF o.op o.o_ o.o_ o.o_ o.op NP., Add ewe o.op o.o_ m.m A.m o.op m.m o.m mm.o m-~m.m~ mo: o.o_ o.o_ o.op o.o_ o.o_ o.op m.m NF._ Add ewe e.m o.o_ m.m A.m o.op ~.m o.a m~.o ~-Nm2m~ mo: o.o_ 0.0. o.o_ o.o. 0.0, o.op o.op ~_.F Au“ one m.e o.o_ m.~ o.m o.o_ A." m.o m~.o _-~mpmm mo: mmecm mmmcm , mmmcmnmcu mmmgmamcu pmppps Pwmpxom Prepxom Am;\mxv amcowpmfizsgom xcwum 4:92.me mmLmJ swooEm OmOLa cmmgw 30—. Fm> mummoo xoze :opcmso: Emop aucmm upmumppw: acmeummch mmmcwpmc Pmamw> .couap mace VP vogue new mm>mm— omega ca 039 um; mommmcm ems: umvpaae mmpam mo: mo meowumpaecom moss» we mcoppmuPFaae mucmmcmsmumoa saw: mommmcm ~m=c=m mo Foncou .N open» 29 Table 3. Visual ratingsa of longspine sandbur control in asparagus after foliar applications Of phenoxy-phenoxy herbicides.b Treatment Dosage DAT Chemical (kthg) _l_8__ _5_6_ 1_0_0 Diclofop-methyl 1.68 1.7 0.3 0.0 3.36 3.3 2.7 5.7 HOE 29152 0.84 6.7 6.7 6.3 1.68 8.3 8.7 9.0 a 0 = no control, 7.5 = acceptable control, 10 = complete control. b Longspine sandbur had two to three leaves at time of treatment. Experiment conducted on a Russo sandy loam. .mm xmcmm woven u mummpmw mo: .omx Poamcmw towns u Nummpmm mo: .cowumpssgow vgmucmam u Pummpmm mo: u .comumuPuwcm u o_ .xczwcw mpamuamuum u m.~ .xcsncw o: u o a .mm>mmp one» Lao» um; cowco .pcmEpmmcu we mm>mmp was“ 03» op one on; scene pamuxm mucepa ~F< a N.o ~.e A., ~.e o.e A.m N.m NF.F Adm ewe A.o 0.0 o.o o.o o.o o.o o.o m~.o m-~mpmm mo: “.0 A.m o._ m.e A.e ~.m o.m ~_._ Aum emv o.o o.o o.o o.o 0.0 o.o o.o m~.o ~-Nm_m~ mo: o.o A.o o.o o._ 0.2 o._ o._ NF.F Adm emv 0.0 o.o o. o.o o.o o.o o.o mN.o _-~mpa~ mo: cowco mmmnnmu poccmu mozpumo Longsuzo :mmn oposo» Am:\mxv ocowpmpaeLou amcm daemon xoze copsmso: Emo_lxvcmm mpmum_ppz acmEpemc» ammcwmmc szmw> .Lepe, when e_ amped nee emmeepeudm FFeEm ea eeepdde mmpmm mo: mo mcowum~=ELom omega mo mcopumowpnam mocmmcmemumoa gpwz «coco mpaoummm> on Anzac“ .e mpnmh 31 tomato, snap bean, cucumber, lettuce and cabbage (Table 3). Onion and carrot were tolerant which might be attributed to their leaf morphology allowing less herbicide uptake by the foliage. Combinations of HOE 29152 with acifluorfen or methazole (Table 5) caused greater onion foliar injury than any Of the herbicides applied alone. However, this injury did not result in a decrease in onion yield. The lack of antagonism in annual grass control indicates that these compounds do not interact like phenoxy compounds do with diclofop-methyl, or that HOE 29152 does not exhibit the same type Of antagonism as diclofop-methyl. The use of tank-mixed non-ionic acetylenic adjuvants increased the activity of HOE 29152 (36 EC) on annual grasses (Table 6). Acceptable control was achieved with 0.13 kg/ha, with control being greater than with either of the 24 EC formulations. As these treatments were not applied to crops it is not known if the increased annual grass control would be accompanied with increased crop phytotoxicity, as was observed with the 24 EC formulations of HOE 29152 (Table 4). The rate used for the tank-mixed adjuvants (0.5%, v/v) was higher than that associated with increased wettability of the leaf (9). Therefore, it could not be determined what function the adjuvant served i.e., wettability, penetration, humectant. The field studies suggest that HOE 29152 has a greater potential for controlling a wider range Of annual grasses at a lower dosage than diclofop-methyl. Formulations with additional surfactants tend to increase grass control at a lower rate, but also decrease crop selectivity. Although the 24 EC formulations did not Offer any 32 .cowumuwvmcm aogu Lo _ocpcou com: mumpaeou u oF .Pocpcou now: mFDmuamoum u m.n .xgzwcw coco mpnmuamoom n m.N .pocpcoo vow: co means? coco o: n o .mm>omp m . mmmcmccmxcgea .mm>omp Lao» op mco . mmmcmnmco mmcmp .cmumEewu soc? macs» . mcmpmcaa coseou .mm>mmp czom . cowco "ucmspmmcu be carota mo mmmum m mm.o Fecaxoeocn o.o_ o.o_ n.m ~.o + om.o + Nmpmm mo: em.o epo~eeeue 0.9, o.op o.m m.F + em.o + mmpmm mo: N..P eueoeeee o.o_ o.op o.e m.o + em.o + ~m_m~ moz- mud cmdzo: crow o.o_ “.9 O.“ ~.N + m~.o + mmpmm mo: o.o o.o o.o o.o Am.o Add ewe .eeaxo5oem o.o o.a N.m o.o em.o Ad: mfiv u_ONeeeuz o.o o.o A.“ N.o ~_._ Adz omv eueeeeez 0.2 o.o A.“ A., m~.o Add «NV euceo=_ceu< o.o_ o.op o.o o.o em.o Adm one Nmqu mo: mmecmugeaccem , mmecmmmtu.muce4 mcmpmcaaucoeeou. cowco ammummv Peursmgu np daemon acmEummCA m.xc=n=p copco new Pocpcoo wow: so mwv_uvncmg mempumocn mocmmcmemumoa chm>om new: umpmm mo: mo cowuumcmch .m mpam» 33 .pocpcoo mpmpaeoo u o_ .Pocucoo mpnmpgmoum u m.~ .Pocucoo o: u o m o.o_ o.op o.o~ o.op o.op o.op o.o— o.m o.m o.m m.m m.¢ N.“ o.o m.n m.m n.m o.“ o.m o.m mmacmnmcu poppwa ppmuxom .Fmgxom spoosm omega goose zoFFm> mmmcwumL Pmamw> >\> am.o u vcwpm + ee\mx e_.o + Add emv P-Nm_mu mo: >\> am.o mfi-mm-eo_-m + e;\mx ep.o + Adm one _-~mpm~ mo: ee\mx e_.o Au“ ewe m-~m_mm mo: ee\m¥ ep.o Adm ewe ~-~mpm~ mo: ee\mx ep.o Add one _-Nmpm~ mo: daemon Fmowemgu ucmsummch .cmump mzeu ep noun; ucm mm>emp moss» on o:» we; mommmcm cog: umppqqe mmpmu mo: ;u_z nmstrxcmu mu=e>snum owcopxpmum upcowucoc do meowumowpnam mucmmcmemumoa saw; mommmcm Fmaccm do Focpcou .c mpneh 34 particular advantage over the 36 EC formulation, the addition of other adjuvants did improve activity with little loss in selectivity. Combinations Of HOE 29152 with the postemergence herbicides tested do not appear to be antagonistic, however, initial activity was greater with chemicals formulated as emulsifiable concentrates such as acifluorfen. Adjuvant Study. The interaction of adjuvant type and con- centration was significant (Table 7). Renex 36 and Genapol X80 when tank-mixed at 0.125% v/v, with either diclofop-methyl or HOE 29152, gave greater fresh weight reduction of barnyardgrass than the acetylenic adjuvants. At 0.25% v/v and 0.5% v/v all adjuvants were equal in response across rate. The greatest fresh weight reduction occurred at 0.5% v/v. These data suggest that Optimum adjuvant concentration is between 0.25 and 0.50% v/v and that under greenhouse conditions adjuvants increased the activity of the phenoxy-phenoxy herbicide on barnyardgrass. These data agree with previous reports (10,12) which Showed increased wild oat control with the phenoxy-phenoxy herbicides when adjuvants were added to the Spray solution. Site of Uptake. Uptake of postemergence applications of phenoxy- phenoxy herbicides occurred primarily through the foliage of the plants (Table 8). Although the greatest toxicity occurred when both the soil and foliage were treated, minimal effect occurred with soil application only. There were no differences between the soil types or plant species. 35 Table 7. Influence of adjuvant type and concentration on post- emergence activity Of phenoxy-phenoxy herbicides on the fresh weight of barnyardgrass 14 days after treatment. Adjuvant Typea Concentration Blend C 104-ES-75 Renex 36 Genapol X80 -——-% (v/v)-—» ——-—————-fresh weight (% of control) 0.125 58 40 18 27 0.250 17 15 16 18 0.500 15 10 6 13 LSD (.05) (type x concentration) = 9.5 a Control was an equivalent chemical spray without the adjuvant. Adjuvants were not toxic at concentrations used. Data are means of diclofop-methyl and HOE 29152, which were not different. 36 Table 8. Site of uptake of foliarly-applied diclofop-methyl and HOE 29152 in barnyardgrass and yellow foxtail grown on various soils.a Site of Chemical Dosagea application fresh weight (% of untreated)c $011 94.3 Foliage 42.4 Soil + foliage 12.1 LSD (.05) = 6.2 a Experiment conducted with greenhouse potting soil (1:1:1, z/v/v), Hogghton muck (80% 0.M.) and Miami loamy soil 1.2% 0.M. . b Dosage (kg/ha) was: diclofop-methyl at 0.3 on barnyardgrass and 0.6 on yellow foxtail, HOE 29152 at 0.15 on barnyardgrass and 0.3 on yellow foxtail. c Values are averaged over soil type, grass species, and chemical. 37 Root uptake of diclofop-methyl (5,6,7,18) and HOE 29152 (7), can occur but diclofop-methyl does not readily move in the soil (21). When covering the soil with 1 cm of vermiculite, the base of the grass plants which sheaths the meristem, is protected from direct chemical application. The meristematic regions Of wild oats have (been shown to be primary Sites Of activity (19). Toxicity could be reduced due to the partial protection of the meristem. These studies show that the foliage is the primary site Of uptake for HOE 29152 and diclofop-methyl. Increasing the adjuvant content in formulations of HOE 29152 increased weed control and decreased crop tolerance. Combinations with other herbicides increased initial crop injury but did not influence final yield Of onions. Field studies are needed to evaluate the role of stresses, such as water and nutrition on activity. Elucidating the site of action within the plant would provide insight on how, when and where to apply the phenoxy-phenoxy herbicides for optimum effectiveness. 10. 11. LITERATURE CITED American Hoechst Corp. 1976. HOE 23408, technical information bulletin. American Hoechst Corp., Agric. Chem. Dept. SomerVTlle, NJ. pp. 7. Anderson, R. N. 1976. Response of monocotyledons to HOE 22870 and HOE 23408. Weed Sci. 24: 266-269. Boldt, P. F. and A. R. Putnam. 1976. Onion and weed response to several newer herbicides. Proc. North Cent. Weed Contr. Conf. 31: 107. Boldt, P. F. and A. R. Putnam. 1977. Annual grass control in vegetable crops with HOE 29152. Proc. North Cent. Weed Contr. Conf. 32: 25. Chow, P. N. P. 1978. Selectivity and site Of action in relation to field performance of diclofop. Weed Sci. 26: 352- 358. Crowley, J., J. T. O'Donovan, and G. N. Prendeville. 1978. Phytotoxicity gf soil-applied dichlorfop-methyl and its effect on uptake of 4 Ca in wild oats, barley and wheat. Can. J. Plant Sci. 58: 395-399. Dekker, J. H., W. F. Meggitt, P. F. Boldt and T. Malefyt. 1978. Soil herbicidal activity from postemergence applications of HOE 29152 and diclofop. Proc. North Cent. Weed Contr. Conf. 33: 115. Friesen, H. A., P. A. O'Sullivan and W. H. Vanden Born. 1976. HOE 23408, a new selective herbicide for wild oats and green foxtail in wheat and barley. Can. J. Plant Sci. 56: 567-578. Holly, K. 1976. Selectivity in relation to formulation and application methods. Pages 249-275 in L. J. Audus, ed. Herbicides-Physiology. Biochemistry, Ecology. Vol. 11. Academic Press, London. Nalewaja, J. 0., K. A. Adamezewski, L. Garcia-Torres, E. Pacholak and S. 0. Miller. 1976. Factors affecting HOE 23408 phytotoxicity. Proc. North Cent. Weed Contr. Conf. 31: 132- 134. Olson, W. A. and J. D. Nalewaja. 1976. HOE 23408 combinations with MCPA and desmidipham. Proc. North Cent. Weed Contr. Conf. 31: 134-136. 38 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 39 O'Sullivan, P. A., H. A. Friesen, and W. H. Vanden Born. 1977. Influence Of herbicides for broad-leaved weeds and adjuvants with dichlorfop-methyl on wild oat control. Can. J. Plant Sci. 57: 117-125. ' Putnam, A. R., A. P. Love and R. P. Rice, Jr. 1974. Control of annual grasses in vegetable crops with HOE 23408 and HOE 22870. Proc. North Cent. Weed Contr. Conf. 29: 74. Rao, 5. A. and R. 0. Sweet. 1977. Weed and crOp response to methyl 2-(4-(2,4-dich1orOphenoxy)phenoxy)propanoate (HOE 23408). II. Possible causes for differential responses Of grasses. Abstr. Weed Sci. Soc. Am. pp. 43. Richardson, W. G. and C. Parker. 1976. The activity and postemergence selectivity of some recently developed herbicides: HOE 22870, HOE 23408, flamprOp-methyl, metamitron and cyperquat. Technical Report Agricultural. Research Council Weed Research Organization, 39. pp. 50. Richardson, W. G. and Parker, C. 1977. The activity and post- emergence selectivity Of some recently developed herbicides: KUE 2079 A, HOE 29152, RH 2915, triclopyr, and Dowco 290. Technical Report Agricultural. Research Council Weed Research Organization, 42. pp. 53. Schreiber, M. M., G. F. Warren and P. L. Orwick. 1976. Effect Of wetting agent, stage Of growth and species on differential selectivity Of HOE 23408. Proc. North Cent. Weed Contr. Conf. 31: 134. Todd, B. G. and E. H. Stobbe. 1977. Selectivity of dichlofop- methyl among wheat, barley, wild oat (Avena fatua) and green foxtail (Setaria viridis). Weed Sci. 25: 382-385. Walter, H. and F. Bischof. 1976. Effectiveness of new post- emergence herbicides against wild oats (Avena fatua L.) in relation to application site. Z. Pflanzenknankheiten 83: 338-351. Walter, H., F. Muller, and W. Koch. 1977. Interaction of diclOfOp-methyl with other post-emergence herbicides. Z. Pflanzenkrankheiten, Sanderh. VIII. 389-402. Wu, C. H. and P. W. Santelman. 1976. Phytotoxicity and soil activity of HOE 23408. Weed Sci. 24: 601-604. Young, F. L. and D. L. Wyse. 1977. Evaluation of HOE 29152 for quackgrass control in soybeans. Proc. North Cent. Weed Contr. Conf. 32: 32-33. CHAPTER 3 SELECTIVITY MECHANISMS T0 FOLIAR APPLICATIONS OF DICLOFOP-METHYL. I. RETENTION, ABSORPTION, TRANSLOCATION AND VOLATILITY ABSTRACT Retention, absorption, translocation and volatility Of foliarly- applied diclofop-methyl [methyl 2-[4-(2,4-dichlorophenoxy)phenoxy] propanoate] were compared in barnyardgrass [Echinochloa crus-galli (L.) Beauv.] - a susceptible (S) grass, proso millet (Panicum miliaceum L.) - a moderately susceptible (MS) grass, longspine sandbur [Cenchrus longispinus (Hack.) Fern.] a tolerant (T) grass, soybean [Glycine max (L.) Merr. 'Hark'] and cucumber (Cucumis sgtjyg_L. 'Green Star') - both tolerant (T) broadleaf plants. On a u9/plant basis the order of diclofop-methyl retention was cucumber > soybean > proso millet > longspine sandbur = barnyardgrass. On a ug/mg dry weight basis proso millet retained 3 to 10 times more diclofop-methyl than all other species. One day after treatment (DAT) absorption Of 14C diclofop-methyl was 14 to 18% less in longspine sandbur than the other species, 3 DAT absorption in cucumber was 8 to 14% greater than the other species, and 5 DAT absorption in soybean was 3 to 12% less than other Species. Translocation of 14C diclofop-methyl did not differ between species with 98% Of the applied radioactivity located in the treated leaf. LOSS of radioactivity applied to the surface of intact living plants and 40 41 excised dead plants of cucumber, soybean, and barnyardgrass as well as glass cover slips showed an average loss Of 11% of the applied radioactivity. None of the parameters measured showed differences large enough to be implicated as primary selectivity mechanisms. 42 INTRODUCTION Very few herbicides are available which selectively control emerged annual grasses in crops. With the recent introduction of diclofop-methyl, a phenoxy-phenoxy herbicide, selective, post- emergence control Of many annual grassy weeds in cereal and broadleaf crops became possible (l,2,5,7,9,13). However, several problem grasses such as large crabgrass [Digitaria sanguinalis (L.) Scop.], sandbur (Cenchrus spp.) and proso millet have not been controlled with this herbicide (3,4,13). Selectivity to foliarly-applied herbicides can be a function of differential spray retention (12), chemical absorption or translocation within the plant (14) among other_factors. In studies with wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), wild oat (Avena fatua L.), and green foxtail [Setaria viridis (L.) Beauv.], green foxtail retained and absorbed more 14C diclofop-methyl than the other species (17). Brezeanu et al. (8) found 95% of the applied diclofOp-methyl remained in the treated zone of both wheat, a tolerant grass, and wild oat, a susceptible grass. Limited symplastic and apoplastic translocation of diclofop-methyl occurred in wheat or wild oat (15). Retention, absorption and translocation studies with dicots have not been reported to date. Loss Of diclofop-methyl via volatilization from the leaf surfaces of wheat, wild oat, barley and green foxtail was not significant over a 192 h treatment period (17). However, in preliminary metabolic studies investigating the fate Of 14C 43 diclofop-methyl in cucumbers the total radioactivity applied to the foliage could not be accounted for 5 DAT]. The rates of foliarly-applied diclofop-methyl which reduced growth by 50% in barnyardgrass (S), proso millet (MS), longspine sandbur (T), cucumber (T), and soybean (T) were: 0.1, 0.7, 1.5, 83 and 110 kg/ha, respectively (4). The purpose of this study was to evaluate the role of differential spray retention, chemical absorption, translocation and/or volatility from leaf surfaces as possible selectivity mechanisms in the preceding plant species. MATERIALS AND METHODS Cultural Practices. All plants were grown in a greenhouse at 30 C - day, 20 C - night; 30 to 60% relative humidity with supplemental metal halide lighting. The light intensity was 842 uE m'ZS”1 with a 16 h photoperiod. In the retention, absorption and volatility studies, seeds of the test species were planted in 20 cm styrofoam pots filled with a sandzpeatzsoil (1:1:1, v/v/v) potting media. The plants were watered by surface and subirrigation on alternate days until treatment after which only subirrigation was used. Two hundred ml Of a water soluble fertilizer solution (22.5% N - 22.5% P205 - 22.5% K20, 2 g/L) was applied to each pot on a weekly basis. I Boldt, P. F. unpublished data, 1978. 44 At the time of treatment, all plants were 10 to 14 days Old. Stages Of growth were: annual grasses - three leaves, soybean - unifoliate leaves, and cucumber - first true leaf. 'RetentiOn Study. A spray solution containing formulated diclofop-methyl (36 EC) and a sulfonine red 38 dye (2 g/0.5 L, Sandoz) was applied to the plants using a moving belt sprayer delivering 234 L/ha at a pressure of 2.1 kg/cmz. The rate of diclofop-methyl was 1 kg/ha. Immediately after spraying, the foliar portion of the plants was removed, placed in a test tube with 25 m1 of distilled water and shaken for 30 sec on a test tube mixer. All solutions were adjusted to equal volumes, and an aliquot removed for spectrophotometric analysis at 640 nm (Beckman Spectrophotometer Model DB-G). A standard curve was established for color intensity vs. ug Of diclofop-methyl in the solution. After washing the plant, foliage was dried at 60 C for 48 h and dry weights determined. The amount (pg) Of diclofop-methyl retained per plant or per mg of dry weights was calculated. Absorption Study. Formulated diclofop-methyl (36 EC) and 14C diclofop-methyl (Specific activity - 2 uCi/uM), uniformly labelled in the 2,4-dichlorophenoxy ring, were mixed with distilled water to make a 11.5 uM solution. With a microsyringe, 25 ul of this solution, containing 0.03 pCi, was applied as 0.5 pl droplets to the adaxial leaf surface. Prior to application of the radioactive mixture, the plants were sprayed with diclOfOp-methyl (36 EC) as previously described. One, 3 and SDAT the plant surfaces were washed with 50% methanol (3 x 7 m1). A 1 m1 aliquot of the combined 45 leaf washes was added to 15 ml Of ACS (Amersham), a prepared liquid scintillation cocktail for aqueous samples. The radioactivity in the samples was quantitated by liquid scintillation spectrometry (Packard Model 3003 Tri-Carb). All samples were corrected for quenching as determined by external standardization and background radiation. The washed foliage was wet combusted in an oxidizer (Harvey 0X-200) with the 14C02 trapped and quantified in 15 m1 Of Permaflour V:Carbosorb 11 (2:1, v/v - Packard). The percent absorption was calculated by the following formula: combusted foliage (dpm) absorption = x 100 leaf wash (dpm) + combusted foliage (dpm) Translocation Study. Seeds were germinated in a dark incubator at 30 C. After 72 h, the seedlings were removed from the germinating media, rinsed and transferred to 200 ml polystyrene specimen cups covered with aluminum foil and containing 180 m1 of one-half strength modified Hoagland's solution (11). After 48 h, all solutions were changed to full strength with additional minor elements and iron for the annual grass species. The solutions were changed every 48 h until treatment after which solution was added as needed during the 5 day treatment period. From a solution Of labelled and formulated diclOfOp-methyl prepared as previously described; a 10 pl droplet, containing 0.04 uCi of radioactivity, was applied with a microsyringe to each of the test Species. The drOplet was placed on the mid-portion of the adaxial surface of the second leaf of the annual grasses, one Of the unifoliate leaves of soybean and the first leaf of cucumber. 46 One, 3 and 5 DAT the plants were harvested and divided into four parts: the treated leaf, plant foliar parts formed above the treated leaf, plant foliar parts formed below the treated leaf and the roots. The treated leaf was washed with 50% methanol and the various plant parts were wet combusted as previously described. Radioactivity was determined as previously described, for a 1 ml aliquot of the nutrient solution and the leaf wash. Data are presented as a percent Of absorbed radioactivity. Volatility Study. Volatility of diclofop-methyl from barnyard- grass, soybean, cucumber, and a glass surface were compared by establishing three treatments: intact and living plant, intact and lyophilized leaf, and a glass surface. The fully expanded third leaf from barnyardgrass, the first true leaf of cucumber and the unifoliate leaves of soybean were excised from their respective plants, taped to a card and placed in a freeze dryer (Virtis) for 24 h. The adaxial leaf surfaces were not touched so as not to disturb epicuticular wax formations, trichomes and other surface structures. Ten ul of solution of 14 C diclofop-methyl and formulated diclofop-methyl prepared as previously described was applied to the dried leaf sections, to identical leaves on intact plants and to thin glass cover Slips. One, 3 and 5 DAT the various treatments were combusted, the 14 C02 trapped and quantitated by liquid scintillation spectrometry as previously described. Loss of radioactivity was determined by the following formula: 47 _ combusted samples 1, 3 and 5 DAT (dpm) Loss ' combusted samples 0 DAT (dpm) x 100 StatiStiCal'ConSiderations. The experimental design, for all experiments was a completely randomized block with four blocks. The experiments were a 3 x 5 factorial for the absorption study, 3 x 5 x 5 factorial for the translocation study and a 3 x 3 x 3 factorial for the volatility study. All studies were repeated and the data was subjected to analysis of variance with mean separation using Fishers protected LSD at the 5% level. RESULTS AND DISCUSSION Retention Study. On a whole plant basis, the order of diclofop-methyl retention among species was cucumber (T) > soybean (T) > proso millet (MS) > longspine sandbur (T) = barnyardgrass (S) (Table l). Expressed on a ug/mg dry weight or ug/cmz'leaf area basis, proso millet (MS) retained more diclofop- methyl than the other species. Visual inspection showed proso millet to be more hirsute which could result in greater spray retention, which could in part be responsible for the moderate susceptibility of proso millet to postemergence sprays Of diclofop-methyl. However, differential retention does not appear to be a primary selectivity mechanism since soybean and cucumber (T), retained the same amounts of diclofop-methyl on a dry weight basis as barnyardgrass (S). 48 Absorption Study. Differential absorption Of foliarly-applied 14C diclofop-methyl occurred among species over time (Table 2). I Longspine sandbur (T) had 14 to 18% less uptake 1 DAT than the other species. Three and 5 DAT all species had less uptake than cucumber (T), and 5 DAT soybean (T) had less uptake than cucumber (T), proso millet (MB) and longspine sandbur (T). It has been proposed that rapid deesterification Of diclofop- methyl to diclofop, and slow conjugation rates result in greater injury to the plant (16). Slower absorption in longspine sandbur and soybean (T) may help these species maintain lower levels of diclofop in the plant and minimize the initial rapid injury. Rapid conjugation occurs in longspine sandbur but not soybeans (5). From the retention and uptake data it can be computed that the tolerant species could theoretically have up to 18 times more diclofop-methyl absorbed on a plant basis than barnyardgrass (S). This would suggest a very efficient detoxication mechanism. Longspine sandbur is usually found growing on sandy soils which are subject to greater water stress (6). Sandbur may have the morphological characteristics Of a xeromorphic plant to survive the stresses imposed on it by its environment. For example, sandbur may have a_thicker cuticle which influences the initial penetration of diclofop-methyl. Diclofop-methyl absorption continued throughout the time course of the experiment. Similar results occurred with wheat, barley, wild oat and green foxtail over 192 h (17). Gorbach et al.(10) indicated that after 16 days only 3% of the radioactivity applied as 14C 49 Table l. Retention of foliarly-applied diclofop-methyl (36 EC) by five species measured immediately after application. Diclofop-methyl retained Total plant Leaf area Dry wt. Species (mg) (ug/cmz) (u97mg) Barnyardgrass (S)a 5.5 0.7 0.2 Proso millet (MS) 37.0 5.7 2.1 Longspine sandbur (T) 9.3 1.8 0.3 Soybean (T) 70.0 2.2 0.4 Cucumber (T) _9_1__._3_ _3_._5_ 9_._§_ LSD (.05) 17.4 2.1 1.0 a S = susceptible, MS = moderately susceptible and T = tolerant species to foliar applications of diclofop-methyl. 50 Table 2. Percent absorption of foliarly-applied 14C diclofop-methyl, l, 3 and 5 DAT by five species. DAT Species _l_ 3 _5_ -——-]4C absorption (%) ——— Barnyardgrass (S)a 55 65 71 Proso millet (MS) 53 69 75 Longspine sandbur (T) 37 64 75 Soybean (T) 51 63 68 Cucumber (T) 53 77 80 LSD .05 (species x time) = 6.2 a S susceptible, MS = moderately susceptible and T tolerant species to foliar applications of diclofop-methyl. 51 diclofop-methyl could be rinsed from the foliage of wheat. 14 TranleCati n Stddy. Movement of foliarly-applied C diclofop- methyl in barnyardgrass, proso millet, longspine sandbur, soybean and cucumber was limited with 98.1% Of the applied radioactivity remaining within the treated leaf Of all species (Table 3). These results agree with previous studies (8,15) which have found limited movement in wheat or wild oats. Although no translocation differences on a macro scale were found between the species in this study, chemical movement at the cellular level may be different among species and contribute to selectivity by preferential exclusion of the chemical from the site of action. Volatility_Study. Loss Of applied radioactivity from the leaf surfaces, either living or dead, and a glass surface, averaged 10.7% and did not differ among the surface tested (Table 4). Also no differences at 1, 3 and 5 DAT, or among species, were detected. Contrary to Todd and Stobbe (17), these results indicate that limited volatility did occur. This may have been due to differences in growing conditions. The former study was conducted in a growth chamber with fluorescent lighting and ours was in a greenhouse with supplemental metal halide lighting. Possibly leaf surface temperatures are higher with the metal halide lighting than under fluorescent lamps, which are relatively COOl. The elevated leaf temperature under metal halide lighting, which would be more comparable to field conditions, would favor the loss Of the chemical by volatility since the vapor pressure of diclofop-methyl Changes from 3 x 10"7 mm Hg 6 at 20 C to 5 x 10' mm Hg at 40 C (l). 52 Table 3. Distribution Of foliarly-applied 14C diclofop- methyl in 5 Species over time.a Portion Tested % of absorbed 14C Treated leafb 98.1 Leaves above treated leaf 1.5 Leaves below treated leaf 0.3 Roots 0.1 Nutrient solution 0.0 a There was no significant difference for Species or time so data was averaged over time (1,3,5 DAT) and species (barnyardgrass, proso millet, longspine sandbur, soybean, cucumber). b Treated leaf was second leaf on annual grasses, unifoliate leaf of soybean and first true leaf of cucumber. 53 Table 4. Loss of foliarly-applied 14C diclofop-methyl from plant and glass surfaces.a lyophilyzed intact glass tissue plagt_ surface % Of applied 14C Barnyardgrass (S)b ll 11 10 Soybean (T) 13 8 - Cucumber (T) 13 9 - a F test for main effects and interactions not significant at .05 level. b S = susceptible and T = tolerant species to foliar applications Of diclofop-methyl. 54 Loss of diclofop-methyl by volatility maytbe important when the rate applied is marginal. From this investigation it would appear that differential retention, absorption, translocation and volatility contribute little to the selectivity mechanisms for diclofop-methyl in plants. Although differences were measured such as the increased retention in proso millet, these differences do not explain the 100 fold difference in tolerance between barnyardgrass and soybean. The magnitude of tOlerance exhibited by broadleaf plants and selected grasses suggests that they possess an efficient detoxication mechanism. The next paper in this series explores the metabolic fate of diclofop-methyl in the five species. 10. 11. 12. LITERATURE CITED American Hoechst Corp. 1976. HOE 23408, technical information bulletin. American Hoechst Corp. Agric. Chem. Dept., Somerville, NJ! pp. 7. Anderson, R. N. 1976. Control Of volunteer corn and giant foxtail in soybeans. Weed Sci. 24: 253-256. Anderson, R. N. 1976. Response Of monocotyledons to HOE 22870 and HOE 23408. Weed Sci. 24: 266-269. Boldt, P. F. and A. R. Putnam. 1980. Response of selected annual grasses and broadleaf species to phenoxy-phenoxy herbicides. Weed Sci., in preparation. Boldt, P. F. and A. R. Putnam. 1980. Selectivity mechanisms to foliar applications Of diclofop-methyl. II. Metabolism. Weed Sci., in preparation. Brady, N. C. 1974. Nature and properties of soils. Macmillan Publishing Co., NY. pp. 639. Brewster, B. 0., A. P. Appleby and R. L. Spinney. 1977. Control Of Italian ryegrass and wild oats in winter wheat with HOE 23408. Agron. J. 69: 911-913. Brezeanu, A. G., D. G. Davis and R. H. Shimabukuro. 1976. Ultrastructural effects and translocation of methyl-2-(4- (2,4-dich1orophenoxy)phenoxy)propanoate in wheat (Triticum aestivum) and wild oat (Avena fatua). Can. J. Bot 54: 2038- 2048. Friesen, H. A., P. A. O'Sullivan and W. H. Vanden Born. 1976. HOE 23408, a new selective herbicide for wild oats and green foxtail in wheat and barley. Can. J. Plant Sci. 56: 567-578. Gorbach, S. G., K. Kuenzler and J. Asshauer. 1977. On the metabolism of HOE 23408 OH in wheat. J. Agric. Food Chem. 25: 507-511. Hoagland, D. R. and D. I. Arnon. 1950. The water culture method for growing plants without soil. Calif. Agric. Expt. Sta. Circ. 347. 32 pp. Holly, K. 1976. Selectivity in relation to formulation and application methods. Pages 249-275 in L. J. Audus, ed. Herbicides-Physiology. Biochemistry, Ecology. Vol. II. Academic Press, London. 55 13. 14. 15. 16. 17. 56 Putnam, A. R., A. P. Love and R. P. Rice, Jr. 1974. Control of annual grasses in vegetable crops with HOE 23408 and HOE 22870. Proc. North Cent. Weed Control Conf. 29:74. Sargent, J. A. 1976. Relationship of selectivity to uptake and movement. Pages 303-312 in L. J. Audus, ed. Herbicides- Physiology. Biochemistry, Ecology. Vol. II. Academic Press, London. Shimabukuro, R. H., W. C. Walsh and R. A. Hoerauf. 1979. Metabolism and selectivity of diclofop-methyl in wild oat and wheat. J. Agric. Food Chem.: in press. Todd, B. G. and E. H. Stobbe. 1976. Selectivity of diclofop- methyl among wheat, barley, wild oat and green foxtail. Proc. North Cent. Weed Control Conf. 31: 140. Todd, B. G. and E. H. Stobbe. 1977. Selectivity Of dichlofop- methyl among wheat, barley, wild oat (Avena fatua) and green foxtail (Setaria viridis). Weed Sci. 25: 382-385. CHAPTER 4 SELECTIVITY MECHANISMS T0 FOLIAR APPLICATIONS OF DICLOFOP-METHYL. II. METABOLISM ABSTRACT The fate of foliarly—applied 14 C diclofop-methyl [methyl 2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate] in intact plants Of barnyardgrass [Echinochloa crus-galli (L.) Beauv.] - a susceptible (S) grass, proso millet (Panicum miliaceum L.) - a moderately susceptible (MS) grass, longspine sandbur [Cenchrus longispinus (Hack.) Fern.] - a tolerant (T) grass, soybean [Glygine max (L.) Merr. 'Hark'], cucumber (Cucumis sativus L. 'Green Star') - both tolerant (T) broadleaf plants, 1, 3 and 5 days after treatment (DAT) was determined. More than 95% of the radioactivity washed from the leaf surface from all species was diclofop-methyl. Plant extracts contained diclofop-methyl, diclofop and water-soluble conjugates with higher levels of diclofop-methyl and diclofop found in barnyardgrass, proso millet and soybean. Higher levels of water- SOluble conjugates were present in cucumber and longspine sandbur. Acid hydrolysis of the water-soluble conjugates yielded high amounts of diclofop in barnyardgrass and proso millet, and high amounts of ring-OH diclofop in longspine sandbur, soybean and cucumber. ' Alkaline hydrolysis of the non-extracted plant residue yielded diclofop as the major component in all species. These studies Show both tolerant and susceptible plants hydrolyze diclofop-methyl to diclofop. Tolerant broadleaf and grass species may hydroxylate diclofop 57 58 in the aryl ring with subsequent glucosidic conjugation. Susceptible species may form conjugates with the propanoate side chain which are readily reversible. Soybean may possess an additional mechanism or mechanisms for detoxication. 59 INTRODUCTION Diclofop-methyl, applied postemergence, controls a wide spectrum Of annual grassy weeds in horticultural and agronomic crops (1,2,6,9). Tolerant species include wheat (Triticum aestivum L.), barley '(Hordeum vulgarg_L.), quackgrass (Agropyron repens L.), longspine sandbur, annual bluegrass (Ega_gggga_L.) and all dicots. Selectivity to foliar sprays can be a function Of differential spray retention, absorption, translocation and/or metabolism (8,10,16). Some differences in retention and absorption of diclofop-methyl have been reported (4,15), however, they do not account fully for the wide differences in selectivity among species. Translocation of diclofop- methyl is limited and does not differ between susceptible and tolerant species (4,5,12). Metabolism studies with wheat, a tolerant species, and wild oat (Avena fatua L.), a susceptible species, Show that diclofop- methyl is rapidly hydrolyzed to diclofop (12,14) with both chemicals being herbicidally active (11). In wheat, diclofop is aryl- hydroxylated then conjugated to an aryl glucoside (7,12). In wild oat, diclofop is conjugated to a neutral glucosyl ester (12). ShimabukUro et al. (12) suggest that the primary selectivity mechanism between the two species is the irreversible aryl hydroxylation of diclofop by wheat but not wild oat. To date, metabolism studies with dicots have not been reported. 60 Previous studies (3) indicated that rates of foliarly-applied diclofop-methyl which caused 50% growth reduction in barnyardgrass (S), proso millet (MS), longspine sandbur (T), cucumber (T), and soybean were: 0.1, 0.7, 1.5, 83 and 112_kg/ha, respectively. The purpose of these investigations was to determine if the differential response of the above species to foliarly-applied diclofop-methyl is a function of differential metabolism. MATERIALS AND METHODS Cultural Practices. Seeds were planted in 20-Cm styrofoam pots filled with a sand:peat:soil (1:1:1, v/v/v) potting media and placed in a greenhouse. Temperatures were 25 C - day, 20 C - night. Natural light was supplemented with metal halide lighting, with a 16 h photoperiod and an intensity of 842 DE m'zs']. Relative humidity ranged from 30 to 60%. All plants were watered overhead and by subirrigation on alternate days except after chemical application when only subirrigation was utilized. Two hundred 0 - ml of a water-soluble fertilizer solution (22.5% N - 22.5% P2 5 22.5% K20; 2 g/L) was applied to each pot on a weekly basis. Chemical Treatment. Plants were treated, 10 to 14 days after seeding, at the following growth stages: annual grasses - 3 leaves, soybean - unifoliate leaves, and cucumber - 1 leaf. All plants were sprayed with diclofop-methyl (36 EC) at 1.0 kg/ha using a moving belt sprayer delivering 234 L/ha at a pressure of 2.1 kg/cmz. Unlabelled diclofop-methyl and ‘4 C diclofop-methyl (specific activity - 2 uCi/uM), uniformly labelled in the 2,4—dichlorophenoxy ring, were mixed with distilled water to make an 11.5 uM solution. 61 Immediately after spraying, 25 ul of this solution, containing 0.12 uCi, was applied as 0.5 ul droplets at random over the plant leaf surfaces. Two annual grass plants or one broadleaf plant per treatment were used for analysis. Sample Handling and Extraction. Plants were harvested 1, 3 and 5 DAT. Since diclofop-methyl has limited translocation in the plant (4,5,12), only the foliar portion Of the plants were used in these studies. Analytical procedures were a modification of the techniques employed by Gorbach et al. (7) and Shimabukuro et al. (12). The foliage was washed with 50% methanol (3 x 7 ml), the washes combined and a 1 ml aliquot assayed for radioactivity (Figure 1, A). The solution (20 ml) was evaporated to dryness under vacuum at 30 C and the residue dissolved in acetone (0.5 m1) and separated by thin layer chromatography (TLC) (Figure 1, B). For TLC procedure refer to the following section. Duplicate plant samples were combusted to determine volatility losses (4). The 14C in the combusted sample minus the 14 C in the foliar wash equaled the total 14C absorbed and available for extraction. The washed foliage was placed in glass vials, frozen in a dry ice-acetone bath, lyophilized, and stored at -4 C then was homogenized and extracted with 80% methanol (15 ml) (Figure l, C). The extract was filtered and the methanol removed by evaporation under vacuum at 30 C. The aqueous extract was partitioned with chlorofOrm (3 x 10 m1) (Figure l, D). A 1 m1 aliquot was removed 62 foliage A surface wash with _ 50% CflfH [lashed foliagq I309; 0430?] C evaporation extraction with 80% CH30H - B evaporation CH3OH] «1:53:33; m H20 solution NaOH_ 0 digestion partition with CHCI3 H ' I ‘ combustion ’ 119909 I quantitatuon _ _ Ego phasil '73}de [CHCI3 phase] quantitatian HCl hydrolysis G E TLC and TLCond quantitation quantitation Figure 1. Flow diagram for extraction, separation and quantification of 4C diclofop methyl and its metabolites in five plant species. ' 63 from the chloroform fraction for radioactivity assay and the remainder reduced in volume under N2 gas to 0.5 m1. This fraction was separated by TLC (Figure 1, E). The aqueous fraction was assayed for radioactivity; removed to 0.5 ml under N2 gas and subjected to TLC (Figure 1, F). In one experiment, the remaining solution was hydrolyzed with 6 N HCl for 24 h in a water bath at 100 C. The solution was reduced in volume to 0.5 ml under N2 gas, and aliquots removed for TLC (Figure l, G). The plant residue was divided into two portions; one being combusted to determine the total non-extracted radioactivity (Figure 1, H) and the second refluxed with l N NaOH (25 ml) for 6 h, acidified to pH 2 and partitioned with ethyl acetate (3 x 25 ml). The ethyl acetate fraction was evaporated to dryness under vacuum and the residue dissolved in acetone (0.5 ml). Aliquots of this solution were separated by TLC (Figure l, I). All extracts containing methanol were processed within 1 h after addition of methanol, to minimize methylation of diclofop as described by Smith (13). Percent recovery of applied radio- activity ranged from 80 to 90% depending on species, time or experiment.. A RadioactivityyQuantification. Quantification Of radioactivity was by liquid scintillation spectrometry. All samples were corrected for quenching as determined by external standardization and for background radiation. Fifteen m1 of the following scintillants were used: ACS (Amersham) for water and methanol samples, ACS + H20 (3 ml) for Silica gel from TLC plates with water-soluble 64 fractions; PPO:POPOP:toluene (5 9:50 mg:l L) for chloroform, ethyl acetate, and silica gel from TLC plates with non-polar fractions. 14 CO2 produced from combustion of solid residues was trapped and counted in 15 ml Of Permaflour V:Carbosorb II (2:1, v/v, Packard). Thin Layer Chromatography. A microsyringe was used to spot 25 to 50 ul of extract to pre-coated silica gel G plates (250 um, Analab). All unknowns were co-chromatographed with known standards Of 14C diclofop-methyl and 14 C diclofop. Plates were developed 15 cm in one of the following solvent systems: 1) benzene:methanol: acetic acid (85:10:5, v/v/v) or 2) benzene:acetic acid (25:4, v/v). RF values for compounds detected in these solvent systems are indicated in Table l. Plates were scanned with a TLC scanner (Berthold L8 276) for locating radioactive spots, which were in turn scraped and quantitated as described previously. Statistical Considerations. The experimental design was a randomized complete block with four blocks with the treatments in a 3 x 5 factorial. The experiment was repeated three times. All data were subjected to analysis of variance with mean separation using Fishers protected LSD at the 1% level. RESULTS AND DISCUSSION Leaf Wash. Averaged over time, the 50% methanol leaf washes indicated a species differential with cucumber (T) and longspine sandbur (T), having 14% less absorption than barnyardgrass (S), or soybean (T) (Table 2). This is in agreement with earlier work (4) 65 Table 1. Rf values for 14C diclofOp-methyl and its metabolites in two solvent systems.a Solvent System .1L. ._9_ _____.Rf_____. Diclofop-methylb 0.7 0.6 Diclofopb 0.5 0.5 Ring-OH-diclofOpc 0.3 0.3 Conjugates (water soTubTe)c 0.0 0.0 a A = benzene:methanol:acetic acid (85:10:5, v/v/v); B = benzene:acetic acid (25:4, v/v). b Identified by co-chromatography with prepared standards and from published results (7,12). 0 Identified from published results (7,12). 66 Table 2. Percentage of total applied radioactivity in the leaf wash, plant extract and plant residue.a TotaT applied radioactivity b Plant extract Leaf Plant Plant plus plant Species wash extract residue residue c % Barnyardgrass (s)d 29.9 34.5 35.5 70.1 Proso millet (MS) 22.3 47.0 30.7 77.7 Longspine sandbur (T)l7.9 36.8 45.3 82.1 Soybean (T) 30.8 52.7 16.5 59.2 Cucumber (T) 16.8 50.7 32.5 83.2 a See Figure l. b Data averaged over time (1,3 and 5 DAT). C Plant extract plus plant residue = % radioactivity absorbed. d S = susceptible, MS = moderately susceptible and T = tolerant species to foliar applications of diclofop-methyl. 67 which suggested differential absorption may partially explain selectivity in soybean but not cucumber or longspine sandbur. TLC analysis of the 50% methanol leaf washes showed barnyardgrass (S) and proso millet (MS) had less diclofop-methyl and more diclofop, averaged over time, than the tolerant species (Table 3). Over all species, 94% of the detected radioactivity was as diclofop-methyl. This is in agreement with Gorbach et a1. (17), who found the majority of radioactivity in the methylene chloride leaf wash of wheat, 14C diclofop-methyl, to be parent compound. 'Clearly, treated with little metabolism occurs on the leaf surface. Diclofop-methyl and diclofop accounted for greater than 99% Of the total washed radioactivity. Averaged over species, diclofop levels were lower at l DAT than 3 or 5 DAT. The converse was true for diclofop. It could be hypothesized that hydrolysis of diclofop-methyl to diclofop 0n the leaf surface may have been due to surface micro- organisms. However, Shimabukura et al. (12) have shown hydrolysis of diclofop-methyl by microbial contamination is very minimal. A more likely possibility is the susceptible plants hydrolyzed diclofop-methyl more rapidly, the diclofop caused cuticular or cellular disruptions (l4), and allowed more of the absorbed diclofop to leak into the methanolic leaf wash than did the tolerant plants. Plant Extract. The chloroform phase of the 80% methanol extract contained diclofop-methyl and diclofop as determined by TLC (Table 4). Averaged over time, longspine sandbur (T) and cucumber (T) had 50 to 60% less diclofop-methyl and diclofop than 68 Table 3. TLC separation of radioactivity in 50% methanol leaf wash on five species, 1, 3 and 5 DAT with 14C diclofOp-methyl.a Radioactivity in leaf wash Spggigs Diclofop-methyl Diclofop % Barnyardgrass (s)b 92.8 7.2 Proso millet (MS) 92.7 7.3 Longspine sandbur (T) 95.8 4.2 Soybean (T) 96.1 3.9 Cucumber (T) g§;§_ 4:§_ LSD (.01) 2.5 2.5 PAT. 1 92.6 7.4 3 95.9 4.1 s 99.2 5...: LSD (.01) 1.9 1.9 a Figure 1, A. b 5 = susceptible, MS = moderately susceptible and T = tolerant species to foliar applications Of diclofop- methyl. 69 Table 4. TLC separation Of radioactivity in 80% methanol extract of five Species, 1, 3 and 5 DAT with 14C diclofop-methyl. Total radioactivity in extract CHC13 phase Water phase ' % Diclofop- §pggig§_ methyl Diclofop, Combined Conjugates Barnyardgrass (S)a 47.3 27.9 75.2 24.2 Proso millet (MB) 48.1 20.2 68.3 32.1 Longspine sandbur (T) 15.9 12.9 29.8 71.3 Soybean (T) 45.9 20.4 66.3 33.6 Cucumber (T) ll_._6_ _5_.4 _l_7_.0 _8_4_.2 LSD (.01) 9.1 7.9 8.4 8.4 DAT. 1 36.6 22.1 58.7 42.3 3 30.7 17.8 48.5 51.1 s 991 122. 9.5.9 59.9. LSD (.01) n.s. 5.9 5.5 5.5 a S = susceptible, MS = moderately susceptible and T = tolerant species to foliar applications of diclofop-methyl. 70 the other species. Diclofop-methyl, as a percent of the extracted radioactivity, did not change with time, however, the percent of diclofop decreased from 1 to 5 DAT, suggesting that the initial diclofop-methyl which is absorbed is hydrolyzed more rapidly than the diclofop-methyl absorbed later. The highest concentration Of diclofop- methyl penetrating the plant occurs during the initial 24 h after treatment (4,15) when diclofop is present in the greatest amount. The water phase of the 80% methanol extract contained water- soluble conjugates (12). Longspine sandbur (T) and cucumber (T) had greater amounts of conjugates than all other species while soybean (T) had greater amounts of conjugates than barnyardgrass (S) or proso millet (MS). For all species, conjugate content increased with time. . Increased conjugation corresponded with a decrease in levels of diclofop. It has been previously proposed that diclofop is either: 1) hydroxylated in the 2,4-dichlor0phenoxy ring and forms a glucosidic conjugate or 2) directly forms a glucosyl ester (7,12). The data supports this proposed pathway, however, ring-0H diclofop was not detected possibly due to its transitory nature. The radioactivity present in the 80% methanol extract Of the various species as a percent of the total applied 14 C diclofop-methyl is Shown in Table 2. Barnyardgrass (S) and longspine sandbur (T) had a lower amount of extracted radioactivity than the other species. This is probably due to extraction procedure rather than differential chemical binding. It was observed that the tissue of barnyardgrass and longspine sandbur were not homogenized as completely as the 71 other species due to differences in the amount of fibers present. Acid Hydrolysis. Acid hydrolysis of the water phase of the plant extracts yielded conjugates, ring-OH diclofop, diclofop and unknown compounds (Table 5). Longspine sandbur (T), soybean (T) and cucumber (T), had greater amounts of conjugates, ring-OH diclofop and unknown compounds and lesser amounts Of diclofop than barnyardgrass (S) and prOso millet (MS). Increased conjugation and hydroxylation and reduced diclofop concentration correlated closely with increasing tolerance of the species. Soybean (T) had an appreciable amount in the unidentified fraction. AS previously noted, ring-OH diclofop was not found in the plant extract. However, upon acid hydrolysis ring-0H diclofop was detected supporting the idea Of its being a transitory compound. The higher amounts of ring-OH diclofop in the tolerant species would indicate that conjugation occurred in the aryl ring and these species metabolize diclofop-methyl in a manner similar to wheat (7). The higher amounts of diclofop in the susceptible species after hydrolysis indicates that conjugation occurred in the propanoate side chain similar to wild oats (12). Solid Residue. Combustion of the solid residue remaining after 14 extraction showed soybean (T) had less non-extracted C than the other species, while proso millet (MS) and cucumber (I) had less than either barnyardgrass (S) or longspine sandbur (T) (Table 6). (Non-extractable radioactivity increased from 1 to 3 or 5 DAT. 72 Table 5. TLC separation of radioactivity in the 6 N HCl hydrolyzed water phase of the 80% methanol extract of absorbed 14C diclofOp-methyl.a Total radioactivity in wateryphaseb Ring-0H Spggigs Conjugates diclofop Diclofop Unidentified % Barnyardgrass (S)c 5.8 8.6 84.1 1.5 Proso millet (M5) 9.2 16.1 71.3 3.4 Longspine sandbur (T) 10.6 25.7 55.8 7.9 Soybean (T) 22.3 23.1 41.4 13.2 Cucumber (T) 18.8 43.5 32.7 5.0 a See Figure l. b Data for 5 DAT. C S = susceptible, MS = moderately‘susceptible and T = tolerant species to foliar applications Of diclofop-methyl. 73 Table 6. Absorbed 14C diclofop-methyl not extracted with 80% methanol in five species, 1, 3 and 5 DAT as determined by combustion.a Spggjg§_ Absorbed radioactivity % Barnyardgrass (S)b 50.7 Proso millet (MS) 39.5 Longspine sandbur (T) 55.2 Soybean (T) 23.8 Cucumber (T) 39.1 LSD (0.1) 6.9 _l_)_A_T 1 33.6 3 43.6 5 1L2. LDS (.01) 5.4 a See Figure l, H. b S = susceptible, MS = moderately susceptible and T = tolerant species to foliar applications of diclofop-methyl. 74 TLC analysis of the l NaOH hydrolyzate of the solid residue indicated the presence of conjugates, diclofop and diclofop-methyl (Table 7). Averaged over species and time, more than 90% of the radioactivity was detected as diclofop. Cucumber (S) had less diclofop and more conjugates than the other species. Barnyardgrass (S) had a lower conjugate content than the three tolerant species. There were no differences among DAT as to compounds present or relative amounts. From balance sheet calculations it was determined that less than 1% of the absorbed radioactivity from all species remained as a bound residue. With this study, we have attempted to relate the selectivity between: 1) annual grass species and 2) annual grass and broadleaf species to differential metabolism of diclofop-methyl on a whole plant basis. Previous metabolism studies have investigated the . metabolic fate of diclofop-methyl in excised tissues of tolerant and susceptible annual grass species (12,15) but not in whole plants or in broadleaf species. The results of this study support the hypothesis differential metabolism provides the basis for selectivity to foliarly-applied diclofop-methyl. However, both rate of chemical absorption and metabolism may play an important role with some species, especially soybean (T). Previous studies (4) have shown diclofop-methyl uptake to be slower in soybean than in the other species. The higher amounts of diclOfOp and diclofop-methyl in the 80% methanol extract Of soybean (Table 4) suggest that the metabolism rate might be slower in this 75 Table 7. TLC separation of radioactivity removed from plant residue by l N NaOH hydrolysis in five species, 1, 3 and 5 DAT. Radioactivity in residue Spggigsl Conjugates Diclofop Diclofoo-methyl % Barnyardgrass (S)a 2.7 93.4 3.8 Proso millet (MS) 4.7 92.9 2.4 Longspine sandbur (T) 6.9 88.5 4.5 Soybean (T) 5.2 91.4 3.4 Cucumber (T) _1_§._2_ _7_9_:_9_ ]_._7_ LSD (.01) 3.8 5.1 n.s. a S = susceptible, M5 = moderately susceptible and T = tolerant species to foliar applications of diclofop-methyl. 76 species. The metabolic pathway appears to be the same for other tolerant species as evidenced by products from acid hydrolysis of the water-soluble conjugates (Table 5). Further studies need to be conducted which eliminate the chemical absorption variable between species. The metabolic fate of diclofop-methyl in the five species chosen far this study appear to follow the proposed scheme of Shimabukuro et al. (12) for tolerant and susceptible grasses. In addition, soybean may possess an additional mechanism or mechanisms for detoxication. 10. 11. LITERATURE CITED Anderson, R. N. 1976. Control of volunteer corn and giant foxtail in soybeans. Weed Sci. 24: 253-256. Anderson, R. N. 1976. Response of monocotyledons to HOE 22870 and HOE 23408. Weed Sci. 24: 266-269. Boldt, P. F. and A. R. Putnam. 1980. Response of selected annual grasses and broadleaf species to phenosy-phenoxy herbicides. Weed Sci., in preparation. Boldt, P. F. and A. R. Putnam. 1980. Selectivity mechanisms to foliar applications of diclofop-methyl. I. Retention, uptake, translocation and volatility. Weed Sci., in preparation. Brezeanu, A. G., D. G. Davis and R. H. Shimabukuro. 1976. Ultrastructural effects and translocation of methyl-2-(4- (2,4-dich10rophenoxy)phen0xy)propanoate in wheat (Triticum aestivum) and wild oat (Avena fatua). Can. J. Bot. 54: 2038- 048. 2 Friesen, H. A., P. A. O'Sullivan and W. H. Vanden Born. 1976. HOE 23408, a new selective herbicide for wild oats and green foxtail in wheat and barley. Can. J. Plant Sci. 56: 567-578. Gorbach, S. G., K. Kuenzler and J. Asshauer. 1977. On the metabolism of HOE 23408 OH in wheat. J. Agric. Food Chem. 25: 507-511. Holly, K. 1976. Selectivity in relation to formulation and application methods. Pages 249-275 in L. J. Audus, ed. Herbicides-Physiology. Biochemistry, Ecology. Vol. II. Academic Press, London. Putnam, A. R., A. P. Love and R. P. Rice,Jr. 1974. Control of annual grasses in vegetable crops with HOE 23408 and HOE 22870. Proc. North Cent. Weed Control Conf. 29: 74. Sargent, J. A. 1976. Relationship Of selectivity to uptake and movement. Pages 303-312 in L. J. Audus, ed. 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