Capsella bursa-pastoris (L.) Medik. has evolved resistance to ALS-inhibiting herbicides on a large scale. Previous studies primarily focused on the target-site resistance (TSR), and the non-TSR (NTSR) is not well characterized. In this study, pre-treatment with the cytochrome P450 monooxygenase (P450) inhibitor malathion clearly reduced the tribenuron-methyl resistance in the resistant (R) population. After tribenuron-methyl treatment, the glutathione S-transferase (GST) activity of R plants was significantly higher than that of susceptible (S) plants. The higher tribenuron-methyl metabolism in R plants was also confirmed by using LC–MS/MS analysis. Isoform sequencing (Iso-Seq) combined with RNA sequencing (RNA-Seq) was used to identify candidate genes involved in non-target metabolic resistance in this population. A total of 37 differentially expressed genes were identified, 11 of them constitutively upregulated in R plants, including three P450s, one GST, two glycosyltransferases, two ATP-binding cassette transporters, one oxidase, and two peroxidases. This study confirmed the metabolic tribenuron-methyl resistance in C. bursa-pastoris, and the transcriptome data obtained by Iso-Seq combined with RNA-Seq provide gene resources for understanding the molecular mechanism of NTSR in C. bursa-pastoris.

Aegilops tauschii Coss. is a malignant weed in wheat fields in China, its herbicide resistance has been threatening crop production. This study identified one mesosulfuron-methyl-resistant­(R) population, JJMHN2018-05 (R), without target resistance mutations. To fully understand the resistance mechanism, non-target site resistance was investigated by using transcriptome sequencing combined with a reference genome. Results showed that the cytochrome P450 monooxygenase (P450) inhibitor malathion significantly increased the mesosulfuron-methyl sensitivity in R plants, and greater herbicide-induced glutathione S-transferase (GST) activity was also confirmed. Liquid chromatography with tandem mass spectrometry analysis further supported the enhanced mesosulfuron-methyl metabolism in R plants. Gene expression data analysis and qRT-PCR validation indicated that eight P450s, six GSTs, two glycosyltransferases (GTs), four peroxidases, and one aldo-keto reductase (AKRs) stably upregulated in R plants. This research demonstrates that the P450s and GSTs involved in enhanced mesosulfuron-methyl metabolism contribute to mesosulfuron-methyl resistance in A. tauschii and identifies potential contributors from metabolic enzyme families.

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Amaranthus retroflexus L. is a highly competitive broadleaf weed of corn–soybean rotation in northeastern China. In recent years, the herbicide(s) resistance evolution has been threatening its effective management in crop fields. One resistant A. retroflexus (HW-01) population that survived the protoporphyrinogen oxidase (PPO) inhibitor fomesafen and acetolactate synthase (ALS) inhibitor nicosulfuron applied at their field-recommended rate was collected from a soybean field in Wudalianchi City, Heilongjiang Province. This study aimed to investigate the resistance mechanisms of fomesafen and nicosulfuron and determine the resistance profile of HW-01 to other herbicides. Whole plant dose–response bioassays revealed that HW-01 had evolved resistance to fomesafen (50.7-fold) and nicosulfuron (5.2-fold). Gene sequencing showed that the HW-01 population has a mutation in PPX2 (Arg-128-Gly) and a rare mutation in ALS (Ala-205-Val, eight/twenty mutations/total plants). In vitro enzyme activity assays showed that ALS extracted from the HW-01 plants was less sensitive to nicosulfuron (3.2-fold) than ST-1 plants. Pre-treatment with the cytochrome P450 inhibitors malathion, piperonyl butoxide (PBO), 3-amino-1,2,4-triazole (amitrole), and the GSTs inhibitor 4-chloro-7-nitrobenzofurazan (NBD-Cl) significantly increased fomesafen and nicosulfuron sensitivity in the HW-01 population compared with that of the sensitive (S) population ST-1. Moreover, the rapid fomesafen and nicosulfuron metabolism in the HW-01 plants was also confirmed via HPLC-MS/MS analysis. Furthermore, the HW-01 population showed multiple resistance (MR) to PPO, ALS, and PSII inhibitors, with resistance index (RI) values ranging from 3.8 to 9.6. This study confirmed MR to PPO-, ALS-, and PSII-inhibiting herbicides in the A. retroflexus population HW-01, as well as confirming that the cytochrome P450- and GST-based herbicide metabolic along with TSR mechanisms contribute to their multiple resistance to fomesafen and nicosulfuron.