Sulfometuron-resistant Amaranthus retro¯exus:cross-resistance and molecular basis for resistanceto acetolactate synthase-inhibiting herbicides

M SIBONY*, A MICHEL , H U HAAS , B RUBIN* & K HURLE *Institute of Plant Science and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel,

and  Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany

Received 16 December 2000

Revised version accepted 19 April 2001

Summary

A biotype of Amaranthus retro¯exus L. is the ®rst weed in Israel to develop resistance to

acetolactate synthase (ALS)-inhibiting herbicides. The resistant biotype (Su-R) was collected

from Ganot, a site that had been treated for more than 3 consecutive years with sulfometuron-

methyl + simazine. On the whole-plant basis, the resistance ratio (ED50 Su-R)/(ED50 Su-S) was

6±127 for sulfonylureas, 4±63 for imidazolinones, 20±35 for triazolopyrimidines and 11 for

pyrithiobac-sodium. Similar levels of resistance were found also when the herbicides were applied

before emergence. Based on a root elongation bioassay, Su-R was 3240-fold more resistant to

sulfometuron-methyl than Su-S. In vitro studies have shown that the Su-R biotype was resistant

at the enzyme level to all ALS inhibitors tested. The nucleotide sequences of two ampli®ed

regions between the Su-S and the Su-R di�ered in only one nucleotide. One substitution has

occurred in domain A, cytosine by thymine (CCC to CTC) at position 248, that confers an

exchange of the amino acid proline in the susceptible to leucine in the Su-R1 . The proline to

leucine change in domain A is the only di�erence in the amino acid primary structure of the

regions sequenced, indicating that it is responsible for the ALS-inhibitor resistance observed.

Keywords: ALS inhibitors, herbicide resistance, cross-resistance, Amaranthus retro¯exus2

.

Introduction

Sulfometuron-methyl, a sulfonylurea herbicide, is the most widely used herbicide in young forest,

roadsides and industrial sites in Israel. Its low mammalian toxicity, high potency (Levitt et al.,

1981), broad-spectrum weed control and long residual activity make it a very desirable herbicide

for these purposes. The herbicide inhibits acetolactate synthase (ALS), the enzyme that catalyses

the condensation step of two molecules of pyruvate to form acetolactate in the biosynthetic

pathway of branched-chain amino acids leucine, isoleucine and valine (De Felice et al., 1974).

Correspondence: Prof. B Rubin, Institute of Plant Science and Genetics, Faculty of Agricultural, Food and Environmental

Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. Tel: (+972) 8 948 9248; Fax: (+972) 8 936 2083;

E-mail: rubin@agri.huji.ac.il

Ó Blackwell Science Ltd Weed Research 2001 41, 509±522 509

This enzyme is indispensable for plants and is inhibited by four herbicide groups: sulfonylurea,

imidazolinone, triazolopyrimidine and pyrimidinylthiobenzoate.

Worldwide, ALS herbicide resistance has been observed in 64 weed species (Devine

& Eberlein, 1997; Heap, 2000), including Israel (Sibony et al., 1992, 1999; Sibony & Rubin,

1996). The evolution of resistant weed populations was attributed generally to continuous

application of sulfonylurea or imidazolinone herbicides for more than 3 years (Rubin, 1996). The

lack of cross-resistance, observed in some cases, provides evidence that the di�erent groups of

herbicides have slightly di�erent sites on the acetolactate synthase molecule (Saxena & King,

1988, 1990; Eberlein et al., 1997; Wright et al., 1998).

The ®rst sulfometuron-methyl-resistant weed, Amaranthus retro¯exus L. (redroot pigweed),

was discovered in 1992 at Ganot junction in the coastal plain of Israel (Sibony et al., 1992), after

successive application for 3 years of sulfometuron-methyl combined with simazine. Meanwhile,

ALS herbicide resistance has been reported for A. rudis J. Sauer (common waterhemp) (Hinz

& Owen, 1997; Foes et al., 1998) and for the multiple resistant A. blitoides S. Watson (prostrate

pigweed) (Sibony et al., 1992, 1999).

The ALS genes of several plant species have been sequenced. Sequencing of the ALS genes of

resistant mutants led to the identi®cation of speci®c mutations corresponding to the resistance

trait. In most cases, a single nucleotide di�erence resulting in a substitution of an amino acid of

the ALS enzyme was observed in at least one of ®ve highly conserved regions of the ALS gene.

These regions are 12±57 bp long and have been designated as domains A, B, C, D and E

(Boutsalis et al., 1999). Each domain contains a variable amino acid (underlined in the following

peptide sequences) that, when changed, confers ALS-inhibitor resistance. The mutations

of domain A (AITGQVPRRMIGT) and B (QWED) seem to be most widespread (Wiersma

et al., 1989; Guttieri et al., 1992) but, in addition, mutations in domain C

(VFAYPGGASMEIHQALTRS), D (AFQETP) and E (IPSGG) were also observed, each

leading to a speci®c pattern of resistance to di�erent ALS inhibitors (Subramanian et al., 1996;

Devine & Eberlein, 1997).

In domain A, all the potential substitutions of the nucleotide that encodes for proline

(proline®serine, leucine, glutamine, alanine, threonine, arginine or histidine) were observed and

led to resistance to ALS inhibitors (Guttieri et al., 1992; Devine & Eberlein, 1997). In addition,

Boutsalis et al. (1999) described a resistant biotype of Sisymbrium orientale L. with mutations of

the ®rst two nucleotides of the proline codon of domain A, resulting in the substitution of proline

by isoleucine. A di�erent situation was observed in domain B. Until now, only mutations of the

tryptophane codon leading to an exchange of tryptophane with leucine have been observed

(Bernasconi et al., 1995; Woodworth et al., 1996a).

Because all ®ve domains were located in two distinct parts of the ALS gene, Wright et al.

(1998) and Foes et al. (1998, 1999) developed two primer pairs useful for a speci®c polymerase

chain reaction (PCR) ampli®cation of two short regions, which encompasses the ®ve known

mutation sites. One region contained domains A, C and D and the second domains B and E.

Sequencing of these PCR products with subsequent comparison of the sequences of the resistant

and the susceptible biotype leads to the identi®cation of mutations conferring resistance to ALS

inhibitors.

The aim of the studies presented here was to characterize the degree of resistance and cross-

resistance of the resistant A. retro¯exus (Su-R) biotype to di�erent classes of ALS inhibitors. We

also attempted to identify the mechanism of resistance and its molecular basis.

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Materials and methods

Resistant A. retro¯exus (Su-R) seeds were collected during the summer of 1995 from Ganot,

Israel. The seeds were collected from many plants that survived an annual treatment with a

mixture of sulfometuron-methyl (Oust, 75% a.i., Dupont, USA) at 75 g a.i. ha±14 and simazine

(Simanex, 50% a.i., Agan, Israel) at 2.5 kg a.i. ha±1 that had been applied in the autumn for 3

successive years. Seeds of the sensitive A. retro¯exus population (Su-S) were collected from a

nearby location (Kfar Shmuel) that had not been treated previously with any herbicide.

Experiments were conducted in Israel and Germany; the plants were grown in Israel in a net

house in the summer and in Germany in a greenhouse. In Germany, all the treatments were

performed in a spraying chamber (Aro, Langental, Switzerland) equipped with a ¯at-fan nozzle

(8004) at 250 kPa calibrated to deliver 400 L ha±1. In Israel, a motorized laboratory sprayer was

used that was equipped with a ¯at-fan nozzle (8001 E) at 245 kPa calibrated to deliver 300 L ha±1.

Plant culture

Seeds were sown 0.5-cm-deep in a box (20 cm ´ 10 cm ´ 3 cm), containing sterilized compost

soil treated with a fungicide-solution (Fonganil-Neu, 240 g a.i. L±1 metalaxyl-M, Novartis).

They were placed in a cold chamber (4 °C, 48 h) and then transferred to a greenhouse (24/18 °Cday/night). Mercury halogen lamps were used to provide 300 lE m±2 s±1 for a 16-h photoperiod.

After development of the ®rst true leaf, four plants were transplanted to 10-cm-paper pots

(Humulus, Hartmann, Germany) ®lled with sterilized compost soil.

In Israel, the seeds were placed in a Petri dish containing wet ®lter paper in a cold chamber

(4 °C, 48 h) and were then planted in 200-mL-plastic pots containing sandy soil and peat mixture

(2 : 1, V/V) and 1 g kg±1 of a slow release fertilizer (Osmocote 14±14±14, Sierra, Holland). The

seedlings were irrigated and grown in a net-house. After emergence, the seedlings were thinned to

four plants per pot with 4±6 pots per treatment.

Pot experiments

Su-R and Su-S A. retro¯exus plants were treated with di�erent ALS-inhibitor herbicides either

before or after emergence (Table 1). The pre-emergence application was commenced

immediately after sowing and the seedlings were not thinned; post-emergence treatments were

applied at the three- to ®ve-leaf stage on uniform seedlings. Herbicide rates (10±12) were chosen

in a way to achieve plant reactions from `no damage' to `complete kill' (Table 1). The e�ect of

the treatment was evaluated 2±3 weeks after application by measuring the shoot dry weight. The

herbicide rate that caused 50% shoot growth inhibition (ED50) was established for each herbicide

and population.

Root elongation bioassay

Square plastic Petri dishes (90 mm ´ 90 mm ´ 15 mm) were ®lled with 200 g of washed and

oven-dried sand (16 mm mesh). Herbicide solution (25 mL) containing di�erent concentrations

of commercial sulfometuron-methyl at 10)1±106 nM was thoroughly mixed in the sand before

sowing. Twenty seeds of Su-R or Su-S biotypes were sown in each dish. The dish was then

covered, sealed with tape to prevent moisture loss, and placed tilted upright so the roots would

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grow down on the dish surface. The Petri dishes were maintained for 48 h in a cold chamber

(4 °C) in the dark and then transferred to another growth chamber at 27 °C. The root length was

measured 10 d after planting. The herbicide concentration causing 50% inhibition in root growth

(ED50) was determined.

ALS crude enzyme extraction and assay

ALS response to herbicides was determined in vivo using crude enzyme extracts. ALS enzyme

was isolated from Su-R and Su-S young seedlings grown in a net-house under the summer

conditions. The extraction and assay were based on the method described by Ray (1984) with

modi®cations. All bio-chemicals were purchased from Sigma, Israel, and the procedures were

conducted on ice or in the cold room (4 °C). Fresh leaves (1±5 g), cut and frozen, were ground

under liquid nitrogen with a chilled mortar and pestle. The powder was homogenized and 10%

polyvinylpolypyrrolidone (PVPP) [wt/V] and 0.2 mM dithiothreitol (DTT) were added just

before extraction. Three volumes of extraction bu�er [100 mM potassium phosphate (pH 7.5),

10 mM sodium pyruvate, 5 mMMgCl2, 0.5 mM thiamine pyrophosphate (TPP), 100 lM ¯avine

adenine dinucleotide (FAD) and 10% glycerol (V/V)] were added. The homogenate was ®ltered

through eight layers of cheesecloth and centrifuged at 25 000 g for 20 min. The supernatant was

then 50% saturated with (NH4)2SO4 on ice and covered. After stirring for 60 min, the solution

was centrifuged at 27 000 g for 20 min. The supernatant was discarded and the pellets were

suspended in 1±6 mL of an elution bu�er [50 mM potassium phosphate bu�er (pH 7.0), 20 mM

sodium pyruvate and 0.5 mM MgCl2]. Total protein content was measured using the Bradford

method (Bradford, 1976). ALS activity was assayed after diluting the extract to 1.5 mg protein

mL±1. Protein (200 lL) and 100 lL of herbicide solution or water was added to 700 lL of

Table 1 Herbicides and rates applied post-emergence on A. retro¯exus at 3±5 leaf stage for establishing

dose±response experiments

Rate range (g a.i. ha)1)

Group  Herbicide Commercial formulation % a.i. Su-S (Kfar Shmuel) Su-R (Ganot)

SU Amidosulfuron1 Gratil, 20 0.50±75 0.50±75

Chlorimuron-ethyl2 Classic, 75 0.30±80 0.30±80

Chlorsulfuron2* Glean, 75 0.01±640 0.01±640

Metsulfuron-methyl2 Gropper, 60 0.03±8 0.13±32

Nicosulfuron2 Accent, 75 0.06±60 0.47±480

Rimsulfuron2 Titus, 25 0.05±12.5 0.38±160

Sulfometuron-methyl2* Oust, 75 0.08±640 0.10±640

Thifensulfuron-methyl2 Harmony, 25 0.06±60 0.47±480

Triasulfuron3 Amber, 75 0.38±75 0.38±75

Tribenuron-methyl2* Pointer, 75 0.50±240 0.50±960

Tri¯usulfuron-methyl2 Debut, 50 0.47±480 0.47±480

IMI Imazamethabenz4 Assert, 30 23.50±6000 23.50±6000

Imazamethapyr4 Cadre, 24 1.50±48 24.00±1536

Imazapyr4* Arsenal, 24 6.25±1600 6.25±1600

Imazethapyr4 Pursuit, 10 6.20±800 6.20±800

TP Metosulam5 Sinal, 10 0.06±60 0.12±120

Flumetsulam5* DE-498, 80 1.56±1600 6.25±1600

PY Pyrithiobac-sodium2 Staple, 85 0.50±676 2.64±676

 SU, Sulfonylurea; IMI, Imidazolinone; TP, Triazolopyrimidine; PY, Pyrimidinylthiobenzoate.1Aventis; 2DuPont; 3Novartis; 4American Cyanamid; 5DowElanco.

*Denotes herbicides that were tested pre- and post-emergence.

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reaction bu�er [50 mM potassium phosphate (pH 7.0), 100 mM sodium pyruvate, 1 mM TPP,

10 mM MgCl2, 10 lM FAD]. The assay mixture was incubated for 60 min at 37 °C. The

reaction was stopped by adding 100 lL of 6 N H2SO4, followed by incubation for 30 min at

60 °C to form acetoin. Next, 100 lL of 50% NaOH, 300 lL of a solution of creatine (0.9%) plus

a-napthol (9%) in 2.5 N NaOH (wt/V) were added to each tube. Tubes were incubated for

30 min at 60 °C and centrifuged at 10 000 g for 2 min. The acetoin content was measured at

530 nm by the method of Westerfeld (1945). Steps involving a-napthol were conducted in the

dark. The herbicide rate that caused 50% inhibition of ALS activity (ED50) was established for

each herbicide and population.

Molecular studies

DNA-extraction: DNA was extracted from leaves according to the method described by Lassner

et al. (1989). Each leaf sample (100 mg) taken from a single plant was frozen in liquid nitrogen,

ground to a ®ne powder and transferred to 0.8 mL of DNA-extraction bu�er [140 mM sorbitol,

220 mM Tris-HCl, (pH 8.5), 22 mM EDTA, 800 mM NaCl, 0.8% CTAB, 1% lauroylsarcosine].

Samples were then extracted for 5 min at 55 °C with 0.32 mL of chloroform-isoamylalcohol

(24:1). DNA was precipitated by adding 1.2 volumes of isopropanol. Reaction tubes were placed

for at least 30 min on ice, and DNA was collected as a pellet by centrifugation (10 min, 20 800 g

at 4 °C). The pellet was washed twice with 70% ethanol, and resuspended in 100 lL of 0.1 ´ TE

(1 mM Tris-HCl, 0.1 mM EDTA, pH 8.5) and used as a template in PCR reactions.

Ampli®cation of speci®c regions of the ALS gene

Two primer pairs were designed using the software LASERGENE, module PRIMERDESIGN

(DNASTAR, Madison, WI, USA) to amplify the regions containing the conserved domains

A-E. They were based on the published ALS sequence of Amaranthus spp. (Woodworth et al.,

1996b) and synthesized by Carl Roth (Karlsruhe, Germany). The primers Up1 and Low1 were

located at 330±350 nt and 638±619 nt respectively. They were used to amplify the 308-bp

fragment region 1, which contains the domains A, C and D. The primers Up2 and Low2 were

located at 1656±1676 nt and 2018±1998 nt respectively. These primers ampli®ed region 2, a

362-bp fragment spanning domains B and E. Region 1: Up1: 5¢ > GTC-TTT-GCT-TAC-CCT-

GGT-GG < 3¢; Low1: 5¢ > GAT-CGT-GTT-ACC-TCA-ACT-AT < 3¢. Region 2: Up2:

5¢ > GGT-AGA-GAA-TCT-CCT-GGT-TAA-A < 3¢; Low2: 5¢ > CCA-ACT-AAT-AAG-

CCC-TTC-TTC-C < 3¢.

PCR ampli®cation

PCR was conducted using 50-lL reactions containing 20 ng genomic DNA, 1x PCR bu�er,

0.2 mM of each dNTP, 1.25 U Taq-polymerase (all from Roche Diagnostics, Mannheim,

Germany) and 1 lM of each primer. The PCR was performed on a Progene II thermal-cycler

with the following conditions: denaturation at 94 °C for 3 min, ramp to 58 °C at 1.5 °C s±1,

annealing at 58 °C for 1.5 min, ramp to 72 °C at 2 °C s±1, elongation at 72 °C for 1 min, ramp

to 94 °C at 2 °C s±1, for 45 cycles. The speci®city of the reaction was checked by agarose-gel-

electrophoresis. If only one fragment of the expected length was ampli®ed, the PCR products

were cleaned using the High-Pure-PCR-Product-Puri®cation-Kit (Roche Diagnostics). Puri®ed

products were sent together with the corresponding primers to GATC GmbH (Konstanz,

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Germany) for sequencing. The resulting sequences of the resistant and the susceptible biotype

were compared for the identi®cation of mutations.

Statistical analysis

All experiments were arranged in a randomized complete block design with 4±6 replicates and

repeated at least twice. Dose±response experiments were analysed by a nonlinear regression using

the model of Streibig (1988):

y � C � Dÿ C1� eb�ln�x�ÿln�ED50��

8

where D � upper limit, C � lower limit, x � dose, b � slope, ED50 � e�ective dose, causing

50% reduction.

For calculation of the dose±response curves, the dry weight of the plants, root length and ALS

activity were taken as relative to the untreated control. Dose±response curves were calculated

using the non-linear regression analysis procedure5 of SAS (SAS Institute, Cary, NC, USA). An

F-test (P � 0.01) was used to test signi®cant di�erences of the regression parameter. Resistance

factors were calculated from the ED50 values (ED50R/ED50S).

Results

Whole-plant studies

The dose±response experiments revealed di�erences in the reaction of the biotypes to the tested

herbicides, as shown in Fig. 1 for sulfometuron-methyl. By comparing the ED50 values of the

post-emergence treated Su-R to the Su-S biotype the resistance levels were calculated (Table 2).

The resistance ratio was 6:127 for sulfonylureas, 4:63 for imidazolinones and 20:35 for

triazolopyrimidines. The resistant biotype was 11 times less susceptible to pyrimidinylthio-

benzoate than the Su-S biotype. Su-R is highly resistant especially to chlorsulfuron (R-ratio 127)

and thifensulfuron (R-ratio 126).

Similar levels of resistance were also found when the herbicides were applied before emergence

(Table 3). The R-ratio varied from 14 to 118 for the sulfonylureas, 3 for imazapyr and 58 for

¯umetsulam.

The root elongation bioassay revealed that sulfometuron-methyl inhibits root growth in soil

to a greater extent than shoot growth. The ED50 value for sulfometuron-methyl in Su-R biotype

was found to be 3240-fold higher than that of Su-S biotype (Fig. 2). The root elongation

bioassay revealed that sulfometuron-methyl, like chlorsulfuron (Rubin & Casida, 1985), inhibits

root growth in soil to a greater extent than shoot growth.

Isolated ALS studies

In the ALS studies, the speci®c activity of the crude enzyme without herbicide was 0.28 and

0.20 lM acetolactate mg±1 protein h±1 for Su-S and Su-R respectively. In vitro studies have

shown that the level of resistance of ALS isolated from the Ganot population was 11- to 14-fold

higher for sulfonylureas, 25±116 for imidazolinones, 46 for the triazolopyrimidine ¯umetsulam

and 39 for the pyrimidinylthiobenzoate pyrithiobac-sodium (Table 4). These data are consistent

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Fig. 1 E�ect of sulfometuron-methyl applied post-emergence at the 3- to 5-leaf stage on the shoot weight

of resistant (Su-R) and susceptible (Su-S) A. retro¯exus biotypes.

Table 2 ED50 values (herbicide rate

causing 50% reduction of shoot dry

weight) of resistant (Su-R) and

susceptible (Su-S) A. retro¯exus

biotypes. The herbicides were

applied post-emergence at the 3- to

5-leaf stage

Herbicide

ED50 (Su-R)

(g a.i. ha)1)

ED50 (Su-S)

(g a.i. ha)1)

Resistance

ratio

Amidosulfuron 38.8 (4.1) 3.55 (0.04) 11

Chlorimuron-ethyl 36.9 (5.5) 2.70 (0.07) 13

Chlorsulfuron 167.7 (6.5) 1.32 (0.09) 127

Metsulfuron-methyl 0.6 (0.1) 0.03 (0.01) 19

Nicosulfuron 9.5 (1.4) 0.75 (0.05) 13

Rimsulfuron 2.3 (0.7) 0.41 (0.07) 6

Sulfometuron-methyl 5.1 (0.8) 0.30 (0.04) 17

Thifensulfuron-methyl 29.1 (5.0) 0.23 (0.01) 126

Triasulfuron 40.8 (2.1) 2.80 (0.40) 15

Tribenuron-methyl 28.2 (5.0) 2.82 (0.88) 10

Tri¯usulfuron-methyl 11.5 (1.1) 1.43 (0.11) 8

Imazamethabenz 300.0 (4.5) 25.00 (0.80) 12

Imazamethapyr 425.0 (20.8) 6.80 (0.40) 63

Imazapyr 41.0 (1.2) 10.50 (0.60) 4

Imazethapyr 675.0 (44.8) 35.00 (2.31) 19

Metosulam 40.0 (2.5) 2.00 (0.35) 20

Flumetsulam 1.8 (0.1) 0.05 (0.001) 35

Pyrithiobac-sodium 5.4 (0.3) 0.50 (0.02) 11

Values in parentheses are standard errors of ED50.

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with the results of the post-emergence treatment of the whole plants in the dose±response

experiments.

Nucleotide and amino acid sequence of region 1 and region 2

PCR with the primer pairs Up1 and Low1 as well as Up2 and Low2 produced a single band of

expected length of 308 bp for region 1 and 362 bp for region 2. Sequencing of both fragments

and comparison of the sequences enabled the proof of all mutations known to confer ALS-

inhibitor resistance in plants.

Herbicide

ED50 (Su-R)

(g a.i. ha)1)

ED50 (Su-S)

(g a.i. ha)1)

Resistance

ratio

Chlorsulfuron 4.7 (0.2) 0.04 (0.01) 118

Sulfometuron-methyl 5.3 (0.1) 0.26 (0.07) 20

Tribenuron-methyl 5.2 (0.1) 0.37 (0.06) 14

Imazapyr 49.0 (1.3) 18.00 (0.90) 3

Flumetsulam 43.1 (1.7) 0.75 (0.08) 58

Values in parentheses are standard errors of ED50.

Table 3 ED50 values (herbicide rate

causing 50% reduction of shoot dry

weight) of resistant (Su-R) and

susceptible (Su-S) A. retro¯exus

biotypes. The herbicides were

applied pre-emergence

Fig. 2 E�ect of sulfometuron-methyl on root elongation of Su-R and Su-S A. retro¯exus seedlings,

germinating in a Petri dish ®lled with sand.

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Region 1

The nucleotide sequences of region 1 di�ered between the Su-S and the Su-R biotype in only one

nucleotide. In domain A, a substitution of cytosine by thymine (susceptible: CCC; resistant:

CTC) at position 248 has occurred (Fig. 3), that confers an exchange of the amino acid proline in

the susceptible to leucine6 in the resistant biotype. This change has been shown previously to

confer resistance to sulfonylureas in Kochia scoparia (L.) Schrad. (Guttieri et al., 1995).

Region 2

In region 2, no di�erences in the nucleotide sequence were observed between the Su-S and the

Su-R biotype (Fig. 4). The amino acid sequence of domain E that was described by various

authors with the amino acids IPSGG di�ered in both A. retro¯exus-biotypes with IPSGA. The

same amino acids were also observed in an Amaranthus species by Woodworth et al. (1996b).

Because alanine occurred in the resistant as well as in the susceptible biotype, it seems not to be

involved in resistance to ALS inhibitors. These results indicate that Domain E is not as conserved

as assumed in that a substitution of the second glycine by alanine does not lead to resistance to

ALS inhibitors.

Discussion

Whole-plant experiments have shown that A. retro¯exus collected from Ganot (Su-R) is highly

resistant to sulfonylurea, and moderately resistant to other ALS inhibitors.

The fact that resistance evolved shortly after the introduction of sulfometuron-methyl into the

market suggests that the initial frequency of the resistant mutant in certain weeds is relatively

high. The rapid evolution of the resistant weed population was attributed to lack of herbicide

rotation as well as the strong selection pressure imposed by the long residual activity of the ALS-

inhibiting herbicide. Because simazine was applied in a tank-mix with sulfometuron-methyl each

autumn, the data indicate that simazine was not a part of the selection pressure imposed on

A. retro¯exus during its germination. In the spring, the residues of the autumn-applied simazine

in the soil are probably not su�cient to control the germinating A. retro¯exus. Sulfometuron-

methyl on the other hand, is more residual than simazine, therefore, allowing the survival of

resistant seedlings only.

Whole-plant and in vitro dose±response experiments demonstrated that the resistance is based

on an altered target site as exhibited by the response of Su-R plants to all ALS inhibitors tested.

Unpublished results in our laboratory have shown that pretreatment of both biotypes with the

organophosphate insecticide malathion immediately before sulfometuron-methyl application did

Table 4 Response of ALS crude

enzyme extracted from Su-S and

Su-R biotypes of A. retro¯exus to

di�erent herbicides

Herbicide

ED50 (Su-R)

(nM)

ED50 (Su-S)

(nM)

Resistance

ratio

Chlorsulfuron 418.2 (21.7) 3.6 (0.28) 114

Rimsulfuron 1.4 (0.1) 0.1 (0.05) 11

Sulfometuron-methyl 2718.7 (117.0) 18.5 (5.07) 147

Tribenuron-methyl 7.0 (0.5) 0.2 (0.06) 35

Imazapyr 1344.0 (340.0) 53.0 (13.6) 25

Imazethapyr 11.6 (0.3) 0.1 (0.02) 116

Flumetsulam 925.5 (30.2) 20.0 (1.70) 46

Pyrithiobac-sodium 122.0 (9.0) 3.1 (0.27) 39

Values in parentheses are standard errors of ED50.

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Fig. 3 Alignment of the nucleotide sequence of region 1 of the Su-S and the Su-R biotype. Dots indicate the same

nucleotide as in the reference sequence. The di�erence is indicated by a T. The sequence of the primers Up1

and Low1 are underlined. The double underlined sequence indicates the domains A, C or D. Bold print in the

amino acid sequence indicates sites where mutations confer resistance to ALS inhibitors. The boxed codon

indicates the Pro-codon of the Su-S changed to leucine in the Su-R biotype.

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Fig. 4 Alignment of the nucleotide sequence of region 2 of the Su-S and the Su-R biotype. Dots indicate the

same nucleotide as in the reference sequence. The sequence of the primers Up2 and Low2 are underlined. The

double underlined sequences indicate the domains B or E. Bold print in the amino acid sequence indicates sites

where mutations confer resistance to ALS inhibitors.

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not change the response of the plants to the herbicide (M Sibony and B Rubin, unpubl. obs.).

Malathion, known as a P450 inhibitor, did not increase sulfometuron-methyl toxicity for both

A. retro¯exus biotypes, hence providing further evidence that the resistance is not caused by

enhanced metabolism. This is in contrast to the results in Lolium rigidum Gaud. (Christopher

et al., 1994) and in maize (Zea mays L.) (Kreuz & Fonne-P®ster, 1992) where malathion

pretreatment signi®cantly altered the response of the resistant biotypes to the herbicide.

These results are consistent with our hypothesis that sulfometuron-methyl is not metabolized

faster in the Su-R Amaranthus plants and it is a target-site resistance.

The pattern of cross-resistance varied within and between the di�erent herbicide classes in

whole-plant studies as well as at enzyme level. This is an important result for the comparison of

resistance ratios of di�erent experiments, and in any attempt to manage resistant populations in

the ®eld. Furthermore, newer compounds (e.g. rimsulfuron) lead to a lower resistance ratio than

the older ones (e.g. chlorsulfuron).

The PCR ampli®cation of regions 1 and 2 revealed a very speci®c ampli®cation, resulting in a

single PCR fragment of the expected size. The same PCR primers were also used, with the same

speci®c results, for the ampli®cation of regions 1 and 2 of A. blitoides, A. rudis and Conyza

canadensis (L.) Cronq. (Michel, 2000). This indicates that these primers could be useful for a

wide variety of di�erent plant species.

Sequencing of the two speci®c regions of the ALS gene enabled the identi®cation of the point

mutation in the proline codon of domain A. This mutation has been shown previously to confer

resistance to ALS inhibitors with a comparable level of resistance and cross-resistance

(Subramanian et al., 1996; Devine & Eberlein, 1997). Because no other di�erences were found

between Su-R and Su-S, it can be concluded that this point mutation is responsible for the

resistance of the Su-R biotype.

The main advantage of the identi®cation of the point mutations leading to resistance is that

the resistance can be classi®ed as target-site resistance without any additional tests. Furthermore,

knowing the mutation and its site seems to facilitate a forecast on the expected levels of resistance

and cross-resistance to other ALS inhibitors.

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft, as a part of the Trilateral

German±Israeli±Palestinian Research Programme: Alternative Practices in Near East

Agriculture for Environmental Conservation. We also thank the support given by the Chief

Scientist Fund of the Ministry of Agriculture, Israel.

References

BERNASCONI P, WOODWORTH AR, ROSEN BA, SUBRAMANIAN MV & SIEHL DL (1995) A naturally occurring

point mutation confers broad range tolerance to herbicides that target acetolactate synthase. Journal of

Biological Chemistry 270, 17381±17385.

BOUTSALIS P, KAROTAM J & POWLES SB (1999) Molecular basis of resistance to acetolactate synthase-

inhibiting herbicides in Sisymbrium orientale and Brassica tournefortii. Pesticide Science 55, 507±516.

BRADFORD M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein

utilizing the principles of protein-dye binding. Annals of Biochemistry 72, 248±254.

CHRISTOPHER JT, PRESTON C & POWLES SB (1994) Malathion antagonizes metabolism-based chlorsulfuron

resistance in Lolium rigidum. Pesticide Biochemistry and Physiology 49, 172±182.

520 M Sibony et al.

Ó Blackwell Science Ltd Weed Research 2001 41, 509±522

DE FELICE M, GUARDIOLA J, ESPOSITO B & LACCARINO M (1974) Structural genes for a new recognized

acetolactate synthase in Escherichia coli K-12. Journal of Bacteriology 120, 1068±1077.

DEVINE MD & EBERLEIN CV (1997) Physiological, biochemical and molecular aspects of herbicide

resistance based on altered target sites. In: Herbicide Activity: Toxicology, Biochemistry and

Molecular Biology (eds RM Roe, JD Burton & RJ Kuhr), 159±186. IOS Press, Amsterdam, The

Netherlands.

EBERLEIN CV, GUTTIERI MJ, MALLORY-SMITH CA & THILL DC (1997) E�ects of mutation for ALS-

inhibitor resistance on ALS activity in resistant and susceptible near-isonuclear Lactuca-lines. In: Weed

and Crop Resistance to Herbicides (eds R De Prado, J Jorrin & L Garcia-Torres), 191±196. Kluwer

Academic Publishers, Dordrecht, The Netherlands.

FOES MJ, LIU LX, TRANEL PJ, WAX LM & STOLLER EW (1998) A biotype of common waterhemp

(Amaranthus rudis) resistant to triazine and ALS herbicides. Weed Science 46, 514±520.

FOES MJ, LIU LX, VIGUE G, STOLLER EW, WAX LM & TRANEL PJ (1999) A kochia (Kochia scoparia)

biotype resistant to triazine and ALS-inhibiting herbicides. Weed Science 47, 20±27.

GUTTIERI MJ, EBERLEIN CV, MALLORY SMITH CA, THILL DC & HOFFMAN DL (1992) DNA sequence

variation in domain A of the acetolactate synthase genes of herbicide-resistant and -susceptible weed

biotypes. Weed Science 40, 670±676.

GUTTIERI MJ, EBERLEIN CV & THILL DC (1995) Diverse mutations in the acetolactate synthase gene confer

chlorsulfuron resistance in kochia (Kochia scoparia) biotypes. Weed Science 43, 175±178.

HEAP I (2000) International survey of herbicide resistant weeds. Annual Report Internet. http:/

www.weedscience.com/November2000.

HINZ JRR & OWEN MDK (1997) Acetolactate synthase resistance in a common waterhemp (Amaranthus

rudis) population. Weed Technology 11, 13±18.

KREUZ K & FONNE-PFISTER R (1992) Herbicide±insecticide interaction in maize: malathion inhibits

cytochrome P450-dependent primisulfuron metabolism. Pesticide Biochemistry and Physiology 43, 232±

240.

LASSNER MW, PETERSON P & YODER JI (1989) Simultaneous ampli®cation of multiple DNA fragments by

polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Molecular Biology

Reporter 7, 116±128.

LEVITT G, PLOEG GL, WEIGEL RC & FITZGERALD DJ (1981) 2-Chloro-N-[4-methoxy-6-methyl-1,3,5-

triazine-2-yl] amonocarbonyl benzenesulfonamide, a new herbicide. Journal of Agricultural and Food

Chemistry 29, 416±424.

MICHEL A (2000) Untersuchungen von Amaranthus spp. und Conyza canadensis (L.) Cronq. gegen

verschiedene ALS-Inhibitoren. PhD thesis, University of Hohenheim, Stuttgart, Germany.

RAY TB (1984) Site of action of chlorsulfuron inhibition of valine and isoleucine biosynthesis in plants.

Plant Physiology 75, 827±831.

RUBIN B (1996) Herbicide-resistant weeds ± the inevitable phenomenon: mechanisms, distribution and

signi®cance. Zeitschrift fur P¯anzenkrankheiten und P¯antzenschutz XV, 17±32.

RUBIN B & CASIDA JE (1985) R-25788 e�ects on chlorsulfuron injury and acetohydroxyacid synthase

activity. Weed Science 33, 462±468.

SAXENA PK & KING J (1988) Herbicide resistance in Datura innoxia. Cross-resistance of sulfonylurea-

resistant cell lines to imidazolinones. Plant Physiology 86, 863±867.

SAXENA PK & KING J (1990) Lack of cross-resistance of imidazolinone-resistant cell lines of Datura innoxia

P. Mill. to chlorsulfuron. Evidence for separable sites of action on the target enzyme. Plant Physiology 94,

1111±1115.

SIBONY M, BENYAMINI Y & RUBIN B (1992) Resistance of Amaranthus retro¯exus and A. blitoides to

sulfonylurea herbicides. Phytoparasitica 20, 335.

SIBONY M, LIOR E, YOSSEF T, YAACOBY T, NIR A & RUBIN B (1999) Distribution of ALS-resistant weeds in

Israel ± update. Phytoparasitica 27, 123±124.

SIBONY M & RUBIN B (1996) Multiple resistance to ALS inhibitors and triazine herbicides in Amaranthus

species. In: Proceedings 1995 International Symposium on Weed and Crop Resistance to Herbicides,

Cordoba, Spain, 112±114.

Sulfometuron-resistant Amaranthus retro¯exus 521

Ó Blackwell Science Ltd Weed Research 2001 41, 509±522

STREIBIG JC (1988) Herbicide bioassay. Weed Research 28, 479±484.

SUBRAMANIAN MV, BERNASCONI P & HESS FD (1996) In: Proceedings Second International Weed Control

Congress, Copenhagen, Denmark, 447±453.

WESTERFELD WW (1945) A colorimetric determination of blood acetoin. Journal of Biological Chemistry

161, 495±502.

WIERSMA PA, SCHMIEMANN MG, CONDIE JA, CROSBY WL & MOLONEY MM (1989) Isolation, expression

and phylogenetic inheritance of an acetolactate synthase gene from Brassica napus.Molecular and General

Genetics 219, 413±420.

WOODWORTH AR, BERNASCONI P, SUBRAMANIAN MV & ROSEN BA (1996a) A second naturally occurring

point mutation confers broad based tolerance to acetolactate inhibitors. Plant Physiology 111, 415.

WOODWORTH AR, ROSEN BA & BERNASCONI P (1996b) Broad range resistance to herbicides targeting

acetolactate synthase (ALS) in a ®eld isolate of Amaranthus sp. is confered by a Trp to Leu mutation in

ALS gene. Plant Physiology 111, 1353.

WRIGHT TR, BASCOMB NF, STURNER SF & PENNER D (1998) Biochemical mechanism and molecular basis

for ALS-inhibiting herbicide resistance in sugarbeet (Beta vulgaris) somatic cell selections. Weed Science

46, 13±23.

522 M Sibony et al.

Ó Blackwell Science Ltd Weed Research 2001 41, 509±522