The failure of PRE and POST applications of atrazine to control Palmer amaranth in recent field studies prompted further investigation to determine whether this population had evolved resistance to multiple herbicide sites of action, including glyphosate (Group 9), thifensulfuron (Group 2), and atrazine (Group 5). Greenhouse and laboratory experiments were conducted to: (1) confirm the presence of resistance to glyphosate, an ALS inhibitor (thifensulfuron), and atrazine in a single Palmer amaranth population; and (2) establish the molecular basis for resistance to these herbicide sites of action. In the greenhouse, glyphosate thifensulfuron atrazine at 1.26 kg ae ha^-1 0.0044 kg ai ha^-1 1.12 kg ai ha^-1 provided 55% control of the suspected multiply resistant (MR) Palmer amaranth population and 93% control of the known susceptible population (S). The decreased sensitivity of the MR population compared with the S population at labeled use rates of these herbicides indicated that this population was likely resistant to three different herbicide site of action groups. The RF values for POST applications of glyphosate, thifensulfuron, and atrazine were 12.2, 42.9, and 9.3 times, respectively, for the MR Palmer amaranth population relative to the S population. The RF value for atrazine PRE for the MR population was 112.2 times. Laboratory experiments confirmed that the mechanisms for resistance to ALS-inhibiting herbicides and glyphosate in the MR Palmer amaranth population were target-site based, via amino acid substitution and amplified EPSPS copy number, respectively. There was a Pro to Leu substitution at site 197 in the ALS inhibitor—resistant plants, and there was a greater than 50-fold increase in EPSPS copy number in the glyphosateresistant plants. There were no nucleotide changes in the psbA gene; therefore, atrazine resistance in this population was not target-site mediated. The evolution of this multiple herbicide-resistant Palmer amaranth population poses significant management challenges to Michigan farmers.

Nomenclature: Atrazine; glyphosate; thifensulfuron; Palmer amaranth; Amaranthus palmeri S. Wats.