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Multiple herbicide resistance in diverse weed species endowed by enhanced herbicide detoxification or degradation is rapidly growing into a great threat to herbicide sustainability and global food safety. Although metabolic resistance is frequently documented in the economically damaging arable weed species shortawn foxtail ( Alopecurus aequalis Sobol.), relevant molecular knowledge has been lacking. Previously, we identified a field population of A. aequalis (R) that had evolved metabolic resistance to the commonly used acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron-methyl. RNA sequencing was used to discover potential herbicide metabolism-related genes, and four cytochrome P450s ( CYP709C56 , CYP71R18 , CYP94C117 , and CYP94E14 ) were identified with higher expressions in the R vs. susceptible (S) plants. Here the full-length P450 complementary DNA transcripts were each cloned with identical sequences between the S and R plants. Transgenic Arabidopsis overexpressing CYP709C56 became resistant to the sulfonylurea herbicide mesosulfuron-methyl and the triazolo-pyrimidine herbicide pyroxsulam. This resistance profile generally but does not completely in accordance with what is evident in the R A. aequalis . Transgenic lines exhibited enhanced capacity for detoxifying mesosulfuron-methyl into O -demethylated metabolite, which is in line with the detection of O -demethylated herbicide metabolite in vitro in transformed yeast. Structural modeling predicted that mesosulfuron-methyl binds to CYP709C56 involving amino acid residues Thr-328, Thr-500, Asn-129, Gln-392, Phe-238, and Phe-242 for achieving O -demethylation. Constitutive expression of CYP709C56 was highly correlated with the metabolic mesosulfuron-methyl resistance in A. aequalis . These results indicate that CYP709C56 degrades mesosulfuron-methyl and its up-regulated expression in A. aequalis confers resistance to mesosulfuron-methyl.

Gene mutations conferring herbicide resistance are hypothesized to have negative pleiotropic effects on plant growth and fitness, which may in turn determine the evolutionary dynamics of herbicide resistance alleles. We used the wide-spread, annual, diploid grass weed Alopecurus aequalis as a model species to investigate the effect of two resistance mutations–the rare Pro-197-Tyr mutation and the most common mutation, Trp-574-Leu–on acetolactate synthase (ALS) functionality and plant growth. We characterized the enzyme kinetics of ALS from two purified A. aequalis populations, each homozygous for the resistance mutation 197-Tyr or 574-Leu, and assessed the pleiotropic effects of these mutations on plant growth. Both mutations reduced sensitivity of ALS to ALS-inhibiting herbicides without significant changes in extractable ALS activity. The 197-Tyr mutation slightly decreased the substrate affinity (corresponding to an increased Km for pyruvate) and maximum reaction velocity (Vmax) of ALS, whereas the 574-Leu mutation significantly increased these kinetics. Significant decrease or increase in plant growth associated, respectively, with the 197-Tyr and 574-Leu resistance mutations was highly correlated with their impact on ALS kinetics, suggesting more likely persistence of the 574-Leu mutation than the 197-Tyr mutation if herbicide application is discontinued.

The resistance to fenoxaprop-P-ethyl, a herbicide that inhibits acetyl-CoA carboxylase (ACCase), has emerged in shortawn foxtail (Alopecurus aequalis Sobol.) since the 1990s, presenting a considerable challenge to wheat (Triticum aestivum L.) production in China. One of the primary mechanisms responsible for this high-level resistance is the presence of mutations at codons 1781, 2041, and 2078 in the ACCase gene. However, the conventional methods used to detect these mutations, such as polymerase chain reaction (PCR) and gene sequencing, are time-consuming and labor-intensive. To address this issue and enable the prompt and effective detection of these common ACCase mutations in A. aequalis, a loop-mediated isothermal amplification (LAMP) strategy was developed. The LAMP assay specifically targets the Ile-1781-Leu and Asp-2078-Gly mutations within the ACCase gene of A. aequalis. Through the optimization of primers, systems, and conditions, the LAMP assay enables rapid differentiation between wild-type individuals and mutants of A. aequalis carrying either of these two mutations. Including SYBR Green I dye in the final reaction mixtures enables detection of the target mutation through a noticeable color change that can be observed with the naked eye. It is noteworthy that the sensitivity of the LAMP assay was approximately 104-fold greater than that of conventional PCR methods. Additionally, a derived cleaved amplified polymorphic sequence (dCAPS) assay was established for each mutation to distinguish between homozygous and heterozygous mutants. Overall, the developed LAMP assay could efficiently detect the Ile-1781-Leu and Asp-2078-Gly mutations in the ACCase gene of A. aequalis, offering significant advantages for the monitoring and management of fenoxaprop-P-ethyl resistance.

Non-target-site resistance (NTSR) to herbicides is a worldwide concern for weed control. However, as the dominant NTSR mechanism in weeds, metabolic resistance is not yet well-characterized at the genetic level. For this study, we have identified a shortawn foxtail ( Sobol.) population displaying both TSR and NTSR to mesosulfuron-methyl and fenoxaprop- -ethyl, yet the molecular basis for this NTSR remains unclear. To investigate the mechanisms of metabolic resistance, an RNA-Seq transcriptome analysis was used to find candidate genes that may confer metabolic resistance to the herbicide mesosulfuron-methyl in this plant population. The RNA-Seq libraries generated 831,846,736 clean reads. The transcriptome assembly yielded 95,479 unigenes (averaging 944 bp in length) that were assigned putative annotations. Among these, a total of 29,889 unigenes were assigned to 67 GO terms that contained three main categories, and 14,246 unigenes assigned to 32 predicted KEGG metabolic pathways. Global gene expression was measured using the reads generated from the untreated control (CK), water-only control (WCK), and mesosulfuron-methyl treatment (T) of R and susceptible (S). Contigs that showed expression differences between mesosulfuron-methyl-treated R and S biotypes, and between mesosulfuron-methyl-treated, water-treated and untreated R plants were selected for further quantitative real-time PCR (qRT-PCR) validation analyses. Seventeen contigs were consistently highly expressed in the resistant plants, including four cytochrome P450 monooxygenase (CytP450) genes, two glutathione S-transferase (GST) genes, two glucosyltransferase (GT) genes, two ATP-binding cassette (ABC) transporter genes, and seven additional contigs with functional annotations related to oxidation, hydrolysis, and plant stress physiology. These 17 contigs could serve as major candidate genes for contributing to metabolic mesosulfuron-methyl resistance; hence they deserve further functional study. This is the first large-scale transcriptome-sequencing study to identify NTSR genes in that uses the Illumina platform. This work demonstrates that NTSR is likely driven by the differences in the expression patterns of a set of genes. The assembled transcriptome data presented here provide a valuable resource for biology, and should facilitate the study of herbicide resistance at the molecular level in this and other weed species.

Shortawn foxtail (Alopecurus aequalis Sobol.) is an obligate wetland plant that is widely distributed throughout Europe, temperate Asia, and North America. In China, it is widespread in the middle and lower reaches of the Yangtze River as a noxious weed in winter cropping fields with a rice (Oryza sativa L.) rotation. The acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron-methyl has been widely used to control annual grass and broadleaf weeds, including A. aequalis, in wheat (Triticum aestivum L.) fields, leading to the selection of herbicide-resistant weeds. In this study, an A. aequalis population, AHFT-4, that survived mesosulfuron-methyl at the field-recommended rate (9 g ai ha–1) was collected in Anhui Province. Single-dose testing confirmed that the suspected resistant AHFT-4 had evolved resistance to mesosulfuron-methyl. Target gene sequencing revealed a resistance mutation of Pro-197-Ala in ALS1 of the resistant plants, and a derived cleaved amplified polymorphic sequence marker was developed to specifically detect the mutation. A relative expression assay showed no significant difference in ALS expression between AHFT-4 and a susceptible population without or with mesosulfuron-methyl treatment. Whole-plant dose–response bioassays indicated that AHFT-4 had evolved broad-spectrum cross-resistance to ALS-inhibiting herbicides of all five chemical families tested, with GR50 resistance index (RI) values ranging from 21 to 206. However, it remained susceptible to the photosystem II inhibitor isoproturon. Pretreatment with the cytochrome P450 inhibitor malathion or the glutathione S-transferase inhibitor 4-chloro-7-nitrobenzoxa-diazole had no significant effects on the resistance of AHFT-4 to mesosulfuron-methyl. To our knowledge, this study reports for the first time the ALS gene Pro-197-Ala substitution conferring broad-spectrum cross-resistance to ALS-inhibiting herbicides in A. aequalis.

QYM201, 1-(2-chloro-3-(3-cyclopropyl-5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-6-(trifluoromethyl)phenyl)piperidin-2-one), is a newly developed HPPD- (4-hydroxyphenylpyruvate dioxygenase; EC 1.13.11.27) inhibiting herbicide for weed control. Experiments were carried out to determine the effect of QYM201 on weeds and its safety for wheat in the glasshouse and field. The results indicated that at doses of 90 and 135 g active ingredient (a.i.) ha −1 QYM201 was highly effective against both grass and broadleaf weeds, such as Alopecurus aequalis Sobol., Alopecurus japonicus Steud, and Capsella bursa-pastoris Medic. In a wheat hybrid tolerance experiment, QYM201 showed a high level of safety for most of the 17 tested wheat hybrids, and the SI values reached ≥5.7 in the selectivity index study. To determine application rules for QYM201, field experiments were conducted in 2016 and 2017. During this time, 90 to 270 g a.i. ha −1 post-emergence herbicide application (POST) was sufficient to supply satisfactory all-season control of Alopecurus aequalis Sobol., Descurainia sophia [L.] Schur., and Malachium aquaticum (L.) Fires. No damage to wheat plants was observed. In order to increase wheat yield and deliver effective weed control, a dosage of 90 to 180 g a.i. ha −1 is suggested. In conclusion, the herbicide QYM201 is safe to use in wheat fields to control winter weeds.

Severe infestations of (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in gene copy numbers among accessions were revealed. Two copies, and , were conserved in all accessions, while some carried two additional copies, and . A single-base deletion in and further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the or gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of genes in will aid future analysis of the mechanism of resistance.

Shortawn foxtail is an invasive grass weed infesting winter wheat and canola production in China. A better understanding of the germination ecology of shortawn foxtail would help to develop better control strategies for this weed. Experiments were conducted under laboratory conditions to evaluate the effects of various abiotic factors, including temperature, light, pH, osmotic stress, salt concentration, and planting depth, on seed germination and seedling emergence of shortawn foxtail. The results showed that the seed germination rate was greater than 90% over a wide range of constant (5 to 25 C) and alternating (15/5 to 35/25 C) temperatures. Maximum germination occurred at 20C or 25/15C, and no germination occurred at 35 C. Light did not appear to have any effect on seed germination. Shortawn foxtail germination was 27% to 99% over a pH range of 4 to 10, and higher germination was obtained at alkaline pH values ranging from 7 to 10. Seed germination was sensitive to osmotic potential and completely inhibited at an osmotic potential of -0.6 MPa, but it was tolerant to salinity: germination even occurred at 200mM NaCl (5%). Seedling emergence was highest (98%) when seeds were placed on the soil surface but declined with the increasing burial depth. No seedlings emerged when seeds were buried 6-cm deep. Deep tillage could be an effective measure to limit seed germination from increased burial depth. The results of this study will lead to a better understanding of the requirements for shortawn foxtail germination and emergence and will provide information that could contribute to its control. Nomenclature: Shortawn foxtail, Alopecurus aequalis Sobol.; canola, Brassica napus L.; wheat, (Triticum aestivum L.).

Understanding the mechanism of herbicide resistance is fundamental for designing sustainable weed control strategies and exploiting herbicides rationally. Shortawn foxtail is a problem grass weed infesting several important crops in China. The repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has resulted in herbicide resistance in this weed. The ACCase gene of resistant individuals of a shortawn foxtail population (JSLS-1) has an Ile-2041-Thr mutation. F2 generation seeds, originated from the same heterozygous plant, were harvested, and two homozygous mutant (JSLS-1RR) and wild (JSLS-1SS) populations for the Ile-2041-Thr mutation were obtained. In whole plants, the JSLS-1RR population conferred high resistance to fenoxaprop and clodinafop, moderate resistance to haloxyfop, low resistance to pinoxaden, and no obvious resistance to clethodim and sethoxydim, compared with JSLS-1SS and a proven susceptible population (HNXY-1). A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to rapidly detect the rare Ile-2041-Thr mutation in the shortawn foxtail population. This is the first report of the cross-resistance pattern of Ile-2041-Thr mutation, and the robust dCAPS marker could quickly detect this mutation in shortawn foxtail. Nomenclature: Clethodim, clodinafop, fenoxaprop, haloxyfop, pinoxaden, sethoxydim, shortawn foxtail, Alopecurus aequalis Sobol.

Cinmethylin is a potential pre-emergence herbicide that could be used to control grass weeds in winter cereals. To determine the herbicidal activity of cinmethylin against common gramineous weeds in wheat fields in China and its level of safety on wheat, we conducted the following experiments: (i) assessing the efficacy of cinmethylin against 11 grass weeds and (ii) determining its safety against 19 wheat varieties. The results showed that cinmethylin had good herbicidal efficacy against annual bluegrass (Poa annua L.), shortawn foxtail (Alopecurus aequalis Sobol.), slender meadow foxtail (Alopecurus myosuroides Huds.), Japanese foxtail (Alopecurus japonicus Steud.), Italian ryegrass (Lolium multiflorum Lam.), British timothy (Phleum paniculatum Huds.), Asia Minor bluegrass (Polypogon fugax Nees ex Steud.), Helictotrichon tibeticum (Roshev.) Holub., and wild oat (Avena fatua L.), with a GR50 (the herbicide dose resulting in 50% growth inhibition) value of 4.50–99.21 g a.i. ha−1 in plant height and 1.43–70.34 g a.i. ha−1 in fresh weight. However, cinmethylin cannot control Japanese brome (Bromus japonicus L.) or Tausch’s goatgrass (Aegilops tauschii Coss.) at a dose of 200 g a.i. ha−1. Different wheat varieties varied in their phytotoxicity to cinmethylin. Overall, there is a phytotoxicity risk when using cinmethylin on wheat, mainly to wheat roots, with a reduction in root length of 40.81–64.09% at a dose of 400 g a.i. ha−1. These findings indicate that the pre-emergence herbicide cinmethylin provides good efficacy against most grass weeds and may possess potential for weed management in wheat fields. However, attention should be given to the application dosage and the sensitivity of wheat varieties when using cinmethylin in wheat fields.