Metabolism-Based Nontarget-Site Mechanism Is the Main Cause of a Four-Way Resistance in Shortawn Foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis Sobol. is a predominant grass weed in Chinese winter wheat fields, posing a substantial threat to crop production owing to its escalating herbicide resistance. This study documented the initial instance of an A. aequalis population (AHFT-3) manifesting resistance to multiple herbicides targeting four distinct sites: acetyl-CoA carboxylase (ACCase), acetolactate synthase, photosystem II, and 1-deoxy-d-xylulose-5-phosphate synthase. AHFT-3 carried an Asp-to-Gly mutation at codon 2078 of ACCase, with no mutations in the remaining three herbicide target genes, and exhibited no overexpression of any target gene. Compared with the susceptible population AHFY-3, AHFT-3 metabolized mesosulfuron-methyl, isoproturon, and bixlozone faster. The inhibition and comparison of herbicide-detoxifying enzyme activities indicated the participation of cytochrome P450s in the resistance to all four herbicides, with glutathione S-transferases specifically linked to mesosulfuron-methyl. Three CYP72As and a Tau class glutathione S-transferase, markedly upregulated in resistant plants, potentially played pivotal roles in the multiple-herbicide-resistance phenotype.

Target-site and non-target-site resistance mechanisms confer mesosulfuron-methyl resistance in Alopecurus aequalis.

BACKGROUND: Shortawn foxtail (Alopecurus aequalis Sobol.) is a noxious weed in China. The resistance of A. aequalis developed rapidly due to the long-term application of acetolactate synthase (ALS)-inhibiting herbicides. Here, a suspected mesosulfuron-methyl-resistant A. aequalis population, Aa-R, was collected from a wheat field in China. RESULTS: A dose‒response test showed that the Aa-R population has evolved a high level of resistance to mesosulfuron-methyl, and its growth was suppressed by imazamox, pyroxsulam and bispyribac-sodium. ALS gene sequence analysis revealed that a known resistance-related mutation (Pro-197-Thr) was present in the Aa-R population. Moreover, ALS gene overexpression was detected in the Aa-R population. The mesosulfuron-methyl resistance could be reversed by cytochrome P450 monooxygenase (CYP450) and glutathione S-transferase (GST) inhibitors. In addition, enhanced metabolism of mesosulfuron-methyl was detected in the Aa-R population compared with the susceptible population. NADPH-cytochrome P450 reductase and GST activities were strongly inducible in the Aa-R population. One CYP450 gene, CYP74A2, and one GST gene, GST4, were constitutively upregulated in the Aa-R population. Molecular docking results showed the binding affinity of CYP74A2 and GST4 for the tested ALS-inhibiting herbicides, respectively. CONCLUSION: This study confirmed that target-site resistance and non-target-site resistance involving CYP450 and GST were the main mechanisms involved in resistance in the mesosulfuron-methyl-resistant A. aequalis population.

Loop-mediated isothermal amplification for detecting the Ile-2041-Asn mutation in fenoxaprop-P-ethyl-resistant Alopecurus aequalis.

BACKGROUND: Shortawn foxtail (Alopecurus aequalis Sobol.), a competitive grass weed severely infesting overwintering crops worldwide, has evolved resistance to the highly efficient acetyl-CoA carboxylase (ACCase)-inhibiting herbicide fenoxaprop-P-ethyl. The Ile-to-Asn substitution at codon position 2041 of ACCase is a dominant resistance mutation that has been associated with fenoxaprop-P-ethyl resistance in A. aequalis. However, its detection based on conventional methods such as polymerase chain reaction (PCR) and gene sequencing is rather labor- and time-consuming. RESULTS: In order to facilitate its detection in field populations of A. aequalis, a simple and efficient method with high sensitivity to the Ile-2041-Asn mutation was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed to target a 244-bp fragment of ACCase comprising codon position 2041. Using the special primers and genomic DNA of A. aequalis, the concentrations of reaction components, temperature and time each were optimized. The LAMP reaction for the detection of the Ile-2041-Asn mutation was processed at 65 °C for 45 min followed by 80 °C for 10 min to stop the reaction. The LAMP method developed was 1000-fold more sensitive than the conventional PCR method, and the detection was also practicable when using crude DNA of A. aequalis as a template. CONCLUSION: The low cost, simplicity and high sensitivity of the developed LAMP assay make the detection of the Ile-2041-Asn mutation easier and quicker, which may contribute to the monitoring and management of resistance development to fenoxaprop-P-ethyl in A. aequalis. © 2022 Society of Chemical Industry.

Cytochrome P450 CYP709C56 metabolizing mesosulfuron-methyl confers herbicide resistance in Alopecurus aequalis.

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.

Comparative analysis of fungal communities between herbicide-resistant and -susceptible Alopecurus aequalis.

Introduction: Alopecurus aequalis is a grass species invading Chinese canola and wheat fields. An A. aequalis KMN-R population surviving mesosulfuron-methyl treatment with recommended rates was acquired from wheatland. Here, we aimed to confirm the resistance profiles of KMN-R to acetolactate synthetase (ALS) inhibiting herbicides and explore the possible resistance mechanisms to mesosulfuron-methyl in this weed population. Methods: The dose-response tests performed in our study were used to test the toxicity of A. aequalis to ALS-inhibiting herbicides. Sanger sequencing was used to analyze the ALS gene of mesosulfuron-methyl -resistant and -susceptible A. aequalis. RNA sequencing analysis was used to find candidate genes that may confer metabolic resistance to the mesosulfuron-methyl in resistant A. aequalis population. Mesosulfuron-methyl -resistant and -susceptible A. aequalis populations fungal composition was measured via Illumina MiSeq Sequencing. Results: Dose-response results indicated that KMN-R population evolved resistance to mesosulfuron-methyl and other tested ALS-inhibiting herbicides. Known resistance-conferring Trp-574-Leu gene mutation in A. aequalis ALS was detected in the KMN-R population. Pretreatment with 4-chloro-7-nitrobenzoxadiazole reversed mesosulfuron-methyl resistance in KMN-R. Glutathione S-transferases (GST) gene GSTZ2 and GSTT3 were highly expressed in KMN-R population. In addition, we evaluated the alpha diversity in A. aequalis, centering on OTU abundance, equality, and multiplicity, and found that the fungal community composition had more unexplained variance between KMN-R and KMN-S A. aequalis. We also observed higher abundances of specific fungi in KMN-R A. aequalis. Discussion: The results proved that resistance to mesosulfuron-methyl in A. aequalis KMN-R population is probably caused by target site- and non-target site-based relating GST and provided the basis for further research between fungal interaction and herbicide resistance.

Unravelling the effect of two herbicide resistance mutations on acetolactate synthase kinetics and growth traits.

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 widespread, 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.

Target site mutations and cytochrome P450s confer resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl in Alopecurus aequalis.

BACKGROUND: Shortawn foxtail (Alopecurus aequalis Sobol.) is a competitive grass weed infesting winter wheat- and canola-growing fields in China. In May 2016, a suspected A. aequalis resistant population AHTC-06 that survived fenoxaprop-P-ethyl and mesosulfuron-methyl applied at their field-recommended rates was collected from a wheat field in Jinji County, Anhui Province, China. This study aimed to determine the resistance profile of this AHTC-06 population to ACCase- and ALS-inhibitors, and to investigate its mechanisms of resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl. RESULTS: Two mutations, a common Ile-2041-Asn (ACCase gene) and a very rare Pro-197-Tyr (ALS1 gene), were both identified in resistant individual plants. The homozygous subpopulation AHTC-06F1 for the two mutations was generated, and it showed broad-spectrum resistance to APPs, DENs, and ALS-inhibiting herbicides of all five chemical families tested, with resistance index (RI) values that ranged from 2.2 to 36.5. In vitro ALS activity assays showed the ALS from the resistant population was insensitive to all the tested ALS inhibitors, with RI values ranging from 3.10 to 22.51. Pre-treatment with piperonyl butoxide (PBO) and malathion significantly (P < 0.05) reversed the weed's resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl, respectively. Two P450 genes, c21190_g1 and c43350_g3, were constitutively overexpressed and mesosulfuron-methyl-induced upregulated in resistant plants, for which c43350_g3 was also fenoxaprop-P-ethyl-induced upregulated. CONCLUSION: This study confirms the first case of a grass weed featuring broad-spectrum resistance to ALS-inhibiting herbicides due to a Pro-197-Tyr mutation in the ALS gene. Fenoxaprop-P-ethyl and mesosulfuron-methyl resistances in AHTC-06 plants were conferred by target site mutations and P450s-based metabolism. © 2018 Society of Chemical Industry.

Characterization of the complete plastome of Alopecurus aequalis (Poaceae), a widespread weed.

Alopecurus aequalis is a predominant weed species that distributes widely in North temperate regions. The complete plastome of A. aequalis is reported here. It is a circular molecular of 136,382 bp in length and consists of a large single-copy region (LSC: 80,455 bp), a small single-copy region (SSC: 12,849 bp), and two inverted repeats regions (IRs: 21,539 bp). GC content is 38.3%. This plastome encodes 112 unique genes, including 78 protein-coding genes, 30 tRNAs, and 4 rRNAs. Phylogenetic tree shows that A. aequalis is sister to Poa annua.

Acetolactate Synthase Overexpression in Mesosulfuron-Methyl-Resistant Shortawn Foxtail ( Alopecurus aequalis Sobol.): Reference Gene Selection and Herbicide Target Gene Expression Analysis.

Severe infestations of shortawn foxtail ( Alopecurus aequalis Sobol.), a noxious weed in wheat and canola cropping systems in China, remain standing even after the application of the herbicides, fenoxaprop- P-ethyl and mesosulfuron-methyl. Analysis of gene expression in weed plants subjected to herbicide treatment is a key step toward more mechanistic studies. Since such an analysis often involves quantitative real-time PCR (qRT-PCR), endogenous reference genes with stable expression are required. Herein, we identified specific gene sets, suitable as references for qRT-PCR data normalization in A. aequalis plants under different experimental conditions, using geNorm, NormFinder, BestKeeper, and RefFinder software. Additionally, the reliability of reference genes was verified by analyzing the expression of genes encoding two major herbicide target enzymes: acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS). Furthermore, functional ALS gene amplification was likely present in resistant plants, although it may make no contribution to the resistant phenotypes.

Fenoxaprop-P-ethyl and mesosulfuron-methyl resistance status of shortawn foxtail (Alopecurus aequalis Sobol.) in eastern China.

Resistance to the acetyl-coenzyme A carboxylase (ACCase)- and acetolactate synthase (ALS)- inhibiting herbicides in shortawn foxtail (Alopecurus aequalis) has been reported in wheat fields of eastern China. To better understand the distribution of the resistant populations and the occurrence of the target-site mutations, 74 populations collected from Anhui, Jiangsu and Shandong province were surveyed, and the ACCase and ALS gene fragments, encompassing all the documented mutant codon positions, were amplified and sequenced. Plants from 37 and 34 populations survived fenoxaprop-P-ethyl and mesosulfuron-methyl treatment at 62.1 g a.i. ha-1 and 9 g a.i. ha-1 respectively, with different survival rates. Twenty-seven populations exhibited multiple resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl. Whole-plant dose-response experiments showed that the resistance index ranged from 6.2 to 167.8 for fenoxaprop-P-ethyl, and from 7.8 to 139.5 for mesosulfuron-methyl. Four ACCase (I1781L, I2041N, I2041T and D2078G) and four ALS (P197R, P197S, P197T and W574 L) resistance mutations were detected respectively. Individuals containing two amino acid substitutions were also found. D2078G and W574 L were predominant ACCase and ALS gene mutations respectively. This study has shown that fenoxaprop-P-ethyl and mesosulfuron-methyl resistance was prevalent in A. aequalis in eastern China, and target site mutations in the ACCase and ALS gene were one of the most common mechanisms.

Multiple resistance to ACCase and AHAS-inhibiting herbicides in shortawn foxtail (Alopecurus aequalis Sobol.) from China.

Shortawn foxtail (Alopecurus aequalis) is a troublesome grass weed infesting winter wheat and oilseed rape productions in China. Fenoxaprop-p-ethyl and mesosulfuron-methyl failed to control shortawn foxtail of AHSX-1 population collected from a wheat field in Shou County, Anhui province. Molecular analyses revealed that Asp2078Gly mutation of ACCase and Trp574Leu mutation of AHAS were present in plants of the AHSX-1 population. The homozygous plants were isolated and cultured until seed maturity. Whole-plant herbicide bioassays were conducted in the greenhouse using the purified seeds of F1 generation. Dose-response experiments showed that the AHSX-1 population has evolved a very high level resistance to fenoxaprop-p-ethyl (RI = 275) and mesosulfuron-methyl (RI = 788). To determine the sensitivity to other herbicides, assays were conducted at the single recommended rate of each herbicide. Based on the results, the AHSX-1 population was considered to be highly resistant to clodinafop-propargyl, pyroxsulam and flucarbazone-sodium, moderately or highly resistant to quizalofop-p-ethyl, clethodim, sethoxydim and pinoxaden, and susceptible to isoproturon and chlorotoluron. This is the first report of Asp2078Gly mutation in shortawn foxtail and the two robust dCAPS markers designed could quickly detect Asp2078 and Trp574 mutations in ACCase and AHAS gene of shortawn foxtail, respectively.

Molecular basis of ALS- and/or ACCase-inhibitor resistance in shortawn foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis, a predominant weed species in wheat and oilseed rape fields, can no longer be controlled by mesosulfuron-methyl application after continuous use over several years. Based on dose-response studies, the putative resistant populations, JTJY-1 and JHHZ-1, were found to be resistant to mesosulfuron-methyl, with resistance index values of 5.5 and 14, respectively. Sensitivity assays of the mesosulfuron-methyl-resistant populations to other herbicides revealed that the JTJY-1 population had moderate or high cross resistance to sulfonylureas (SUs) and triazolopyrimidines (TPs), but displayed a low level resistance to imidazolinones (IMIs). JTJY-1 also had high multi-resistance to ACCase inhibitors, but remained susceptible to photosystem II inhibitors. The JHHZ-1 population was resistant to all ALS inhibitors tested, but was sensitive to ACCase inhibitors and photosystem II inhibitors. To clarify the molecular basis of resistance in JTJY-1 and JHHZ-1 population, the ALS and ACCase gene were sequenced. Two ALS mutations (Pro-197-Thr or Trp-574-Leu) were detected in the mesosulfuron-methyl-resistant plants. The ACCase gene analysis revealed that the resistant JTJY-1 population had an Ile-1781-Leu mutation. Furthermore, the presence of two different target site resistance (TSR) mechanisms (ALS and ACCase mutations) existing simultaneously in individual A. aequalis was firstly documented in the presented study.

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.).

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b5 in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.