Introduction
Horseweed is an annual weed that is native throughout most of North and Central America (Weaver Reference Weaver2001). Horseweed does not have dormancy requirements, which allows for germination and establishment shortly after seed release from the mother plant (Buhler and Owen Reference Buhler and Owen1997). Fall-germinated seeds develop a winter annual growth pattern, forming rosettes that overwinter, followed by stem elongation in the spring. Seeds that germinate in the spring bypass the rosette stage, and stem elongation commences shortly after emergence (Weaver Reference Weaver2001). Horseweed’s extended and variable emergence pattern makes control difficult, because weed size at application is variable (Buhler and Owen Reference Buhler and Owen1997; Kruger et al. Reference Kruger, Davis, Weller and Johnson2010a). Horseweed has been reported to produce as many as 230,000 seeds/plant (Weaver Reference Weaver2001). The seeds germinate close to the soil surface with a maximum seedling recruitment depth of 0.5 cm, making this weed well adapted to no-till cropping systems (Nandula et al. Reference Nandula, Eubank, Poston, Koger and Reddy2006). Horseweed seed is wind dispersed with the majority of seed falling within 100 m of the source (Dauer et al. Reference Dauer, Mortensen and VanGessel2007).
Glyphosate-resistant (GR) horseweed was fist reported in 2001 in Delaware, in the United States (VanGessel Reference VanGessel2001). In Ontario, it was first identified in 2010 in Essex County, and as of 2015, it has been found across southern Ontario (Budd et al. Reference Budd, Soltani, Robinson, Hooker, Miller and Sikkema2017; Byker et al. Reference Byker, Soltani, Robinson, Tardif, Lawton and Sikkema2013a). GR horseweed can interfere with soybean growth and development and substantially reduce yield. Both Bruce and Kells (Reference Bruce and Kells1990) and Byker et al. (Reference Byker, Soltani, Robinson, Tardif, Lawton and Sikkema2013b) reported soybean yield reductions of >90% with horseweed densities of 100 to 200 plants per square meter. Herbicides applied POST do not provide adequate control of GR horseweed in GR soybean (Byker et al. Reference Byker, Soltani, Robinson, Tardif, Lawton and Sikkema2013c). Therefore, GR horseweed control should be accomplished using preplant (PP) herbicides with residual activity (Bruce and Kells Reference Bruce and Kells1990). Burndown/residual herbicides applied prior to soybean emergence is recommended for control of fall- and spring-emerged GR horseweed (Budd et al. Reference Budd, Soltani, Robinson, Hooker, Miller and Sikkema2016; Davis and Johnson Reference Davis and Johnson2008).
Pyraflufen-ethyl/2,4-D is a preformulated herbicide sold under the trade name BlackHawk® that consists of pyraflufen-ethyl (6.1 g ai L−1) and 2,4-D ester (473 g ae L−1; Anonymous 2019). Pyraflufen-ethyl/2,4-D is registered for emerged broadleaf weed control prior to emergence of soybean (Anonymous 2019). Susceptible plants often display foliar necrosis prior to plant death (Mabuchi et al. Reference Mabuchi, Miura and Ohtsuka2002). Only a limited amount of published research exists on GR horseweed control with pyraflufen-ethyl/2,4-D; one study reported inconsistent and generally unsatisfactory control (60%) of GR horseweed in a soybean crop (Soltani et al. Reference Soltani, Shropshire and Sikkema2020a). As a member of the phenoxy carboxylic acids, 2,4-D is a commonly used synthetic auxin, or growth regulator, herbicide for broadleaf weed control. Eubank et al. (Reference Eubank, Poston, Nandula, Koger, Shaw and Reynolds2008) indicated that at 4 wk after application (WAA), 2,4-D + glyphosate (840 + 860 g ha−1) controlled GR horseweed by 99% in 2005 and by 95% in 2006. Kruger et al. (Reference Kruger, Davis, Weller and Johnson2010a) found that 2,4-D controlled 30-cm-tall horseweed by 90% to 97%. In Ontario, 2,4-D (500 g ha−1) applied PP to soybean provided variable GR horseweed control (from 53% to 92%) at 8 WAA (Byker et al. Reference Byker, Soltani, Robinson, Tardif, Lawton and Sikkema2013d; Soltani et al. Reference Soltani, Shropshire and Sikkema2020b). Glyphosate/2,4-D choline applied PP at 1,720 and 3,440 g ha−1 provided up to 79% and 100% GR horseweed control (8 WAA) in corn, respectively (Ford et al. Reference Ford, Soltani, McFadden, Nurse, Robinson and Sikkema2014).
Metribuzin is classified as a photosystem II–inhibitor. Metribuzin is commonly used in combination with other herbicides at a PP timing in soybean at a rate of 400 g ai ha−1 (Budd et al. Reference Budd, Soltani, Robinson, Hooker, Miller and Sikkema2016; Soltani et al. Reference Soltani, Jhala, Shropshire and Sikkema2016). The co-application of metribuzin with saflufenacil; glufosinate; pyraflufen-ethyl/2,4-D; S-metolachlor/dicamba; and 2,4-D was shown to provide >80% control of GR horseweed in soybean (Budd et al. Reference Budd, Soltani, Robinson, Hooker, Miller and Sikkema2016; Eubank et al. Reference Eubank, Poston, Nandula, Koger, Shaw and Reynolds2008; Soltani et al. Reference Soltani, Shropshire and Sikkema2020a). Soltani et al. (Reference Soltani, Shropshire and Sikkema2020a) indicated that 2,4-D ester (500 g ha−1) or pyraflufen-ethyl/2,4-D (532 ai ha−1) mixed with metribuzin (400 g ha−1) can control GR horseweed by 91% and 95%, respectively.
Little published information exists on the biological activity of pyraflufen-ethyl/2,4-D, applied by itself, or in mixture with metribuzin for GR horseweed control. The purpose of this research was to assess the biologically effective dose (BED) of pyraflufen-ethyl/2,4-D applied by itself, and in mixture with metribuzin for GR horseweed control in soybean.
Materials and Methods
Five experiments (in a randomized complete block design) were established in Ontario over 2 yr (2019, 2020) in commercial fields with confirmed GR horseweed populations. Plots were 2.25 m by 8 m (three soybean rows 0.75 m apart). To determine the BED, pyraflufen-ethyl/2,4-D was applied at doses of 66, 132, 264, 527, 1,054, and 2,108 g ha−1; and to determine the dose of pyraflufen-ethyl/2,4-D plus metribuzin, pyraflufen-ethyl/2,4-D was applied at the aforementioned doses with metribuzin at 400 g ha−1. Pyraflufen-ethyl/2,4-D rates were selected to include a range of doses surrounding the current label rate in Ontario of 527 g ai ha−1. Saflufenacil + metribuzin (25 + 400 g ai ha−1) and glyphosate/dicamba + saflufenacil (1,200/600+ 25 g ai ha−1) were included as the current industry standards for the control of GR horseweed in identity-preserved and GR soybean and glyphosate/dicamba-resistant soybean, respectively (Budd et al. Reference Budd, Soltani, Robinson, Hooker, Miller and Sikkema2016a; Hedges et al. Reference Hedges, Soltani, Robinson, Hooker and Sikkema2018). Glyphosate (900 g ha−1) was applied with all treatments at the time of application to eliminate all other weed species and non-GR horseweed plants. A CO2-pressurized backpack sprayer was used for all herbicide treatment applications. The sprayer was set to 260 kPa to deliver a water volume of 200 L ha−1. The spray boom was equipped with four ULD 11002 nozzles spaced 50 cm apart producing a 2-m spray width. Treatments were applied once the diameter or height of GR horseweed had reached 10 cm. Herbicides were applied PP with soybean being planted 1 to 10 d after herbicide applications. Glyphosate/dicamba-resistant soybean (DKB 12-16) was planted using a no-till planter at a rate of 416,000 seeds ha−1. Experiment details are presented in Table 1.
Year, location, application information, crop information, and soil characteristics for five field experiments.
a Abbreviation: SOM, soil organic matter of the upper 15 cm of the soil profile.
b Size measured as height or diameter of plant on day of application. Mean of two measurements per replication.
c Mean density at time of application based on eight stand counts per experiment in the non-treated control.
Soybean injury was assessed visually at 1, 2, and 4 wk after soybean emergence (WAE) on a rating scale of 0% to 100% (where 0% = no injury; 100% = plant death). GR horseweed control was assessed visually on a 0% to 100% scale (where 0% = no control; 100% = complete control) at 2, 4, and 8 WAA. Horseweed density and shoot dry weight (biomass) were collected at 8 WAA and expressed as plants per square meter and grams per square meter, respectively. Horseweed density was determined by counting GR horseweed plants within two 0.25-m2 quadrats per plot, drying to a constant moisture and weighing. Soybean yield was collected upon harvest maturity by combining the center two rows of each plot. Soybean moisture and weight were recorded. Soybean moisture content was adjusted to 13.5%.
Nonlinear Regression—Exponential to Maximum
Data from the dose-response bioassay of pyraflufen-ethyl/2,4-D and pyraflufen-ethyl/2,4-D + metribuzin were analyzed using nonlinear regression (PROC NLIN) in SAS software, version 9.4 (SAS Institute, Cary, NC). Root mean square error and modeling efficiency coefficient were calculated and used to determine the goodness of fit of the models as suggested by Sarangi et al. (Reference Sarangi, Irmak, Lindquist, Knezevic and Jhala2016) for nonlinear regressions. An exponential to maximum model (Equation 1) was used to regress the control of GR horseweed at 2, 4, and 8 WAA, and soybean yield against pyraflufen-ethyl/2,4-D and pyraflufen-ethyl/2,4-D + metribuzin. Similarly, weed density and shoot dry weight were regressed using an inverse exponential model (Equation 2). The parameters derived from each regression analysis were used to calculate the BED of pyraflufen-ethyl/2,4-D and pyraflufen-ethyl/2,4-D + metribuzin that resulted in 50%, 80%, and 95% control in density reduction, shoot dry weight reduction, and soybean yield reduction (R50, R80, R95, respectively). Where a dose was calculated to be greater than the highest dose evaluated in this study (>2,108 g ha−1) it was reported as not estimable.
Exponential to maximum
$Y = a - b (e ^{-c\ Dose}) $
where a is the upper asymptote, b is the magnitude, and c is the slope.
Inverse exponential
$Y = a + b (e ^{-c\ Dose}) $
where a is the lower asymptote, b is the reduction in y from intercept to asymptote, and C is the slope.
Least-Square Means Comparisons
The data were averaged for all five site-years for analysis to compare pyraflufen-ethyl/2,4-D and pyraflufen-ethyl/2,4-D + metribuzin to the current industry standards (saflufenacil + metribuzin and glyphosate/dicamba + saflufenacil) in Ontario, Canada. The GLIMMIX procedure in SAS 9.4 was used to analyze data with least-square means being separated using a Tukey-Kramer multiple-range test (α = 0.05). Herbicide was considered the fixed effect. Environment, replication within environment, and the treatment-by-environment interaction were considered the random effects. The Shapiro-Wilks test of normality along with visual assessments of studentized residuals and scatterplots were used to confirm the assumptions of normality. All visible GR horseweed control and yield followed a normal distribution. The shoot dry weight and density data followed a log-normal distribution and was back transformed for presentation using the omega method for back-transformation.
To determine the interactions of pyraflufen-ethyl/2,4-D and metribuzin, expected values for control, density, and shoot dry weight were calculated using the Colby Equations (Colby Reference Colby1967). The TTEST procedure in SAS 9.4 was used to compare the observed and expected values. If the observed and expected values were not significantly different at P < 0.05 or P < 0.01, the interaction was considered additive. The interaction was considered antagonistic or synergistic if there was a significant difference between the expected and observed values at P < 0.05 or P < 0.01.
Colby’s Equation – Visible Weed Control
$E= X+Y-(XY/100) $
where E = expected control with pyraflufen-ethyl/2,4-D + metribuzin; X = observed control with pyraflufen-ethyl/2,4-D; and Y = observed control with metribuzin.
Colby’s Equation: Density and Shoot dry weight
$E = (XY/Z) $
where E = expected density or shoot dry weight with pyraflufen-ethyl/2,4-D + metribuzin; X = observed density or shoot dry weight pyraflufen-ethyl/2,4-D; Y = observed density or shoot dry weight of metribuzin; and Z = density or shoot dry weight of the control.
Results and Discussion
Soybean Injury
None of the treatments evaluated caused >15% visible injury. Pyraflufen-ethyl/2,4-D (2,108 g ha−1) caused the greatest soybean injury at 13% and 9% at 1 and 4 WAE, respectively. The co-application of metribuzin to pyraflufen-ethyl/2,4-D did not increase soybean injury. The registered rate of pyraflufen-ethyl/2,4-D (527 g ha−1) resulted in 5% and 1% soybean injury at 1 and 4 WAE, respectively. Similarly, pyraflufen-ethyl/2,4-D + metribuzin at 527 + 400 g ha−1 resulted in 3% and 1% injury at 1 and 4 WAE, respectively. Soybean injury consisted of stunting, twisting of the stems, and leaf strapping consistent with typical 2,4-D injury.
Biologically Effective Dose of Pyraflufen-Ethyl/2,4-D and Pyraflufen-Ethyl/2,4-D + Metribuzin for the Control of GR Horseweed
Pyraflufen-ethyl/2,4-D
The calculated doses of pyraflufen-ethyl/2,4-D to provide 50%, 80%, and 95% control of GR horseweed control were 185, 568, and >2,108 g ha−1, respectively, at 2 WAA (Table 2). At 4 and 8 WAA, there was an increase in the calculated doses required for 50%, 80%, and 95% control (Table 2). At 8 WAA, the calculated doses for a 50%, 80%, and 95% decrease in density were 876, 1,793, and > 2,108 g ha−1, and shoot dry weight was 934, 1,851, and >2,108 g ha−1, respectively. The calculated doses of <66, 516, and 988 g ha−1 were required to provide soybean yield of 50%, 80%, and 95% relative to the industry standard, respectively.
Parameter estimates and calculated effective doses of pyraflufen-ethyl/2,4-D and pyraflufen-ethyl/2,4-D plus metribuzin for 50%, 80%, and 95% glyphosate-resistant horseweed control, reduction in shoot dry weight and density, and yield from five experiments.e
a Regression parameters: Y = a − b (e–C Dose); a is the upper asymptote, b is the magnitude, and c is the slope.
b Regression parameters: Y = a + b (e–C Dooe); a is the lower asymptote, b is the reduction in y from intercept to asymptote, and c is the slope.
c Greater than the highest dose evaluated in this study (>2,108 g ai ha−1) or could not be calculated by the model.
d Expressed as percent of yield in the industry standard (dicamba + saflufenacil (1,800 + 25 g ai ha−1)) among replications.
e Abbreviation: Non-est., not estimable.
Pyraflufen-ethyl/2,4-D is a relatively new preformulated herbicide mixture; however, similar rates of pyraflufen and 2,4-D, when applied alone, have provided comparable control of GR horseweed. Based on the concentrations of pyraflufen (6.1 g L−1) and 2,4-D ester (473 g ae L−1) in BlackHawk®, the calculated dose of 1,148 g ha−1 for 80% GR horseweed control consists of 15 g ha−1 of pyraflufen and 1,133 g ha−1 of 2,4-D ester. Pyraflufen (8 g ha−1) was shown to provide acceptable GR horseweed control in Europe (Tahmasebi et al. Reference Tahmasebi, Alebrahim, Roldán-Gómez, da Silveira, de Carvalho, Alcántara-de la Cruz and De Prado2018). Kruger et al. (Reference Kruger, Davis, Weller and Johnson2010a) calculated the dose of 2,4-D ester to control GR horseweed by 90% to range from 316 to 684 g ha−1. Byker et al. (Reference Byker, Soltani, Robinson, Tardif, Lawton and Sikkema2013d) found that 2,4-D ester + glyphosate (500 + 900 g ha−1) controlled GR horseweed by 78% to 92% 4 WAA, depending on location. Soltani et al. (Reference Soltani, Shropshire and Sikkema2020a, Reference Soltani, Shropshire and Sikkema2020b) found that 2,4-D ester applied at 500 g ha−1 can control GR horseweed by 53% to 68% at 8 WAA.
At 8 WAA, the calculated doses of pyraflufen-ethyl/2,4-D for 50% control of GR horseweed and 50% reduction in GR horseweed density and shoot dry weight were 390, 876, and 934 g ha−1, respectively. There was regrowth of GR horseweed following application of pyraflufen-ethyl/2,4-D, which may have contributed the higher calculated dose for shoot dry weight reduction. Regrowth of injured plants after 2,4-D application was noted by Zimmer et al. (Reference Zimmer, Young and Johnson2018); surviving plants in some scenarios were able to produce seed (Kruger et al. Reference Kruger, Davis, Weller and Johnson2010b). The calculated doses of pyraflufen-ethyl/2,4-D that provided 80% and 95% soybean yield compared to the industry standard were 516 and 988 g ha−1, respectively (Table 2).
Pyraflufen-ethyl/2,4-D + Metribuzin Dose Response
Metribuzin mixed with pyraflufen-ethyl/2,4-D improved GR horseweed control relative to pyraflufen-ethyl/2,4-D alone. At 2 WAA, the calculated doses of pyraflufen-ethyl/2,4-D + metribuzin to provide 50%, 80%, and 95% GR horseweed control were 17, 42, and 145 g ha−1, respectively (Table 2). At 8 WAA, the calculated doses of pyraflufen-ethyl/2,4-D + metribuzin increased to 19, 46, and 201 g ha−1 for 50%, 80%, and 95% control, respectively. The calculated doses of pyraflufen-ethyl/2,4-D to provide 50%, 80%, and 95% decrease in density and shoot dry weight were 15, 34, and 64 g ha−1; and 26, 62, and 115 g ha−1, respectively. The doses of 1 and 2 g ha−1 of pyraflufen-ethyl/2,4-D + metribuzin were needed to provide soybean yields that were 80% and 95% of the industry standard, respectively.
Pyraflufen-Ethyl/2,4-D, Metribuzin, Pyraflufen-Ethyl/2,4-D + Metribuzin vs. the Industry Standards
GR horseweed was controlled by 59% to 69% with pyraflufen-ethyl/2,4-D at 527 g ai ha−1 and by 75% to 78% with metribuzin (Table 3). At 8 WAA, metribuzin alone controlled GR horseweed by 75%, which was greater than 59% control provided by pyraflufen-ethyl/2,4-D. Soltani et al. (Reference Soltani, Shropshire and Sikkema2020a) found that pyraflufen-ethyl/2,4-D applied at 532 g ha−1 provided up to 63% GR horseweed control. Pyraflufen-ethyl/2,4-D decreased GR horseweed density by 39%; there was no effect on shoot dry weight. Metribuzin applied alone decreased GR horseweed density by as much as 90% but it had no effect on shoot dry weight. At 2, 4, and 8 WAA, pyraflufen-ethyl/2,4-D (527 g ai ha−1) + metribuzin gave similar GR horseweed control (97% to 100%) as the industry standards. Pyraflufen-ethyl/2,4-D + metribuzin, glyphosate/dicamba + saflufenacil, and saflufenacil + metribuzin decreased GR horseweed density and shoot dry weight similarly (≥97%). Soybean seed yield was decreased up to 60% in the nontreated control due to GR horseweed competition (Table 3). Soybean yield was not different between herbicide treatments evaluated (Table 3). Other studies have shown similar soybean seed yield losses from interference of GR horseweed (Budd et al. Reference Budd, Soltani, Robinson, Hooker, Miller and Sikkema2016; Soltani et al. Reference Soltani, Shropshire and Sikkema2020a).
Glyphosate-resistant horseweed control, density and shoot dry weight reduction, and soybean yield from five field experiments. a
a Means followed by the same letter within a column are not statistically different at α = 0.05 according to Tukey-Kramer’s multiple range test.
b Included Merge (0.5% vol/vol).
c Abbreviaton: WAA, weeks after application.
Interaction: Pyraflufen-Ethyl/2,4-D, Metribuzin vs. Pyraflufen-Ethyl/2,4-D + Metribuzin
Additive and synergistic interactions were observed with the co-application of pyraflufen-ethyl/2,4-D and metribuzin for control and density and shoot dry weight reduction (Table 4). There was a synergistic improvement in GR horseweed percent control when pyraflufen-ethyl/2,4-D at 66, 264, 527, and 1,054 g ha−1 was mixed with metribuzin at 2 WAA; when pyraflufen-ethyl/2,4-D was applied at 264, 527, and 1,054 g ha−1 with metribuzin at 4 WAA; and when pyraflufen-ethyl/2,4-D was applied at 66, 264, 527, and 1,054 g ha−1 with metribuzin at 8 WAA. There were similar synergistic decreases in density and shoot dry weight of GR horseweed with the co-application of pyraflufen-ethyl/2,4-D plus metribuzin. All other interactions were additive (Table 4). Han et al. (Reference Han, Picoli, Guo, Yu and Powles2020) found that sequential applications of 2,4-D amine followed by metribuzin caused a synergistic interaction with increased metribuzin movement to the newly developed leaves in wild oats [Avena sterilis (L.)]. In corn, the co-application of 2,4-D with atrazine, a photosystem II–inhibitor, resulted in a synergistic improvement in the control of prostrate knotweed [Polygonum aviculare (L.); Ludwig Reference Ludwig1973].
Glyphosate-resistant horseweed control at 2, 4, and 8 wk after application, plant density, and shoot dry weight reduction at 8 WAA with metribuzin, pyraflufen-ethyl/2,4-D and pyraflufen-ethyl/2,4-D + metribuzin applied pre-plant from five field experiments conducted in2019 and 2020. a,b,c
* and ** denote a significant difference of P < 0.05 or P < 0.01, respectively, between observed and expected values based on a t-test, indicating synergistic interactions of pyraflufen/2,4-D + metribuzin.
a Expected values for herbicide combinations are shown in parentheses following observed values based on Colby’s equation (Equation 3).
b Expected values for herbicide combinations are shown in parentheses following observed values based on Colby’s equation (Equation 4).
c Means followed by the same letter within a column are not statistically different at α = 0.05 according to Tukey-Kramer’s multiple range test.
d Abbreviation: WAA, weeks after application.
This study concludes that the calculated doses of pyraflufen-ethyl/2,4-D for 50%, 80%, and 95% control of GR horseweed at 8 WAA were 390, 1,148, and >2,108 g ai ha−1, respectively. Metribuzin (400 g ha−1) mixed with pyraflufen-ethyl/2,4-D reduced the calculated doses for 50%, 80%, and 95% GR horseweed control to 19, 46, and 201 g ha−1, respectively, at 8 WAA. Pyraflufen-ethyl/2,4-D (527 g ha−1) provided 59% control of GR horseweed, and metribuzin (400 g ha−1) provided 75% control of GR horseweed in soybean. The co-application of pyraflufen-ethyl/2,4-D + metribuzin (527 + 400 g ha−1) controlled GR horseweed 97% at 8 WAA, which was comparable to the industry standards. Based on Colby’s Equations there was an additive or synergistic improvement in GR horseweed control when pyraflufen-ethyl/2,4-D was combined with metribuzin. Overall, this study concludes that GR horseweed control with pyraflufen-ethyl/2,4-D + metribuzin, applied PP, is comparable to the current industry standards. Growers should consider adding metribuzin (400 g ai ha−1) to pyraflufen-ethyl/2,4-D or use saflufenacil + metribuzin in identity-preserved and GR soybean and glyphosate/dicamba + saflufenacil in dicamba-resistant soybean for the control of GR horseweed.
Acknowledgments
We thank Chris Kramer for his technical support and Dr. Michelle Edwards for her advice on the statistical analysis. Funding for this research was provided by Grain Farmers of Ontario and the Canadian Agricultural Partnership program. No conflicts of interest have been declared.
