Mechanisms of Resistance to Pyroxsulam and ACCase Inhibitors in Japanese Foxtail (Alopecurus japonicus)

Japanese foxtail is a predominant tetraploid grass weed in wheat and oilseed rape fields in eastern China. In China, pyroxsulam is mainly used to manage annual grass weeds, especially those resistant to acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides. Using dose–response studies, a pyroxsulam-resistant population, ACTC-1, was identified with a resistance index value of 58. Additionally, ACTC-1 was cross-resistant to sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates, and sulfonylaminocarbonyl-triazolinones and multiresistant to ACCase and photosystem II inhibitors. Sequence analysis revealed four gene fragments encoding acetolactate synthase (ALS) from ACTC-1, and three from JNXW-1, a pyroxsulam-sensitive population. An Asp-376-Glu substitution was found in ALS1;2 and an Ile-2041-Asn in Acc1;1, which may be responsible for its resistance to pyroxsulam and ACCase inhibitors, respectively. In vitro assays of ALS activity revealed that in ACTC-1, the sensitivity of ALS to pyroxsulam was lower, and the basal ALS activity was twofold higher than that of sensitive population JNXW-1. Additionally, the combined application of pyroxsulam with malathion or piperonyl butoxide increased the sensitivity of ACTC-1 to pyroxsulam, although it could not completely overcome the resistance. It was inferred that both target-site-based resistance and nontarget-site-based resistance may be involved in the resistance to pyroxsulam.

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Confirmation and characterization of cyhalofop-butyl–resistant Chinese sprangletop (Leptochloa chinensis) populations from China

Weed Science ◽

10.1017/wsc.2020.15 ◽

2020 ◽

Vol 68 (3) ◽

pp. 253-259

Author(s):

Yajun Peng ◽

Lang Pan ◽

Ducai Liu ◽

Xiaomei Cheng ◽

Guolan Ma ◽

...

Keyword(s):

Resistance Index ◽

Resistance Mechanisms ◽

The Other ◽

Susceptible Population ◽

Acetyl Coenzyme A ◽

Target Site Resistance ◽

High Level ◽

Acetyl Coenzyme A Carboxylase ◽

Grass Weed

AbstractChinese sprangletop [Leptochloa chinensis (L.) Nees] is one grass weed severely affecting rice (Oryza sativa L.) growth in paddies in China. Cyhalofop-butyl is the main herbicide used to control grass weeds in Chinese paddy fields, especially for controlling L. chinensis; however, L. chinensis has evolved resistance to cyhalofop-butyl due to continuous and extensive application. To investigate cyhalofop-butyl resistance levels and mechanisms in L. chinensis in some of the Chinese rice areas, 66 field populations were collected and treated with cyhalofop-butyl. Of these tested populations, 10 showed a high level of resistance to cyhalofop-butyl; the 50% effective dose ranged within 108.4 to 1,443.5 g ai ha−1 with resistance index values of 9.1 to 121.8 when compared with the susceptible population. Acetyl-coenzyme A carboxylase genes (ACCase) of susceptible and all 10 resistant populations were amplified and sequenced. Among them, Ile-1781-Leu, Trp-2027-Cys, Trp-2027-Ser, and Ile-2041-Asn mutations were found in five resistant populations. No known resistance-related mutations were found in the other five resistant populations, indicating that resistance to cyhalofop-butyl in these populations was likely to be endowed by non–target site resistance mechanisms. Notably, the Ile-1781-Leu and Trp-2027-Cys substitutions have previously been reported, but this is the first report of Trp-2027-Ser and Ile-2041-Asn mutations in L. chinensis. Furthermore, three derived cleaved amplified polymorphic sequence methods were developed to rapidly detect these mutations in L. chinensis.

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Resistance to ACCase inhibitors in Eleusine indica from Brazil involves a target site mutation

Planta Daninha ◽

10.1590/s0100-83582012000300025 ◽

2012 ◽

Vol 30 (3) ◽

pp. 675-681 ◽

Cited By ~ 11

Author(s):

M.D. Osuna ◽

I.C.G.R. Goulart ◽

R.A. Vidal ◽

A. Kalsing ◽

J.P. Ruiz Santaella ◽

...

Keyword(s):

Amino Acid Position ◽

Target Site ◽

In Vitro Assays ◽

Site Mutation ◽

Eleusine Indica ◽

Herbicide Resistant ◽

Frequent Use ◽

Target Site Resistance ◽

Grass Weed

Eleusine indica (goosegrass) is a diploid grass weed which has developed resistance to ACCase inhibitors during the last ten years due to the intensive and frequent use of sethoxydim to control grass weeds in soybean crops in Brazil. Plant dose-response assays confirmed the resistant behaviour of one biotype obtaining high resistance factor values: 143 (fenoxaprop), 126 (haloxyfop), 84 (sethoxydim) to 58 (fluazifop). ACCase in vitro assays indicated a target site resistance as the main cause of reduced susceptibility to ACCase inhibitors. PCR-generated fragments of the ACCase CT domain of the resistant and sensitive reference biotype were sequenced and compared. A point mutation was detected within the triplet of aspartate at the amino acid position 2078 (referred to EMBL accession no. AJ310767) and resulted in the triplet of glycine. These results constitute the first report on a target site mutation for a Brazilian herbicide resistant grass weed.

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Point Mutations and Cytochrome P450 Can Contribute to Resistance to ACCase-Inhibiting Herbicides in Three Phalaris Species

Plants ◽

10.3390/plants10081703 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1703

Author(s):

José G. Vázquez-García ◽

Joel Torra ◽

Candelario Palma-Bautista ◽

Ricardo Alcántara-de la Cruz ◽

Rafael De Prado

Keyword(s):

Point Mutations ◽

Resistance Mechanisms ◽

Target Site ◽

In Vitro Assays ◽

Cross Resistance ◽

Northern Iran ◽

Target Site Resistance ◽

Acetyl Coenzyme A Carboxylase ◽

Selection Of

Species of Phalaris have historically been controlled by acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides; however, overreliance on herbicides with this mechanism of action has resulted in the selection of resistant biotypes. The resistance to ACCase-inhibiting herbicides was characterized in Phalaris brachystachys, Phalaris minor, and Phalaris paradoxa samples collected from winter wheat fields in northern Iran. Three resistant (R) biotypes, one of each Phalaris species, presented high cross-resistance levels to diclofop-methyl, cycloxydim, and pinoxaden, which belong to the chemical families of aryloxyphenoxypropionates (FOPs), cyclohexanediones (DIMs), and phenylpyrazolines (DENs), respectively. The metabolism of 14C-diclofop-methyl contributed to the resistance of the P. brachystachys R biotype, while no evidence of herbicide metabolism was found in P. minor or P. paradoxa. ACCase in vitro assays showed that the target sites were very sensitive to FOP, DIM, and DEN herbicides in the S biotypes of the three species, while the R Phalaris spp. biotypes presented different levels of resistance to these herbicides. ACCase gene sequencing confirmed that cross-resistance in Phalaris species was conferred by specific point mutations. Resistance in the P. brachystachys R biotype was due to target site and non-target-site resistance mechanisms, while in P. minor and P. paradoxa, only an altered target site was found.

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Response of a Chlorsulfuron-Resistant Biotype ofKochia scopariato Sulfonylurea and Alternative Herbicides

Weed Science ◽

10.1017/s0043174500057659 ◽

1993 ◽

Vol 41 (1) ◽

pp. 100-106 ◽

Cited By ~ 19

Author(s):

Lyle F. Friesen ◽

Ian N. Morrison ◽

Abdur Rashid ◽

Malcolm D. Devine

Keyword(s):

Field Experiments ◽

Good Control ◽

Acetolactate Synthase ◽

In Vitro Assays ◽

Shoot Biomass ◽

Wheat Crop ◽

Enzyme Assays ◽

Dry Matter Accumulation ◽

Severe Stunting

Kochia growing on an industrial site where chlorsulfuron was applied repeatedly over several seasons was confirmed to be resistant to chlorsulfuron and several other acetolactate synthase (ALS) -inhibiting herbicides. In growth room experiments, resistant (R) plants were 2 to >180 times more resistant to five sulfonylurea herbicides and one imidazolinone herbicide (imazethapyr) than susceptible (S) plants, as measured by the ratio of dosages required to inhibit shoot dry matter accumulation by 50% (GR50R/S). Similarly, in vitro assays of ALS activity indicated that from 3 to 30 times more herbicide was required to inhibit the enzyme from R plants than from S plants. Results of ALS enzyme assays indicated that R kochia was approximately equally resistant to metsulfuron, triasulfuron, and thifensulfuron, and 2.5 times more resistant to tribenuron than thifensulfuron. However, the response of R kochia growing in a spring wheat crop in the field was not consistent with results of the ALS enzyme assays. In field experiments, thifensulfuron at 32 g ai ha−1had little effect on R kochia. In contrast, metsulfuron, triasulfuron, and tribenuron at 8 g ha−1did not reduce R kochia seedling densities, but caused severe stunting such that 2 mo after treatment the shoot biomass of plants in untreated plots was four times greater than in sprayed plots. Herbicides with alternative modes of action including fluroxypyr, bromoxynil/MCPA ester, dichlorprop/2,4-D ester, and 2,4-D ester provided good control of R kochia in the field. Quinclorac did not reduce kochia densities, but surviving plants were stunted. To delay or avoid development of ALS inhibitor-resistant kochia populations, these alternative herbicides applied alone or in tank mixtures could be incorporated into a herbicide rotation.

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Cross-Resistance of Eclipta (Eclipta prostrata) in China to ALS Inhibitors Due to a Pro-197-Ser Point Mutation

Weed Science ◽

10.1017/wsc.2017.16 ◽

2017 ◽

Vol 65 (5) ◽

pp. 547-556 ◽

Cited By ~ 5

Author(s):

Dan Li ◽

Xiangju Li ◽

Huilin Yu ◽

Jingjing Wang ◽

Hailan Cui

Keyword(s):

Gene Sequence ◽

Resistance Index ◽

Acetolactate Synthase ◽

Cross Resistance ◽

Eclipta Prostrata ◽

Whole Plant ◽

Plant Assays ◽

Als Inhibitors ◽

Als Inhibitor

Eclipta, widespread in tropical, subtropical, and temperate regions, is one of the main malignant broadleaf weeds and thrives in moist and dryland fields. Field rates of acetolactate synthase (ALS) inhibitors have failed to control eclipta in some farmlands in China. One ALS inhibitor–resistant population (R) collected from Jiangsu province in China was confirmed in the greenhouse in our preliminary work. Whole-plant assays revealed that this R population was highly resistant to four sulfonylureas (pyrazosulfuron-ethyl, 134-fold; bensulfuron-methyl, 172-fold; metsulfuron-methyl, 30-fold; and tribenuron-methyl, 195-fold), two triazolopyrimidines (pyroxsulam, 98-fold; penoxsulam, 30-fold), and one pyrimidinylthio-benzoate (bispyribac-sodium, 166-fold) and was moderately resistant to two imidazolinones (imazethapyr, 10-fold; imazapic, 19-fold). ALS enzyme-activity assays showed insensitivity of the ALS from the R population (resistance index values ranged from 12 to 293) to all of the above ALS inhibitors in vitro. Chromatograms fromALSgene sequence analysis detected a homozygous Pro-197-Ser amino acid substitution in the R population. These results confirmed that the Pro-197-Ser substitution results in broad-spectrum cross-resistance to ALS inhibitors in the eclipta R population. To our knowledge, this study is the first to report broad cross-resistance to ALS inhibitors in eclipta and to obtain the full-lengthALSgene sequence.

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Synthesis, Molecular Docking, and Antimalarial Activity of Hybrid 4-Aminoquinoline-pyrano[2,3-c]pyrazole Derivatives

Pharmaceuticals ◽

10.3390/ph14111174 ◽

2021 ◽

Vol 14 (11) ◽

pp. 1174

Author(s):

Mohd Asyraf Shamsuddin ◽

Amatul Hamizah Ali ◽

Nur Hanis Zakaria ◽

Mohd Fazli Mohammat ◽

Ahmad Sazali Hamzah ◽

...

Keyword(s):

Molecular Docking ◽

Lactate Dehydrogenase ◽

Mammalian Cells ◽

Antimalarial Activity ◽

Resistance Index ◽

Selective Inhibition ◽

Docking Analysis ◽

Hybrid Molecules ◽

Index Value

Widespread resistance of Plasmodium falciparum to current artemisinin-based combination therapies necessitate the discovery of new medicines. Pharmacophoric hybridization has become an alternative for drug resistance that lowers the risk of drug–drug adverse interactions. In this study, we synthesized a new series of hybrids by covalently linking the scaffolds of pyrano[2,3-c]pyrazole with 4-aminoquinoline via an ethyl linker. All synthesized hybrid molecules were evaluated through in vitro screenings against chloroquine-resistant (K1) and -sensitive (3D7) P. falciparum strains, respectively. Data from in vitro assessments showed that hybrid 4b displayed significant antiplasmodial activities against the 3D7 strain (EC50 = 0.0130 ± 0.0002 μM) and the K1 strain (EC50 = 0.02 ± 0.01 μM), with low cytotoxic effect against Vero mammalian cells. The high selectivity index value on the 3D7 strain (SI > 1000) and the K1 strain (SI > 800) and the low resistance index value from compound 4b suggested that the pharmacological effects of this compound were due to selective inhibition on the 3D7 and K1 strains. Molecular docking analysis also showed that 4b recorded the highest binding energy on P. falciparum lactate dehydrogenase. Thus, P. falciparum lactate dehydrogenase is considered a potential molecular target for the synthesized compound.

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Multiple herbicide resistance in a glyphosate-resistant rigid ryegrass (Lolium rigidum) population

Weed Science ◽

10.1614/ws-04-007r1 ◽

2004 ◽

Vol 52 (6) ◽

pp. 920-928 ◽

Cited By ~ 41

Author(s):

Paul Neve ◽

Jemma Sadler ◽

Stephen B. Powles

Keyword(s):

Root Growth ◽

Dose Response ◽

Population Level ◽

Acetolactate Synthase ◽

Low Levels ◽

Rigid Ryegrass ◽

Moderate Resistance ◽

Inhibition Studies ◽

Acetyl Coenzyme A Carboxylase

Surviving rigid ryegrass plants were collected from a cropping field at Pindar, Western Australia (population WALR 50), after inadequate control by glyphosate applied at the normal field rate. Plants were grown to maturity in pots and seeds were collected. Glyphosate dose–response experiments with known susceptible and resistant control populations confirmed the resistant status of the WALR 50 population. The glyphosate rate resulting in 5% mortality (LD50) and GR50(the glyphosate rate required to reduce mean growth of individuals to 50% of the untreated control) values for this population were 1,069 and 217 g ae ha−1, respectively, corresponding to R:S ratios of 3.4 and 1.9 for mortality and growth. In addition, a novel root growth–based assay of glyphosate resistance was developed and validated, giving a root growth GR50R:S ratio of 3.4. A resistance profile was established by assessing population-level survival of WALR 50 after applications at recommended rates of a range of herbicides commonly used for rigid ryegrass control in Australia. High levels of resistance to the acetolactate synthase (ALS)–inhibiting sulfonylurea herbicides chlorsulfuron and sulfometuron, moderate resistance to the acetyl coenzyme A carboxylase (ACCase)–inhibiting herbicide diclofop, and low levels of resistance to the imidazilinone herbicide imazethapyr were found. More detailed dose–response experiments confirmed resistance to chlorsulfuron, sulfometuron, and diclofop. In vitro enzyme-inhibition studies demonstrated that ALS resistance in WALR 50 is due to an insensitive target enzyme and that ACCase resistance is due to a nontarget site–based mechanism. WALR 50 is the first glyphosate-resistant weed population with confirmed resistance to ACCase- and ALS-inhibiting herbicides.

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Multiple resistance to PPO and ALS inhibitors in redroot pigweed (Amaranthus retroﬂexus)

Weed Science ◽

10.1017/wsc.2019.61 ◽

2019 ◽

pp. 1-8

Author(s):

Hao Wang ◽

Hengzhi Wang ◽

Ning Zhao ◽

Baolin Zhu ◽

Penglei Sun ◽

...

Keyword(s):

Resistance Mechanism ◽

Resistance Index ◽

Acetolactate Synthase ◽

Amaranthus Retroflexus ◽

Target Site ◽

Multiple Resistance ◽

Redroot Pigweed ◽

First Case ◽

Als Inhibitors

Abstract A redroot pigweed (Amaranthus retroﬂexus L.) population (HN-02) collected from Nenjiang County, Heilongjiang Province, exhibited multiple resistance to fomesafen and nicosulfuron. The purposes of this study were to characterize the herbicide resistance status of an HN-02 population for both acetolactate synthase (ALS) and protoporphyrinogen oxidase (PPO) inhibitors and the response to other herbicides and to investigate the target site-based mechanism governing fomesafen and nicosulfuron resistance. Three mutations, Ala-205-Val and Trp-574-Leu mutations in the ALS gene and an Arg-128-Gly mutation in the PPX2 gene, were identified in individual resistant plants. An HN-02F1-1 subpopulation homozygous for the Ala-205-Val and Arg-128-Gly mutations was generated, and whole-plant experiments confirmed multiple resistance to PPO inhibitors (fomesafen, fluoroglycofen-ethyl, and acifluorfen) and ALS inhibitors (imidazolinones [IMI], sulfonylureas [SU], and triazolopyrimidines [TP]) in the HN-02F1-1 plants, which presented resistance index values ranging from 8.3 to 110; however, these plants were sensitive to flumioxazin, fluroxypyr-meptyl, and 2,4-D butylate. In vitro ALS enzyme activity assays revealed that, compared with ALS from susceptible plants, ALS from the HN-02F1-1 plants was 15-, 28- and 320-fold resistant to flumetsulam, nicosulfuron, and imazethapyr, respectively. This study confirms the first case of multiple resistance to PPO and ALS inhibitors in A. retroﬂexus and determines that the target-site resistance mechanism was produced by Ala-205-Val and Arg-128-Gly mutations in the ALS gene and PPX2 gene, respectively. In particular, the Ala-205-Val mutation was found to endow resistance to three classes of ALS inhibitors: TP, SU, and IMI.

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Non–target site based resistance to the ALS-inhibiting herbicide mesosulfuron-methyl in American sloughgrass (Beckmannia syzigachne)

Weed Science ◽

10.1017/wsc.2019.22 ◽

2019 ◽

Vol 67 (05) ◽

pp. 527-533 ◽

Cited By ~ 1

Author(s):

Mingliang Wang ◽

Bingqi Liu ◽

Yihui Li ◽

Xiaoyong Luo ◽

Lingxu Li

Keyword(s):

Gene Sequence ◽

Acetolactate Synthase ◽

In Vitro Assays ◽

Resistance Level ◽

Chromatography Tandem Mass Spectrometry ◽

Gene Sequence Analysis ◽

Herbicide Metabolism ◽

Liquid Chromatography Tandem Mass ◽

Als Inhibitors

AbstractAmerican sloughgrass [Beckmannia syzigachne (Steud.) Fernald] is one of the most predominant and troublesome weeds in wheat (Triticum aestivum L.) fields rotated with rice (Oryza sativa L.) in China. Mesosulfuron-methyl is one of the main herbicides used to selectively control B. syzigachne in winter wheat fields in China. After many years of application, mesosulfuron-methyl failed to control B. syzigachne in Yutai County. The objectives of this study were to determine the resistance level to mesosulfuron-methyl and other acetolactate synthase (ALS) inhibitors in the B. syzigachne population collected from Yutai County (R) and identify the mechanism of resistance. The results indicated that the R population was 4.1-fold resistant to mesosulfuron-methyl and was cross-resistant to pyroxsulam (600-fold), imazethapyr (4.1-fold), flucarbazone (12-fold), and bispyribac-sodium (12-fold). In vitro assays revealed that ALS in the R population was as sensitive as that in a susceptible (S) population. Gene sequence analysis identified no known resistant mutations in the ALS gene of the R population. Furthermore, real-time quantitative reverse transcriptase PCR experiments indicated that the expression level of the ALS gene in the R population was not different from that of the S population. However, the cytochrome P450 inhibitor malathion reversed the R population's resistance to mesosulfuron-methyl. The result of ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS-MS) spectral analysis indicated that the metabolic rates of mesosulfuron-methyl in the R population were significantly faster than in the S population. Therefore, non-target resistance to mesosulfuron-methyl has been demonstrated in the R population. The resistance was very likely caused by enhanced herbicide metabolism.

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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