Target-site and non-target-site based resistance to clodinafop-propargyl in wild oats (Avena fatua L.).

Wild oat (Avena fatua L.) is a common and problematic weed in wheat fields in China. In recent years, farmers found it increasingly difficult to control A. fatua using acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. The purpose of this study was to identify the molecular basis of clodinafop-propargyl resistance in A. fatua. In comparison to the S1496 population, whole dose response studies revealed that the R1623 and R1625 populations were 71.71- and 67.76-fold resistant to clodinafop-propargyl, respectively. The two resistant A. fatua populations displayed high resistance to fenoxaprop-p-ethyl (APP) and low resistance to clethodim (CHD) and pinoxaden (PPZ), but they were still sensitive to the ALS inhibitors mesosulfuron-methyl and pyroxsulam. An Ile-2041-Asn mutation was identified in both resistant individual plants. The copy number and relative expression of the ACCase gene in the resistant population were not significantly different from those in the S1496 population. Under the application of 2160 g ai ha -1 of clodinafop-propargyl, the fresh weight of the R1623 population was reduced to 74.9%; however, pretreatment with the application of the cytochrome P450 inhibitor malathion and the GST inhibitor NBD-Cl reduced the fresh weight to 50.91% and 47.16%, respectively, which proved the presence of metabolic resistance. This is the first report of an Ile-2041-Asn mutation and probable metabolic resistance in A. fatua, resulting in resistance to clodinafop-propargyl.

Novel aryloxyphenoxypropionate derivates containing benzofuran moiety: Design, synthesis, herbicidal activity, docking study and theoretical calculation.

A series of novel aryloxyphenoxypropionate (APP) herbicides containing benzofuran moiety were designed, synthesized and tested for herbicidal activity. The bioassay results indicated that most of target compounds possessed moderate to good herbicidal activity against monocotyledonous weeds. Compounds 5a-5d and 6a-6d showed 100% control efficiency against crabgrass (Digitaria sanguinalis) and barnyard grass (Echinochloa crus-galli) in both pre-emergence and post-emergence treatments at the dosage of 1500 g a.i. ha-1. Compound 6c was the most promising, with herbicidal activity better than clodinafop-propargyl. Molecular docking for compound 6c and its hydrolysis acid 1c were performed. ACCase activities of some compounds were also tested. Theoretical calculations for corresponding hydrolysis products 1a-1ewere carried out. Based on the results of molecular docking, enzyme activity test and theoretical calculation, the potential mechanism for herbicidal activity of these compounds was evaluated.

Transcriptomics analysis of the flowering regulatory genes involved in the herbicide resistance of Asia minor bluegrass (Polypogon fugax).

BACKGROUND: Asia minor bluegrass (Polypogon fugax, P. fugax), a weed that is both distributed across China and associated with winter crops, has evolved resistance to acetyl-CoA carboxylase (ACCase) herbicides, but the resistance mechanism remains unclear. The goal of this study was to analyze the transcriptome between resistant and sensitive populations of P. fugax at the flowering stage. RESULTS: Populations resistant and susceptible to clodinafop-propargyl showed distinct transcriptome profiles. A total of 206,041 unigenes were identified; 165,901 unique sequences were annotated using BLASTX alignment databases. Among them, 5904 unigenes were classified into 58 transcription factor families. Nine families were related to the regulation of plant growth and development and to stress responses. Twelve unigenes were differentially expressed between the clodinafop-propargyl-sensitive and clodinafop-propargyl-resistant populations at the early flowering stage; among those unigenes, three belonged to the ABI3VP1, BHLH, and GRAS families, while the remaining nine belonged to the MADS family. Compared with the clodinafop-propargyl-sensitive plants, the resistant plants exhibited different expression pattern of these 12 unigenes. CONCLUSION: This study identified differentially expressed unigenes related to ACCase-resistant P. fugax and thus provides a genomic resource for understanding the molecular basis of early flowering.

Metabolism of the14C-labeled herbicide clodinafop-propargyl in plant cell cultures of wheat and tobacco.

The metabolism of 14C-clodinafop-propargyl (CfP) was examined in cell cultures of wheat (Triticum aestivum L. cv. 'Heines Koga II') and tobacco (Nicotiana tabacum L.). Besides the non-transgenic tobacco culture, cultures transformed separately with cDNA of human cytochrome P450-monooxygenases (P450s) CYP1A1, CYP1A2, CYP3A4, CYP2B6 and CYP2C19 were examined. Experiments with wheat were executed in the presence and absence of safener cloquintocet-mexyl (CqM). After 48 h of incubation, only about 10% of applied 14C was found in media (both tobacco and wheat). Non-extractable residues of 14C-CfP in wheat cells were 16.54% (without CqM) and 30.87% (with CqM). In all tobacco cultures, 82.41-92.46% of applied radioactivity was recovered in cell extracts. In contrast to wheat, non-extractable residues amounted only to 1.50-2.82%. As determined by radio-thin layer chromatography (TLC) and -high-performance liquid chromatography (HPLC), the parent CfP was not found in the cell extracts of wheat; in tobacco cell extracts, only traces of CfP were detected. After a hydrolysis of assumed carbohydrate conjugates of CfP derived polar 14C-labeled compounds, TLC and HPLC analysis showed that in wheat, a more complex pattern of metabolites of CfP were observed as compared to all tobacco cultures. In hydrolysates resulting from wheat, the identity of three primary products was confirmed by means of GC-EI-MS: free acid clodinafop (Cf), hydroxy-Cf hydroxylated at the pyridinyl moiety, and 4-(5-chloro-3-fluoropyridin-2-yloxy)phenol. In hydrolysates derived from all tobacco cultures, main metabolite was Cf besides only traces of further unidentified products. Differences among the different tobacco cultures (non-transgenic, transgenic) did not emerge. According to kinetics of disappearance of primary metabolite Cf as well as formation of polar soluble products and non-extractable residues, metabolization of CfP proceeded at a noticeably higher rate in wheat cells treated with safener CqM than in cells without CqM treatment. Thus, these results indicated a stimulation of CfP's metabolism by CqM, although metabolic profiles observed in CqM treated and non-treated cells (after hydrolysis) were qualitatively similar. The findings obtained from all tobacco cultures suggested that with the exception of ester cleavage to Cf, CfP cannot be metabolized by tobacco itself or by the human P450s examined.

Resistance of American sloughgrass (Bechmannia syzigachne) populations to ACCase-inhibiting herbicides involves three different target site mutations from China.

American sloughgrass [Beckmannia syzigachne (Steud.) Fernald] is a problematic annual grass weed in winter wheat fields of China, which causes great loss of wheat yield. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides during the last two decades to control this weed has been selected for resistance in American sloughgrass in Jiangsu province. In this study, whole-plant dose-response assays were conducted to investigate the level of resistance in four resistant American sloughgrass populations (LY, JH, BYJ and BYP) to four ACCase-inhibiting herbicides belonging to aryloxyphenoxypropionates, cyclohexanediones, and phenylpyrazolines groups under greenhouse conditions. Based on resistance factor (RF), three populations, LY, BYJ and BYP, were highly resistant to fenoxaprop-P-ethyl, clodinafop propargyl, sethoxydim and pinoxaden. JH plants exhibited resistance to fenoxaprop-P-ethyl and clodinafop propargyl, but showed much lower RF values for sethoxydim and pinoxaden. Molecular analysis of resistance revealed that resistance in all the four populations was target site-based. Results confirmed that substitutions of Ile-1781-Leu, Ile-2041-Asn and Asp-2078-Gly, respectively, in LY, JH and BYJ/BYP, are responsible for diverse sensitivity to different ACCase-inhibiting herbicides in these populations. The substitution at position 1781 had been reported, while it is the first report of Ile-2041-Asn and Asp-2078-Gly mutations that corresponded to resistance in American sloughgrass.

Metabolic fate of the (14)C-labeled herbicide clodinafop-propargyl in a sediment-water system.

The metabolic fate of (14)C-phenyl-labeled herbicide clodinafop-propargyl ((14)C-CfP) was studied for 28 days in lab assays using a sediment-water system derived from a German location. Mineralization was 5.21% of applied (14)C after 28 days exhibiting a distinct lag phase until day 14 of incubation. Portions of radioactivity remaining in water phases decreased at moderate rate to 18.48% after 28 days; 62.46% were still detected in water after 14 days. Soxhlet extraction of the sediment using acetonitrile released 35.56% of applied (14)C with day 28, while 33.99% remained as non-extractable residues. A remarkable increase of bound (14)C was observed between 14 and 28 days correlating with the distinct increase of mineralization. No correlation was found throughout incubation with microbial activity of the sediment as determined by dimethyl sulfoxide reduction. Dissolved oxygen and pH value of water phases remained almost constant for 28 days. Analyses of Soxhlet extracts of the sediment and ethyl acetate extracts of water phases by radio-TLC and radio-HPLC revealed that CfP was rapidly cleaved to free acid clodinafop (Cf), which was further (bio-) transformed. DT50 values (based on radio-HPLC) were below 1 day (CfP) and slightly above 28 days (Cf). Further metabolites were not detected. Fractionation of humic and non-humic components of the sediment demonstrated that CfP's non-extractable residues were predominantly associated with fulvic acids up to 14 days of incubation (3.36%), whereas after 28 days, the majority of radioactivity was found in the humin/mineral fraction (13.30% of applied (14)C). Due to high-performance size-exclusion chromatography of the fulvic acids fraction derived from assays incubated for 28 days, this portion of (14)C was firmly, possibly covalently bound to fulvic acids and did not consist of CfP or Cf. Using an isolation strategy comprising preincubation of sediment with CfP and mineralization of (14)C-CfP as criterion, a microorganism was isolated from the sediment examined. It grew on (14)C-CfP as sole carbon source with evolution of (14)CO2. The bacterium was characterized by growth on commonly used carbon sources and 16S rDNA sequence analysis. Its sequence exhibited high similarity with that of Nocardioides aromaticivorans strain H-1 (98.85%; DSM 15131, JCM 11674).

Resistance to ACCase-inhibiting herbicides in an Asia minor bluegrass (Polypogon fugax) population in China.

Asia minor bluegrass (Polypogon fugax) is a common annual grass weed of winter crops distributed across China. We conducted a study on the resistance level and the mechanism of resistance to ACCase-inhibiting herbicides in a P. fugax population from China. Whole-plant dose-response experiments in greenhouse showed that the resistant P. fugax population was 1991, 364, 269, 157, and 8-fold resistant to clodinafop-propargyl, fluazifop-p-butyl, haloxyfop-R-methyl, quizalofop-p-ethyl and fenoxaprop-p-ethyl relative to the reference susceptible population, which was susceptible to all the five AOPP herbicides. Much lower R/S values of 3.5, 2.4 and 3.5, respectively, were detected for clethodim, sethoxydim and pinoxaden. Molecular analysis of resistance confirmed that the Ile2041 to Asn mutation in the resistant population conferred resistance to AOPP herbicides, but not to CHD and DEN herbicides. This is the first report of a target site mutation that corresponded to resistance to AOPP herbicides in P. fugax. Proper resistance management practices are necessary to prevent ACCase-inhibiting herbicides from becoming ineffective over wide areas.

Dissipation kinetics of co-formulation with two herbicides, clodinafop-propargyl and oxyfluorfen, in/on onion (Allium cepa) samples.

Supervised field trail on dissipation of co-formulation with herbicides clodinafop-propargyl and oxyfluorfen in spring onion showed similar pattern of dissipation during two different seasons. Residues of clodinafop-propargyl reached ≤ limit of quantitation (LOQ, 0.05 mg kg-1) on 3rd day after application at both standard and double dose during both the seasons. Oxyfluorfen residues followed first-order kinetics in both the doses during first season with half-life of 0.81 to 3.14 days. The residues of clodinafop-propargyl were detected in soil at both the doses during first season. However, residues were ≤ LOQ (0.05 mg kg-1) during second season. The residues of oxyfluorfen were detected only in double dose during first season in soil. In all other cases and in onion bulb, residues were ≤ LOQ (0.05 mg kg-1) at the time of harvest. As the residues were either ≤ LOQ (0.05 mg kg-1) on 3rd day or have a half-life of 3.14 days, the co-formulation can be used safely, provided a pre harvest interval (PHI) of 3 days is followed. On the basis of maximum residue limits (MRLs) in other commodities and from the data of present study, a default MRL of 0.05 mg kg-1 is proposed for both the pesticides.

The Ile-2041-Val mutation in the ACCase gene confers resistance to clodinafop-propargyl in American sloughgrass (Beckmannia syzigachne Steud).

BACKGROUND: Exploring the mechanisms of herbicide resistance in weeds is an important part of designing resistance management strategies and rationalizing herbicide use. Beckmannia syzigachne is one of the most important agricultural weeds in China. Long-term use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has led to the evolution of herbicide resistance in B. syzigachne. ACCase-inhibiting herbicides comprise three chemical families: aryloxyphenoxypropionates (APPs), cyclohexanediones (CHDs) and phenylpyraxoline (DENs). RESULTS: Based on whole-plant dose-response experiments, a B. syzigachne population (BS-R) was confirmed to be 12- and 20-fold resistant to the APP herbicides quizalofop-P-ethyl and clodinafop-propargyl, and 2.2-, 2.8- and 2.8-fold resistant to fenoxaprop-P-ethyl, the CHD herbicide sethoxydim and the PPZ herbicide pinoxaden, respectively, compared with its susceptible counterpart (BS-S). Resistance to clodinafop-propargyl in the BS-R population could not be reversed by the known cytochrome P450 (CYP450) inhibitor malathion and the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole. In addition, no difference in CYP450 and GST activity was confirmed between the BS-R and BS-S populations. ACCase gene sequencing revealed an Ile-2041-Val mutation in the BS-R population. A derived cleaved amplified polymorphic sequence marker was developed for rapid detection of the specific Ile-2041-Val mutation. Correlation quantification of resistance in homo- and hetero-resistant versus wild-type plants showed that resistance to clodinafop-propargyl in this population is conferred by the Ile-2041-Val mutation. CONCLUSION: Unlike previous reports on the unique cross-resistance pattern conferred by the 2041 mutation, this study demonstrates that the Ile-2041-Val mutation in BS-R population confers resistance to certain ACCase-inhibiting APP, CHD and PPZ herbicides in B. syzigachne. © 2021 Society of Chemical Industry.

Peer review of the pesticide risk assessment of the active substance clodinafop (variant evaluated clodinafop-propargyl).

The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Greece, and co-rapporteur Member State, Germany, for the pesticide active substance clodinafop-propargyl are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of clodinafop-propargyl as a herbicide on wheat, rye and triticale. The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified. The European Commission mandated EFSA to reconsider the acceptable operator exposure level (AOEL) setting and to update the non-dietary exposure assessment if needed.

Peer review of the pesticide risk assessment of the active substance clodinafop (variant evaluated clodinafop-propargyl).

The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State Greece and co-rapporteur Member State Germany for the pesticide active substance clodinafop-propargyl are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of clodinafop-propargyl as a herbicide on wheat, rye and triticale. The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.