mechanism of sulfonylurea herbicide resistance in the broadleaf

7
Plant Physiol. (1990) 93, 55-61 0032-0889/90/93/0055/07/$01 .00/0 Received for publication September 6, 1989 and in revised form December 14, 1989 Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia Leonard L. Saari,* Josephine C. Cotterman, and Michael M. Primiani Agricultural Products Department, E. I. duPont de Nemours & Co., Inc., Wilmington, Delaware 19880-0402 ABSTRACT Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant 150 to susceptible 150) was 5- to 28-fold, 2- to 6- fold, and 20-fold for sulfonylurea herbicides, imidazolinone her- bicides, and a sulfonanilide herbicide, respectively. No differ- ences were observed in the ALS-specific activities or the rates of ["4C]chlorsulfuron uptake, translocation, and metabolism be- tween susceptible and resistant kochia biotypes. The Km values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme. The loss of effective control by pesticides of weeds, insects, and fungi because of resistance is a well known, established phenomenon (9). Among weeds, triazine resistance was first reported in 1970 (27). Subsequently, resistance to several other herbicides in addition to the triazines has occurred in weeds (2, 10). The continued use of a single control agent is often a common feature in cases of resistance. The sulfonylureas are highly active herbicides that have been in commercial use since 1982. The mode of action of the sulfonylurea (5, 20, 26) as well as the imidazolinone (30) and sulfonanilide (18) herbicides is the inhibition of ALS (see abbreviation list in Table I) (also known as acetohydroxyacid synthase, EC 4.1.3.18), the first enzyme common to the biosynthesis of the branched-chain amino acids, Leu, Ile, and Val. Chlorsulfuron, a sulfonylurea herbicide used in cereals, is wheat (Triticum aestivum L.)-tolerant because the crop metabolizes the chlorsulfuron to nonphytotoxic products more quickly than susceptible weeds (32). This differential selectivity based on metabolism is contrasted by selectivity due to differences in target site sensitivity as obtained by selection or production of sulfonylurea-resistant bacteria (20, 34), yeast (7), and plants (5, 12). ALS isolated from these sulfonylurea-resistant organisms is less sensitive to inhibition by ALS inhibitors such as the sulfonylureas. Kochia (Kochia scoparia [L.] Schrad.) is a major broadleaf weed in cereals (6) in the United States and Canada that is generally well controlled by chlorsulfuron. The singular use of chlorsulfuron or chlorsulfuron/metsulfuron methyl mix- tures over several growing seasons has resulted in the selection of sulfonylurea-resistant biotypes of kochia (25) and prickly lettuce (Lactuca serriola L.) (22) in certain cereal-growing areas. In this paper, the mechanism by which kochia is resistant to the sulfonylurea and imidazolinone herbicides is described. MATERIALS AND METHODS Whole Plant Experiments Plant Materials Kochia (Kochia scoparia [L.] Schrad.) seeds were collected from two sites near Liberal, KS, in October 1987. One site had been treated with chlorsulfuron (17.5 g ai/ha in April 1987; 87.5 g ai/ha from 1983 through 1987). The other site was a nearby untreated area. Seeds from the two collections were sown in plastic trays containing potting medium (Metro Mix 350) and germinated in a growth chamber (12 h photo- period, constant 16 ± 3C and 75% RH ± 10%). One to 2 cm seedlings were individually transplanted to 7.5 cm pots containing potting medium and placed in a greenhouse (14 h photoperiod maintained by metal halide supplemented nat- ural daylight, 27°C day, 20°C night). Pots were watered with tap water as required to maintain good growing conditions. Uniform, vigorous plants with single shoots were selected for herbicide treatment when they were 3 to 5 cm tall. Herbicide Treatment Herbicide treatments were applied to the plant foliage with a laboratory sprayer (traveling belt sprayer; stationary, single flat fan 8002E nozzle; Spraying Systems Co., Wheaton, IL, 207 kPa, 364 L/ha spray volume). Chemicals were applied as commercial herbicide formulations dispersed in water con- taining surfactant (Ortho X-77, 0.25% v/v) or in acetone- based carrier system (acetone:water:glycerin:Tween 20, 91.4:4.2:4.2:0.2, v/v). The range of herbicide rates used to determine GRso values was 0.063 to 62 g ai/ha and 0.25 to 1000 g ai/ha for the susceptible and resistant kochia biotypes, respectively. Treated plants were returned to the greenhouse and watered as necessary to maintain vigorous growth. 55 www.plantphysiol.org on April 8, 2018 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.

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Page 1: Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf

Plant Physiol. (1990) 93, 55-610032-0889/90/93/0055/07/$01 .00/0

Received for publication September 6, 1989and in revised form December 14, 1989

Mechanism of Sulfonylurea Herbicide Resistance in theBroadleaf Weed, Kochia scoparia

Leonard L. Saari,* Josephine C. Cotterman, and Michael M. PrimianiAgricultural Products Department, E. I. duPont de Nemours & Co., Inc., Wilmington, Delaware 19880-0402

ABSTRACT

Selection of kochia (Kochia scoparia) biotypes resistant to thesulfonylurea herbicide chlorsulfuron has occurred through thecontinued use of this herbicide in monoculture cereal-growingareas in the United States. The apparent sulfonylurea resistanceobserved in kochia was confirmed in greenhouse tests. Freshand dry weight accumulation in the resistant kochia was 2- to>350-fold higher in the presence of four sulfonylurea herbicidesas compared to the susceptible biotype. Acetolactate synthase(ALS) activity isolated from sulfonylurea-resistant kochia wasless sensitive to inhibition by three classes of ALS-inhibitingherbicides, sulfonylureas, imidazolinones, and sulfonanilides.The decrease in ALS sensitivity to inhibition (as measured by theratio of resistant 150 to susceptible 150) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone her-bicides, and a sulfonanilide herbicide, respectively. No differ-ences were observed in the ALS-specific activities or the ratesof ["4C]chlorsulfuron uptake, translocation, and metabolism be-tween susceptible and resistant kochia biotypes. The Km valuesfor pyruvate using ALS from susceptible and resistant kochiawere 2.13 and 1.74 mm, respectively. Based on these results, themechanism of sulfonylurea resistance in this kochia biotype isdue solely to a less sulfonylurea-sensitive ALS enzyme.

The loss of effective control by pesticides of weeds, insects,and fungi because of resistance is a well known, establishedphenomenon (9). Among weeds, triazine resistance was firstreported in 1970 (27). Subsequently, resistance to severalother herbicides in addition to the triazines has occurred inweeds (2, 10). The continued use of a single control agent isoften a common feature in cases of resistance.The sulfonylureas are highly active herbicides that have

been in commercial use since 1982. The mode of action ofthe sulfonylurea (5, 20, 26) as well as the imidazolinone (30)and sulfonanilide (18) herbicides is the inhibition ofALS (seeabbreviation list in Table I) (also known as acetohydroxyacidsynthase, EC 4.1.3.18), the first enzyme common to thebiosynthesis of the branched-chain amino acids, Leu, Ile, andVal. Chlorsulfuron, a sulfonylurea herbicide used in cereals,is wheat (Triticum aestivum L.)-tolerant because the cropmetabolizes the chlorsulfuron to nonphytotoxic productsmore quickly than susceptible weeds (32). This differentialselectivity based on metabolism is contrasted by selectivitydue to differences in target site sensitivity as obtained byselection or production of sulfonylurea-resistant bacteria (20,34), yeast (7), and plants (5, 12). ALS isolated from these

sulfonylurea-resistant organisms is less sensitive to inhibitionby ALS inhibitors such as the sulfonylureas.Kochia (Kochia scoparia [L.] Schrad.) is a major broadleaf

weed in cereals (6) in the United States and Canada that isgenerally well controlled by chlorsulfuron. The singular useof chlorsulfuron or chlorsulfuron/metsulfuron methyl mix-tures over several growing seasons has resulted in the selectionof sulfonylurea-resistant biotypes of kochia (25) and pricklylettuce (Lactuca serriola L.) (22) in certain cereal-growingareas. In this paper, the mechanism by which kochia isresistant to the sulfonylurea and imidazolinone herbicides isdescribed.

MATERIALS AND METHODS

Whole Plant Experiments

Plant Materials

Kochia (Kochia scoparia [L.] Schrad.) seeds were collectedfrom two sites near Liberal, KS, in October 1987. One sitehad been treated with chlorsulfuron (17.5 g ai/ha in April1987; 87.5 g ai/ha from 1983 through 1987). The other sitewas a nearby untreated area. Seeds from the two collectionswere sown in plastic trays containing potting medium (MetroMix 350) and germinated in a growth chamber (12 h photo-period, constant 16 ± 3C and 75% RH ± 10%). One to 2cm seedlings were individually transplanted to 7.5 cm potscontaining potting medium and placed in a greenhouse (14 hphotoperiod maintained by metal halide supplemented nat-ural daylight, 27°C day, 20°C night). Pots were watered withtap water as required to maintain good growing conditions.Uniform, vigorous plants with single shoots were selected forherbicide treatment when they were 3 to 5 cm tall.

Herbicide Treatment

Herbicide treatments were applied to the plant foliage witha laboratory sprayer (traveling belt sprayer; stationary, singleflat fan 8002E nozzle; Spraying Systems Co., Wheaton, IL,207 kPa, 364 L/ha spray volume). Chemicals were applied ascommercial herbicide formulations dispersed in water con-taining surfactant (Ortho X-77, 0.25% v/v) or in acetone-based carrier system (acetone:water:glycerin:Tween 20,91.4:4.2:4.2:0.2, v/v). The range of herbicide rates used todetermine GRso values was 0.063 to 62 g ai/ha and 0.25 to1000 g ai/ha for the susceptible and resistant kochia biotypes,respectively. Treated plants were returned to the greenhouseand watered as necessary to maintain vigorous growth.

55

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Page 2: Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf

Plant Physiol. Vol. 93, 1990

Table I. Abbreviations Used in This Paper

ALS, acetolactate synthaseai, active ingredientChlorimuron ethyl, 2-([(4-chloro-6-methoxypyrimidine-2-yl)amino car-

bonyl]-aminosulfonyl)benzoic acid, ethyl esterChlorsulfuron, 2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-

yl)amino]-carbonyl]benzenesulfonamide2,6-dichlorosulfonanilide, 1,2,4-triazolo-(1,5-a-2,4-dimethyl-3-N-sul-

fonyl(2,6-dichloroaniline)-1 ,5-pyrimidineGR50, the rate of herbicide application required to inhibit plant growth

by 50% relative to untreated controls150, herbicide concentration required for 50% inhibition of ALS activitylmazamethabenz, 6-4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)m-

toluic acid and 2-4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)p-toluic acid

Imazapyr, (±)-2-[4,5-dihydro-4-methyl-4-(1 -methylethyl)-5-oxo-1 H-imidazol-2-yl]-3-pyridinecarboxylic acid

Imazethapyr, (±)2-[4,5-dihydro-4-methyl-4-(1 -methylethyl)-5-oxo-1 H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid

LSC, liquid scintillation countingMetsulfuron methyl, 2-[[[[(4-methoxy-6-methyl-1 ,3,5-triazin-2-yl)

amino]carbonyl]amino]sulfonyl]benzoic acid, methyl esterSulfometuron methyl, 2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]car-

bonyl]amino]sulfonyl]benzoic acid, methyl esterThifensulfuron methyl, methyl 3-[[[[(4-methoxy-6-methyl-1 ,3,5-triazin-

2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylateTriasulfuron, 2(2-chloroethoxy)N-[[1 (4-methoxy-6-methyl-1 ,3,5-tria-

zin-2-yl)amino]carbonyl]benzenesulfonamide

Plant Growth Measurements

Growth effects were determined 16 d after treatment bydetermining fresh and dry weight increases ofthe shoot tissue.A reference weight was determined at treatment time andsubtracted from final weights to give a weight increase overthe incubation period. Shoots were harvested at the soilsurface, weighed, placed in drying bags, dried (58°C for 72 h),and weighted for fresh weight and dry weight determinations,respectively. The data were analyzed using a logit procedureto calculate GRso values (3).

Acetolactate Synthase Extraction and Assay

Kochia plants were germinated and grown in Metro Mix350 in the growth chamber. All subsequent operations werecarried out at 4°C unless specified otherwise. ALS was ex-tracted from the aerial portion of 20- to 25-cm-high kochiaplants (approximately 50 g fresh weight of each biotype wasused) with modifications to the procedures described previ-ously (26). The plants used were larger in size than those usedfor herbicide treatments; this was done to increase ALS yield.Identical ALS Iso results were obtained using either size ofplant (data not shown). The shoots were homogenized withextraction buffer (buffer:plant material, 4:1, v/w) in a WaringBlendor. The extraction buffer contained 100 mM potassiumphosphate buffer (pH 7.5), 0.5 mM MgCl2, 1 mM pyruvate,0.5 mM thiamine pyrophosphate, 10 ,uM flavine adenine di-nucleotide, and 10% glycerol (v/v). Prior to homogenization,1 mm dithiothreitol, 0.0165% antifoam A (v/v) (Sigma Chem-ical Co.), and 5% polyvinylpolypyrollidone (w/v) were addedto the extraction buffer and plants, and this solution was

covered and sparged with ultrapure nitrogen for 15 min.Homogenization was performed for 1 min. The homogenatewas filtered through six layers of cheesecloth. The ALS wasprecipitated from the supematant between 25 and 50%(NH4)2SO4 saturation. The final pellet was resuspended inextraction buffer (5 mL/100 g fresh weight) containing(NH4)2SO4 at 55% of saturation and frozen as pellets in liquidN2, and stored at -80°C until needed. To assay the ALS, 5 gof frozen pellets were thawed on ice, centrifuged at 17,000gfor 15 min, and the pellet dissolved in 2 mL of extractionbuffer. A slight amount of insoluble matter was removed bycentrifugation (17,000g for 5 min) before the enzyme wasdesalted on a Sephadex G-25 column (Pharmacia PD-10)equilibrated with extraction buffer.ALS assays were performed as previously outlined (26)

either in microplates or test tubes. Controls containing 24.1,uM chlorimuron ethyl ensured that the activity observed wasdue to ALS and not other acetoin-forming enzymes. In assaysusing microplates, the volumes of all reagents and enzymesolutions were reduced by a factor of 0.2 relative to theprocedure used with test tubes (26). The absorbance valuesused for 15o determinations were read on a microplate reader(Dynatech Laboratories, model MR600) equipped with a 530nm filter. The I50 values were calculated using methods pre-viously described (26) and were the average of at least threedeterminations. Specific activities of ALS and Km (pyruvate)values were calculated using A530 values obtained on a dualbeam spectrophotometer. An extinction coefficient of 6.5mM-' cm-' was used to quantitate the acetolactate formed(20). The Km for pyruvate was determined by using the MLABdata modeling program (19) to fit the equation, v = VS/(K +S), to the data where v is the residual ALS activity, V is themaximal enzymic activity, K is the Michaelis constant forpyruvate, and S is the pyruvate concentration. Protein con-centrations were determined by the method of Peterson (23).

Metabolism Study

Plant Treatment

Shoots were excised from 6-week-old greenhouse-grownsusceptible and resistant kochia plants. Groups ofthree shoots

Table II. Postemergent Herbicide Rates Required for 50%/oReduction of Plant Growth of Sulfonylurea-Susceptible (S) and -Resistant (R) Kochia in Greenhouse Tests

GR5oa

Herbicide Dry Weight Fresh Weight

S R Ratio (R/S) S R Ratio (R/S)g ai/ha

Chlorsulfuron 0.072 31 431 0.065 23 354Metsulfuron 0.64 1.5 2 0.95 1.5 2

methylSulfometuron 0.12 2.4 20 0.10 2.2 22

methylThifensulfuron 0.45 99 220 0.61 160 262

methyla GR50 is defined as the rate of herbicide application in grams active

ingredient/hectare (g ai/ha) required to inhibit plant growth by 50%relative to untreated controls.

56 SAARI ET AL.

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Page 3: Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf

MECHANISM OF SULFONYLUREA RESISTANCE IN KOCHIA

Table IlIl. /50 Values for Sulfonylurea, Imidazolinone, andSulfonanilide Herbicides Using ALS Isolated from Sulfonylurea-Susceptible (S) and -Resistant (R) Kochia

The specific activities of the ALS enzymes isolated from the S andR kochia biotypes were 0.98 and 1.2 nmol acetolactate formed/min/mg protein, respectively.

150Herbicide Ratio (R/S)

S R

nM

Chlorimuron ethyl 5 99 19Chlorsulfuron 22 400 18Metsulfuron methyl 26 130 5Sulfometuron methyl 10 280 28Thifensulfuron methyl 62 780 13Triasulfuron 40 460 122,6-Dichlorosulfonanilide 86 1700 20Imazapyr 6000 38000 6Imazamethabenz 2300 7500 3Imazethapyr 6600 13000 2

were recut under water and placed with the cut ends in vialsof 14 AM [triazine-2-'4C]chlorsulfuron (DuPont NEN Re-search Products, 15.2 ,uCi/mg; 85% radiochemical purity byHPLC) in a lighted growth room (12 h photoperiod; 23°C/20°C day/night temperature). After 2 h, shoots were blottedand transferred to vials of water. Groups of shoots wereharvested 0, 4, or 21 h later by freezing in liquid N2 afterdetermining tissue fresh weight. Sample fresh weights rangedfrom 3.1 to 4.4 g. The tissue was stored at -80°C untilextraction and analysis.

Plant Extraction

The frozen tissue was ground to a fine powder under liquidN2 with a mortar and pestle and returned to -80°C storage.Tissue was extracted with 20 mL 80% (v/v) acetone for 1 hat room temperature with vigorous shaking on a wrist-actionshaker. The extraction mixture was centrifuged at 700g in atabletop centrifuge for 20 min. The supernatant was decantedand saved. The pellet was resuspended and extracted threemore time as above, the supernatants were combined, andaliquots were removed to determine total radioactivity in theextract by LSC (Packard 2000 CA). The combined superna-tants were rotary evaporated to dryness in vacuo at 40°C. Thedry residue was stored at -20°C until just prior to HPLCanalysis.

Analysis of Plant Extract

The extract residues were resuspended in 5 mL HPLC-grade water by shaking vigorously for 1 h. Resuspension ofapproximately 90% of the total extracted radioactivity wasachieved. A 1-mL aliquot of the resuspended extracts wascentrifuged in a microcentrifuge for 15 min. The supernatantswere analyzed by reversed-phase HPLC. Samples (150 AL)were injected on a 4.6 mm i.d. x 250 mm DuPont ZorbaxODS column with a flow rate of 1.4 mL/min and columntemperature of 35°C. Solvents were A = dH20 + 0.1% (v/v)formic acid and B = acetonitrile + 0.1 % (v/v) formic acid inthe following elution method:

Step 1, 20% B, 10-min isocraticStep 2, 20% B to 57% B, 20-min linear gradientStep 3, 57% B to 100% B, 5-min linear gradient

For plant samples, 1-min fractions were collected, andradioactivity in each fraction was determined by LSC. Thepercentage of the total sample radioactivity present in thefractions corresponding to the elution time of chlorsulfuron(determined by absorbance of chlorsulfuron standard at 250nm) was calculated and used as a measure of metabolism of['4C]chlorsulfuron over time.For comparison, the same procedures were used to measure

the metabolism of ["4C]chlorsulfuron in excised leaves of 8-d-old growth chamber-grown spring wheat (cv Era), which isknown to be chlorsulfuron-tolerant due to rapid metabolicinactivation of the herbicide (32). Groups of40 excised wheatleaves were harvested 0, 2, 4, 6, and 21 h after transfer towater. Extraction and analysis were as described for kochia.

Uptake and Translocation Study

A 10 ,uL drop of ['4C]chlorsulfuron solution (1,000 dpm/,gL) with 0.25% (v/v) Ortho X-77 surfactant was gently wipedonto the adaxial surface of a single leaf midway up the stemof 5- to 7-cm-tall greenhouse-grown susceptible and resistantkochia plants. Plants were held in the lighted growth room,and five replicate plants of each biotype were harvested 1, 3,6, 24, and 48 h after ['4C]chlorsulfuron application.

N_<R2R1-SO2NIICONI1 --<\ X

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Figure 1. Structures of sulfonylurea herbicides used in this study.

57

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Page 4: Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf

Plant Physiol. Vol. 93, 1990

INIAZANI Elil'AIIE,NZ

0NC113

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I NIAZEI''IIA,PYRl

2,6-D) IC II 1,010SULFO()N ANI EI)E,

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100 I -

80 -

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Figure 2. Structures of three imidazolinone and one sulfonanilideherbicide used in this study.

100

I-P

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80

60

40

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0 0.5 1 1.5 2 2.5 3

CHLORSULFURON CONCENTRATION, ,tM

Figure 3. Inhibition of ALS activity isolated from sulfonylurea-sus-ceptible and -resistant kochia by chlorsulfuron.

Harvested plants were separated into four sections: shootabove the treated leaf, treated leaf, shoot below the treatedleaf, and roots. Roots were rinsed in water three times toremove soil. ['4C]Chlorsulfuron which had not been absorbedby the treated leaf was rinsed off by vigorously swirling(Vortex-Genie) the leaf in 10 mL 80% (v/v) acetone for 1

min. Radioactivity in the rinsate was determined by LSC andused to calculate the percentage of the applied radioactivityabsorbed by the plants.The plant sections were dried in a vacuum oven at 45°C.

Radioactivity in individual plant sections was determined byLSC following tissue combustion by a sample oxidizer (Pack-ard 306). Percent of applied radioactivity absorbed was cal-culated for each plant as:

2(dpm in plant sections) x 100(2[dpm in plant sections])+ (dpm in treated leaf rinse)

Figure 4. Metabolism of [14C]chlorsulfuron by sulfonylurea-suscep-tible and -resistant kochia and wheat. Excised shoots were allowedto take up [14C]chlorsulfuron for 2 h and incubated for various timesas described in "Materials and Methods". Tissue was then extractedand analyzed by HPLC and LSC to determine the percentage ofunmetabolized [14C]chlorsulfuron remaining. Data were normalized to85% radiochemical purity of the [14C]chlorsulfuron.

Ofthe applied radioactivity absorbed, the percent translocatedout of the treated leafwas calculated for each plant as:

2(dpm in plant sections other than treated leaf) 100.

2(dpm in plant sections including treated leaf)

RESULTS AND DISCUSSION

This work was preformed to determine the mechanism bywhich a certain biotype of kochia is resistant to the sulfonyl-urea herbicide, chlorsulfuron. Several laboratories have pre-

viously obtained sulfonylurea-resistant organisms by varioustechniques but this kochia biotype along with a prickly lettucebiotype (22, 25) represents the first instance of sulfonylurearesistance occurring as a result of commercial sulfonylureaherbicide use. Kochia not controlled by chlorsulfuron wasisolated from a wheat field that had received five applicationsof chlorsulfuron over a 5 year period. This biotype was ableto tolerate higher doses of cereal-tolerant (chlorsulfuron, met-sulfuron methyl, and thifensulfuron methyl) and nonselective(sulfometuron methyl) sulfonylurea herbicides than the sus-

ceptible biotype (Table II). The resistant biotype requiredgreater than 350-fold postemergent rates of chlorsulfuron ingreenhouse tests to suppress its growth as compared to thesensitive biotype when changes in dry and fresh weight were

used as the criteria. The increase in resistance (as measuredby the ratio of resistant to sensitive GRso values) was also very

large for thifensulfuron methyl (>200-fold) but less for met-sulfuron methyl (2-fold) and sulfometuron methyl (20-22-fold). Estimates of growth reduction by visual methodsshowed chlorsulfuron-resistant kochia to be cross-resistant toother sulfonylurea and imidazolinone herbicides whereas her-bicides with modes of action different for ALS inhibitioncontrolled the two biotypes with equal efficacy (25).The sulfonylurea resistance was due to a change in the

0 SUSCEPTIBLE KOCHIA

A RESISTANT KOCHIA0 WHEAT

0

IS a

58 SAARI ET AL.

cooii If CIIICII.1

C11.0 12 cil.,N N

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Page 5: Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf

MECHANISM OF SULFONYLUREA RESISTANCE IN KOCHIA

20-fold less effective in inhibiting resistant kochia ALS asA compared to the ALS from the susceptible biotype.

Imidazolinones (Fig. 2) are another chemical class of ALS-|_ - inhibiting herbicide (30). Resistant kochia is cross-resistant to

-.'/ I ' the imidazolinone herbicide, imazapyr, at the whole plantlevel (25), and ALS activity isolated from the resistant biotypewas less inhibited by the imidazolinone herbicides than theALS activity from the susceptible kochia (Table III). It is

O SUSCEPTIBLE interesting that the decrease in ALS sensitivity (as measured_ RESISTANT by the ratio of the resistant ALS 150/susceptible ALS I50) to

inhibition by the imidazolinones was generally less than withl the sulfonylurea or sulfonanilide herbicides. The ratio of the

resistant to susceptible ALS I50 values was 2- to 6-fold for theB | imidazolinones whereas the ratio was 5- to 28-fold for the

sulfonylureas (Table III). All three classes of ALS inhibitors,I sulfonylureas, imidazolinones, and sulfonanilides, compete

v ! j ifor a common binding site in the ALS molecule (29). Thedifferences between sulfonylureas and imidazolinones withrespect to the degree of ALS insensitivity are possibly due toslightly different binding domains in the common bindingsite of the protein. That is, if a single amino acid change in

0 SUSCEPTIBLE the ALS enzyme is responsible for the increased insensitivityc^ RESISTANT_ | to inhibition, then one binding domain might be affected

differently than another domain.0 10 20 30 40 50 There was a positive qualitative correlation between the

TIME, h inhibition of plant growth and inhibition of ALS activity ofsulfonylurea-susceptible and -resistant biotypes by sulfonyl-

lptake (A) and translocation (B) of ['4C]chlorsulfuron by urea herbicides (Tables II and III). However, the relative-susceptible and -resistant kochia. The percentage of increase in resistance at the whole plant level was not alwaysDactivity taken up was determined by combustion and reflected by a quantitative increase in enzyme insensitivity to

LSC analysis after an 80% acetone (v/v) rinse of the treated leaf. Ofthe radioactivity taken up, the percent translocated out of the treatedleaf was determined by combustion and LSC analysis of the rinsedtreated leaf and the remaining plant sections.

sensitivity of the ALS enzyme to inhibition by ALS-inhibitingherbicides (Table III), including sulfonylureas (Fig. 1), imi-dazolinones, and sulfonanilides (Fig. 2). The inhibition ofALS activity isolated from susceptible and resistant kochia bychlorsulfuron is shown in Figure 3. At the highest concentra-tion of 2.8 AM chlorsulfuron, the ALS activity from thesusceptible biotype was completely inhibited whereas the ALSfrom the resistant kochia still retained 29% of the uninhibitedcontrol activity. The I50 values for chlorsulfuron with thesusceptible and resistant ALS enzymes were 22 and 400 nM,respectively (Table III). The ALS from the resistant biotypewas inhibited less effectively than the susceptible ALS byevery sulfonylurea and sulfonanilide herbicide studied as evi-denced by the higher 150 values (Table III). Similar wholeplant and ALS results have been obtained for sulfonylurea-resistant biotypes of Russian thistle (Salsola iberica Sennen& Pau) and common chickweed (Stellaria media [L.] Vill.)(data not shown).

Several diverse structural types of sulfonylurea herbicideswere represented in these tests (Fig. 1). Included were cereal-tolerant (chlorsulfuron, metsulfuron methyl, thifensulfuronmethyl, triasulfuron), soybean-tolerant (chlorimuron ethyl),and nonselective (sulfometuron methyl) sulfonylurea herbi-cides. In addition, a different chemical class of ALS inhibitoras represented by 2,6-dichlorosulfonanilide (18) (Fig. 2) was

0w

N

0

z

C.

0.8

0.6

0.4

0.2

0

0 5 10 15 20 25

[PYRUVATE], mM

Figure 6. Effect of pyruvate concentration on ALS activity isolatedfrom sulfonylurea-susceptible and -resistant kochia. Velocities werenormalized to the respective maximal enzymic velocity for eachenzyme. The equation, v = VS/(K + S), was fit to the compositesusceptible and resistant kochia ALS data as well as the individualdata sets using the MLAB data modeling program (15). Km values forpyruvate using the individual results for susceptible and resistantALS were 2.13 ± 0.19 and 1.74 ± 0.26 mm, respectively. Assayswere performed as described in "Materials and Methods" except thatthe pyruvate concentrations were varied from 0.074 to 22 mm for theKm determination (plotted) and 0.074 to 278 mm for the V determi-nation.

100

0-

zLL

I-

80

60

40

20

0

60

aui

< 4000C,)z

-20

I.-91

0

Figure 5. Usulfonylureaapplied radi(

0 Oo

0

0

/ 0 SUSCEPTIBLE

O RESISTANT

59

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Page 6: Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf

Plant Physiol. Vol. 93,1990

inhibition. A possible reason for the variation is that plantinjury due to ALS-inhibiting herbicides is time- and environ-ment-dependent and the degree of plant injury observed istherefore a function of when and under what conditions theevaluation is made. Also, the dose-response relationship is notlinear for either plant-growth inhibition (data not shown) orALS inhibition (Fig. 3), and this could contribute to the lackof quantitative correlation between the two parameters.Other mechanisms were investigated for their possible role

in contributing to resistance. The metabolism of ['4C]chlor-sulfuron by susceptible and resistant kochia was very similarup to 21 h (Fig. 4). Not only were the rates similar but neitherbiotype metabolized chlorsulfuron to any appreciable extentduring the experiment. In a previous study (32), chlorsulfuronwas metabolized with a half-life of 2 to 3 h to nonphytotoxicproducts by wheat which is chlorsulfuron tolerant. ALS iso-lated from wheat and chlorsulfuron-susceptible weeds havesimilar Io values for chlorsulfuron (26). Metabolism of chlor-sulfuron by wheat was included as a control in this study todemonstrate that the metabolism rate required for chlorsul-furon tolerance in wheat was not approached by either biotypeof kochia (Fig. 4). The rapid metabolism of chlorsulfuron inwheat was apparent by the partial metabolism that took placeduring the uptake period. That is, at time = 0 h (2 h after thebeginning of uptake), 35% (65% intact) of the chlorsulfuronhad been metabolized. From these results, metabolism doesnot appear to contribute to chlorsulfuron resistance in kochia.

Possible differences in the uptake and translocation rates of['4C]chlorsulfuron by susceptible and resistant kochia werealso studied. Both biotypes of kochia took up ['4C]chlorsul-furon at similar rates up to 48 h after application to leaves(Fig. 5A). Of the chlorsulfuron absorbed by the leaf, theamount translocated out of the treated leaf after 24 h was notdifferent between the two biotypes and was only slightlygreater at 48 h for the resistant kochia biotype (Fig. SB). Thisdifference at 48 h was not of sufficient magnitude or correctdirection to be responsible for the resistance in kochia. Re-sistant kochia was only marginally more effective at translo-cating chlorsulfuron at 48 h than susceptible kochia, andsulfonylurea resistance due to differences in translocationmight be expected if translocation were lower, not higher, inthe resistant biotype relative to the susceptible biotype.

If in vitro enzyme assays reflected in vivo enzyme activities,then resistance apparently was not a result of ALS overex-pression in the resistant biotype because the specific activitiesof 0.98 and 1.2 nmol acetolactate/min/mg protein for thesusceptible and resistant kochia ALS enzymes, respectively,were similar. The amount of protein extracted from the twobiotypes was nearly the same (the protein concentrations ofthe preparations used for ALS assay were 0.22 and 0.33 mg/mL/g fresh weight for susceptible and resistant kochia, re-spectively). Also, the Km values for pyruvate of 2.13 ± 0.19mm and 1.74 ± 0.26 mm were similar when ALS isolatedfrom susceptible and resistant kochia, respectively, was used(Fig. 6). These results are of the same magnitude to previouslyreported results for the plant enzyme (14) and suggest thatthe mutation(s) resulting in sulfonylurea resistance has notaffected pyruvate binding and possibly catalysis.

It is interesting to contrast these results with those from

triazine-resistant plants. An amino acid substitution in thebinding domain of the 32 kD quinone binding protein (Qb)of PSII because of a mutation in the chloroplast psbA gene(16) results in both a decreased affinity of the Qb protein fortriazine herbicides and a decreased rate of electron transferfrom the primary electron acceptor of PSII to the secondaryacceptor (1, 4). The resistance mutation is translated into adecreased plant yield (31) and is suggested to be responsiblefor the fitness difference observed between triazine-susceptibleand -resistant plants (1, 17). The fitness difference betweensulfonylurea-susceptible and -resistant kochia is currently un-known; however, the similarity in the Km (pyruvate) and ALSspecific activity results for the two biotypes suggests that anyfitness differences observed will be less than that seen fortriazine-susceptible and -resistant biotypes.The observed sulfonylurea resistance could occur from a

single amino acid change in the ALS molecule. Sulfonylurea-resistant mutants of Salmonella typhimurium (20), Saccha-romyces cerevisiae (7), Nicotiana tabacum (5), Escherichiacoli (34), Arabidopsis thaliana (13), Chlamydomonas rein-hardtii (l1), Datura innoxia (28), and Synechococcus (8)produce ALS with decreased sensitivity to inhibition by sul-fonylurea herbicides. Single nucleotide substitutions in theALS structural gene(s) that confer resistance have been iden-tified in E. coli (34), S. cerevisiae (7), and N. tabacum (21).Interestingly, mutants of C. reinhardtii (33) and D. innoxia(28) selected for chlorsulfuron resistance have cross-resistance(as determined by the ratio of resistant to susceptible ALS 15ovalues) to the imidazolinone herbicide similar to the resultsobserved in this study using ALS isolated from susceptibleand resistant kochia. While it is currently unknown whethera single nucleotide change in the kochia ALS gene results insulfonylurea resistance, the model studies with the organismsnoted indicate that single nucleotide changes in the ALS genecan confer resistance and that cross resistance to structurallyunrelated ALS inhibitors is possible.Mechanisms of resistance other than changes in target site

sensitivity may also result in weeds being resistant to sulfo-nylurea herbicides. A diclofop-methyl-resistant biotype ofannual ryegrass (Lolium rigidum Gaud.) is cross resistant tometsulfuron methyl and chlorsulfuron (15). However, thisbiotype has ALS activity with the same sensitivity to ALS-inhibiting herbicides as ALS from the susceptible biotype (24)suggesting that a more general resistance mechanism is oper-ating in ryegrass such as metabolism of the herbicide tononphytotoxic products. This type of mechanism could ex-plain the cross resistance observed to herbicides with com-pletely different modes of action.The results of this study indicate that the decreased sensi-

tivity of ALS from resistant kochia to inhibition by ALS-inhibiting herbicides accounts for the decreased response atthe whole plant level. This conclusion is supported by theresults that show no significant differences in chlorsulfuronmetabolism, uptake, translocation, Km (pyruvate), or in ALSexpression between the susceptible and resistant kochia bio-types.

ACKNOWLEDGMENTS

The technical assistance of Mr. Wayne S. Hanna and Ms. SharonL. Tomlinson is gratefully acknowledged. Commentary and help

60 SAARI ET AL.

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Page 7: Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf

MECHANISM OF SULFONYLUREA RESISTANCE IN KOCHIA

given by Drs. J. L. Saladini, W. W. John, H. M. Brown, J. V. Schloss,and W. F. Smith are greatly appreciated.

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