Target‐site and non‐target‐site mechanisms confer multiple herbicide resistance in waterhemp ( Amaranthus tuberculatus ) accessions from Wisconsin

BACKGROUND: A preliminary screening identified a multiple herbicide-resistant waterhemp, Amaranthus tuberculatus (Moq.) Sauer, accession (A101) exhibiting resistance to 2,4-D and atrazine despite no prior exposure to these herbicides. Therefore, our objective was to characterize resistance to 2,4-D, atrazine, glyphosate, fomesafen, and mesotrione in A101, along with two additional multiple herbicide-resistant accessions (A75 and A103). RESULTS: A101 exhibited low to medium levels of resistance to all five herbicides evaluated (ranging from 1.8-fold for mesotrione to 8.5-fold for fomesafen). Both A75 and A103 also had multiple resistance to glyphosate and atrazine, with A75 and A103 additionally resistant to 2,4-D and fomesafen, respectively. Amplification of EPSPS and the P106S substitution accounted for some of the glyphosate resistance, and some of the fomesafen resistance was explained by the G210 deletion in the target enzyme. Moreover, the use of cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs) inhibitors indicated that non-target-site resistance (NTSR) mechanisms also contribute to at least some of the resistance traits. CONCLUSION: Metabolic resistance to 2,4-D and atrazine suggests that the use of other herbicides may have contributed to the selection of enhanced P450s and GSTs activity in A101 accession. To our knowledge, this is the first report of P450s associated with atrazine resistance in A. tuberculatus globally. A101 is the first confirmed case of A. tuberculatus resistance to hydroxyphenyl pyruvate dioxygenase inhibitors in Wisconsin, exhibiting a low-level resistance likely associated with P450s and GSTs activity. Our results suggest the coexistence of target-site resistance and NTSR mechanisms associated with glyphosate resistance in A101. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.