Physiological and molecular analysis of glyphosate resistance in non-rapid response Ambrosia trifida from Wisconsin

Abstract

Background: We previously identified a glyphosate-resistant A. trifida phenotype from Wisconsin USA that showed a non-rapid response to glyphosate. The mechanism of glyphosate resistance in this phenotype has yet to be elucidated. We conducted experiments to investigate non-target-site resistance and target-site resistance mechanisms. The roles of glyphosate absorption, translocation, and metabolism in resistance of this phenotype have not been reported previously, nor have EPSPS protein abundance or mutations to the full-length sequence of EPSPS.

Results: Whole-plant dose-response results confirmed a 6.5-level of glyphosate resistance for the resistant (R) phenotype compared to a susceptible (S) phenotype. Absorption and translocation of ¹⁴ C-glyphosate were similar between R and S phenotypes over 72 h. Glyphosate and AMPA concentrations in leaf tissue did not differ between R and S phenotypes over 96 h. In vivo shikimate leaf disc assays confirmed that glyphosate EC₅₀ values were 4.6- to 5.4-fold greater for the R than S phenotype. Shikimate accumulation was similar between phenotypes at high glyphosate concentrations (>1000 μM), suggesting that glyphosate entered chloroplasts and inhibited EPSPS. This finding was supported by results showing that EPSPS copy number and EPSPS protein abundance did not differ between R and S phenotypes, nor did EPSPS sequence at Gly101, Thr102, and Pro106 positions. Comparison of full-length EPSPS sequences found five nonsynonymous polymorphisms that differed between R and S phenotypes. However, their locations were distant from the glyphosate target site and, therefore, not likely to affect enzyme-glyphosate interaction.

Conclusion: The results suggest that a novel mechanism confers glyphosate resistance in this A. trifida phenotype. © 2019 Society of Chemical Industry.

Keywords: 5-enolpyruvylshikimate-3-phosphate synthase; dose-response; glyphosate metabolism; non-target-site resistance; resistance mechanism; shikimate accumulation; target-site resistance.