Non-target-site and target-site resistance to AHAS inhibitors in American sloughgrass (Beckmannia syzigachne)

WANG Jing-jing, LI Xiang-ju, LI Dan, HAN Yu-jiao, LI Zheng, YU Hui-lin, CUI Hai-lan

Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

Abstract American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) is one of the most competitive and malignant weeds in rice-wheat rotation flelds in China. American sloughgrass populations in the Jiangsu Province of China became less sensitive to acetohydroxyacid synthase (AHAS) inhibitors after repeated application for many years in these areas. Two suspected resistant American sloughgrass populations (R1 and R2) collected in the fleld were detected the resistance to inhibitors of AHAS in whole-plant dose-response assays, compared to the susceptible (S) population. These assays indicated that R1 showed low resistance to mesosulfuron-methyl (3.32-fold), imazapic (2.84-fold) and pyroxsulam (1.55-fold), moderate resistance to flazasulfuron (4.67-fold) and pyribenzoxim (7.41-fold), and high resistance to flucarbazone (11.73-fold).However, using a combination of the cytochrome P450 inhibitor, malathion, with mesosulfuron-methyl resulted in a reduction in R1 resistance relative to mesosulfuron-methyl alone. Furthermore, R2 was highly resistant to flazasulfuron (34.90-fold),imazapic (11.30-fold), flucarbazone (49.20-fold), pyribenzoxim (12.94-fold), moderately resistant to mesosulfuron-methyl(9.77-fold) and pyroxsulam (6.26-fold), and malathion had no effect on R2 resistance to mesosulfuron-methyl. The fulllength of AHAS genes was sequenced and the AHAS enzymes were assayed in vitro in order to clarify the mechanism of resistance to AHAS inhibitors in R1 and R2 populations. The results demonstrated that R2 had a Pro-197-Ser mutation in the AHAS gene, and the sensitivity of R2 to the flve AHAS inhibitors was decreased, which may result in R2 resistance to AHAS inhibitors. There was no mutation in the AHAS gene of R1, and there were no signiflcant differences in enzyme sensitivity between susceptible (S) and resistant (R1) populations. An enhanced metabolism may be the main mechanism of R1 resistance to AHAS inhibitors.

Keywords: American sloughgrass, cross-resistance, gene mutation, non-target-site resistance

1. Introduction

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18), also known as acetolactate synthase (ALS, EC 2.2.1.6), is the flrst key enzyme in the biosynthesis of the branched chain amino acids, valine, leucine, and isoleucine in plants and microorganisms (Duggleby and Pang2000). AHAS inhibitors have high biological activity, broad spectrum speciflcity and low toxicity. AHAS inhibitors have been developed rapidly and there are flve major classes: sulfonylureas (SUs)(Chaleff and Mauvais 1984), imidazolinones (IMIs) (Shaneret al. 1984), sulfonylaminocarbonyltriazolinone (SCTs)(Santelet al. 1999), triazolopyrimidines (TPs) (Gerwicket al.1990) and pyramidinylthiobenzoates (PTBs) (Stidhamet al.1991). However, the long-term use and over-reliance on AHAS inhibitors has led to an increase in weed resistance to these herbicides (Powles and Yu 2010) and currently,there are 160 biotypes of AHAS inhibitor-resistant weeds(Heap 2018).

Mechanisms of weed resistance to herbicides are mainly related to mutations in the target genes, such as target site resistance, and metabolic enhancement (Délye 2005).Compared to enhanced metabolism, target gene mutations lead to a higher level of resistance (Yu and Powles 2014),and have been well studied in some weed species. To date,29 amino acid substitutions have been reported to cause resistance to AHAS inhibitors. There are 8AHASsites,Ala122, Pro197, Ala205, Asp376, Arg377, Trp574, Ser653,and Gly654 (Hanet al. 2012; Yu and Powles 2014), and Pro197 and Trp574, are the most common sites at which substitutions have been identifled. The speciflc site of amino acid substitution inAHASis important for its effects on AHAS resistance to inhibitors. InLolium rigidumGaud.,the Pro-197-Ser mutation can confer resistance to SUs, but the Trp-574-Leu can cause resistance to both SUs and IMIs(Yuet al. 2008). In addition, the same mutation inAHASin different weeds can affect AHAS inhibitor-resistance differently. For example, a Pro-197-Ser substitution inRapistrum rugosumL. causes cross resistance to TPs and SUs (Yuet al. 2012); whereas, inPapaver rhoeasL., this substitution enables cross resistance to TPs, SUs, PTBs and IMIs (Kaloumenoset al. 2011). There are few known molecular mechanisms for non-target resistance (NTSR)(Délye 2013), and metabolic enhancement appears to be the most important mechanism of NTSR in many weeds,such as inL.rigidum(Hanet al. 2014),Sinapis arvensisL. (Veldhuiset al. 2000),Echinochloa phyllopogon(Stapf)Koss. (Yasuoret al. 2009),Bromus rigidusRoth. (Owenet al.2012),Bromus tectorumL.(Parket al. 2004),Amaranthus tuberculatusMaq. (Guoet al. 2015),Descurainia sophiaL.(Yanget al. 2016),Sagittaria trifoliaL. (Zhaoet al. 2017)andP.rhoeas(Rey-Caballeroet al. 2017).

American sloughgrass (Beckmannia syzigachne(Steud.) Fernald) is an annual or biennial weed, distributed throughout China, and found abundantly in areas where wheat-rice rotation is employed, especially in the middle and lower reaches of the Yangtze River (Raoet al. 2008).After American sloughgrass was reported to be resistant to fenoxaprop-p-ethyl (Liet al. 2014; Panet al. 2015)and acetyl-CoA carboxylase (ACCase) inhibitors, AHAS inhibitors became the main herbicide of choice. However,AHAS inhibitors can no longer effectively control American sloughgrass in some regions (Liet al. 2015). In a primary study, we found the populations of American sloughgrass had resistance to AHAS inhibitors. The aims of this study were to: (1) detect the cross-resistance of American sloughgrass to different types of AHAS herbicides; (2)determine the effect of the P450 inhibitor, malathion, on American sloughgrass resistance; (3) detect American sloughgrass AHASin vitroactivity in the presence of different AHAS inhibitors; and (4) clarify the mechanism of AHAS inhibitor-resistance by using target enzyme gene characterization.

2. Materials and methods

2.1. Plant materials

Two resistant populations (R1 and R2) of American sloughgrass exposed to long-term AHAS herbicide use and one susceptible population (S) with no history of herbicide use were collected from the Jiangsu Province in China. The R1 population seeds were collected from Huai’an City, and the R2 population seeds were collected from Wuxi City. The seeds were soaked in a gibberellin solution (3 200 mg L–1)for 12–24 h to break dormancy; washed with sterile distilled water, then sown in 10.5-cm diameter plastic pots with a 3:1(v/v) mix of sand and soil (organic content≥15%). The pots were placed in the greenhouses with a (20±5)°C/(15±5)°C day/night cycle. Before the application of herbicides, the pot seedling density was thinned to 10 per pot.

2.2. Dose-response assays to AHAS inhibitors

Seedlings at the 3-leaf stage were treated with mesosulfuronmethyl, flazasulfuron, imazapic, flucarbazone, pyroxsulam and pyribenzoxim using a moving TeeJet?XR8002 flat fan nozzle cabinet sprayer (Compressed Air Cabinet Sprayer 3WPSH-500D, Beijing Research Center for Information Technology in Agriculture, Beijing, China) with a spray volume of 367.5 L ha–1at the pressure of 0.275 MPa. A non-ionic surfactant solution (78% polyoxyethylene dodecyl ether in water) was added to mesosulfuron-methyl and pyroxsulam at the recommended dose. Herbicide dosages were designed as described in Table 1. The aboveground tissue of the plants was harvested 21 d after the treatment and dried at 70°C for 72 h, and the dry biomass was weighed. All treatments in this experiment had three replicates, and this experiment was repeated twice.

2.3. Effect of malathion on mesosulfuron-methyl resistance

Malathion is an insecticide, and it is also known to be an inhibitor of cytochrome P450. It is commonly used to detect metabolic resistance in resistant weeds. In the greenhouse experiments, it was conflrmed that the use of 1 000 g a.i. ha–1of malathion (97% technical, Dezhou Luba Fine Chemical Co., Ltd., Dezhou, China) alone had no effect on the growth of seedlings. The S, R1 and R2 populations were sprayed with mesosulfuron-methyl at 0, 1.25, 2.5, 5, 10, 20, and 40 g a.i. ha–1, 0, 5, 10, 20, 40, 80, and 160 g a.i. ha–1and 0, 10, 20,40, 80, 160, and 320 g a.i. ha–1with malathion, respectively.Malatjion sprayed 1 h before herbicide was applied. The above-ground tissue of the plants was harvested 21 d after the treatment and dried at 70°C for 72 h, and the dry biomass was weighed. All treatments in this experiment had three replicates, and this experiment was repeated twice.

2.4. In vitro assays of AHAS activities

Leaves were harvested from seedlings at the 3–4-leaf stage from each population and stored at –80°C immediately. The AHAS assay was conducted according to Yuet al.(2004),with modiflcations in the enzyme extraction procedure. A total of 3.5 g leaf tissue was ground into flne powder in liquid nitrogen with a mortar and pestle and further homogenized in 8 μL extraction buffer containing the 100 mmol L–1K2HPO4-KH2PO4, and 1 mmol L–1thiamine pyrophosphate (TPP), 0.5 mmol L–1MgCl2, 10% (v/v) glycerol, 0.01 mmol L–1flavine adenine dinucleotide (FAD), 10 mmol L–1sodium pyruvate,1 mmol L–1phenylmethylsulfonyl fluoride (PMSF),1 mmol L–1dithiothreitol (DTT), and 0.5% soluble polyvinylpolypyrrolidone (PVP), pH 7.5. The homogenate flltered by two nylon mesh into 50-mL centrifuge tube and centrifuged at 27 000×g for 15 min at 4°C. The supernatant(6 mL) was brought to 50% (NH4)2SO4saturation and centrifuged at 27 000×g for 30 min at 4°C. The pellet was dissolved in 2.5 mL of resuspension buffer (the same as the extraction buffer without PVP) and desalted on a column of Sephadex G25. The column was equilibrated with reaction buffer containing 50 mmol L–1HEPES (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), pH 7.5, 200 mmol L–1sodium pyruvate, 20 mmol L–1MgCl2, 2 mmol L–1TPP,and 20 μmol L–1FAD (4.5 mL) and was immediately used in the assay. The reaction mixture contained 100 μL of enzyme extract and 100 μL of AHAS inhibitor solution (the herbicide was prepared in water) and was incubated at 37°C for 30 min. The reaction was stopped with 40°C of 100 μL 3 mol L–1H2SO4and incubated at 60°C for 15 min to convert acetolactate to acetoin. Then, 190 μL of creatine solution(0.55%) and 190 μL of α-naphthol solution (5.5% in 5 mol L–1NaOH) were added and the mixture was incubated at 60°C for 15 min. The AHAS enzyme activity was determinedin vitrocolorimetrically (530 nm) from the acetoin formed.The technical-grade AHAS inhibitors, mesosulfuron-methyl,flazasulfuron, imazapic, pyroxsulam, flucarbazone and pyribenzoxim were used at 0, 10–6, 10–5, 10–4, 10–3, 10–2,10–1, 1, 10, and 102μmol L–1for the S and R1 population; 0,10–6, 10–5, 10–4, 10–3, 10–2, 10–1, 1, 10, 102, and 103μmol L–1for the R2 population. Each population had three biological replicates, and each extract were measured twice.

Table 1 The doses of acetohydroxyacid synthase (AHAS) inhibitors for bioassay of cross-resistance

2.5. Statistical analysis

All data from dose-response andin vitroAHAS activity assays were analyzed by a non-linear log-logistic regression modelviaSigmaplot 12.0, according to the following equation (Seefeldtet al. 1995):

Where,Cis the lower limit,Dis the upper response limit,bis the slope of the curve and ED50is the herbicides concentration required for 50% growth reduction (GR50) or AHAS activity inhibition (I50).Yis the response expressed as the percentage of the untreated control at herbicide doseX. The resistant index (RI) was calculated as the ratio of the GR50or I50value of the R population to that of the S population to indicate the level of resistance.

2.6. Detection of AHAS gene amino acid sites

Extraction of genomic DNA and total RNASeedlings of R populations at 2–3-leaf stage were treated with mesosulfuron-methyl at 20 g a.i. ha–1, and those of the S population were treated with water alone. After treatment for 21 d, leaves were collected from surviving plants and stored at –80°C. Fifteen fresh leaves were randomly sampled from each population. Genomic DNA extraction used a novel,rapid plant genomic DNA extraction kit (Tiangen Biotech,Beijing, China), and the extraction method was based on the Plant RNA Extraction Kit (Tiangen Biotech, Beijing, China),according to the manufacturer’s instructions.

AHAS gene amplificationTheAHASgene sequences of R and S populations were registered at the National Center for Biotechnology Information (NCBI). The three pairs of primers (1F-1R, 2F-2R and 3F-3R) (Table 2) were designed according toAHASsequences fromApera spica-ventiL.(JN646110),Alopecurus aequalisSobol. (JQ743908) andAlopecurus myosuroidesHuds. (AJ437300) as reference sequences using Primer Premier 5.0 software (Primer Biosoft International, Palo Altro, CA). The three pairs of primers containing 6 mutations (A122, P197, A205,D376, R377, and W574) associated with AHAS inhibitor resistance were used to amplify theAHASgene from the S and R populations in a 25-μL reaction containing: 1 μL genomic DNA (75 μg L–1), 0.5 μL of each primer (10 μmol L–1; BGI·Tech, Beijing, China), 2.5 μL 10× PCR buffer, 2.0 μL dNTP mixture (2.5 mmol L–1), 0.5 μLrTaqDNA polymerase(5 U μL–1; TaKaRa, China) and 18 μL ddH2O. The PCR was run in an Eppendorf AG-22331 Hamburg Automated Thermal Cycler (Eppendorf, Hamburg, Germany) under the following conditions: denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, annealing temperature (Table 2) for 30 s and an extension at 72°C for a time determined for the amplifled fragment, followed flnally by an extension step of 10 min at 72°C.

3′ RACE PCR3′ RACE PCR (3′ RACE Kit, TaKaRa Biotechnology, Dalian, China) was used to obtain the terminal sequence of theAHASgenes. According to the existing target gene fragment, 3′ RACE adaptor primer was used as the reverse transcription primer with reverse transcriptase M-MMLV (RNase H), and cDNA was obtained by reverse transcription RNA, using TaKaRaLA Taqwith cDNAas the template for nested PCR. Speciflc steps were as following: (1) reverse transcription reaction using the RNA as template with the 3′ RACE adaptor primer for transcription, synthesis of flrst strand cDNA. The reaction system was as follows: 5.5 μL of total RNA, 1 μL of 3′ RACE adaptor (5 μmol L–1), 2 μL of 5× PrimerScript buffer, 1 μL of dNTP mixture (10 mmol L–1each), 0.25 μL of RNase inhibitor(40 U μL–1), 0.25 μL of PrimerScript RTase (200 U μL–1), at 42°C for 60 min and 70°C for 15 min.

Nested PCRSpeciflc primer (3′-GSP1: 5′-CGCTTTACAG GGTTTGAATAC-3′) was designed according to theAHASgene, then the PCR reaction was performed with this primer and the 3′ RACE outer primer. This reaction system contained 3 μL of the reverse transcriptional product, 7 μL of 1× cDNA dilution buffer II, 2 μL of 3′-GSP1 (10 mmol L–1),2 μL of 3′ RACE outer primer (10 mmol L–1), 5 μL of 10×LA PCR buffer II (Mg2+plus), 0.3 μL of TaKaRaLA Taq(5 UμL–1) and 31 μL of ddH2O.

Table 2 Primers designed to amplify the acetohydroxyacid synthase (AHAS) gene of American sloughgrass

AHAS gene walking PCRBased on known sequences,three pairs of primers (5′-SP1, 5′-SP2, 5′-SP3) (Table 2)were designed, to amplify the 5′ end of theAHASgene using three nested PCRs by the Genome Walking Kit (TaKaRa Biotechnology, Dalian, China). After three nested PCRs, the third PCR was purifled and cloned into the pEASY-T1 cloning vector and then transformed intoEscherichia coli Trans1-T1 phage-resistant, chemically competent cells using the pEASY-T1 Cloning Kit (Beijing TransGen Biotech, Beijing,China). Colonies were chosen, and positive recombinants were sequenced.

Cloning of the AHAS gene regionAfter PCR ampliflcation,the full-length sequence of theAHASgene from the S population was conflrmed. According to the above experiments,two pairs of primers: 5′-CGCCTTACCCAAACCTACT-3′(1F) and 5′-AGATGGCTGTGCCTGTCTG-3′ (2R) and the annealing temperature was 58°C; 5′-ATCCCAC CACAATATGCTATCC-3′ (4F) and 5′-TCACAGTTGA CCACACTTC-3′ (4R) and the annealing temperature was 56°C to clone theAHASgene containing eight resistanceendowing amino acid substitutions. All PCR products were detected by 1% agarose gel electrophoresis, then sequenced by BGI·Tech (Beijing, China) and the sequencing results were analyzed by DNAMAN version 5.2.2 software(Lynnon LLC, San Ramon, CA).

3. Results

3.1. Dose-response assays of AHAS inhibitors

The effect of six AHAS inhibitors on American sloughgrass growth reduction was investigated in S, R1 and R2 populations and the RI values for R1 and R2 populations were calculated (Table 3). This cross-resistance assay showed that relative to the susceptible population, R1 had only a low resistance (resistant index (RI)＜4-fold) to SUs-mesosulfuron-methyl, IMIs and TPs herbicides, but it had moderate resistance (4≤RI＜10-fold) to PTBs and SUs-flazasulfuron herbicides and high resistance (RI≥10-fold) to SCTs herbicides. However, the R2 population demonstrated high resistance to SUs, IMIs, SCTs and PTBs,but only a moderate resistance to TPs herbicides (Fig. 1 and Table 3).

3.2. Effect of malathion on mesosulfuron-methyl resistance

Malathion, when used alone, had no signiflcant effect on the growth and biomass of American sloughgrass. However, when used in conjunction with mesosulfuron-methyl, the RI value was decreased in R1 from 3.32- to 1.75-fold,indicating an increase in the susceptibility to mesosulfuronmethyl alone. In contrast, the addition of malathion had no signiflcant effect on the moderate resistance to mesosulfuron-methyl in R2 (Fig. 2 and Table 4).

Table 3 The GR50 values and resistant index (RI) to acetohydroxyacid synthase (AHAS) inhibitors in American sloughgrass populations

Fig. 1 Dose-response curve for shoot dry weight of susceptible (S) and resistant (R1 and R2) populations to different doses of acetohydroxyacid synthase (AHAS) inhibitors. Error bar represents the standard error of mean (n=3).

3.3. In vitro assays of AHAS activity

The data obtained showed that relative to AHAS activity in the S population, AHAS activity in R2 was less sensitive and less reduced by all AHAS inhibitors tested. Compared to the S population, sensitivity of R2, which has I50values 10–100 fold greater, to mesosulfuron-methyl, flazasulfuron,imazapic, pyroxsulam, flucarbazone and pyribenzoxim was reduced. In contrast, the I50values obtained for AHAS activities in the R1 population were not signiflcantly different from that in the S population (Fig. 3 and Table 5).

3.4. AHAS gene amplification and sequencing

The full-length ofAHASgene sequence (1 917-bp) of American sloughgrass was predicted, and the resulting sequences had 96% similarity with documentedAHASgene sequences ofA.spica-venti(JN646110),A.aequalis(JQ743908) andA.myosuroides(AJ437300) and was uploaded to NCBI GenBank (MG891930). Using 1F-2R and 4F-4R primers, the 1 035- and 766-bp fragments containing eight target sites were amplifled, respectively. The comparison of theAHASgenes in the S and R populations revealed a single nucleotide substitution in the R2AHASgene, leading to an amino acid mutation of Pro-197-Ser,whereas no mutation was found in the R1AHASgene.

4. Discussion

Due to the selective pressure from the wide-spread use of AHAS inhibitors in wheat of China, the 197 site mutations ofAHASare the most common reported in gramineae resistant weeds. For instance,Alopecurus japonicusSteud. with Pro-197-Thr was highly resistant to mesosulfuron-methyl(Biet al. 2013),L.rigidumwith Pro-197-Gln mutation was highly resistant to sulfometuron, but moderately resistant to imazapyr (Yuet al. 2008);A.aequaliswith Pro-197-Thr showed moderate resistance to rimsulfuron,flucarbazone, nicosulfuron, pyroxsulam, penoxsulam,imazamox and imazapic (Xiaet al. 2015). In this study,the R2 was identifled with a Pro-197-Ser mutation and had a high level of resistance to mesosulfuron-methyl,flazasulfuron, imazapic, flucarbazone and pyribenzoxim,but moderate-level resistance to pyroxsulam. It has been reported that American sloughgrass in Danyang, with this same mutation, was highly resistant to flucarbazone(20.3-fold) whilst moderately resistant to pyroxsulam (6.0-fold) and mesosulfuron-methyl (7.6-fold), respectively (Liet al. 2015). The activity ofin vitroAHAS ofA.japonicuswith the Pro-197-Thrwassigniflcantly higher than that of susceptible population (Biet al. 2013). In this study, relative to susceptible population, the sensitivity of R2 AHAS to the flve AHAS inhibitors is decreased, so it is speculated the target gene mutation reduces the sensitivity of target enzymes to AHAS inhibitors.

Fig. 2 Dose-response curve for shoot dry weight of susceptible(S) and resistant (R1 and R2) populations to different doses of mesosulfuron-methyl with (open symbols) or without (fllled symbols) malathion (M). Error bars represent the standard error of mean (n=3).

There was no signiflcant difference in the sensitivity of AHAS to bensulfuron-methyl between non-mutagenic resistance and sensitiveS.trifolia(Zhaoet al. 2017), the resistant population showed high resistance to bensulfuronmethyl, pyrazosulfuron-ethyl and ethoxysulfuron, but showed moderate resistance to penoxsulam and bispyribacsodium. In this study, the R1 population identifled without any mutations had a moderate resistance to mesosulfuron-methyl, flazasulfuron, imazapic, pyroxsulam and pyribenzoxim, but high resistance to flucarbazone(Table 3). In addition, the sensitivity of AHAS enzyme activity to AHAS inhibitorsin vitrowas found to be similar in S and R1. Therefore, the resistance in the R1 population is not due to target-site AHAS enzyme insensitivity to herbicides.

Malathion is an organophosphorus insecticide, also known as a cytochrome P450 inhibitor that has been used as an indicator of metabolic resistance mediated by P450s.It has been reported that malathion is an effective inhibitor of P450-mediated herbicide resistance in weed species such asE.phyllopogon(Yasuoret al. 2009),B.rigidus(Owenet al. 2012),S.trifolia(Zhaoet al. 2017) andD.sophia(Yanget al. 2016). In this study, malathion reduced the resistance level of R1 to mesosulfuron-methyl. All the results indicate that P450s may be involved in the resistance of the R1 American sloughgrass population.

Many weeds have been reported to have non-target resistance, some genes associated with non-target resistance have also been reported. The genes related to the metabolic resistance ofLoliumspp. to AHAS inhibitors were P450 (CYP72AandCYP81B1), GST (GSTA) and glycosyltransferase (GTA) (Duhouxet al. 2015). The genes related to the non-target resistance ofA.myosuroidesto AHAS inhibitors were P450 (CYP71A,CYP71B3,CYP71B7andCYP81D), peroxidase (Perox2) and disease-resistant proteins (DP01) (Gardinet al. 2015). The genes of American sloughgrass associated with non-target resistance to ACCase inhibitors include: P450 (CYP90B1,CYP86B1andCYP704B1), peroxidase (Perox 66andPerox 1), GST(GST-T3,GST-U6andGST-U1), glycosyltransferase (UDPGT 73 C1andUDP-GT 85 A2)miRNA and so on (Panet al.2016a, b). There was no report about American sloughgrass with NTSR to AHAS inhibitors, and additional studies are needed to identify further genes associated with NTSR in AHAS inhibitors of American sloughgrass.

Table 4 The GR50 and resistant index (RI) values of susceptible (S) and resistant (R1 and R2) populations treated with mesosulfuronmethyl and malathion at 1 000 g a.i. ha–1

Fig. 3 Dose-response curve of susceptible (S) and resistant (R1 and R2) populations to different doses of acetohydroxyacid synthase (AHAS) inhibitors. Error bar represents the standard error of mean (n=3).

In summary, the metabolic resistance and target resistance play a key role in weed resistance management.Non-target-resistance can lead to unpredictable patterns of resistance, and even resistance to new herbicides and target site resistance through mutation can cause cross-resistance(Petitet al. 2010), which can also be a threat to local wheat production. The current study shows that NTSR and TSR each play a role in developing resistance to AHAS inhibitors in American sloughgrass. More integrated management measures should be developed to sustainably control this resistant weed.

Table 5 The I50 values and resistant index (RI) of acetohydroxyacid synthase (AHAS) in vitro to AHAS inhibitors in susceptible(S) and resistant (R1 and R2) American sloughgrass populations

5. Conclusion

In this work, the full-length ofAHASgene (1 917-bp) of American sloughgrass was sequenced, and R2 population had a Pro-197-Ser mutation in theAHASgene, which may result in R2 resistance to AHAS inhibitors. There was no mutation in theAHASgene of R1, an enhanced metabolism may be the main mechanism of R1 resistance to AHAS inhibitors. In addition, R2 has a higher resistance than R1 to AHAS inhibitors.

Acknowledgements

This work was flnanced by the National Natural Science Foundation of China (31371952) and the Special Fund for Agro-scientiflc Research in the Public Interest of China(201303031).