Weed Science 2011 59:376–379

Acetolactate Synthase Gene Proline (197) Mutations Confer Tribenuron-Methyl Resistance in Flixweed ( sophia) Populations from China Hai Lan Cui, Chao Xian Zhang, Shou Hui Wei, Hong Jun Zhang, Xiang Ju Li, Yan Qiu Zhang, and Gui Qi Wang*

The molecular basis of resistance to tribenuron-methyl, an acetolactate synthase (ALS)–inhibiting herbicide was investigated in four resistant (R) and three susceptible (S) flixweed populations. The resistance level in the R populations was assessed in whole- pot experiments in a greenhouse, and resistance indices ranged from 723 to 1422. The ALS genes of the three S populations and four R populations were cloned and sequenced, and the full coding sequence of the ALS gene of flixweed was 2,004 bp. The sequences of the ALS genes of the three S populations collected from Shaanxi, Gansu, and Tianjin were identical. Comparison of the ALS gene sequences of the S and R populations with Arabidopsis revealed that proline at position 197 of the ALS gene was substituted by leucine in R population SSX-2, by alanine in R population SSX-3, and by serine in R populations TJ-2 and GS-2. In another study of two R flixweed populations from Hebei and Shaanxi, resistance was also related to mutation at position 197 of the ALS gene. Both studies confirmed tribenuron-methyl resistance in flixweed in China, with the resistance mechanism being conferred by specific ALS point mutations at amino acid position 197. Nomenclature: Tribenuron-methyl; flixweed, Descurainia sophia (L.) Webb ex Prantl DESSO; Arabidopsis, Arabidopsis thaliana (L.) Heynh. ARBTH. Key words: ALS inhibitor, ALS gene, flixweed, tribenuron resistance, herbicide resistance.

Flixweed, a cross- and self-pollinated species, is a member chlorsulfuron, iodosulfuron, tribenuron) have been applied of the family (Hickey and King 1981). It is intensively and continuously in wheat in China. Flixweed believed to have originated in Europe, temperate and tropical populations in Shaanxi and Hebei provinces were first Asia, and North Africa (GRIN 2010). Currently, flixweed has reported in 2005 to evolve resistance to tribenuron (Heap become naturalized in North America and is widely 2010). Since then, 21 flixweed populations from eight distributed throughout the temperate zones of the world provinces in China were also found to be moderately or (USDA, NRCS 2010). Flixweed is a prolific producer highly resistant to tribenuron (Cui et al. 2008), indicating that and a strong competitor, and it can substantially reduce crop tribenuron-resistant flixweed populations are widely distrib- yield in winter wheat (Triticum aestivum L.) and oilseed rape uted. In the United States, an ALS-resistant flixweed (Brassica napus L.) in China (Li 1998; Sun et al. 1990; Zhang population was also confirmed in 2006 (Heap 2010). 2003). Resistance to ALS inhibitors is most often a consequence of Tribenuron, an acetolactate synthase (ALS, EC 4.1.3.18)– amino acid substitutions in the ALS enzyme. To date, ALS inhibiting sulfonylurea herbicide, was introduced in China in resistance in field-selected weed populations are conferred by 1988 to selectively control flixweed and other broad-leaved 22 amino acid substitutions at seven sites in the ALS gene, weeds in wheat (ICAMA 1988). Tribenuron soon became a which are alanine 122 (Ala122), proline 197 (Pro197), alanine popular herbicide in wheat fields in China, and about 205 (Ala205), aspartate 376 (Asp376), tryptophan 574 1,492,000 kg of its formulations were applied in wheat fields (Trp574), serine 653 (Ser653), and glycine 654 (Gly654) for broad-leaved weed control in 2009 (personal communi- (Tranel and Wright 2002; Tranel et al. 2010; Whaley et al. cation). ALS is the first enzyme in the biosynthetic pathway of 2007; recently reviewed by Powles and Yu [2010]). Eleven essential branched-chain amino acids, including leucine, substitutions have been reported for Pro197 (Ala, Arg, Gln, isoleucine, and valine. The ALS-inhibiting herbicides inhibit His, Ile, Leu, Lys, Met, Ser, Thr, and Trp) (Beckie et al. 2007; ALS activity, blocking the biosynthesis of branched-chain Cui et al. 2008; Powles and Yu 2010; Tranel and Wright amino acids, finally resulting in plant death. Due to their low 2002; Tranel et al. 2010; Warwick et al. 2008; Yu et al. 2003, use rates, high efficacy, broad spectrum, and excellent crop 2010), and these single substitutions usually result in a high safety, the ALS-inhibiting herbicides have been used world- level of resistance to sulfonylurea herbicides. wide for weed control (Corbett and Tardif 2006; Tranel and Based on our previous study and other reports of ALS gene Wright 2002). mutations conferring ALS resistance, we wondered if any ALS inhibitors are prone to resistance evolution, with 107 other mutations exist in the ALS gene in tribenuron-resistant weed species having evolved resistance to this mode of action flixweed populations. The objective of this research was to worldwide (Heap 2010). ALS-inhibiting herbicides (e.g., determine the molecular basis of ALS resistance in flixweed populations in China. DOI: 10.1614/WS-D-10-00099.1 * First, second, third, and fifth authors: Assistant Professor, Professor, Associate Professor, and Professor, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Key Laboratory of Weed and Rodent Biology and Materials and Methods Management, and State Key Laboratory for Biology of Plant Diseases and Insect Pests, No. 2 West Yuanmingyuan Road, Haidian, Beijing 100193, China; fourth Seed Collection. of seven flixweed populations were and sixth authors: Associate Professor and Professor, Institute for the Control of collected either from separate wheat fields with various Agrochemicals, Ministry of Agriculture, China, No. 22 Maizidian Street, Chaoyang, Beijing 100125, China; seventh author: Professor, Institute of Food histories of repeated tribenuron application, or from remote and Oil, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang hills that had no history of ALS-inhibiting herbicide use in 050031, China. Corresponding author’s E-mail: [email protected] Gansu, Shaanxi, and Tianjin in China (Table 1).

376 N Weed Science 59, July–September 2011 Table 1. Flixweed collection sites and associated herbicide application histories.a Table 2. Primers used to amplify the flixweed ALS gene. Estimated years of Annealing Population tribenuron Amplicon temperture Collection locations code Site applications Primers Sequence (59–39) size (bp) (C)

Gansu Forward 1 TCTATCTCTCGCTCCTCTCC 670 60 Lanzhou County GS-1 Wheat field Never applied Reverse 1 TTACCTCAACAATCGGCGT Linxia County GS-2 Wheat field Over 6 yr Forward 2 CAAGGAGGTGTATTCGCAGC 783 60 Reverse 2 CTTATTCTTCCCAATCTCAGCCG Shaanxi Forward 3 GAAGCCTGTGTTGTATGTTGGT 956 62 Baoji City SSX-1 Remote hill Never applied Reverse 3 CTTTCTTTGTCACCCTCGCC Xianyang City SSX-2 Wheat field Over 12yr Forward 4 GCTATTCCTCCACAGTATGCG 762 56 Xianyang City SSX-3 Wheat field Over 12 yr Reverse 4 CAAACAAACAGCAGTAGCG Tianjin Baodi District TJ-1 Wheat field Never applied Wuqing District TJ-2 Wheat field Over 8 yr and the full coding sequence of the ALS gene of flixweed was a Flixweed seeds of each population were collected from about 10 that determined. made up the seed pools. DNA Amplification and Sequencing. The DNA engine Bio- RAD3 was used to amplify ALS gene fragments from flixweed Whole-Plant Pot Experiments. Dose-response experiments genomic DNA. Each polymerase chain reaction (PCR) 2 were performed as described by Cui et al. (2008). Thirty contained 1 ml of genomic DNA (about 25 ng ml 1 ), 0.5 ml 2 pregerminated flixweed seeds were planted into 12-cm-diam of each primer (25 pmol ml 1), 2.5 mlof103 PCR buffer, pots containing moist loam soil. Pots were placed in the 1.5 ml of 2.5 mM deoxynucleotide triphosphates (dNTPs) 2 greenhouse (temperature maintained at 15 to 22 C) under mixture, and 0.5ml Taq DNA polymerase (5 U ml 1) in a final natural sunlight and watered and fertilized as required. The volume of 25 ml. PCR reactions were subjected to a 3-min greenhouse was located on the site (40u019N, 116u169E) of denaturation at 94 C; 25 cycles of 1 min at 94 C, 1min at X the Institute of Plant Protection, Chinese Academy of C, and 1 min at 72 C; then 3 min at 72 C, where X is the Agricultural Sciences, in Beijing. Flixweed seedlings were annealing temperature for each primer pairs used. Annealing thinned to 10 evenly sized plants per pot prior to herbicide temperatures were 60, 60, 62, and 56 C for primer sets 1, 2, application. 3, and 4, respectively (Table 2). Tribenuron was applied at 0, 0.11, 1.13, 11.25, 112.50, The desired PCR products were cloned with the competent 21 and 1,125.00 g ai ha to flixweed at the four-leaf growth cell (JM109) and plasmid (PMD18-T) for sequencing. Each stage. Tribenuron was applied with a compressed-air, moving- desired fragment was sequenced in forward and reverse 1 nozzle cabinet sprayer equipped with one Teejet XR8003 flat directions, to minimize sequencing errors, by a commercial 2 21 fan nozzle and calibrated to deliver 400 L ha at 0.3 MPa. sequencing company.4 The sequences of five plants of each Treatments were organized as a completely randomized design susceptible population were analyzed first and then compared with four replications per treatment. The experiment was with sequences from resistant populations to determine conducted twice, and results are combined. whether a nucleotide substitution occurred. A minimum of Flixweed shoots were harvested 21 d after treatment and five plants was sequenced for each population examined. dried at 80 C for 48 h; dry weight was then determined. DNA Analyzer5 with the common primers M13F (247) Combined data over the two experimental runs were subjected (59CGCCAGGGTTTTCCCAGTCACGAC39) was used to to ANOVA and dose-response relationships were examined obtain the complementary strand of the sequenced ALS gene using the Probit model: Y 5 b + kx, where Y is probit, b is fragments. Sequences of flixweed and Arabidopsis were intercept, k is the regression coefficient, and x is log10 (dose). assembled and compared using DNAMAN software package Herbicide dose that reduces dry weight by 50% (GR50 values) (Version 5.2.2, Lynnon Biosoft, Canada). were calculated and resistance indices (RI) were determined as GR50 (R)/GR50 (S), where R is resistant and S is susceptible. Results and Discussion Genomic DNA Extraction. All plant populations were grown Whole-Plant Bioassay. The dose-response experiments in the greenhouse and treated with tribenuron at 21 showed that populations GS-1, SSX-1, and TJ-1, collected 11.25 g ai ha at the four-leaf growth stage except the S from untreated areas, were susceptible to tribenuron. The populations SSX-1, TJ-1, and GS-1. Approximately 1 g of GR50 values for these susceptible populations were 21 young shoot tissue of individual surviving plants from all 0.11 g ai ha (Table 3). Comparing the GR50 values populations was harvested and stored at 280 C. DNA was between the susceptible and resistant populations, populations extracted from 100 mg young shoot tissue of each plant using GS-2, SSX-2, SSX-3, and TJ-2 exhibited a high level of the CTAB method (Doyle and Doyle 1990). resistance to tribenuron, with resistance indices (RI) ranging from 723 to 1,472. These results are in accordance with our Oligonucleotide Primers. Four pairs of forward and reverse previous study demonstrating that the RI values for flixweed overlapping primers (Table 2) were designed based on the populations from Hebei (population 7) and Shaanxi (popu- ALS gene sequences of Arabidopsis (Arabidopsis, X51514) lation 29) province were up to 1,400 (Cui et al. 2008). The from the GenBank database to amplify the highly conserved RI for the newly identified flixweed population TJ-2 from region of the ALS gene. The 39 end and 59 end were Tianjin was 1,186 (Table 3), which was much higher than the sequenced by 39 RACE and 59 RACE methods, respectively, RIs of 5 to 44 for Tianjin population reported in our previous

Cui et al.: Tribenuron-methyl resistance in flixweed N 377 a Table 3. Regression equation, relative GR50, and resistance index of tribenuron-susceptible (S) and -resistant (R) flixweed populations in China. 21 b Collection locations Population Regression equation GR50 (g ai ha ) Resistance index Gansu GS-1 (S) y 5 0.41x + 5.87 0.11 GS-2 (R) y 5 0.39x + 4.23 94 854 Shaanxi SSX-1 (S) y 5 0.36x + 6.11 0.11 SSX-2 (R) y 5 0.46x + 4.13 80 723 SSX-3 (R) y 5 0.39x + 4.14 162 1,472 Tianjin TJ-1 (S) y 5 0.49x + 5.78 0.11 TJ-2 (R) y 5 0.40x + 4.15 130 1,186

a Abbreviation: GR50, herbicide dose that reduces dry weight by 50%; S, tribenuron-susceptible; R, tribenuron-resistant. b 21 21 GR50 values for the S populations were less than 0.11 g ai ha , the lowest rate used, and therefore were set at 0.11 g ai ha to calculate resistance indices. study (Cui et al. 2008). Populations GS-2, SSX-2, and SSX-3 inhibiting herbicide-resistant weed species (Tranel et al. were also the newly identified populations with high levels of 2010). It is clear that the resistance mutations found in this tribenuron resistance. study are similar to those reported for ALS-resistant prickly lettuce (Lactuca serriola L.) populations due to the modifica- Molecular Basis of Resistance. The ALS genes of three tion of proline 197 to threonine (Preston et al. 2006). Wild susceptible populations and four resistant populations were radish (Raphanus raphanistrum L.) populations also had point cloned and sequenced, and the full coding sequence of the mutations of proline 197 to four different amino acid ALS gene without any intron was about 2,004 bp. The substitutions: histidine, threonine, alanine, and serine (Yu et sequences of the ALS gene of the three susceptible populations al. 2003; Walsh et al. 2007). Proline 197 substitution by were identical. Comparison of the ALS gene sequences from leucine was reported in ALS inhibitor–resistant stinkweed susceptible and resistant populations with Arabidopsis revealed (Thlaspi arvense L.) populations (Beckie et al 2007). that proline at position 197 of the ALS gene was substituted Nontarget site resistance due to enhanced rates of ALS by leucine in resistant populations SSX-2, by alanine in herbicide metabolism (Preston and Mallory-Smith 2001) has resistant populations SSX-3, and by serine in resistant been reported in ALS-resistant weed species. It is known that populations TJ-2 and GS-2 (Table 4). Together with two resistance to ALS herbicides can be due to nontarget site– resistance mutations identified in our previous study (Cui et based enhanced rates of herbicide metabolism endowed by al. 2008), four different resistance-endowing amino acid P450 enzymes (Christopher et al. 1992; Powles and Yu 2010). substitutions at position 197 in six flixweed populations have The flixweed populations, 7 from Hebei Province and 29 been revealed so far. from Shaanxi Province, were resistant to tribenuron (Cui et al. Target site–based ALS-inhibitor resistance is conferred by 2008). Based on this and the previous studies, the distribution single amino acid substitutions of the ALS gene (Shaner of resistant flixweed populations in China is shown in 1999), which occur at multiple sites within the ALS gene. Figure 1. It is clear that the populations (7, SSX-2, TJ-2, and Twenty-nine ALS-resistant weed species were reported with GS-2) collected from different provinces had same amino acid various substitutions involved (Saari et al. 1992, 1994; Tranel substitution, and the populations (29, SSX-2, and SSX-3) and Wright 2002; Tranel et al. 2008, reviewed by Powles and collected from same county of Shaanxi Province had different Yu 2010). ALS resistance-endowing substitutions at proline amino acid substitutions at position 197 of the ALS gene; 197 have been reported in many weed species (Saari et al. however, flixweed resistance to tribenuron was conferred in all 1994; Tranel and Wright 2002). Proline 197 substitutions by by the point mutation at position 197 of the ALS gene. other amino acids, such as leucine and threonine, are known The evolution of a high level of tribenuron resistance in to confer ALS-inhibitor resistance in 22 of the 107 ALS- flixweed populations conferred by ALS gene point mutations

Table 4. DNA and derived amino acid sequences of ALS gene from susceptible (S) and resistant (R) populations of flixweed. The bold nucleotide bases encode Pro-197 in S populations and the mutations in R populations. Populations The amino acid position, relative sequence of nucleotide and amino acid** 191 192 193 194 195 196 197 198 199 200 201 202 203 A. thaliana GCA ATC ACA GGA CAA GTC CCT CGT CGT ATG ATT GGT ACA Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Sus.* GCC ATT ACA GGT CAA GTC CCT CGT CGG ATG ATT GGT ACA Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr 7 (R)** GCC ATT ACA GGT CAA GTC CTT CGT CGG ATG ATT GGT ACA Leu 29 (R)*** GCC ATT ACA GGT CAA GTC ACT CGT CGG ATG ATT GGT ACA Thr SSX-2 (R) GCC ATT ACA GGT CAA GTC CTT CGT CGG ATG ATT GGT ACA Leu SSX- 3 (R) GCC ATT ACA GGT CAA GTC GCT CGT CGG ATG ATT GGT ACA Ala TJ-2 (R) GCC ATT ACA GGT CAA GTC YCT CGT CGG ATG ATT GGT ACA Pro/Ser GS-2 (R) GCC ATT ACA GGT CAA GTC TCT CGT CGG ATG ATT GGT ACA Ser

Note: *Y5C/T, S5C/G; **Sus.5SSX-1, TJ-1 and GS-1 population, ***population 7 and population 29 were confirmed resistant populations (Cui et al. 2008).

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