Glutathione S-transferase genes PfGSTU6 and PfGSTL1 involved in mesosulfuron-methyl resistance in Polypogon fugax.

TSR and peroxidase genes confer resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl in Alopecurus aequalis.

BACKGROUND Alopecurus aequalis poses severe threat to global wheat production due to evolving resistance to acetyl-CoA carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides. In this study, the resistance mechanisms of a field-evolved resistant population (R) were systematically investigated using dose-response bioassays, target-site gene sequencing, inhibitor assays, antioxidant enzyme activity measurements, RNA sequencing (RNA-seq), quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and yeast functional validation. RESULTS Dose-response results revealed that the R population exhibited moderate resistance to fenoxaprop-P-ethyl (RI = 9.58) and low-level resistance to mesosulfuron-methyl (RI = 3.07). Cross-resistance testing indicated that the R population was resistant to other ACCase-inhibiting herbicides (haloxyfop-P-methyl, clodinafop-propargyl, clethodim, and pinoxaden) and the ALS-inhibiting herbicide rimsulfuron. Target-site sequence analysis identified two mutations in the R population: Ile-1781-Leu (ACCase) and Pro-197-Ser (ALS1). Pretreatment with the cytochrome P450 and GST inhibitor did not reverse resistance to fenoxaprop-P-ethyl or mesosulfuron-methyl. Compared to the susceptible (S) population, the R population had significantly lower H2O2 content and higher activities of peroxidase (POD) and catalase (CAT), indicating an enhanced reactive oxygen species (ROS) scavenging capacity. RNA-seq and qRT-PCR analyses identified three POD-annotated contigs (PODSPC4, POD12-1, POD12-2) that were upregulated in the R population. Yeast heterologous expression validated that AaPOD12-1 and AaPOD12-2 significantly increased yeast resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl. CONCLUSION These results demonstrate that resistance in the R population is co-mediated by target-site mutations and non-target-site resistance involving enhanced ROS scavenging, with AaPOD12-1 and AaPOD12-2 representing the first functionally characterized antioxidant enzyme genes associated with herbicide resistance in A. aequalis. © 2026 Society of Chemical Industry.

Characterization of ALS Inhibitor Resistance in Alopecurus myosuroides : Primary Role of Trp574Leu and Secondary Contributions of Additional Mutations and Metabolism

Blackgrass is a highly competitive weed in wheat fields and has increasingly evolved resistance to acetolactate synthase (ALS)-inhibiting herbicides. Ten field populations were screened, resulting in the selection of one highly resistant population (R-06) and one susceptible population (S-19) for detailed study. Whole-plant bioassays, ALS gene sequencing, molecular docking, metabolic inhibitor assays, glutathione S-transferase (GST) and ALS activity assays, and cross-resistance profiling were conducted to dissect the mechanisms of resistance. ALS sequencing identified three amino acid substitutions in R-06: Pro232Thr (P232T), His363Lys (H363K), and Trp574Leu (W574L). While W574L is a well-characterized ALS resistance-conferring mutation, P232T and H363K are outside major resistance hotspots and may serve secondary or compensatory roles. Molecular docking analyses predicted altered binding of mesosulfuron-methyl in the mutant ALS model, consistent with structural changes in the binding pocket. Metabolic inhibitor assays using malathion, piperonyl butoxide (PBO), and NBD-Cl resulted in modest increases in herbicide sensitivity, with a maximum reduction factor of 2.95 for PBO. GST activity was higher in R-06 at a single sampling time point (3 DAT). In addition, R-06 showed reduced sensitivity to several ALS inhibitors and reduced efficacy against selected herbicides with alternative modes of action. High-level resistance to mesosulfuron-methyl in the R-06 population is primarily associated with the ALS target-site mutation Trp574Leu, while the roles of Pro232Thr, His363Lys, and metabolism appear secondary and remain unresolved. These findings highlight complex resistance patterns in blackgrass and emphasize the need for diversified and integrated weed management strategies.

Expression of cytochrome P450 (CYP96A146) from flixweed (Descurainia sophia) in Saccharomyces cerevisiae and metabolism of model substrates and herbicides

Abstract It is well established that cytochrome P450 monooxygenases (P450s) play a crucial role in herbicide metabolism and resistance evolution in weeds. In most documented cases, P450-mediated resistance is primarily conferred through the overexpression of P450 enzymes. However, the regulatory mechanisms underlying this overexpression remain poorly understood. In insects, amino acid substitutions that enhance P450-mediated metabolic detoxification have been clearly demonstrated as a key mechanism of insecticide resistance. In contrast, their potential role in herbicide resistance in weeds remains unclear. In this study, two CYP96A146 variants from flixweed [Descurainia sophia (L.) Webb ex Prantl], designated CYP96A146-S and CYP96A146-R, were heterologously expressed in Saccharomyces cerevisiae. These variants, which differ by four amino acid residues, were examined for their ability to metabolize model substrates and herbicides. The results indicated that both variants exhibited catalytic activity toward model substrates of p-nitroanisole, methoxyresorufin, ethoxyresorufin, 7-ethoxycoumarin, and benzo[a]pyrene, as well as toward the herbicides tribenuron-methyl, bensulfuron-methyl, and carfentrazone-ethyl. Notably, CYP96A146-R showed significantly higher catalytic activity than CYP96A146-S to both the model substrates (p-nitroanisole, methoxyresorufin, ethoxyresorufin, and 7-ethoxycoumarin) and the herbicides (tribenuron-methyl and carfentrazone-ethyl). These findings suggest that the amino acid substitutions are likely responsible for the enhanced metabolic capability of CYP96A146-R. Such mutations may induce conformational changes of CYP96A146 enzyme, facilitating more frequent molecular collisions between CYP96A146 and the substrates or herbicides, thereby improving catalytic efficiency.

Distinct P450-mediated resistance mechanisms of flupyrimin in neonicotinoid-resistant Nilaparvata lugens

Resistance to protoporphyrinogen oxidase inhibitor herbicides in giant ragweed (Ambrosia trifida) is associated with a novel R98Q target-site mutation in PPO2

Identification and functional validation of glutathione S-transferase genes involved in detoxification of sulfoxaflor, afidopyropen and lambda-cyhalothrin in Aphis glycines.

BACKGROUND Glutathione S-transferases (GSTs) play important roles in the detoxification of insecticides in insects. However, genome-wide identification and functional characterization of the GST gene family in the soybean aphid Aphis glycines have not been performed. RESULTS A total of 17 AgGST genes were identified in the A. glycines genome and classified into five classes. Phylogenetic analysis and chromosomal mapping showed that delta and epsilon class genes experienced significant expansion. Exposure to LC₅₀ concentrations of sulfoxaflor, afidopyropen and lambda-cyhalothrin strongly induced several AgGST genes with AgGSTd5, AgGSTd6 and AgGSTe2 displaying the highest expression levels. RNA interference of AgGSTd5 significantly increased aphid mortality following exposure to all three insecticides. Knockdown of AgGSTd6 significantly elevated mortality under sulfoxaflor, while knockdown of AgGSTe2 significantly increased mortality under both sulfoxaflor and lambda-cyhalothrin. In contrast, silencing of AgGSTt1 and AgGSTt2 showed no significant effect on aphid mortality under the tested insecticides. CONCLUSION This study provides comprehensive characterization of the GST gene family in A. glycines and demonstrates that AgGSTd5 plays a central role in the detoxification of sulfoxaflor, afidopyropen and lambda-cyhalothrin, while AgGSTd6 and AgGSTe2 contribute to tolerance against specific insecticides among the three compounds. These genes represent promising molecular targets for monitoring insecticide detoxification responses and for the development of strategies based on GST inhibitors to enhance insecticide efficacy in integrated pest management. © 2026 Society of Chemical Industry.