Conyza bonariensis resistance to glyphosate and ALS inhibitors involves target and non-target site resistance.

Herbicide resistance in Conyza bonariensis (hairy fleabane) poses a significant challenge to agricultural systems worldwide. The genetic variability and prolific seed production of this species contribute significantly to its adaptative potential and fast spread in the agricultural fields. This study aimed to investigate the mechanisms underlying multiple herbicide resistance to glyphosate and ALS inhibitors in C. bonariensis biotypes from Southern Brazil. Resistance factors exceeded 100 times for chlorimuron-ethyl and 49 times for glyphosate. DNA Sequencing revealed the target-site mutations Pro106Thr in the EPSPS gene conferring glyphosate resistance, and Pro197Arg and Trp574Leu in the ALS gene contributing to chlorimuron-ethyl resistance. Additionally, the resistance factor decreased at least 80 % for resistant biotypes after application of chlorimuron-ethyl following treatment with the P450 inhibitor malathion, which might indicate enhanced metabolism mediated by cytochrome P450 enzymes. Copy number variation and overexpression of ALS and EPSPS genes were not related to resistance. Biotype II carries the Pro197Arg mutation and exhibited cross-resistance to imazethapyr, diclosulam, bispyribac‑sodium, and flucarbazone‑sodium. Biotypes carrying the Trp574Leu mutation were resistant to imazethapyr, diclosulam and flucarbazone‑sodium but demonstrated varying resistance patterns to bispyribac‑sodium, highlighting the complexity of resistance mechanisms. These findings underscore the importance of understanding both target and non-target-site resistance mechanisms to develop effective management strategies, including herbicide rotation and molecular diagnostics, to mitigate the spread of herbicide-resistant C. bonariensis in agricultural systems.