Comprehensive analysis of microtubule-inhibiting herbicides reveals shared subcellular phenotypes and distinct...

Herbicides that disrupt microtubules have historically been classified by the Herbicide Resistance Action Committee (HRAC) into microtubule assembly inhibitors (Group 3) and microtubule organization inhibitors (Group 23); however, this classification may not fully reflect their underlying modes of action.

Using a unified approach combining subcellular phenotyping in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) cells and resistance profiling in Eleusine indica and Chlamydomonas reinhardtii, we show that all tested herbicides produce indistinguishable, dose-dependent subcellular effects, including microtubule depolymerization, multipolar spindle formation, mitotic arrest, and multinucleation. At the resistance level, the α-tubulin T239I mutation confers strong resistance to all microtubule-inhibiting herbicides except the carbamates and propyzamide, whereas several β-tubulin mutations confer resistance to propyzamide.

These findings reveal distinct sites of action across chemical classes and demonstrate that resistance profiles - rather than subcellular phenotypes - provide a more robust basis for classification. Our results support redefining Group 3 as α-tubulin binders and relocating propyzamide to Group 23, now described as microtubule-interference herbicides with an unresolved site of action, strengthening the mechanistic foundation for improved classification and resistance-management strategies. © 2026 BASF Agricultural Solutions Deutschland GmbH. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.