Characterization and diagnostic tests of resistance to acetohydroxyacid synthase inhibitors due to an Asp 376Glu substitution in Amaranthus powellii

Abstract

A population of Amaranthus powellii that had survived imazethapyr did not have any of the documented AHAS mutations causing resistance in this species in Ontario. The goal of this research was to confirm resistance in this population, determine its molecular basis and develop diagnostic tests. Whole plant dose–response experiments showed the resistant population had greater than 25-fold resistance to the selective agent imazethapyr at the whole plant level. Whole plant resistance to other herbicides ranged between 9-fold to flumetsulam and 85-fold to flucarbazone. This was confirmed at the enzyme level with resistance ranging between 25-fold to imazethapyr and 485-fold to thifensulfuron. AHAS from the resistant population had 16% higher specific activity than that of the susceptible population and it was also less sensitive to feedback inhibition by valine, leucine and isoleucine. Genomic DNA was extracted to PCR amplify and sequence the AHAS gene and to determine the applicability of diagnostic tests. Sequencing of the AHAS gene revealed a single point mutation of thymine 1125 to guanine coding for an Asp 376Glu substitution. Two different diagnostic tests, restriction fragment length polymorphism and PCR amplification of specific allele, were able to correctly identify the resistant population from the susceptible control and from other resistant populations. Although this mutation appears to confer higher resistance to pyrithiobac and flucarbazone, two herbicides not registered in Eastern Canada, it endows plants with enough protection to allow survival to imazethapyr, the main selective agent in Ontario.