Mechanism of Fenoxaprop Resistance in an Accession of Smooth Crabgrass (Digitaria ischaemum)

Pesticide Biochemistry and Physiology 64, 112–123 (1999) Article ID pest.1999.2417, available online at http://www.idealibrary.com on

Mechanism of Fenoxaprop Resistance in an Accession of Smooth Crabgrass (Digitaria ischaemum)1 Yong-In Kuk,2 Jingrui Wu, Jeffrey F. Derr,3 and Kriton K. Hatzios Laboratory for Molecular Biology of Plant Stress, Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0330 Received October 30, 1998; accepted April 28, 1999 An accession of smooth crabgrass [Digitaria ischaemum (Schreb.) Muhl] exhibiting resistance to the herbicide fenoxaprop was discovered recently in New Jersey. This accession was highly resistant to fenoxaprop-ethyl (approximate R/S GR50 ratio of 102) and moderately resistant to quizalofop-ethyl (approximate R/S GR50 ratio of 16.3), but exhibited low resistance to cyclohexanediones such as sethoxydim (R/S GR50 ratio of 1.3). The potential mechanism of resistance was investigated by evaluating the effect of fenoxaprop on acetyl-CoA carboxylase (EC 6.4.1.2, ACCase) activity extracted from shoots of resistant and susceptible plants and comparing the absorption, translocation, and metabolism of radiolabeled fenoxaprop in resistant and susceptible plants. The patterns of absorption, translocation, and metabolism of fenoxaprop were similar in resistant and susceptible smooth crabgrass. ACCase activity from susceptible plants was very sensitive to fenoxaprop-ethyl and fenoxaprop acid with I50 values of 2 and 4.9 mM, respectively. ACCase activity from resistant plants was very resistant to fenoxaprop-ethyl (I50 . 182 mM) and moderately resistant to fenoxaprop acid (I50, 29 mM). ACCase activity from resistant smooth crabgrass was 50-fold less sensitive to quizalofop-ethyl than that extracted from susceptible smooth crabgrass. ACCase activity extracted from either resistant or susceptible plants was inhibited strongly by sethoxydim (I50 of 7.4 mM in R and 3.4 mM in S). These results suggest that a less sensitive form of the target enzyme, acetyl-CoA carboxylase, confers a high degree of resistance to smooth crabgrass toward fenoxaprop and moderate resistance to other aryloxyphenoxypropionate herbicides. q1999 Academic Press Key Words: fenoxaprop; quizalofop; herbicide resistance; sethoxydim; smooth crabgrass; acetyl-CoA carboxylase; ACCase inhibitors; cross-resistance.

INTRODUCTION

many grass weeds (1–3). Fenoxaprop-ethyl is a postemergence applied herbicide used for the control of annual and perennial grasses in crops such as soybean, turf, and wheat (2, 3). The target site of AOPP herbicides such as fenoxaprop and quizalofop (Fig. 1) and CHD herbicides such as sethoxydim (Fig. 1) is the biotin-containing enzyme acetyl-coenzyme A

Following their respective introduction in the 1970s and 1980s, aryloxyphenoxypropionate4 (AOPP) and cyclohexanedione (CHD) herbicides have been used widely for the control of 1 Contribution 633 from the Department of Plant Pathology, Physiology and Weed Science, Virginia Agricultural Experiment Station, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0330. 2 Current address: University of Arkansas, 276 Altheimer Drive, Fayetteville, AR 72704. 3 Permanent address: Hampton Roads AREC, Virginia Tech, 1444 Diamond Spring Road, Virginia Beach, VA 23455. 4 Abbreviations used: ACCase, acetyl-CoA carboxylase; AOPP, aryloxyphenoxypropionate herbicides; BSA, bovine serum albumin; CHD, cyclohexandione herbicides; DTT, dithiothreitol; fenoxaprop, (6)-2-[4-[(chloro-2-benzoxazolyl)oxy]phenoxy]propanoic acid; fenoxaprop-ethyl, (6)-2[4-[(chloro-2-benzoxazolyl)oxy]phenoxy]propanoic acid,

ethyl ester; GR50, herbicide dose required for 50% reduction in growth; I50, herbicide concentration required for 50% inhibition of enzyme activity; LSS, liquid scintillation spectrometry; PMSF, phenylmethylsulfonyl fluoride; PVP, polyvinylpyrrolidone; quizalofop, (6)-2-[6-[(chloro-2-quinoxalinyll)oxy]phenoxy]propanoic acid, R/S, resistant/susceptible ratio; sethoxydim, 2-[1-(ethoxyimino)butyl]-5[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one; TLC, thin-layer chromatography; tricine, N-tris(hydroxymethyl)-methylglycine.

112 0048-3575/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.

FENOXAPROP-RESISTANT SMOOTH CRABGRASS

FIG. 1. Chemical structures of the aryloxyphenoxypropionate herbicides fenoxaprop-ethyl and quizalofopethyl and of the cyclohexanedione herbicide sethoxydim.

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carboxylase (EC 6.4.1.2; ACCase) (4–6). ACCase is the first dedicated enzyme in the de novo fatty acid biosynthetic pathway. ACCase catalyzes the ATP-dependent conversion of acetyl-coenzyme A to malonyl-coenzyme A. The establishment of ACCase as the target site for all AOPP and CHD herbicides has been supported by the work of several investigators and the subject has been reviewed recently by Incledon and Hall (6). Dicotyledonous plants contain two forms of ACCase in their cells: a prokaryotic form in the plastids, which is insensitive to AOPP and CHD herbicides, and a herbicide-sensitive eukaryotic form in the cytosol (7). In grass plants, the prokaryotic form is absent and isozymes of the eukaryotic form are found in both the plastids and the cytosol (7). The frequent and widespread use of the AOPP and CHD herbicides has resulted in the development of resistance in biotypes or accessions of several grass weeds, which were detected first outside the United States. Such weeds include annual ryegrass (Lolium rigidum Gaud), wild oat (Avena fatua L.), and winter wild oat (Avena sterilis ssp. ludoviciana Malzew.) in Australia (8–11); wild oat and green foxtail [Setaria viridis (L.) Beauv.] in Canada (12–14); slender foxtail (Alopecurus myosuroides Huds.) in England (15); goosegrass [Eleusine indica (L.) Gaertn.] in Malaysia (16); and littleseed canarygrass (Phalaris minor Retz.) in Israel (17). The occurrence of grass weed resistance to AOPP and CHD herbicides in the United States has been confirmed in recent years. Diclofopresistant biotypes of Italian ryegrass (Lolium multiflorum Lam.) and of wild oat have been confirmed in Oregon (18, 19) and wild oat in the Red River Valley (20). Two biotypes of Johnsongrass [Sorghum halepense (L.) Pers.] resistant to fluazifop and quizalofop have been confirmed in Mississippi (21), whereas a Johnsongrass biotype resistant to quizalofop and sethoxydim has been reported in Virginia (22). Accessions of large crabgrass [Digitaria sanguinalis (L.) Scop.] and giant foxtail (Setaria faberii Herrm.) exhibiting resistance to fluazifop-butyl

and sethoxydim have been confirmed in Wisconsin (23, 24). Finally, a smooth crabgrass accession resistant to fenoxaprop-ethyl was discovered recently in New Jersey (25). The resistance of grass weeds to ACCaseinhibiting herbicides appears to result mainly from alterations of the target ACCase enzyme (6, 26). For example, the resistance of littleseed canarygrass to fenoxaprop (17), red fescue (Festuca rubra L.) to sethoxydim (27), and Italian ryegrass to diclofop (28) has been linked to reduced sensitivity of ACCase to these herbicides. Similarly, the resistance of green foxtail, wild oats, and goosegrass to AOPP and CHD herbicides has been attributed to reduced herbicide sensitivity of ACCase in the resistant biotypes (29–35). While the evidence supporting this hypothesis is convincing, alternative mechanisms of resistance have also been proposed (26). Reduced absorption and translocation have not been implicated as mechanisms for the resistance of grass weeds to AOPP and CHD herbicides (26). However, enhanced herbicide metabolism has been suggested as a mechanism of resistance to ACCase inhibitors in selected biotypes of slender foxtail (36) and annual ryegrass (37). Finally, although a specific mechanism of resistance was not identified, evidence linking grass weed resistance to a differential effect of AOPP and CHD herbicides on the electrogenic potential of plasma membranes has been presented (38, 39). The specific objectives of the present study were to (a) confirm the resistance of smooth crabgrass to fenoxaprop-ethyl, (b) determine the potential cross-resistance of smooth crabgrass to quizalofop-ethyl and sethoxydim herbicides, and (c) determine the mechanism of resistance by evaluating the effects of fenoxaprop on ACCase activity extracted from resistant and susceptible plants and comparing the absorption, translocation, and metabolism of radiolabeled fenoxaprop in resistant and susceptible accessions of smooth crabgrass. MATERIALS AND METHODS

Chemicals Analytical-grade (98% pure) samples of fenoxaprop-ethyl and fenoxaprop acid and

FENOXAPROP-RESISTANT SMOOTH CRABGRASS

radiolabeled samples of fenoxaprop-ethyl (chlorophenyl-U-14C-labeled, sp. act. 1177.8 MBq/g) were provided kindly by Dr. Chris Hall, University of Guelph, Guelph, Ontario, Canada and the AgrEvo Company (Frankfurt, Germany). Analytical-grade quizalofop-P-ethyl (95% pure) and technical sethoxydim (50% pure) were provided by the DuPont (Wilmington, DE) and BASF (Research Triangle Park, NC) companies, respectively. Radiolabeled sodium bicarbonate was obtained from Sigma Chemical Co. (St. Louis, MO). Solvents and other reagents were obtained commercially from either Sigma or Fisher Scientific (Pittsburgh, PA). Plant Material Seeds of fenoxaprop-resistant smooth crabgrass were collected from plants growing in a golf course tee in the southern part of New Jersey, where fenoxaprop-ethyl was the primary herbicide used for crabgrass control for several years (25). Seeds of susceptible smooth crabgrass were collected from plants growing in fields near Virginia Beach, VA, where fenoxaprop-ethyl or other AOPP and CHD herbicides had not been used. Herbicide Treatments and Growth Response Seeds of resistant and susceptible smooth crabgrass were sown 1-cm deep in 450-ml styrofoam cups filled with a commercial soil mixture (Mitro-Mix 360, Scotts-Sirra Horticultural Products Co., Marysville, OH), one seed per cup. The cups were watered daily and placed in a greenhouse with 258C temperature, 16-h photoperiod, and natural plus artificial lighting providing a photon flux density of 650 mmol/m2/s. Seedlings of resistant and susceptible accessions of smooth crabgrass were treated at the two-tiller stage with commercial formulations of fenoxaprop-ethyl (Acclaim 1EC), quizalofopP-ethyl, and sethoxydim using a belt-link sprayer equipped with a single flat fan nozzle (8001E) delivering 233.7 liter ha21. Treatment rates used were 0, 0.03, 0.05, 0.07, 0.14, 0.28, 0.56, 1.12, 2.24, 4.48, 6.72, 8.96, and 11.2 kg/ha for fenoxaprop-ethyl; 0, 0.01, 0.03, 0.07, 0.14, 0.28, 0.56,

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and 0.84 kg/ha for quizalofop-P-ethyl; and 0, 0.05, 0.1, 0.2, and 0.4 kg/ha for sethoxydim. The full range of herbicide doses was applied only to the resistant accessions of smooth crabgrass. Because of their sensitivity, susceptible accessions of smooth crabgrass were treated with herbicide doses equal to or less than the recommended rate. Treated plants were returned to the greenhouse. Two weeks after treatment, the plants were cut at the soil surface and shoot dry biomass was determined after drying in an oven at 608C for 24 h. A randomized design with five replications of each herbicide dose was used and each experiment was duplicated. Dose-response data were analyzed and GR50 doses were determined from the dose-response relationship by probit analysis (40). Absorption, Translocation, and Metabolism Plants were grown in the greenhouse, as described above. After the roots were washed free of the soil mixture, smooth crabgrass seedlings were transferred to aluminum foil-covered glass jars filled with 100 ml of quarter-strength Hoagland’s nutrient solution (pH 6.3) and were acclimated in this medium for 3 days. Hydroponically grown seedlings of resistant and susceptible smooth crabgrass (three-leaf stage) were sprayed with 0.15 kg/ha (approximate field rate) of formulated fenoxaprop-ethyl. Immediately after this treatment, 10 ml of [14C]fenoxapropethyl was applied to the second leaf of the smooth crabgrass seedlings, which had been covered during spraying with nonlabeled herbicide. [14C]fenoxaprop-ethyl was dissolved in acetone and diluted to 5% acetone in an aqueous solution containing 0.5% (v/v) Tween 20 to obtain an approximate radioactivity content of 3700 Bq/ml (0.01mmol). Plants were harvested at 8, 24, 48, and 72 h after treatment and dissected into three parts; the treated leaf, the remaining shoot and leaves, and the roots. The amount of unabsorbed radiolabeled herbicide at each harvest time was determined by agitating the treated leaf for 30 s in 10 ml wash solution (10% ethanol and 0.1% Tween). A 1-ml aliquot

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of the rinsate was mixed with 10 ml scintillation cocktail (ScintiVerse, Fisher, Pittsburgh, PA) and the 14C was quantified by liquid scintillation spectrometry (Beckman LS 5000TA Model, Beckman Instruments, Fullerton, CA). Total absorption of applied radioactivity and translocation of absorbed radioactivity were determined by combustion of the seedling tissues in a biological sample oxidizer (Model B307, Packard Instrument Company, Downers Grove, IL) and liquid scintillation spectrometry. Absorbed radioactivity was expressed as a percentage of applied radioactivity. Distribution of 14 C in plant tissues was expressed as a percentage of absorbed radioactivity. After the surface rinse, the treated leaves of resistant and susceptible smooth crabgrass seedlings were homogenized in 3 ml of acetonitrile and water (7:3, v/v) and centrifuged at 5200g for 3 min. The pellet was combusted in a biological sample oxidizer to determine nonextractable radioactivity. The supernatant was evaporated to dryness under N2 and redissolved in 100 ml methanol. The methanolic extract was spotted on silica gel thin-layer chromatography plates (Silica Gel 60F254 TLC Plates, EM Science, Gibbstown, NJ). Authentic standards of labeled fenoxaprop-ethyl and nonlabeled fenoxaprop acid were cochromatographed with the plant extracts and the plates were developed in a butanol:acetate:water (12:3:5, v/v/v) solvent system. Developed plates were dried (308C for 5 min) and visualized under UV light and by X-ray autoradiography. Each detected fraction was scraped from the plates and placed into scintillation vials and radioactivity was quantified by liquid scintillation spectrometry. Metabolites were separated by their Rf values and the distribution of radioactivity in each metabolite fraction was expressed as a percentage of the absorbed radioactivity recovered in the TLC analysis of the plant extracts. Absorption, translocation, and metabolism experiments were repeated and treatments were replicated three times in each experiment.

ACCase Extraction Enzyme extraction and purification operations were performed according to Maneechote et al. (30) with slight modifications. Three grams of shoot tissue were pulverized in liquid nitrogen with a mortar and pestle and extracted with 15 ml extraction buffer containing 100 mM Tris (pH 8.0), 1 mM EDTA, 10% glycerol, 2 mM isoascorbic acid, 0.5% PVP-40, 0.5% insoluble PVP, 20 mM DTT, and 0.2 mM PMSF. The homogenate was filtered through two layers of Miracloth and the supernatant was centrifuged (27,000g for 15 min) to obtain a partially purified enzyme. The pellet was discarded and the supernatant was slowly brought to 40% ammonium sulfate saturation and kept on ice for 30 min under stirring, then centrifuged at 27,000g for 30 min. The pellet was resuspended in 2 ml of elution buffer [50 mM Tricine (pH 8.0), 2.5 mM MgCl2?6H2O, 50 mM KCl, 1.0 mM DTT] and then desalted on a Sephadex G-25 column equilibrated with the same buffer. ACCase was extracted and eluted from shoot tissues of resistant and susceptible smooth crabgrass in paired experiments. Total protein content in ACCase extracts was determined by the method of Bradford (41). ACCase Assay Enzyme extracts (100 mg) were incubated at 328C in assay buffer [20 mM Tricine–KOH (pH 8.3), 10 mM KCl, 5 mM ATP, 2 mM MgCl2, 0.2 mg (w/v) BSA (fatty acid free), 2.5 mM DTT, 3.7 mM NaHCO3 (including 0.185 MBq of NaH14CO3 and 0, 0.1, 1, 10, and 100 mM concentrations of fenoxaprop-P-ethyl, fenoxaprop acid, quizalofop-P-ethyl, and sethoxydim)]. The reaction was initiated by the addition of 2.5 mM acetyl-CoA (lithium salt) and was stopped after 10 min by the addition of 20 ml concentrated HCl. Under these conditions the assay was linear for 20 min. A 110-ml aliquot of assay solution was transferred to a 2.2-cm filter paper disc and dried by blowing warm air in a fume hood. The acid- and heat-stable products were then quantified by liquid scintillation spectroscopy. Nonenzymatic 14CO2 fixation was

FENOXAPROP-RESISTANT SMOOTH CRABGRASS

assessed in tubes in which a buffer was substituted for acetyl-CoA. Control tubes received buffer instead of herbicide. The concentration of herbicide causing a 50% inhibition of ACCase activity (I50) was calculated from the concentration-response curves. Fenoxaprop-P-ethyl (the active isomer of fenoxaprop), fenoxaprop acid, and quizalofop-P-ethyl were dissolved in acetone (0.1% final concentration) and made up to 100 mM with buffer (100 mM Tricine–KOH, pH 8.3). Sethoxydim was dissolved in the Tricine–KOH buffer (pH 8.3). All dilutions were made with Tricine–KOH to yield final assay concentrations ranging from 0.01 to 100 mM. ACCase assays were conducted with three separate extracts from each smooth crabgrass accession and each treatment was assayed in triplicate. RESULTS AND DISCUSSION

Growth Responses Treatment with fenoxaprop-ethyl, quizalofopethyl, and sethoxydim caused a greater reduction of shoot dry weight in the susceptible than in the resistant smooth crabgrass (Fig. 2). Fenoxapropethyl reduced the shoot dry weight of susceptible plants at rates equal to or lower than the recommended field rate of 0.15 kg/ha (Fig. 2A). The calculated GR50 dose of fenoxaprop-ethyl on susceptible plants was 0.11 kg/ha (Fig 2A). In contrast, fenoxaprop-ethyl reduced only slightly the shoot dry weight of resistant plants at rates as high as 11.2 kg/ha (Fig. 2A). Based on the range of fenoxaprop rates used, an exact GR50 value could not be determined, but it is greater than 11.2 kg/ha. Thus, the resistant smooth crabgrass accession had greater than 102-fold resistance to fenoxaprop-ethyl relative to the susceptible smooth crabgrass.

FIG. 2. Dose-response effects of fenoxaprop-ethyl (A), quizalofop-ethyl (B), and sethoxydim (C) on the shoot dry weight of susceptible (S) and resistant (R) smooth crabgrass at 2 weeks after herbicide treatment. Vertical bars represent standard errors of means (n 5 10) and they are not shown when less than the marker size.

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Data in Figs. 2B and 2C show that the fenoxaprop-resistant smooth crabgrass exhibits moderate resistance to quizalofop-ethyl (approximate R/S GR50 ratio of 16.3), but very low resistance to the cyclohexanedione herbicide sethoxydim (R/S GR50 ratio of 1.3). The morphology and growth of resistant smooth crabgrass seedlings were comparable to those of susceptible smooth crabgrass seedlings grown in the greenhouse. After 20 days of growth, the average shoot dry weight of susceptible smooth crabgrass was 477 6 17 mg/plant, while that of resistant smooth crabgrass was 443 6 70 mg/plant. However, seedlings of resistant smooth crabgrass flowered about 2 weeks later than susceptible smooth crabgrass under greenhouse conditions. Further studies will be needed to determine whether the delayed flowering of the resistant plants is indicative of reduced fitness of this accession under field conditions. Nevertheless, since the resistant and susceptible accessions are not isogenic lines, it may be that these differences are not related to resistance. Wiederholt and Stoltenberg (42, 43) have reported that the development of resistance to AOPP and CHD herbicides in accessions of large crabgrass and giant foxtail was not associated with reduced fitness. Absorption, Translocation, and Metabolism of Fenoxaprop The rate of absorption of radioactivity following exposure to leaf-applied radiolabeled fenoxaprop-ethyl was similar between the two accessions of smooth crabgrass (Table 1). The TABLE 1 Absorption of Radioactivity by Seedlings of Susceptible and Resistant Smooth Crabgrass after 8, 24, 48, and 96 h of Exposure to Leaf-Applied [14C]Fenoxaprop-Ethyl Exposure time (h) 8 24 48 72

Susceptible biotypea 87.0 95.0 98.2 93.8

6 6 6 6

4.8 0.3 2.0 2.0

Resistant biotypea 86.1 94.7 96.0 96.6

6 6 6 6

1.3 1.8 2.0 1.2

C (% of applied). Presented values are means 6 SE (n 5 6). a 14

total amount of radioactivity absorbed by the resistant smooth crabgrass ranged from 86% of applied at 8 h to 96% of applied radioactivity at 48 h after treatment (Table 1). Similarly, the total amount of radioactivity absorbed by the susceptible plants ranged from 87% of applied at 8 h to 98% of applied at 48 h after treatment (Table 1). The majority of the absorbed 14C remained in the treated leaf at all sampling times, indicating that fenoxaprop did not translocate appreciably out of the treated leaf in both accessions of smooth crabgrass (Table 2). Translocation of absorbed radioactivity to shoots was greater in seedlings of resistant smooth crabgrass. However, the percentages of absorbed radioactivity remaining at the treated leaf of resistant and susceptible plants were 99% or greater at all sampling times (Table 2). These results suggest that differential absorption and translocation are not involved in the observed resistance of smooth crabgrass to the herbicide fenoxaprop. Because of the lack of appreciable herbicide translocation, the metabolism of 14C-fenoxaprop-ethyl was examined only in extracts from treated leaves of resistant and susceptible smooth crabgrass seedlings (Table 3). Fenoxaprop-ethyl ester (Rf, 0.91) was deesterified in the treated leaf to the herbicidally active fenoxaprop acid (Rf, 0.81). The parent herbicide and its deesterified metabolite made up about 12% of the radioactivity recovered during TLC analysis at 8 h after treatment but their amount declined to about 2.5% by 72 h (Table 3). At the same sampling times, there was a concomitant increase in the combined fraction of more polar metabolites (Rf, 0.39, 0.53, and 0.69) from 82 to about 90% of absorbed radioactivity. At 72 h after treatment, 9.3% of the radioactivity was recovered as nonextractable in both accessions of smooth crabgrass (Table 3). Previous studies have shown that fenoxapropethyl may be detoxified in plants by conjugation with carbohydrates and glutathione (44, 45). Although the rate of fenoxaprop metabolism by smooth crabgrass was high (Table 3), we made no attempt to identify the polar metabolites or

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TABLE 2 Distribution of Absorbed Radioactivity in Susceptible and Resistant Smooth Crabgrass after 8, 24, 48, and 72 h of Exposure to Leaf-Applied [14C]Fenoxaprop-Ethyl Exposure time (h) 8 24 48 72 a 14

Susceptible biotypea Treated leaf 99.77 99.55 99.44 99.39

6 6 6 6

0.04 0.11 0.02 0.12

Resistant biotypea

Shoot 0.13 0.24 0.32 0.40

6 6 6 6

Roots

0.11 0.14 0.03 0.13

0.10 0.21 0.24 0.21

6 6 6 6

Treated leaf

0.02 0.02 0.11 0.03

99.72 99.42 99.11 98.58

6 6 6 6

0.03 0.22 0.24 0.29

Shoot 0.18 0.41 0.69 1.08

6 6 6 6

0.04 0.12 0.27 0.31

Roots 0.10 0.18 0.20 0.34

6 6 6 1

0.03 0.04 0.04 0.01

C recovered (% of absorbed). Presented values are means 6 SE (n 5 6).

radiolabeled fenoxaprop since there were no differences in the levels of metabolites detected in the two smooth crabgrass accessions. These results indicate that differential metabolism is not a factor contributing to the observed resistance of smooth crabgrass to the herbicide fenoxaprop. Our results are consistent with previous reports demonstrating that the resistance of many grass weeds to AOPP and CHD herbicides is not conferred by altered herbicide absorption, translocation, or metabolism (6, 26). ACCase Sensitivity The specific activity of ACCase extracted from shoot tissues of susceptible and resistant smooth crabgrass was similar, with respective means of 17 and 20 nmol CO2 fixed min21 mg21 protein. Data in Fig. 3A show that ACCase extracted from susceptible plants was 91 times more sensitive to inhibition by fenoxaprop-ethyl

(I50 of 2 mM) than ACCase extracted from resistant smooth crabgrass (I50 of 182 mM). The activity of ACCase extracted from resistant plants was also less sensitive to fenoxaprop acid, quizalofop-P-ethyl, and sethoxydim than that extracted from susceptible plants (Figs. 3B, 3C, and 3D). The respective R/S I50 ratios were 5.9 for fenoxaprop acid (Fig. 3B), 41.3 for quizalofop (Fig. 3C), and 2.2 for sethoxydim (Fig. 3D). These data suggest that a less sensitive form of the target enzyme, acetyl-CoA carboxylase, confers fenoxaprop resistance to the accession of smooth crabgrass discovered in New Jersey. The basis of fenoxaprop resistance in smooth crabgrass appears to be similar to that of biotypes and/or accessions of green foxtail, annual ryegrass, Italian ryegrass, and winter wild oat, whose resistance to AOPP and CHD herbicides has been linked to reduced sensitivity of the target enzyme (28–35). A single dominant or

TABLE 3 Metabolism of [14C]Fenoxaprop-Ethyl in Susceptible and Resistant Smooth Crabgrass 14

Exposure time (h) 8 24 48 72 a

Biotype S R S R S R S R

Fenoxaprop-ethyl 9.9 8.4 3.8 4.8 2.3 2.5 1.4 1.7

6 6 6 6 6 6 6 6

2.4 2.1 0.7 0.3 0.6 0.9 0.2 0.3

Presented values are means 6 SE (n 5 6).

C Recovered in Metabolites (% of absorbed)a Fenoxaprop-acid 2.5 2.9 1.1 1.4 1.4 1.3 0.8 0.9

6 6 6 6 6 6 6 6

0.2 0.5 0.1 0.0 0.4 0.1 0.0 0.0

Metabolites 81.1 83.4 86.4 87.4 90.2 90.8 88.4 88.1

6 6 6 6 6 6 6 6

3.3 2.8 0.8 0.5 1.0 1.4 0.6 1.1

Nonextractable 6.4 5.2 8.6 6.3 6.1 5.4 9.5 9.3

6 6 6 6 6 6 6 6

0.8 1.0 0.5 0.2 0.3 0.4 0.4 0.8

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FIG. 3. Inhibition of ACCase activity extracted from shoot tissues of susceptible (S) and resistant (R) smooth crabgrass following treatment with fenoxaprop-ethyl (A), fenoxaprop acid (B), quizalofop-ethyl (C), and sethoxydim (D). Vertical bars represent standard errors of means (n 5 9) and they are not shown when less than the marker size.

partially dominant nuclear gene (46, 47) controls the target enzyme-based resistance to ACCase inhibitors. This suggest that resistance can be conferred by a number of different point mutations, each of which confers a different pattern of resistance and cross-resistance to AOPP and CHD herbicides. It is apparent that the altered sensitivity of ACCase of the resistant smooth crabgrass results

from a mutation of the target enzyme which does not affect equally the binding of other AOPP and CHD herbicides. The accession of smooth crabgrass discovered in New Jersey is highly resistant to fenoxaprop-ethyl, moderately resistant to quizalofop-ethyl, and slightly resistant to sethoxydim. Similar patterns of resistance and cross-resistance have been reported for biotypes and/or accessions of other grass weeds. Thus, a

FENOXAPROP-RESISTANT SMOOTH CRABGRASS

wild oat biotype showed very high (150-fold) resistance to sethoxydim but much lower resistance to other AOPP and CHD herbicides (12, 32). Similarly, a biotype of giant foxtail from Wisconsin was highly (.145-fold) resistant to sethoxydim and resistant (25-fold) to fluazifopbutyl in greenhouse experiments (23). A commercialized sethoxydim-resistant line of maize is cross-resistant (16-fold) to haloxyfop at the ACCase level (46). Other resistant weed biotypes appear to result from different ACCase mutations, conferring quite different patterns of resistance to the same herbicide. For example, ACCase from a resistant goosegrass biotype was very resistant to fluazifop but much less resistant to sethoxydim, fenoxaprop, and clethodim (33). Similarly, ACCase from a resistant biotype of annual ryegrass was very resistant to diclofop but much less resistant to CHD herbicides (47). In summary, the results of this study confirmed the resistance of a smooth crabgrass accession from New Jersey to fenoxaprop-ethyl. Data obtained in growth-response (GR50) studies (Fig. 2) complemented well the data obtained from the ACCase inhibition (I50) (Fig. 3). It is evident that at both the whole-plant level and the target enzyme level, the resistant accession of smooth crabgrass exhibited a high degree of resistance to fenoxaprop-ethyl, good cross-resistance to quizalofop, but very low resistance to sethoxydim. Altered absorption, translocation, or metabolism did not appear to contribute to the observed fenoxaprop resistance of smooth crabgrass. Instead, a less sensitive form of ACCase confers the resistance of the smooth crabgrass accession from New Jersey to the herbicide fenoxaprop. The nature and the genetic profile of the ACCase mutation in smooth crabgrass remains to be investigated in future studies. ACKNOWLEDGMENTS The authors thank Dr. J. Christopher Hall (University of Guelph, Ontario, Canada) and AgrEvo Company (Frankfurt, Germany) for providing the analytical and radiolabeled samples of fenoxaprop-ethyl and fenoxaprop acid used in this study. The sabbatical leave of Dr. Kuk in Professor Hatzios’ laboratory was supported by the Korean Research Foundation.

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