Variation in mutations providing resistance to acetohydroxyacid synthase inhibitors in Cyperus difformis in China

doi:10.1016/j.pestbp.2020.104571

Pesticide Biochemistry and Physiology

Volume 166, June 2020, 104571

https://doi.org/10.1016/j.pestbp.2020.104571 Get rights and content

Highlights

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Distribution of 16 resistant populations of C. difformis in China were determined.
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The complete sequence of AHAS gene was amplified.
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Pro-197-Ser/Arg/Leu, Asp-376-Glu, and Trp-574-Leu were identified in 16 resistant populations.
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Various mutations in C. difformis endowed different cross-resistance patterns to AHAS inhibitors.

Abstract

Cyperus difformis has evolved resistance to pyrazosulfuron-ethyl and other acetohydroxyacid synthase (AHAS) inhibitors in paddy fields in China. To understand the distribution of resistance and the mutations involved, 38 populations collected were from 7 provinces and compared. Application of pyrazosulfuron-ethyl at 30 g a.i. ha⁻¹ identified 16 populations that survived, demonstrating resistance to this herbicide. Two exons of 498 and 1428 bp in length and a 1228–1233-bp intron of AHAS were cloned by genome walking, and three pairs of primers were designed to amplify eight conserved regions in this gene. In the 16 resistant (R) populations, five different types of mutations in the conserved region of the AHAS gene were identified: Pro-197-Ser, Pro-197-Arg, Pro-197-Leu, Asp-376-Glu, and Trp-574-Leu. Three R populations, YX15-22, YX12-10 and YX15-38, were chosen for in vitro AHAS activity assays, and the results showed that AHAS from YX15–22 carrying the Pro-197 mutation was insensitive to pyrazosulfuron-ethyl (resistance index (RI) = 310.0) and penoxsulam (RI = 10.0), whereas the enzyme from YX12–-10 and YX15-38 was insensitive to pyrazosulfuron-ethyl, penoxsulam, imazapic and bispyribac‑sodium (RI values ranging from 4.3 to 4462.0). AHAS inhibitor cross-resistance bioassays showed that YX12-10 and YX15-38 had cross-resistance to all of the tested herbicides (RI values ranging from 5.8 to 3321.9), while the YX15–22 population only had resistance to pyrazosulfuron-ethyl (RI = 827.4) and penoxsulam (RI = 6.6). This study clarified the distribution of resistant C. difformis in China and the different cross-resistance patterns given by various mutation types of AHAS.

Graphical abstract

Introduction

Acetohydroxyacid synthase (AHAS), also called acetolactate synthase (ALS), is a key enzyme in the biosynthesis of branched chain amino acids, valine, leucine and isoleucine, in plants and microorganisms (Durner et al., 1991; Devine and Shukla, 2000). AHAS catalyzes the reactions of both pyruvic acid to 2-acetolactate, and pyruvic acid and 2-ketobutyrate to 2-aceto-2-hydroxybutyrate (Schloss et al., 1988). AHAS inhibitors block the biosynthesis of the three branched chain amino acids, hindering protein synthesis during cell mitosis and making it difficult for plants to grow normally, thus leading to their consequent death. Since the introduction of AHAS inhibitors, they have been widely used in weed control due to their high control efficacy, wide herbicide spectrum and low environmental impact (Mazur and Falco, 1989). There are five chemical families of AHAS inhibitors: imidazolinones (IMI), sulfonylureas (SU), triazolopyrimidines (TP), pyrimidinylthio-benzoates (PTB) and sulfonylamino‑carbonyltriazolinones (SCT), and all act on the target enzyme of AHAS (Chaleff and Mauvais, 1984; Shaner and Studham, 1984; Gerwick et al., 1990; Stidham, 1991; Santel et al., 1999).

The extensive use of AHAS inhibitors has led to the rapid development of herbicide resistance. As early as 1982, Australia reported the first case of the evolved resistance of Lolium rigidum to AHAS inhibitors (Heap and Knight, 1986). Until now, 162 weed species have developed resistance to AHAS inhibitors globally, mostly due to point mutations in the AHAS gene that reduce the sensitivity of AHAS to herbicides (Heap, 2019). To date, 29 resistance-endowing amino acid substitutions were identified at 8 positions (Ala-122, Pro-197, Ala-205, Asp-376, Arg-377, Trp-574, Ser-653, and Gly-654) in the AHAS enzyme (Heap, 2019). Cross-resistance among AHAS-inhibiting herbicides can occur and is related to the mutation types of the target sites. For example, the Ala-122, Ser-653 and Gly-654 mutations confer resistance to IMI but not SU herbicides. The Pro-197 mutation confers resistance to SU and TP herbicides, while the Asp-376 or Trp-574 mutations provided resistance to all five families of AHAS-inhibiting herbicides (Pang et al., 2002; McCourt et al., 2005; Beckie and Tardif, 2012). However, the exact pattern of resistance due to specific amino acid substitutions can vary between species (Deng et al., 2016).

Cyperus difformis, also known as smallflower umbrella sedge, is an annual weed of the Cyperaceae family, a problematic weed in paddy fields with high levels of self-pollination (Merotto et al., 2009). C. difformis is widely distributed in central, east, north and northeast of China and has a serious impact on rice production. One hundred plants of C. difformis grown in 1 m² can lead to a significant reduction in rice yield of 49–81% (Swain et al., 1975). AHAS inhibitors have been frequently applied to control C. difformis in paddy fields since the 1980s and there have been some reports of resistance of C. difformis. In some areas, C. difformis has developed cross-resistance to AHAS inhibitors, with reduced or no chemical control effects. A study in Korea found that C. difformis exhibited varying degrees of cross-resistance to imazosulfuron, bensulfuron-methyl, pyrazosulfuron-ethyl, bispyribac‑sodium, and imazapyr (Kuk et al., 2004). Furthermore, cross-resistance to AHAS inhibitors has also been found in Spain, America and Italy (Busi et al., 2006). Resistance mutations at Pro-197 (substituted by Ala, Ser or His) were also identified in C. difformis, conferring resistance to the AHAS inhibitors, azimsulfuron, halosulfuron-methyl, and bispyribace‑sodium (Ntoanidou et al., 2016; Tehranchian et al., 2015). The AHAS inhibitors used to control C. difformis in paddy fields in China are mainly pyrazosulfuron-ethyl, penoxsulam, bensulfuron-methyl, and halosulfuron-methyl. Pyrazosulfuron-ethyl and bensulfuron-methyl are very important herbicides to control C. difformis in paddy fields of China, and they have been commercialized since 1980s. However, the control effect of them is decreasing significantly recently. To date, there is limited information about resistance to AHAS inhibitors in C. difformis in China and the mutations conferring resistance.

The aims of this research were: 1) confirm the distribution of resistance to pyrazosulfuron-ethyl in C. difformis populations collected from paddy fields in 7 provinces of China; 2) sequence the AHAS gene and elucidate the mechanism of resistance to AHAS inhibitors; 3) detect the biochemical basis of resistant C. difformis based on the AHAS in vitro activity in the presence of different AHAS inhibitors; and 4) determine the cross-resistance patterns conferred by different AHAS mutations.

Section snippets

Plant material

A total of 38C. difformis populations were collected in paddy fields from 2011 to 2015 from the provinces of Heilongjiang, Liaoning, Hubei, Hunan, Anhui, Jiangsu and Jiangxi. YX11, YX12, YX13 and YX15 represent the populations of C. difformis collected in 2011, 2012, 2013 and 2015, respectively.

Soil from an area without an herbicide application history was sifted and mixed with nutrient soil in a 4:1 ratio (organic matter content >15% and total nitrogen, phosphorus and potassium contents >0.83%

Effect of pyrazosulfuron-ethyl on C. difformis populations

At 21 DAT with pyrazosulfuron-ethyl at the recommended dose of 30 g ha⁻¹, the survival rates of all C. difformis populations were determined and are shown in Table 4. Among the 38 tested populations, 22 were dead and 16 still had surviving plants, 12 of which showed complete survival and 4 of which showed partial survival. The R populations were found in the provinces of Hunan, Jiangxi, Jiangsu and Anhui (Fig. 1).

Sequences comparison and identification of resistance mutation in AHAS

By comparing the differences between DNA and cDNA amplification products, we found

Discussion

Pyrazosulfuron-ethyl and bensulfuron-methyl were successfully developed in the late 1980s as highly efficient, broad-spectrum and low toxicity herbicides in paddy fields and have been popularly applied for weed control (Yamamoto et al., 1994). Previous studies have shown that AHAS inhibitors have a tendency to select for resistance rapidly (Powles and Yu, 2010). Until now, 26 species of weeds, such as Monochoria korsakowii, Lindernia micrantha, C. difformis and Sagittaria trifolia, have been

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31972281) and the National Key Research and Development Program of China (2016YFD0200500). The authors thank all of the workers for their assistance in conducting this research. No conflicts of interest have been declared.

References (46)

J. Ashigh et al.
Characterization and diagnostic tests of resistance to acetohydroxyacid synthase inhibitors due to an Asp 376 Glu substitution in Amaranthus powellii
Pestic. Biochem. Physiol.
(2009)
H.J. Beckie et al.
Herbicide cross resistance in weeds
Crop Prot.
(2012)
M.M. Bradford
A rapid and sensitive method for quantization of microgram quantities of protein utilizing the principle of protein binding
Anal. Biochem.
(1976)
W. Deng et al.
Cross-resistance pattern to four AHAS-inhibiting herbicides of tribenuron-methyl-resistant flixweed (Descurainia sophia) conferred by Asp-376-Glu mutation in AHAS
J. Integr. Agric.
(2016)
W. Deng et al.
Cross-resistance patterns to acetolactate synthase (ALS)-inhibiting herbicides of flixweed (Descurainia sophia L.) conferred by different combinations of ALS isozymes with a Pro-197-Thr mutation or a novel Trp-574-Leu mutation
Pestic. Biochem. Physiol.
(2017)
M.D. Devine et al.
Altered target sites as a mechanism of herbicide resistance
Crop Prot.
(2000)
Z. Huang et al.
Target-site basis for resistance to imazethapyr in redroot amaranth (Amaranthus retroflexus L.)
Pestic. Biochem. Physiol.
(2016)
S. Ntoanidou et al.
Molecular basis of Cyperus difformis cross-resistance to ALS-inhibiting herbicides
Pestic. Biochem. Physiol.
(2016)
S.S. Pang et al.
Crystal structure of yeast acetohydroxyacid synthase: a target for herbicidal inhibitors
J. Mol. Biol.
(2002)
J.M. Rosario et al.
White mustard (Sinapis alba) resistance to ALS-inhibiting herbicides and alternative herbicides for control in Spain
Eur. J. Agron.
(2011)

X. Xu et al.

Mutation at residue 376 of ALS confers tribenuron-methyl resistance in flixweed (Descurainia sophia) populations from Hebei province, China

Pestic. Biochem. Physiol.

(2015)

Q. Yu et al.

Tolerance to acetolactate synthase and acetyl-coenzyme a carboxylase inhibiting herbicides in Vulpia bromoides is conferred by two co-existing resistance mechanisms

Pestic. Biochem. Physiol.

(2004)

B. Zhao et al.

Non-target-site resistance to ALS-inhibiting herbicides in a Sagittaria trifolia L. population

Pestic. Biochem. Physiol.

(2017)

R. Busi et al.

Patterns of resistance to ALS herbicides in smallflower umbrella sedge (Cyperus difformis) and rice field bulrush (Schoenoplectus mucronatus)

Weed Technol.

(2006)

R.S. Chaleff et al.

Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants

Science

(1984)

M.J. Christoffers et al.

Target-site resistance to acetolactate synthase inhibitors in wild mustard (Sinapis arvensis)

Weed Sci.

(2006)

W. Deng et al.

Target-site resistance mechanisms to tribenuron-methyl and cross-resistance patterns to ALS-inhibiting herbicides of catchweed bedstraw (Galium aparine) with different ALS mutations

Weed Sci.

(2019)

J. Durner et al.

New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin

Plant Physiol.

(1991)

B.C. Gerwick et al.

Mechanism of action of the 1, 2, 4-triazolo [1, 5-a] pyrimidines

Pestic. Sci.

(1990)

I. Heap

The International Survey of Herbicide Resistant Weeds

I. Heap et al.

The occurrence of herbicide cross-resistance in a population of annual ryegrass, Lolium rigidum, resistant to diclofop-methyl

Aust. J. Agric. Res.

(1986)

K. Itoh

Sulfonylurea resistance in Lindernia micrantha, an annual paddy weed in Japan

Weed Res.

(1999)

Y.I. Kuk et al.

Cross-resistance pattern and alternative herbicides for Cyperus difformis resistant to sulfonylurea herbicides in Korea

Pest Manag. Sci.

(2004)

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An Asp376Glu substitution and P450s-involved metabolism endow resistance to ALS inhibitors in an Ammannia auriculata Willd. Population
2024, Pesticide Biochemistry and Physiology
Ammannia auriculata Willd. is a noxious broadleaf weed, commonly infesting rice ecosystems across southern China. A putative resistant A. auriculata population (AHSC-5) was sampled from a rice field of Anhui Province, where bensulfuron-methyl (BM) was unable to control its occurrence. This study aimed to determine the sensitivities of the AHSC-5 population to common-use herbicides, and to investigate the underlying resistance mechanisms. The bioassays showed that the AHSC-5 population was 138.1-fold resistant to BM, compared with the susceptible population (JSGL-1). Pretreatment of malathion reduced the resistance index to 19.5. ALS sequencing revealed an Asp376Glu substitution in the AHSC-5 population, and in vitro ALS activity assays found that 50% activity inhibition (I₅₀) of BM in AHSC-5 was 75.4 times higher than that of JSGL-1. Moreover, the AHSC-5 population displayed cross-resistance to pyrazosulfuron-ethyl (10.6-fold), bispyribac‑sodium (3.6-fold), and imazethapyr (2.2-fold), and was in the process of evolving multiple resistance to synthetic auxin herbicides fluroxypyr (2.3-fold) and florpyrauxifen-benzyl (3.1-fold). This study proved the BM resistance in A. auriculata caused by the Asp376Glu mutation and P450-regulated metabolism. This multi-resistant population can still be controlled by penoxsulam, MCPA, bentazone, and carfentrazone-ethyl, which aids in developing targeted and effective weed management strategies.
Effect of mutations on acetohydroxyacid synthase (AHAS) function in Cyperus difformis L.
2024, Journal of Integrative Agriculture
Cyperus difformis L. is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase (AHAS) inhibitors. It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis. However, the effect of different mutations on AHAS function is not clear in Cyperus difformis. To confirm the effect of mutations on AHAS function, six biotypes were collected, including Pro197Arg, Pro197Ser, Pro197Leu, Asp376Glu, Trp574Leu and wild type, from Hunan, Anhui, Jiangxi and Jiangsu provinces, China and the function of AHAS was characterized. The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl, in which the I₅₀ (the half maximal inhibitory concentration) of wild type AHAS was 0.04 μmol L^–1 and Asp376Glu, Pro197Leu, Pro197Arg, Pro197Ser and Trp574Leu mutations were 3.98, 11.50, 40.38, 38.19 and 311.43 μmol L^–1, respectively. In the determination of enzyme kinetics parameters, the Km and the maximum reaction velocity (Vmax) of the wild type were 5.18 mmol L^–1 and 0.12 nmol mg^–1 min^–1, respectively, and the Km values of AHAS with Asp376Glu, Trp574Leu, Pro197Leu and Pro197Ser mutations were 0.38–0.93 times of the wild type. The Km value of the Pro197Arg mutation was 1.14 times of the wild type, and the Vmax values of the five mutations were 1.17–3.33-fold compared to the wild type. It was found that the mutations increased the affinity of AHAS to the substrate, except for the Pro197Arg mutation. At a concentration of 0.0032–100 mmol L^–1 branched-chain amino acids (BCAAs), the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased, except for the Pro197Arg mutation. This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.
Target gene mutations endowed cross-resistance to acetolactate synthase-inhibiting herbicides in wild Brassica juncea
2023, Pesticide Biochemistry and Physiology
Wild Brassica juncea is a troublesome weed that infests wheat fields in China. Two suspected wild B. juncea populations (19–5 and 19–6) resistant to acetolactate synthase (ALS) inhibitors were collected from wheat fields in China. To clarify their resistance profiles and resistance mechanism, the resistance levels of populations 19–5 and 19–6 to ALS-inhibiting herbicides and their underlying target-site resistance mechanism were investigated. The results showed that the 19–5 population exhibited resistance to tribenuron-methyl, pyrithiobac‑sodium and florasulam, while the 19–6 population was resistant to tribenuron-methyl, pyrithiobac‑sodium, imazethapyr and florasulam. Using the homologous cloning method, two ALS genes were identified in wild B. juncea, with one gene (ALS1) encoding 652 amino acids and the other (ALS2) encoding 655 amino acids. Pro-197-Arg mutation on ALS2 and Trp-574-Leu mutation on ALS1, together with the combination of these two mutations in a single plant, were observed in both 19–5 and 19–6 populations. ALS2 enzymes carrying the Pro-197-Arg mutation were cross-resistant to tribenuron-methyl, pyrithiobac‑sodium, imazerthapyr and florasulam, with resistance index (RI) values of 6.23, 32.81, 7.97 and 1162.50, respectively. Similarly, ALS1 enzymes with Trp-574-leu substitutions also displayed high resistance to these four herbicides (RI values ranging from 132.61 to 3375.00). In addition, the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations increased the resistance level of the ALS enzyme to ALS inhibitors, with its RI values 3.83–214.19, 6.88–37.34, 1.91–31.82 and 2.03–5.90-fold higher than a single mutation for tribenuron-methyl, pyrithiobac‑sodium, imazerthapyr and florasulam, respectively. Collectively, Pro-197-Arg mutation on ALS2, Trp-574-Leu mutation on ALS1 and the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations in wild B. juncea could endow broad-spectrum resistance to ALS inhibitors, which might provide guides for establishing effective strategies to prevent or delay such resistance evolution in this weed.
Multiple herbicide resistance in a Cyperus difformis population in rice field from China
2023, Pesticide Biochemistry and Physiology
Herbicide resistance is rapidly emerging in Cyperus difformis in rice fields across China. The response of a C. difformis population GX-35 was tested against five acetolactate synthase (ALS)-inhibiting herbicides, auxin herbicide MCPA and photosynthesis II (PSII)-inhibitor bentazone. Population GX-35 evolved multiple resistance to ALS-inhibiting herbicides (penoxsulam, bispyribac‑sodium, pyrazosulfuron-ethyl, halosulfuron-methly and imazapic) and auxin herbicide MCPA, with resistance levels of 140-, 1253-, 578-, 18-, 13-, and 21-fold, respectively, compared to the susceptible population. In this population, ALS gene expression was similar to that of the susceptible population. However, an Asp376Glu mutation in ALS gene was observed, leading to reduced inhibition of in-vitro ALS activities by five ALS-inhibiting herbicides. Furthermore, CYP71D8, CYP77A3, CYP78A5 and three ABC transporter genes (cluster-14412.23067, cluster-14412.25321, and cluster-14412.24716) over-expressed in absence of penoxsulam. On the other hand, an UGT73C1 and an ABC transporter (cluster-14412.25038) were induced by penoxsulam. Additionally, both over-expression and induction were observed for CYP74, CYP71A1, UGT88A1 and an ABC transporter (cluster-14412.21723). The GX-35 population has indeed evolved multiple herbicide resistance in China. Therefore, a diverse range of weed control tactics should be implemented in rice field.
Target and nontarget mechanisms of AHAS inhibitor cross-resistance patterns in Cyperus difformis
2023, Pesticide Biochemistry and Physiology
Weed resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides has been a critical issue for rice growers worldwide since the early 1990's. In California, resistance to bensulfuron-methyl was first detected in Cyperus difformis in 1993. Since then, populations of most major weeds of rice in California have been reported to show resistance to at least one AHAS inhibitor. We sought to describe the magnitude and mechanisms of AHAS inhibitor cross-resistance in California populations of C. difformis. Sixty-two populations were collected and screened for cross-resistance to bensulfuron-methyl (BEN), halosulfuron-methyl (HAL), bispyribac‑sodium (BIS), and penoxsulam (PEN), revealing six major patterns of cross-resistance. Representative C. difformis populations from each cross-resistance pattern were then subjected to dose-response, cytochrome P450 inhibition, AHAS gene sequencing, and metabolic studies with the same herbicides as in the screening. Dose-response confirmed the detected resistances in the representative populations, and suggested that the majority of observed resistance was dose-dependent. Cytochrome P450 inhibition via malathion revealed evidence of increased metabolic activity in resistant populations to BEN, BIS, and PEN. AHAS gene sequencing revealed amino acid substitutions in five of six populations: R3 (Pro197-Ser), R4 (Pro97-His), R10 (Asp376), R41 (Ala122-Asn), and R18 (Trp574-Leu). Metabolic studies confirmed evidence of increased activity of cytochrome P450s in all populations. Metabolic BEN and HAL analysis did not yield similar results to malathion inhibition, suggesting different P450's or other pathways. Taken together, the results of the studies confirm the complexity of AHAS inhibitor cross-resistance in C. difformis, and the presence of both target-site and metabolic resistance in most of the representative populations underscores the importance of proper herbicide selection, rotation, and scouting in fields.
Comparative analysis of resistance to ALS-inhibiting herbicides in smallflower umbrella sedge (Cyperus difformis) populations from direct-seeded and puddled-transplanted rice systems
2022, Weed Science

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Variation in mutations providing resistance to acetohydroxyacid synthase inhibitors in Cyperus difformis in China

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Plant material

Effect of pyrazosulfuron-ethyl on C. difformis populations

Sequences comparison and identification of resistance mutation in AHAS

Discussion

Acknowledgments

Pestic. Biochem. Physiol.

Crop Prot.

Anal. Biochem.

J. Integr. Agric.

Pestic. Biochem. Physiol.

Crop Prot.

Pestic. Biochem. Physiol.

Pestic. Biochem. Physiol.

J. Mol. Biol.

Eur. J. Agron.

Pestic. Biochem. Physiol.

Pestic. Biochem. Physiol.

Pestic. Biochem. Physiol.

Patterns of resistance to ALS herbicides in smallflower umbrella sedge (Cyperus difformis) and rice field bulrush (Schoenoplectus mucronatus)

Weed Technol.

Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants

Science

Target-site resistance to acetolactate synthase inhibitors in wild mustard (Sinapis arvensis)

Weed Sci.

Target-site resistance mechanisms to tribenuron-methyl and cross-resistance patterns to ALS-inhibiting herbicides of catchweed bedstraw (Galium aparine) with different ALS mutations

Weed Sci.

New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin

Plant Physiol.

Mechanism of action of the 1, 2, 4-triazolo [1, 5-a] pyrimidines

Pestic. Sci.

The International Survey of Herbicide Resistant Weeds

The occurrence of herbicide cross-resistance in a population of annual ryegrass, Lolium rigidum, resistant to diclofop-methyl

Aust. J. Agric. Res.

Sulfonylurea resistance in Lindernia micrantha, an annual paddy weed in Japan

Weed Res.

Cross-resistance pattern and alternative herbicides for Cyperus difformis resistant to sulfonylurea herbicides in Korea

Pest Manag. Sci.