Triallate Resistance inAvena fatuaL. Is Due to Reduced Herbicide Activation

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO.

56, 163–173 (1996)

0070

Triallate Resistance in Avena fatua L. Is Due to Reduced Herbicide Activation ANTHONY J. KERN, DWIGHT M. PETERSON, ERICA K. MILLER, COREY C. COLLIVER, AND WILLIAM E. DYER Department of Plant, Soil, and Environmental Sciences, Montana State University, Bozeman, Montana 59717-0312 Received October 22, 1996; accepted February 1997 Extensive use of triallate, a preemergence herbicide used for wild oat (Avena fatua L.) control in cereal crops, has selected for resistant (R) wild oat populations. Triallate is thought to be activated via metabolic sulfoxidation to create the more potent triallate sulfoxide. Treatment of R and susceptible (S) wild oat lines with [1-14C]triallate showed that triallate is metabolized to the same primary endproduct, 2,3,3-trichloropropene sulfonic acid, in both types. However, the rate of triallate metabolism was more than 12-fold slower in R than in S plants. Dose–response studies indicated that although R plants were 6- to 20-fold more resistant than S plants to triallate treatment, both types were equally sensitive to in vitro synthesized triallate sulfoxide. In addition, [1-14C]triallate sulfoxide was metabolized to the same endproducts and at the same rate in R and S plants. The data indicate that resistance is conferred by a reduced rate of triallate sulfoxidation and represent the first documented case of herbicide resistance in plants conferred by reduced metabolism. q 1996 Academic Press

INTRODUCTION

Extensive herbicide use in agricultural and industrial settings has selected for resistant weed populations. In 1970, Ryan reported the first case of herbicide resistance to the photosynthesis inhibitor simazine (6-chloro-N,N *diethyl-1,3,5-triazine-2,4-diamine) in common groundsel (Senecio vulgaris L.) (1), and during the next 25 years over 125 instances of herbicide resistance have been documented, representing many weedy plant species and nearly every herbicide class used in agriculture (2). Several diverse physiological mechanisms that confer herbicide resistance have been described. Reduced uptake and translocation have been shown to confer resistance to the nonselective bipyridilium herbicide paraquat (1,1*-dimethyl-4,4*-bipyridinium dichloride) in certain biotypes of Erigeron (3) and Conyza bonariensis L. (4). However, most cases of weed resistance are caused by an alteration in the enzyme target site or an increase in the rate of herbicide metabolism. Elevated levels of cytochrome P450-mediated aryl-hydroxylation, aliphatic-hydroxylation,

and N-dealkylation reactions have been proposed as mechanisms of plant resistance to simazine (5), chlorsulfuron (6), chlortoluron (7), and other herbicides. Metabolism-based herbicide resistance could theoretically be conferred by a decreased rate of herbicide metabolism (8). For example, since diclofop-methyl activity is often enhanced by hydrolysis of the methyl ester (9), a lack or reduced rate of deesterification could confer resistance by reducing formation of the herbicidally active metabolite. However, this kind of herbicide resistance has not previously been documented in field- or laboratory-selected plants. Triallate is a selective thiocarbamate herbicide used to control wild oats in barley (Hordeum vulgare L.) and spring wheat (Triticum aestivum L.). The thiocarbamates are usually applied so that the herbicide becomes incorporated in the top 1–2 cm of soil. Herbicide uptake occurs primarily through the coleoptile of germinating wild oats, and susceptible seedlings often do not emerge. Crop selectivity may be achieved by lesser elonga-

163 0048-3575/96 $18.00 Copyright q 1996 by Academic Press All rights of reproduction in any form reserved.

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tion of the coleoptilar node in wheat and barley than in wild oats (10). Repeated use of triallate has selected for resistant (R) wild oat populations which previous studies showed to be 10- to 20-fold more resistant to triallate than susceptible (S) lines (11, 12). Preliminary characterization showed that uptake and translocation patterns were reduced about 30% in R seedlings compared to S seedlings (12); however, the differences were not judged sufficient to confer field levels of resistance. Triallate and other thiocarbamates are thought to require sulfoxidation to exert their phytotoxic effects. Casida et al. (13) showed that the sulfoxide derivatives of butylate (S-ethyl bis(2methylpropyl)carbamothioate), cycloate (S-ethyl cyclohexylethylcarbamothioate), EPTC (S-ethyl dipropylcarbamothioate), molinate (S-ethylhexahydro-1H-azepine-1-carbothioate), pebulate (Spropyl butylethylcarbamothioate), and vernolate (S-propyl dipropylcarbamothioate) were more toxic to a number of grasses and broadleaf weeds than their respective parent compounds. In all systems studied to date, sulfoxidation appears to be part of the major catabolic pathway for this herbicide family (14–16). Triallate sulfoxidation (Fig. 1) may be mediated by enzymes with cytochrome P450-like oxygenase activity. Sulfoxidation of triallate in mouse hepatic microsomal systems was oxygen- and NADPH-dependent (17), and triallate was metabolized extensively in NADPH-fortified rat liver microsome systems (14). Although the reaction has not been directly examined in planta, sulfoxidation of the related S-alkyl thiocarbamate EPTC occurred under aerobic conditions in maize microsomes in a NADPH-independent reaction (18). However, because pretreatment with the cytochrome P450 inhibitor piperonyl butoxide did not affect sulfoxidation, the exact nature of the enzyme system(s) responsible for thiocarbamate sulfoxidation in plants remains unclear. It is important to note that while the sulfoxide derivatives of the S-chloroallyl thiocarbamates (such as triallate) have not been

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detected in plant extracts due to their proposed instability (19), their presence as intermediates is inferred from studies using the S-ethyl thiocarbamates, which produce more stable sulfoxide derivatives (17). While it is accepted that the first reaction in the metabolism of triallate is oxidation to triallate sulfoxide (Fig. 1, 2), subsequent reactions appear to vary, perhaps according to the system tested. Following oxidation of triallate to form triallate sulfoxide, Mair and Casida (16) identified the sulfenic acid derivative (3) formed via conjugation of triallate sulfoxide with glutathione and subsequent cleavage and oxidation. Alternatively, Nadeau et al. (15) proposed that triallate sulfoxide is hydrolyzed to a transient thiol derivative (4) or further oxidized to the sulfone derivative (5). Regardless of which of these intermediates (3, 4, or 5) triallate sulfoxide metabolism proceeds by, Nadeau et al. (1993) and Hackett et al. (1993) have identified 2,3,3-trichloropropene sulfinic acid (6) as the major metabolite produced in rat liver microsomes. Subsequent oxidation of 6 leads to 2,2,3-trichloropropene sulfonic acid (TCPSA, 7), a breakdown product of triallate in common pea (Pisum sativum L.) (A. G. Hackett, personal communication). The objectives of these studies were to (i) determine the mechanism of triallate resistance in R wild oats, (ii) compare the metabolic pathway and fate of triallate and triallate sulfoxide in R and S wild oats; and (iii) compare the phytotoxicity of triallate sulfoxide with triallate in R and S wild oats. MATERIALS AND METHODS

Plant Material Susceptible wild oat seeds used for experiments were collected from field-grown populations of the nondormant inbred line SH430 (20) grown at the Arthur H. Post Research Farm near Bozeman, Montana, in 1993. Resistant seeds were collected in August, 1993 from fields near Fairfield, Montana, in which triallate had been used annually for 15 to 22 years (21) from plants surviving treatment

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FIG. 1. In vivo triallate metabolism scheme and TCPSA formation as proposed by Hackett et al. (1993). Proposed transient intermediates are in brackets. Position of radiolabel is denoted by an asterisk. 1, triallate; 2, triallate sulfoxide; 3, 2,3,3-trichloro-2-propenesulfenic acid; 4, 2,3,3-trichloro-2-propenethiol; 5, triallate sulfone; 6, 2,3,3-trichloro-2-propenesulfinic acid; 2,3,3-trichloro-2-propenesulfonic acid (TCPSA).

with 1.1 kg ha01 triallate the preceeding spring. The field collections were tested in greenhouse screening experiments to confirm

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resistance as described previously (12). One of these collections, FG93R22, contained about 80% R individuals and was used to de-

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velop the inbred R line used in all subsequent experiments. Approximately 200 FG93R22 plants showing a greater than 8-fold increase in triallate resistance were grown to maturity, selfed, and their progeny were subjected to an identical triallate treatment. The surviving individuals (about 1000 plants) were again grown, selfed, and their progeny were used for the experiments reported here. The selected R line was shown to be 17-fold more resistant to triallate treatment than the S line (12). Triallate and Triallate Sulfoxide Dose Response Triallate sulfoxide was synthesized from technical grade triallate (Monsanto, 98.9% pure) using the method of Schuphan et al. (19) with the following modifications. An emulsion of 800 mg ml01 triallate was made in 25 ml distilled water containing 7% (v/v) octyl phenoxy polyethoxyethanol surfactant (Triton X-100, Sigma Cat. No. T9284). Meta-chloroperoxy benzoic acid (Sigma Cat. No. C9416; mCPBA) was added in a 1.4-fold molar excess, the mixture was vortexed for 30 sec, and the emulsion was incubated for 1 hr on ice. Upon completion of the reaction (ú95% conversion as determined by HPLC), the emulsion was diluted in deionized water containing 7% surfactant. Caryopses of R and S wild oats were surface sterilized by shaking for 15 min in 15% (v/v) commercial bleach and vigorously rinsed for 10 min in sterile deionized water. The caryopses were incubated at 22 { 27C on one sheet of Whatman No. 4 filter paper moistened with 3.5 ml of sterile deionized, distilled water in 25 1 100mm petri dishes in the dark. After 4 days, uniform seedlings with 1-cm long shoots were treated by adding 0.5 ml triallate or triallate sulfoxide solution on the filter paper to give treatment concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, or 100 mg ml01 for triallate sulfoxide or 0, 0.1, 0.5, 1, 5, 10, 50, 100, or 150 mg ml01 for triallate. Triallate and control treatment solutions also contained 7% surfactant. Shoot lengths (within or beyond the coleop-

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tile) were recorded after 7 days. There were two replicate petri dishes for each treatment containing six seedlings as subsamples, and the experiment was repeated once. Data are reported as the means of the two experiments. Triallate Metabolism R and S seedlings grown as before in petri dishes were treated by spotting 10 mg l01 technical grade triallate plus 1 ml of [1-14C]triallate (1.67 1 103 Bq; sp act 3.42 1 106 Bq/mg) dissolved in methanol on the shoot apex. This concentration was shown in previous studies to have no effect on the growth of R and S plants (data not shown). Shoots were harvested 1, 3, 6, 9, 12, 24, 36, 48, 60, 72, or 96 hr after treatment and rinsed for 10 sec in 60% (v/v) acetone to remove unabsorbed triallate. Then, five shoots were homogenized in a 2.0ml Broeck glass homogenizer in 0.5 ml 60% (v/v) acetone. The crude homogenate was centrifuged at 16,000g for 4 min at 47C and the supernatant filtered through 0.22-mm nylon mesh in microspin centrifuge filters at 5000g for 5 min at 47C. The filtrates were standardized by dilution with 60% acetone to yield 0.5-ml extracts containing 4.16 1 102 Bq each and subjected to HPLC analysis. There were three replications of five seedlings for each time point, and the experiment was repeated once. Data are reported as the means of both experimental repeats. HPLC Analyses HPLC analyses of nonpolar herbicide metabolites used a C18 reverse-phase column in a 4.6 1 250-mm format. The mobile phase consisted of a linear gradient from 95% water: 5% acetonitrile to 50% water:50% acetonitrile over 10 min and then to 100% acetonitrile over the next 2 min, which was held for an additional 5 min. The gradient programmer and HPLC pump apparatus delivered solvent at 2.0 ml min01, and separated radioactive compounds were detected using an in-line scintillation counter. Radioactivity present in

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individual peaks was quantified using a dedicated integrator. HPLC analyses of highly charged metabolites used a 4.6 1 125-mm weak anion exchange (WAX) column. The mobile phase consisted of a linear gradient from 100% water to 90% water:10% 0.75 M ammonium phosphate buffer (pH 3.2) over 10 min. The HPLC apparatus and gradient programmer delivered solvent at 1.0 ml min01, and retained radioactive metabolites were quantified using an inline scintillation counter as above. Triallate Sulfoxide Metabolism For in vivo metabolism studies, triallate sulfoxide was synthesized as above with the following modifications. A 23 mM solution of 1-14C-labeled triallate (1.67 1 103 Bq) was oxidized in distilled water containing 0.1% (v/v) X-77 nonionic surfactant (Valent USA, Walnut Creek, CA), 0.41 MS salts (22), and 5 mM mCPBA and incubated on ice. After 1 hr, conversion to triallate sulfoxide was verified by HPLC and 35 ml of the solution was added to individual wells of a 96-well microtiter plate which was sealed with Parafilm. Shoots from 4-day-old R and S etiolated seedlings were excised under water, and four shoots were placed into each well through holes in the Parafilm. After 3 hr, the reaction solution was replaced with an equal volume of 0.41 MS salts. After 3, 6, or 24 hr, shoots were harvested and homogenized as described above, and the extracts were subjected to reverse-phase HPLC. There were two replications of four shoots for each time point, and the experiment was repeated once. Data are presented as the means of both experimental repetitions. RESULTS AND DISCUSSION

Triallate Dose Response In petri dishes, R wild oats were more tolerant to triallate treatment than S lines, with estimated GR50 values (the treatment rate at which growth is reduced by 50%) of 150 and

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FIG. 2. Growth inhibition of R (triangles) and S (circles) wild oat seedlings 7 days after treatment with triallate in petri dishes. Vertical bars are standard errors of means.

25 mg ml01, respectively, representing a sixfold increase in triallate resistance levels (Fig. 2). While this increase in resistance is less than the 12- to 20-fold increase previously reported in greenhouse dose–response studies (11, 12), triallate was likely more toxic in petri dishes because of root uptake and increased tissue exposure in a closed system. Even though triallate studies are typically done using shoot (11, 12) or vapor phase (23) uptake, we tested the response of R and S wild oats to root-absorbed triallate in order to confirm the applicability of this treatment method for later triallate sulfoxide studies. Triallate Metabolism R and S wild oat seedlings were treated with [1-14C]triallate to compare herbicide metabolite profiles after 24 hr (Fig. 3). S seedlings metabolized triallate extensively, forming one major (Peak I) and two minor (Peaks II and III) metabolites under our experimental conditions. Peak IV was identified as unmetabolized triallate, based on cochromatography with authentic [1-14C]triallate. For confirmation, Peak IV was collected from fractions eluted 16.9 to 17.1 min after injection and rechromatographed using a 65:35 acetonitrile:water isocratic mobile phase, in which

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FIG. 3. Reverse-phase HPLC chromatograms from S (top) and R (bottom) wild oats harvested 24 hr after treatment with [1-14C]triallate. Peak I, TCPSA; Peaks II and III, unknown; Peak IV, triallate.

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Peak IV coeluted with the [1-14C]triallate standard at 6.6 min (data not shown). The elution time of Peak I in Fig. 3 was 1.9 min, indicating that this highly polar metabolite was not retained on the column. Therefore, a fraction of Peak I eluting 1.8 to 2.0 min after injection was collected and rechromatographed under WAX HPLC conditions. Elution time of Peak I was identical to that of TCPSA under two different WAX protocols (data not shown), thus confirming its identity. In addition, a subsample of Peak I spiked with the TCPSA standard resulted in a quantitative increase in peak area under both WAX protocols (data not shown). Peaks II and III eluted at 8.1 and 8.9 min, respectively, under these HPLC conditions. Several approaches were taken to purify and identify Peaks II and III. However, these efforts were unsuccessful, due in part to coelution of matrix polymers (14). As shown in Fig. 1, R seedlings appeared to metabolize triallate at a substantially slower rate than S seedlings. Because R and S seedlings ultimately metabolized triallate to the same endproducts (Peaks I–III; data not shown), extracts were analyzed by HPLC at several times after treatment and the integrated peak areas quantified to compare metabolism rates between the two types (Fig. 4). In S plants, more than 50% of absorbed triallate was metabolized into TCPSA and other compounds by 9 hr after treatment, with total disappearance of the parent molecule after 48 hr. Metabolites other than TCPSA accounted for 2 to 15% of the radioactivity up to 96 hours after treatment. In contrast, about 50% of the absorbed triallate remained unmetabolized in R seedlings 96 hr after treatment. Half-life values estimated from these data for triallate metabolism in R and S wild oats were greater than 96 and 8 hr, respectively, indicating that the rate of triallate metabolism was more than 12-fold slower in R plants than in S plants. Thus, a substantially reduced rate of triallate sulfoxidation would allow R plants to effectively avoid rapid exposure to high in vivo concentrations of triallate sulfoxide. Ap-

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FIG. 4. Distribution of [14C]triallate and its metabolites in S (top) and R (bottom) wild oat seedlings at 1 to 96 hr after treatment. LSD Å 9.3.

parently, R plants are able to further metabolize the low levels of triallate sulfoxide that are generated into TCPSA and other nontoxic metabolites. We did not detect triallate sulfoxide in plant extracts at any time after triallate treatment, verifying previous reports that this intermediate is extremely labile in vivo (17). These results may help explain our previous studies showing that triallate uptake and translocation rates were slightly reduced in R seedlings (24), since slower removal of triallate from R coleoptile and shoot cells would effectively reduce the formation of a triallate concentration gradient across the plasmalemma. The magnitude of difference in metabolism rates agrees closely with the 12- to 20-fold greater resistance exhibited by R plants in greenhouse studies (11, 12).

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FIG. 5. Distribution of [14C]triallate sulfoxide and its metabolites in S (top) and R (bottom) wild oat seedlings at 3 to 24 hr after treatment. LSD Å 5.4.

oxide proceeded equally in both types. These data further support the idea that the biochemical lesion conferring triallate resistance occurs prior to the conversion of triallate sulfoxide to additional metabolites. TCPSA was the major triallate sulfoxide metabolite detected in R and S seedlings, with additional breakdown products observed at very low levels. These minor products had the same retention times as the triallate metabolites Peaks II and III in Fig. 4, indicating that triallate sulfoxide was converted to the same endproducts as triallate in R and S seedlings. Thus, the data support the participation of triallate sulfoxide as an intermediate of the triallate metabolism pathway. Triallate sulfoxide is known to be very labile in vitro (19) and we estimated its halflife to be 12 hr under our plant uptake conditions. In vitro breakdown products of triallate sulfoxide consisted of two compounds, one of which coeluted with Peak II in Fig. 4 (but was not identified). However, TCPSA was only detected in plant extracts, and its presence thus confirmed that triallate sulfoxide was indeed being taken up and metabolized by R and S seedlings. Triallate Sulfoxide Dose Response

Triallate Sulfoxide Metabolism To further test the hypothesis that a reduced rate of triallate sulfoxidation was responsible for herbicide resistance, [1-14C]triallate sulfoxide was synthesized and applied to R and S seedlings. Radioactive metabolites were extracted, separated by HPLC, and quantified 3, 6, and 24 hr after treatment (Fig. 5). Triallate sulfoxide was rapidly metabolized in R and S wild oats, with 50% of the absorbed compound metabolized into TCPSA and other metabolites by 6 hr after treatment, and nearly total disappearance of the sulfoxide by 24 hr after treatment. No significant differences in the rate of triallate sulfoxide metabolism were detected between R and S plants, indicating that enzymatic detoxification of triallate sulf-

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The whole plant phytotoxicity of triallate sulfoxide to R and S wild oats was tested by treating seedlings in petri dish dose–response assays (Fig. 6). Triallate sulfoxide was equally phytotoxic to R and S plants, with GR50 values estimated at 7 and 6 mg ml01, respectively. Triallate sulfoxide was also more phytotoxic than triallate to both R and S plants, with estimated 21- and 4-fold increases in phytotoxicity, respectively. These data agree with previous studies showing that other thiocarbamate sulfoxides were more phytotoxic than their parent compounds (13) and help confirm that the sulfoxide derivative of triallate is the more herbicidally active compound. The fact that R and S plants were equally sensitive to triallate sulfoxide further supports the hypothesis that

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FIG. 6. Growth inhibition of R (triangles) and S (circles) wild oat seedlings 7 days after treatment with triallate sulfoxide in petri dishes. Vertical bars are standard errors of means.

R plants are deficient in triallate sulfoxidase activity. We have been unable to detect the putative triallate sulfoxidase activity in seedling extracts using NADPH and oxygen as cofactors in either microsomal or soluble fractions using standard methodologies for assaying cytochrome P450 activities (26). Although cytochrome P450-dependent oxidases are responsible for thiocarbamate sulfoxidation in mammalian systems (16) and for metabolic detoxification of many other herbicides in plants (26, 27), their role in thiocarbamate sulfoxidation is not clear. We tested the cytochrome P450 inhibitors tetcyclacis and piperonyl butoxide (28) for their effects on triallate metabolism patterns in R and S seedlings. Neither inhibitor altered either triallate metabolism or its phytotoxic effects to R and S seedlings (data not shown), indicating that a cytochrome P450 may not be involved in tri-

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allate sulfoxidation. It has been suggested that EPTC sulfoxidation in maize is carried out by a hydroperoxide-dependent peroxygenase (29), and similar activities may be responsible for triallate sulfoxidation in wild oats. Because we have been unable to assay the putative triallate sulfoxidase activity, we do not know if lower activity in R plants is due to lower enzyme levels, an altered substrate specificity, or an increase in triallate sequestration into the cell wall or vacuole. Although sequestration has been proposed as a mechanism of resistance to paraquat (4), we have no direct or indirect evidence supporting such a phenomenon for triallate resistance in wild oats. Furthermore, because our inheritance studies showed that triallate resistance is controlled by two recessive nuclear genes (30), we speculate that two enzymes are required for triallate sulfoxidation, and that mutations in both respective genes are required for resis-

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tance. The true identities of these enzymes and their substrate specificities await further characterization. This report marks the first documented case of herbicide resistance in plants conferred by a reduced rate of metabolic activation. In addition, we know of no other examples of resistance to insecticides, fungicides, or antibiotics due to a similar lack of activation. Reduced amounts of the insecticidally active metabolite of parathion, termed paraoxon, have been documented in parathion-resistant greenbug (Schizaphis graminum). However, this decrease was due to elevated metabolic detoxification of paraoxon and not a decrease in the rate of activation (31). Documentation of this type of resistance mechanism illustrates the highly variable ability of organisms to respond to strong selection pressure and will likely have implications on resistance management strategies in several types of pest control situations. ACKNOWLEDGMENTS This work was partially supported by the Montana Agricultural Experiment Station, the Monsanto Company, and the Montana Noxious Weed Trust Fund. We thank Dr. Amy Hackett and the Monsanto Co. for the kind gifts of [14C]triallate and 14C-labeled TCPSA. The excellent technical assistance of Tracey Myers is much appreciated.

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