Target-site EPSPS Pro-106-Ser mutation in Conyza canadensis biotypes with extreme resistance to glyphosate in Ohio and Iowa, USA.

Documenting the diversity of mechanisms for herbicide resistance in agricultural weeds is helpful for understanding evolutionary processes that contribute to weed management problems. More than 40 species have evolved resistance to glyphosate, and at least 13 species have a target-site mutation at position 106 of EPSPS. In horseweed (Conyza canadensis), this p106 mutation has only been reported in Canada. Here, we sampled seeds from one plant (= biotype) at 24 sites in Ohio and 20 in Iowa, screened these biotypes for levels of resistance, and sequenced their DNA to detect the p106 mutation. Resistance categories were based on 80% survival at five glyphosate doses: S (0×), R1 (1×), R2 (8×), R3 (20×), or R4 (40×). The p106 mutation was not found in the19 biotypes scored as S, R1, or R2, while all 25 biotypes scored as R3 or R4 had the same proline-to-serine substitution at p106. These findings represent the first documented case of target-site mediated glyphosate resistance in horseweed in the United States, and the first to show that this mutation was associated with very strong resistance. We hypothesize that the p106 mutation has occurred multiple times in horseweed and may be spreading rapidly, further complicating weed management efforts.

No evidence for early fitness penalty in glyphosate-resistant biotypes of Conyza canadensis: Common garden experiments in the absence of glyphosate.

Strong selection from herbicides has led to the rapid evolution of herbicide-resistant weeds, greatly complicating weed management efforts worldwide. In particular, overreliance on glyphosate, the active ingredient in RoundUp®, has spurred the evolution of resistance to this herbicide in ≥40 species. Previously, we reported that Conyza canadensis (horseweed) has evolved extreme resistance to glyphosate, surviving at 40× the original 1× effective dosage. Here, we tested for underlying fitness effects of glyphosate resistance to better understand whether resistance could persist indefinitely in this self-pollinating, annual weed. We sampled seeds from a single maternal plant ("biotype") at each of 26 horseweed populations in Iowa, representing nine susceptible biotypes (S), eight with low-level resistance (LR), and nine with extreme resistance (ER). In 2016 and 2017, we compared early growth rates and bolting dates of these biotypes in common garden experiments at two sites near Ames, Iowa. Nested ANOVAs showed that, as a group, ER biotypes attained similar or larger rosette size after 6 weeks compared to S or LR biotypes, which were similar to each other in size. Also, ER biotypes bolted 1-2 weeks earlier than S or LR biotypes. These fitness-related traits also varied among biotypes within the same resistance category, and time to bolting was inversely correlated with rosette size across all biotypes. Disease symptoms affected 40% of all plants in 2016 and 78% in 2017, so we did not attempt to measure lifetime fecundity. In both years, the frequency of disease symptoms was greatest in S biotypes and similar in LR versus ER biotypes. Overall, our findings indicate there are no early growth penalty and possibly no lifetime fitness penalty associated with glyphosate resistance, including extremely strong resistance. We conclude that glyphosate resistance is likely to persist in horseweed populations, with or without continued selection pressure from exposure to glyphosate.

High Levels of Glyphosate Resistance in Conyza canadensis from Agricultural and Non-Agricultural Sites in Ohio and Iowa.

Glyphosate is an important herbicide worldwide, but its efficacy has been compromised where weed species have evolved glyphosate resistance. To better understand evolutionary outcomes of continued and strong selection from glyphosate exposure, we characterized variation in resistance in self-pollinating Conyza canadensis (horseweed) in Ohio and Iowa, where glyphosate resistance was first reported in 2002 and 2011, respectively. In 2015, we collected seeds from a total of 74 maternal plants (biotypes) from no-till soybean fields vs. non-agricultural sites in each state, using one representative plant per site. Young plants from each biotype were sprayed with glyphosate rates of 0x, 1x (840 g ae ha-1), 8x, 20x, or 40x. Resistant biotypes with at least 80% survival at each dosage were designated as R1 (1x), R2 (8x), R3 (20x), or R4 (40x). Nearly all Ohio agricultural biotypes were R4, as were 62% of biotypes from the non-agricultural sites. In Iowa, R4 biotypes were clustered in the southeastern soybean fields, where no-till agriculture is more common, and 45% of non-agricultural biotypes were R1-R4. Our results show that resistance levels to glyphosate can be very high (at least 40x) in both states, and that non-agricultural sites likely serve as a refuge for glyphosate-resistant biotypes.

Effects of over-expressing a native gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) on glyphosate resistance in Arabidopsis thaliana.

Widespread overuse of the herbicide glyphosate, the active ingredient in RoundUp®, has led to the evolution of glyphosate-resistant weed biotypes, some of which persist by overproducing the herbicide's target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the shikimic acid pathway for biosynthesis of aromatic amino acids, lignin, and defensive compounds, but little is known about how overproducing EPSPS affects downstream metabolites, growth, or lifetime fitness in the absence of glyphosate. We are using Arabidopsis as a model system for investigating phenotypic effects of overproducing EPSPS, thereby avoiding confounding effects of genetic background or other mechanisms of herbicide resistance in agricultural weeds. Here, we report results from the first stage of this project. We designed a binary vector expressing a native EPSPS gene from Arabidopsis under control of the CaMV35S promoter (labelled OX, for over-expression). For both OX and the empty vector (labelled EV), we obtained nine independent T3 lines. Subsets of these lines were used to characterize glyphosate resistance in greenhouse experiments. Seven of the nine OX lines exhibited enhanced glyphosate resistance when compared to EV and wild-type control lines, and one of these was discarded due to severe deformities. The remaining six OX lines exhibited enhanced EPSPS gene expression and glyphosate resistance compared to controls. Glyphosate resistance was correlated with the degree of EPSPS over-expression for both vegetative and flowering plants, indicating that glyphosate resistance can be used as a surrogate for EPSPS expression levels in this system. These findings set the stage for examination of the effects of EPSPS over-expression on fitness-related traits in the absence of glyphosate. We invite other investigators to contact us if they wish to study gene expression, downstream metabolic effects, and other questions with these particular lines.