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.
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.
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.
Premise of research. Strong environmental selection pressures can lead to rapid adaptation and the opportunity to study evolutionary dynamics in real time. A prime example is the recent evolution of resistance to the herbicide glyphosate, the active ingredient in Roundup, in more than 35 weed species. Mechanisms for glyphosate resistance include gene amplification and overproduction of its target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), but little is known about whether these genetic changes are associated with differing fitness in glyphosate-free environments. Such fitness effects could have major implications for anticipated changes in the frequency of resistance traits without continued exposure to the selective pressure.Methodology. We used transgenic Arabidopsis thaliana as a model system to test for the effects of overproducing EPSPS on plant growth and reproduction. In a previous study, we developed six independent transgenic lines that overexpress a native EPSPS gene driven by the CaMV35S promoter (designated OX) and seven independent empty vector lines (designated EV). Here, we compared phenotypic traits among these lines and their wild-type parents in greenhouse experiments.Pivotal results. Two of the OX lines produced 23%-37% more seeds per plant than the wild-type line, respectively, and none showed evidence of a fitness penalty. In contrast, the performance of the EV lines was similar to, or somewhat worse than, that of the wild-type line. Despite considerable variation among lines, the OX lines had greater fecundity than the wild-type or EV lines overall. Conclusions.Our results suggest that overproduction of EPSPS in Arabidopsis does not have a fitness cost and might confer a fitness benefit under the examined growth conditions. Further basic research on how surplus EPSPS affects plant growth is warranted. We hypothesize that similar effects could occur in weed species that overproduce EPSPS, but the few studies that address this question have shown mixed results and no evidence for a fitness benefit to date.
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.
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