Introduction
Multiple herbicide–resistant (MHR) waterhemp [Amaranthus tuberculatus (Moq.) Sauer] interference can cause substantial corn (Zea mays L.) yield losses and reduce net returns for producers in the United States and Canada. Amaranthus tuberculatus has been reported in 19 states of the United States and 3 Canadian provinces (Heap Reference Heap2020). Amaranthus tuberculatus is a dioecious weed species that exhibits a rapid growth rate, high reproductive rate, delayed emergence, and an extended emergence pattern, all of which facilitate the evolution and spread of herbicide resistance among populations (Costea et al. Reference Costea, Weaver and Tardif2005; Hartzler et al. Reference Hartzler, Buhler and Stoltenberg1999; Liu et al. Reference Liu, Davis and Tranel2012). A dioecious reproductive system allows A. tuberculatus to stack traits that confer resistance to multiple herbicide mechanisms of action (MOAs) (Jhala et al. Reference Jhala, Norsworthy, Ganie, Sosnoskie, Beckie, Mallory-Smith, Liu, Wei, Wang and Stoltenberg2020; Sarangi et al. Reference Sarangi, Tyre, Patterson, Gaines, Irmak, Knezevic, Lindquist and Jhala2017).
Herbicide resistance was first identified in A. tuberculatus in 1993 in a population that exhibited resistance to acetolactate synthase (ALS) inhibitors (Heap Reference Heap2020). Resistance to six herbicide MOAs, including the synthetic auxins and ALS, photosystem II (PS II), protoporphyrinogen oxidase (PPO), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors was reported in a Missouri MHR A. tuberculatus population in 2015 (Shergill et al. Reference Shergill, Barlow, Bish and Bradley2018). MHR A. tuberculatus is one of the most problematic weed species in row-crop production in North America and was the first to evolve resistance to HPPD inhibitors (Jhala et al. Reference Jhala, Sandell, Rana, Kruger and Knezevic2014; McMullan and Green Reference McMullan and Green2011). Populations of MHR A. tuberculatus resistant to ALS, PS II, EPSPS, and PPO inhibitors have since been reported in Ontario, Canada (Benoit et al. Reference Benoit, Soltani, Hooker, Robinson and Sikkema2019a). MHR A. tuberculatus continues to evolve resistance to herbicides that are extensively used for weed management in corn. When left uncontrolled, MHR A. tuberculatus can reduce grain corn yield by up to 74% (Steckel and Sprague Reference Steckel and Sprague2004). Yield losses of up to 48% have been reported in Ontario (Soltani et al. Reference Soltani, Vyn and Sikkema2009).
Herbicides that inhibit HPPD are widely used for weed management in corn production due to their broad-spectrum weed control and excellent crop safety. The HPPD inhibitors include isoxaflutole, mesotrione, topramezone, tembotrione, bicyclopyrone, and tolpyralate (Mitchell et al. Reference Mitchell, Bartlett, Fraser, Hawkes, Holt, Townson and Wichert2001; USEPA 2007, 2015; van Almsick et al. Reference van Almsick, Ahrens, Lange, Muller, Rosinger and Willms2013; Williams et al. Reference Williams, Boydston, Peachey and Robinson2011). They control many annual grass and broadleaf weed species, including herbicide-resistant biotypes (Grossman and Ehrhardt Reference Grossman and Ehrhardt2007; Pallett et al. Reference Pallett, Cramp, Little, Veerasekaran, Crudace and Slater2001; Schulz et al. Reference Schulz, Ort, Beyer and Kleinig1993). HPPD inhibitors can be applied PRE or POST in corn, permitting greater flexibility to various corn-cropping systems.
HPPD inhibitors and atrazine are often co-applied due to their complementary activity (Abendroth et al. Reference Abendroth, Martin and Roeth2006; Mitchell et al. Reference Mitchell, Bartlett, Fraser, Hawkes, Holt, Townson and Wichert2001; Woodyard et al. Reference Woodyard, Bollero and Riechers2009). Applying herbicides with distinct MOAs in a tank mixture can result in antagonistic, additive, or synergistic interactions. Colby’s equation [E = X + Y −(XY)/100] (Equation 1), where E represents expected level of control of the products in combination, and X and Y represent the level of control of each herbicide applied independently, is used to determine the response of two herbicides in a tank mix (Colby Reference Colby1967). When the herbicides are applied in combination and the observed level of control is less, equal, or greater than expected, the interaction can be classified as antagonistic, additive, or synergistic, respectively.
The HPPD- and PS II-inhibitor MOAs are distinct and complementary. Inhibition of the HPPD enzyme indirectly leads to a shortage of α-tocopherols, β-tocopherols, plastoquinone, and carotenoids (Collakova and DellaPenna Reference Collakova and DellaPenna2001; Fritze et al. Reference Fritze, Linden, Freigang, Auerbach, Huber and Steinbacher2004; Lindblad et al. Reference Lindblad, Lindstedt and Lindstedt1970). The PS II inhibitors readily displace plastoquinone from the QB binding site of the D1 protein in PS II, effectively blocking electron transport and causing reactive oxygen species (ROS) to accumulate (Hankamer et al. Reference Hankamer, Barber and Boekema1997; Hess Reference Hess2000). Enhanced weed control from the co-application of an HPPD and a PS II inhibitor is due to (1) the lack of plastoquinone, which increases the binding efficiency of the PS II inhibitor to the D1 protein; and (2) enhanced levels of ROS due to lack of α-tocopherols, β-tocopherols, plastoquinone, and carotenoids, which quench ROS. Overall, this leads to photo-oxidative destruction of chlorophyll and destruction of photosynthetic membranes, giving rise to the characteristic white bleaching, or chlorosis, of young plant tissue followed by necrosis (Lee et al. Reference Lee, Prisbylla, Cromartie, Dagarin, Howard, Provan, Ellis, Fraser and Mutter1997).
Complementary activity between HPPD inhibitors and PS II inhibitors has been reported for the control of susceptible and triazine-resistant redroot pigweed (Amaranthus retroflexus L.), Palmer amaranth (Amaranthus palmeri S. Watson), and A. tuberculatus (Hugie et al. Reference Hugie, Bollero, Tranel and Riechers2008; Kohrt and Sprague Reference Kohrt and Sprague2017; Sutton et al. Reference Sutton, Richards, Buren and Glasgow2002; Woodyard et al. Reference Woodyard, Bollero and Riechers2009). Woodyard et al. (Reference Woodyard, Bollero and Riechers2009) reported excellent control of a susceptible A. tuberculatus biotype and synergy between mesotrione and atrazine or bromoxynil applied POST. Visible control of a susceptible A. palmeri biotype from Nebraska was improved with the co-application of mesotrione + atrazine, bromoxynil, or metribuzin applied POST (Abendroth et al. Reference Abendroth, Martin and Roeth2006). Other studies have reported excellent control of MHR A. tuberculatus with HPPD + atrazine tank mixtures (Benoit et al. Reference Benoit, Soltani, Hooker, Robinson and Sikkema2019b; Schryver et al. Reference Schryver, Soltani, Hooker, Robinson, Tranel and Sikkema2017; Vyn et al. Reference Vyn, Swanton, Weaver and Sikkema2006). Complementary activity between HPPD inhibitors and other PS II inhibitors such as bentazon and linuron has not been documented for control of Amaranthus species. Tank mixtures of HPPD inhibitors and PS II inhibitors are effective for control of MHR A. tuberculatus and MHR A. palmeri; however, reported interactions between HPPD inhibitors and PS II inhibitors can be specific to the HPPD inhibitor, Amaranthus species, and biotype resistance profile (Chahal and Jhala Reference Chahal and Jhala2018; Hugie et al. Reference Hugie, Bollero, Tranel and Riechers2008; Kohrt and Sprague Reference Kohrt and Sprague2017; Woodyard et al. Reference Woodyard, Bollero and Riechers2009).
Current MHR A. tuberculatus control programs rely heavily on the mixture of HPPD inhibitors and atrazine applied PRE, POST, or PRE fb POST in corn. Of great concern to weed management practitioners is the restricted use of atrazine in some geographic areas. A comprehensive study on the mixture of HPPD inhibitors with alternative PS II inhibitors, applied PRE or POST, is needed to determine the effect on MHR A. tuberculatus control. Given the ability of A. tuberculatus to rapidly evolve resistance, rapidly reproduce, and competitively interfere with corn production, other PS II inhibitors complementary with the HPPD inhibitors need to be identified. It is hypothesized that PRE or POST herbicide tank mixtures containing HPPD inhibitors + PS II inhibitors will result in season-long control of MHR A. tuberculatus in corn. The objectives of these studies were to evaluate the interaction between HPPD inhibitors and a series of PS II inhibitors, applied PRE or POST, on MHR A. tuberculatus control in Ontario corn production.
Materials and Methods
Experimental Methods
Ten field trials were conducted in Canada during 2 yr (2019, 2020) at sites (S) on Walpole Island, ON (S2, S5) (42.561492°N, 82.501487°W), near Cottam, ON (S1, S3, S4) (42.149076°N, 82.683687°W), and near Port Crewe, ON (S6) (42.192390°N, 82.215453°W) (Table 1). The PRE study was conducted at S1, S2, S4, S5, and S6, and the POST study was conducted at S1, S2, S3, S4, and S5 (Table 1). Populations of MHR A. tuberculatus resistant to ALS, PS II, EPSPS, and PPO inhibitors were confirmed at all sites by treating separate quadrats with a POST application of imazethapyr (BASF Canada, 100 Milverton Drive Mississauga, ON, Canada) (100 g ai ha−1) + Agral® 90 (Syngenta Canada, 140 Research Lane, Research Park, Guelph, ON, Canada) (0.2% v/v) + urea ammonium nitrate (UAN 28-0-0, Sylvite, 3221 North Service Road, Burlington, ON, Canada) (2.5% v/v), atrazine (Syngenta Canada) (1,500 g ai ha−1) + Assist® oil concentrate (BASF Canada) (1% v/v), glyphosate (Bayer CropScience, 160 Quarry Park Boulevard SE, Calgary, AB, Canada) (900 g ae ha−1), or fomesafen (Syngenta Canada) (240 g ai ha−1), respectively, when plants were 10 cm in height. The percentage of resistant plants was calculated for each MOA by dividing the number of surviving A. tuberculatus plants in each quadrat at 3 wk after application (WAA) by the number of plants before herbicide application.
Table 1. Soil characteristics and multiple herbicide–resistant (MHR) Amaranthus tuberculatus resistance profile of six field sites where 4-hydroxyphenylpyruvate dioxygenase (HPPD), photosystem II (PS II), and HPPD + PSII inhibitors were applied PRE and POST in Ontario, Canada, in 2019 and 2020. a
a Abbreviations: OM, organic matter; ALS, acetolactate synthase inhibitor; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase inhibitor; PPO, protoporphyrinogen oxidase inhibitor.
b Mean number of surviving plants at 3 wk after application divided by the number of plants treated within eight quadrats per mode of action.
Sites were cultivated twice in the spring to prepare the trial area for planting. Glyphosate- and glufosinate-resistant corn was seeded in rows spaced 0.75 m apart at approximately 83,000 seeds ha−1 to a depth of 4 cm in late May to late June. The PRE study was designed as a two by four factorial. Factor 1 consisted of two levels of HPPD inhibitor: nontreated control and isoxaflutole, and Factor 2 consisted of four levels of PS II inhibitor: nontreated control, atrazine, metribuzin, and linuron. The POST study was designed as a four by four factorial. Factor 1 consisted of four levels of HPPD inhibitor: nontreated control, mesotrione, tolpyralate, and topramezone, and Factor 2 consisted of four levels of PS II inhibitor: nontreated control, atrazine, bromoxynil, and bentazon. PRE and POST trials were arranged in a randomized complete block design with four replications separated by a 2-m alley. Plots were 8-m long and 2.25-m (3 corn rows) wide. The entire trial area was sprayed with glyphosate (450 g ae ha−1), applied POST to 5 cm A. tuberculatus, including nontreated controls, to remove the confounding effect of EPSPS inhibitor– susceptible MHR A. tuberculatus biotypes and other weed species.
Herbicide treatments (Table 2) were applied using a CO2-pressurized backpack sprayer calibrated to deliver 200 L ha−1 at 240 kPa. The sprayer was equipped with four 120-02 ultra-low drift nozzles (Pentair, 375 5th Avenue NW, New Brighton, MN, USA) spaced 50 cm apart, producing a spray width of 2 m. The PRE treatments were applied in mid-May to late June between corn planting and corn emergence. All PRE treatments received sufficient rainfall within 2 WAA for activation. POST treatments were applied when MHR A. tuberculatus reached an average 10 cm in height in mid-June to late July. POST trials at S1 and S3 were in the same field; therefore, they were temporally separated by applying herbicide treatments 2 d apart.
Table 2. Herbicide active ingredient, trade name, and manufacturer for the study of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PS II) inhibitors applied PRE and POST for the control of multiple herbicide–resistant (MHR) Amaranthus tuberculatus in Ontario, Canada, in 2019 and 2020.
a Herbicide treatments with mesotrione included Agral® 90 (Syngenta Canada Inc., 140 Research Lane, Research Park, Guelph, ON, Canada) (0.2% v/v); with tolpyralate included methylated seed oil (MSO Concentrate®) (Loveland Products, 3005 Rocky Mountain Avenue, Loveland, CO, USA) (0.5% v/v) and urea ammonium nitrate (UAN 28-0-0) (Sylvite, 3221 North Service Road, Burlington, ON, Canada) (2.5% v/v); and with topramezone included Merge® (BASF Canada Inc., 100 Milverton Drive, Mississauga, ON, Canada) (0.5% v/v).
Data were collected on visible MHR A. tuberculatus control, density and biomass, visible corn injury, grain corn moisture content, and grain corn yield. Visible MHR A. tuberculatus control was evaluated on a 0% to 100% scale as a visual estimation of weed control based on plant chlorosis, necrosis, and reduction in height compared with the nontreated control within each replication, where 0% represented no herbicide injury and 100% represented complete plant death (Metzger et al. Reference Metzger, Soltani, Raeder, Hooker, Robinson and Sikkema2018). Visible MHR A. tuberculatus control was evaluated at 2, 4, 8, and 12 WAA for the PRE trial and at 4, 8, and 12 WAA for the POST trial. Density and biomass of MHR A. tuberculatus were determined at 4 WAA by counting and harvesting the plants within two randomly placed 0.25-m2 quadrats in each plot. The aboveground biomass of the plants within each quadrat was determined by cutting the MHR A. tuberculatus at the soil surface; the plants were placed inside paper bags, kiln-dried for 3 wk to a consistent moisture, and then weighed using an analytical balance to obtain MHR A. tuberculatus biomass (g m−2). Visible corn injury was assessed on a 0% to 100% scale at 1, 2, and 4 wk after emergence for the PRE trial and 1, 2, and 4 WAA for the POST trial; 0% represented no visible injury and 100% represented complete plant death. Grain corn yield (kg ha−1) and moisture (%) were determined by harvesting two rows of each plot at maturity using a small-plot combine. Grain yields were adjusted to 15.5% moisture before statistical analysis.
Statistical Analysis
The PRE and POST field studies were analyzed separately. Data were subjected to variance analysis using the PROC GLIMMIX procedure in SAS v. 9.4 (SAS Institute, Cary, NC, USA). An initial mixed model analysis was conducted to evaluate site by treatment interactions for all parameters to determine whether data could be pooled across years and locations. The fixed effect for this analysis was herbicide treatment, and the random effects were replication, replication within site, and site by treatment. To pool data, sites were grouped when site by treatment interactions were nonsignificant. Site groups were then subject to a second mixed model analysis to evaluate the effect of herbicide treatment on MHR A. tuberculatus control, density, biomass, corn injury, and grain yield. The random effect for this analysis was replication, and the fixed effects were HPPD inhibitor (Factor One), PS II inhibitor (Factor Two), and the two-way interaction of HPPD by PS II inhibitor. The Shapiro-Wilk test and plots of studentized residuals were used to confirm the assumptions of variance analysis (residuals are homogenous, have a mean of zero, and are normally distributed). Visible MHR A. tuberculatus control, visible corn injury, and corn yield data were analyzed using a normal distribution. To satisfy the assumptions of variance analysis, a lognormal distribution was used to analyze MHR A. tuberculatus density and biomass data. Least-square means for main effects (HPPD inhibitor or PS II inhibitor) were compared when interaction between HPPD inhibitors and PS II inhibitors was nonsignificant. Simple effects were analyzed when the interaction between HPPD inhibitors and PS II inhibitors was significant. Simple effect least-square means were separated using Tukey-Kramer’s multiple range test. To present the data, MHR A. tuberculatus density, and biomass least-square means and standard errors were back-transformed from the log-scale using the omega method (M Edwards, Ontario Agricultural College Statistician, University of Guelph, personal communication). Colby’s equation (Equation 1) was used to calculate expected visible MHR A. tuberculatus control and corn injury means for each replication using the observed means for HPPD inhibitor alone (A) and PS II inhibitor alone (B).
${\rm Expected} = (A + B) - [(A \times B)/100] $
Expected mean MHR A. tuberculatus density and biomass were calculated for each replication using the modified Colby’s equation and corresponding nontreated control mean (W) (Equation 2).
${\rm Expected} = [(A \times B)/W] $
Observed and expected values were compared using a t-test. If observed and expected values were similar, the interaction was considered additive, and if the values were significantly different, the interaction was classified as either antagonistic or synergistic. A significance level of α = 0.05 was used for data analyses; however, significance levels of α = 0.01 were noted.
Results and Discussion
HPPD Inhibitors and PS II Inhibitors Applied PRE
Site by treatment interactions were significant for MHR A. tuberculatus control, density, biomass, and grain yield with no differences between S1, S4, and S6 or S2 and S5; therefore, data for S1, S4, and S6 were combined and data for S2 and S5 were combined for analysis. Lower MHR A. tuberculatus control at S1, S4, and S6 can be attributed to greater MHR A. tuberculatus density and biomass in the nontreated control of 890 plants m−2 and 171.8 g m−2 compared with 35 plants m−2 and 68.9 g m−2 at S2 and S5 (Tables 3 and 4). Vyn et al. (Reference Vyn, Swanton, Weaver and Sikkema2006) reported similar differences in MHR A. tuberculatus control, which they attributed to variation in A. tuberculatus density, biomass, and the resistance profiles of each population. MHR A. tuberculatus control can vary due to the resistance profile of each population (Benoit et al. Reference Benoit, Soltani, Hooker, Robinson and Sikkema2019a; Hager et al. Reference Hager, Wax, Bollero and Simmons2002; Hausman et al. Reference Hausman, Tranel, Riechers, Maxwell, Gonzini and Hager2013; Vyn et al. Reference Vyn, Swanton, Weaver and Sikkema2006). The number of individual MHR A. tuberculatus resistant to each MOA varied by site, with greater resistance to ALS, PS II, and PPO inhibitors at S1, S4, and S6 compared with S2 and S5 (Table 1).
Table 3. Least-square means and significance of main effects and interaction for multiple herbicide–resistant (MHR) Amaranthus tuberculatus control at 2, 4, 8 and 12 wk after PRE application (WAA) in corn treated with isoxaflutole, PS II inhibitors, and isoxaflutole + PS II inhibitors applied PRE across five field sites in 2019 and 2020 in Ontario, Canada.
a Visible MHR A. tuberculatus control was evaluated based on plant chlorosis, necrosis, and reduction in plant height relative to the nontreated control, where 0% represented no herbicide injury, and 100% represented complete plant death.
b Standard error of the mean.
*Significant (P < 0.05).
**Significant (P < 0.01).
NS, nonsignificant (P > 0.05).
Table 4. Least-square means and significance of main effects and interaction for multiple-herbicide-resistant (MHR) Amaranthus tuberculatus density and biomass 4 weeks after PRE application (WAA) and corn grain yield in corn treated with isoxaflutole, photosystem II (PS II inhibitors, and isoxaflutole + PS II inhibitors applied PRE across five field sites in 2019 and 2020 in Ontario, Canada. a
a Means within same main effect and same column followed by the same letter are not significantly different according to Tukey-Kramer multiple range test (P < 0.05).
b Standard error of the mean.
*Significant (P < 0.05).
**Significant (P < 0.01).
NS, nonsignificant (P > 0.05).
MHR Amaranthus tuberculatus Control
The interaction of HPPD inhibitor by PS II inhibitor, applied PRE, was significant for MHR A. tuberculatus control at 2, 4, 8, and 12 WAA (Table 3); therefore, the simple effects are presented (Table 5). At S1, S4, and S6, atrazine, metribuzin, and linuron controlled MHR A. tuberculatus 45% to 69%, 69% to 85%, and 82% to 98%, respectively. Linuron controlled MHR A. tuberculatus better than atrazine. Isoxaflutole controlled MHR A. tuberculatus 58% to 76% across 2, 4, 8, and 12 WAA. The co-application of isoxaflutole with atrazine, metribuzin, or linuron controlled MHR A. tuberculatus 80% to 100%. At 8 WAA, the addition of metribuzin or linuron to isoxaflutole increased MHR A. tuberculatus control 27% and 33%, respectively, compared with isoxaflutole alone. At 12 WAA, the addition of atrazine, metribuzin, or linuron to isoxaflutole improved MHR A. tuberculatus control 22%, 30%, and 34%, respectively. Conversely, the addition of isoxaflutole to atrazine improved MHR A. tuberculatus control 24% to 35% at 2, 8, and 12 WAA, and the addition of isoxaflutole to metribuzin improved control 19% at 12 WAA when compared with metribuzin alone. At S1, S4, and S6, the control of MHR A. tuberculatus in all herbicide treatments decreased during the course of the growing season, which can be attributed to late-emerging cohorts (Steckel and Sprague Reference Steckel and Sprague2004).
Table 5. Multiple herbicide–resistant (MHR) Amaranthus tuberculatus control at 2, 4, 8 and 12 wk after PRE application (WAA), density, biomass, and grain yield in corn treated with isoxaflutole, photosystem II (PS II) inhibitors, and isoxaflutole + PS II inhibitors applied PRE across five field sites in 2019 and 2020 in Ontario, Canada.
a Within site groupings, means within column followed by the same letter (a–c) or means within row followed by the same letter (Y or Z) are not significantly different according to Tukey-Kramer multiple range test (P < 0.05).
b Standard error of the mean.
c Visible MHR A. tuberculatus control was evaluated based on plant chlorosis, necrosis, and reduction in plant height relative to the nontreated control, where 0% represented no herbicide injury and 100% represented complete plant death.
d Values in parentheses represent expected values calculated from Colby’s analysis.
e Interaction was negligible; therefore, only treatment means and results from Colby’s analysis are shown.
At S2 and S5, atrazine, metribuzin, and linuron controlled MHR A. tuberculatus 87% to 100% and isoxaflutole controlled MHR A. tuberculatus 95% to 98% across 2, 4, 8, and 12 WAA. The co-application of isoxaflutole with atrazine, metribuzin, or linuron controlled MHR A. tuberculatus 99% to 100%. These results are consistent with those of Hausman et al. (Reference Hausman, Tranel, Riechers, Maxwell, Gonzini and Hager2013), who reported 8% to 58% control of HPPD inhibitor–resistant A. tuberculatus 4 WAA with PRE-applied atrazine (1,680 g ha−1). In contrast, Hager et al. (Reference Hager, Wax, Bollero and Simmons2002) and Hausman et al. (Reference Hausman, Tranel, Riechers, Maxwell, Gonzini and Hager2013) reported metribuzin (420 g ha−1) and linuron (840 g ha−1) applied PRE controlled MHR A. tuberculatus up to 92% and up to 58% in soybean [Glycine max (L.) Merr.], respectively. Greater A. tuberculatus control in this study may reflect the higher application rates of metribuzin (560 g ha−1) and linuron (2,160 g ha−1) compared with Hager et al. (Reference Hager, Wax, Bollero and Simmons2002) and Hausman et al. (Reference Hausman, Tranel, Riechers, Maxwell, Gonzini and Hager2013), and the more competitive nature of corn.
Results from Colby’s equation indicated observed and expected MHR A. tuberculatus control were similar for all HPPD- and PS II-inhibitor tank mixtures at all sites and suggests the interaction between isoxaflutole and atrazine, metribuzin, or linuron, applied PRE, is additive for MHR A. tuberculatus control. In previous studies, isoxaflutole + atrazine (105 + 1,063 g ha−1) resulted in greater than 97% control of MHR A. tuberculatus at 10 WAA (Vyn et al. Reference Vyn, Swanton, Weaver and Sikkema2006) and 90% at 12 WAA (Benoit et al. Reference Benoit, Soltani, Hooker, Robinson and Sikkema2019b). Chahal and Jhala (Reference Chahal and Jhala2018) reported variable control of HPPD and PS II inhibitor–resistant A. palmeri with isoxaflutole + atrazine that ranged from 14% to 85%; in that study, the interaction between isoxaflutole and atrazine was additive. Additionally, O’Brien et al. (Reference O’Brien, Davis and Riechers2018) reported an additive interaction between isoxaflutole (105 g ha−1) and metribuzin (191 g ha−1) applied POST for control of an HPPD- and PS II-resistant A. tuberculatus population from Nebraska.
MHR Amaranthus tuberculatus Density and Biomass
Averaged across PS II inhibitors, isoxaflutole reduced MHR A. tuberculatus density 70% at S1, S4, and S6 (Table 4), which is similar to the 75% density reduction at 4 WAA reported by Hausman et al. (Reference Hausman, Tranel, Riechers, Maxwell, Gonzini and Hager2013). In contrast, there was an interaction between isoxaflutole and PS II inhibitors for MHR A. tuberculatus density at S2 and S5 and biomass at all sites (Table 4); therefore, the simple effects are presented (Table 5). At S2 and S5, atrazine, metribuzin, and linuron reduced MHR A. tuberculatus density 74%, 100%, and 99%, respectively. Isoxaflutole reduced MHR A. tuberculatus density 97%, and isoxaflutole + atrazine reduced plant density by 26 percentage points more than atrazine alone.
At S1, S4, and S6, isoxaflutole, metribuzin, and linuron reduced MHR A. tuberculatus biomass 78%, 87%, and 97%, respectively; atrazine did not reduce A. tuberculatus biomass. The addition of metribuzin or linuron to isoxaflutole reduced MHR A. tuberculatus biomass 20% and 21% more than isoxaflutole alone, respectively. The addition of isoxaflutole to atrazine or metribuzin reduced MHR A. tuberculatus biomass 42% and 11% more than atrazine or metribuzin alone, respectively. At S2 and S5, atrazine, metribuzin, and linuron reduced MHR A. tuberculatus biomass 93% to 100%. Isoxaflutole reduced MHR A. tuberculatus biomass 99% and was not improved with the addition of a PS II inhibitor. In contrast, the addition of isoxaflutole to atrazine resulted in 6% greater reductions in MHR A. tuberculatus biomass compared with atrazine alone. These results complement those of Vyn et al. (Reference Vyn, Swanton, Weaver and Sikkema2006), who reported a greater reduction in ALS- and PS II-resistant A. tuberculatus density and biomass with isoxaflutole + atrazine that ranged from 92% to 95%, compared with atrazine alone. Results of Colby’s analysis indicated additive interactions between isoxaflutole and PS II inhibitors for MHR A. tuberculatus density and biomass.
Corn Injury and Grain Yield
Corn injury was not observed with any treatment (data not shown). The lack of corn injury is likely due to the safener, cyprosulfamide, present in the isoxaflutole formulation used in these studies (Robinson et al. Reference Robinson, Soltani, Shropshire and Sikkema2013; USEPA 2015). There was an interaction between isoxaflutole and PS II inhibitors for corn grain yield at S1, S4, and S6 (Table 4); therefore, the simple effects are presented (Table 5). Season-long MHR A. tuberculatus interference reduced grain corn yield up to 43% or 3,900 kg ha−1. Grain yield reductions at S1, S4, and S6 can be attributed to greater MHR A. tuberculatus interference due to greater weed density, biomass, and a greater number of herbicide-resistant individuals compared with S2 and S5, where there was no corn yield loss due to MHR A. tuberculatus interference. This is consistent with Vyn et al.’s (Reference Vyn, Swanton, Weaver and Sikkema2006, Reference Vyn, Swanton, Weaver and Sikkema2007) report that corn grain yield differences between herbicide treatments occurred only at sites with greater MHR A. tuberculatus pressure and resistance to both ALS inhibitors and PS II inhibitors.
HPPD and PS II Inhibitors Applied POST
Site by treatment interactions were significant for MHR A. tuberculatus control, density, biomass, and corn grain yield, with no differences between S1 and S4, or S2, S3, and S5; therefore, for purposes of analysis, data for S1 and S4 were combined and data for S2, S3, and S5 were combined. Lower MHR A. tuberculatus control at S1 and S4 can be attributed to greater plant density and biomass in the nontreated control of 463 plants m−2 and 69.6 g m−2 compared with 54 plants m−2 and 50.3 g m−2 at S2, S3, and S5 (Table 6). Vyn et al. (Reference Vyn, Swanton, Weaver and Sikkema2006) reported similar differences in MHR A. tuberculatus control, which they attributed to variation in plant density, biomass, and the resistance profiles of the populations under evaluation. The number of individual MHR A. tuberculatus plants resistant to each MOA varied by site, with greater resistance to ALS, PS II, and PPO inhibitors at S1, S3, and S4 compared with S2 and S5 (Table 1). Site by treatment interactions were significant for corn injury. Similar corn injury was observed at S1, S2, and S5; therefore, corn injury data were combined for S1, S2, and S5 for analysis. In contrast, corn injury was not observed at S3 and S4.
Table 6. Least-square means and significance of main effects and interaction for multiple herbicide–resistant (MHR) Amaranthus tuberculatus control at 4, 8 and 12 wk after POST application (WAA) in corn treated with 4-hydroxyphenylpyruvate dioxygenase (HPPD), photosystem II (PS II), and HPPD + PS II inhibitors applied POST across five field sites in 2019 and 2020 in Ontario, Canada.
a Means within same main effect and same column followed by the same letter are not significantly different according to Tukey-Kramer multiple range test (P < 0.05).
b Visible MHR A. tuberculatus control was evaluated based on plant chlorosis, necrosis, and reduction in plant height relative to the nontreated control where 0% represented no herbicide injury and 100% represented complete plant death.
c Herbicide treatments with mesotrione included Agral® 90 (Syngenta Canada Inc., 140 Research Lane, Research Park, Guelph, ON, Canada) (0.2% v/v); with tolpyralate included methylated seed oil (MSO Concentrate®) (Loveland Products, 3005 Rocky Mountain Avenue, Loveland, CO, USA) (0.5% v/v) and urea ammonium nitrate (UAN 28-0-0) (Sylvite, 3221 North Service Road, Burlington, ON, Canada) (2.5% v/v); and with topramezone included Merge® (BASF Canada Inc., 100 Milverton Drive, Mississauga, ON, Canada) (0.5% v/v).
d Standard error of the mean.
*Significant (P < 0.05).
**Significant (P < 0.01).
NS, nonsignificant (P > 0.05).
MHR Amaranthus tuberculatus Control
There was no interaction between HPPD inhibitors and PS II inhibitors for MHR A. tuberculatus control at 8 and 12 WAA at S1 and S4 (Table 6); therefore, the main effects are presented. When averaged across PS II inhibitors, mesotrione, tolpyralate, and topramezone controlled MHR A. tuberculatus 79% to 80%, 69%, and 53%, respectively at 8 and 12 WAA. When averaged across HPPD inhibitors, atrazine, bromoxynil, and bentazon controlled MHR A. tuberculatus 59%, 63 to 64%, and 56 to 57%, respectively, at 8 and 12 WAA.
There was an interaction between HPPD inhibitors and PS II inhibitors for MHR A. tuberculatus control at S1 and S4 at 4 WAA and at S2, S3, and S5 at 4, 8, and 12 WAA (Table 6); therefore, the simple effects are presented (Tables 7 and 8). At S1 and S4, the HPPD inhibitors mesotrione, tolpyralate, and topramezone controlled MHR A. tuberculatus 54%, 61%, and 45%, respectively, and the PS II inhibitors atrazine, bromoxynil, and bentazon controlled MHR A. tuberculatus 31%, 23%, and 16%, respectively, at 4 WAA. Unacceptable MHR A. tuberculatus control with POST-applied PS II inhibitors is consistent with previous studies that report <80% control of MHR A. tuberculatus and MHR A. palmeri (Anderson et al. Reference Anderson, Roeth and Martin1996; Chahal and Jhala Reference Chahal and Jhala2018; Corbett et al. Reference Corbett, Askew, Thomas and Wilcut2004; Foes et al. Reference Foes, Tranel, Wax and Stoller1998; Hausman et al. Reference Hausman, Tranel, Riechers and Hager2016; Kohrt and Sprague Reference Kohrt and Sprague2017; Vyn et al. Reference Vyn, Swanton, Weaver and Sikkema2006). The addition of atrazine, bromoxynil, or bentazon to mesotrione improved MHR A. tuberculatus control 29%, 34%, and 22%, respectively, when compared with mesotrione alone. Similarly, the addition of bromoxynil to tolpyralate increased control 20% when compared with tolpyralate alone. Control of MHR A. tuberculatus was not improved with the addition of PS II-inhibiting herbicides to topramezone. Kohrt and Sprague (Reference Kohrt and Sprague2017) reported synergistic responses occur more frequently with tank mixtures of atrazine plus triketone HPPD inhibitors, such as mesotrione and tembotrione, compared with the pyrazole HPPD inhibitor topramezone.
Table 7. Least-square means and significance of main effects and interaction for multiple herbicide–resistant (MHR) Amaranthus tuberculatus density, biomass and corn injury and corn grain yield in corn treated with 4-hydroxyphenylpyruvate dioxygenase (HPPD), photosystem II (PS II), and HPPD + PS II inhibitors applied POST across five field sites in 2019 and 2020 in Ontario, Canada.
a Means within same main effect and same column followed by the same letter are not significantly different according to Tukey-Kramer multiple range test (P < 0.05).
b WAA, weeks after POST application.
c Herbicide treatments with mesotrione included Agral® 90 (Syngenta Canada Inc., 140 Research Lane, Research Park, Guelph, ON, Canada) (0.2% v/v); with tolpyralate included methylated seed oil (MSO Concentrate®) (Loveland Products, 3005 Rocky Mountain Avenue, Loveland, CO, USA) (0.5% v/v) and urea ammonium nitrate (UAN 28-0-0) (Sylvite, 3221 North Service Road, Burlington, ON, Canada) (2.5% v/v); and with topramezone included Merge® (BASF Canada Inc., 100 Milverton Drive, Mississauga, ON, Canada) (0.5% v/v).
d Standard error of the mean.
*Significant (P < 0.05).
**Significant (P < 0.01).
NS, nonsignificant (P > 0.05).
Table 8. Multiple herbicide–resistant (MHR) Amaranthus tuberculatus control at 4, 8, and 12 wk after POST application (WAA), density, biomass, and corn grain yield in corn treated with HPPD, PSII, and HPPD + PS II inhibitors applied POST across five field sites in 2019 and 2020 in Ontario, Canada.
a Within site groupings, means within column followed by the same letter (a–c) or means within row followed by the same letter (X–Z) are not significantly different according to Tukey-Kramer multiple range test (P < 0.05).
b Visible MHR A. tuberculatus control was evaluated based on plant chlorosis, necrosis, and reduction in plant height relative to the nontreated control, where 0% represented no herbicide injury and 100% represented complete plant death.
c Herbicide treatments with mesotrione included Agral® 90 (Syngenta Canada Inc., 140 Research Lane, Research Park, Guelph, ON, Canada) (0.2% v/v); with tolpyralate included methylated seed oil (MSO Concentrate®) (Loveland Products, 3005 Rocky Mountain Avenue, Loveland, CO, USA) (0.5% v/v) and urea ammonium nitrate (UAN 28-0-0) (Sylvite, 3221 North Service Road, Burlington, ON, Canada) (2.5% v/v); and with topramezone included Merge® (BASF Canada Inc., 100 Milverton Drive, Mississauga, ON, Canada) (0.5% v/v).
d Interaction was negligible; therefore, only treatment means and results from Colby’s analysis are shown.
e Values in parentheses represent expected values calculated from Colby’s analysis.
f Standard error of the mean.
*Significant difference of P < 0.05 between observed and expected values based on a two-sided t-test.
**Significant difference of P < 0.01 between observed and expected values based on a two-sided t-test.
Based on Colby’s analysis, the improvement in MHR A. tuberculatus control with the addition of a PS II inhibitor to a HPPD inhibitor was either additive or synergistic, except topramezone + atrazine, which was antagonistic. These results are consistent with those of Hausman et al. (Reference Hausman, Singh, Tranel, Riechers, Kaundun, Polge, Thomas and Hager2011, Reference Hausman, Tranel, Riechers and Hager2016) and Woodyard et al. (Reference Woodyard, Bollero and Riechers2009), who reported additive or synergistic responses with mesotrione + atrazine and mesotrione + bromoxynil for control of non–herbicide resistant and MHR A. tuberculatus. Additionally, Kohrt and Sprague (Reference Kohrt and Sprague2017) reported additive or synergistic responses with mesotrione + atrazine, tembotrione + atrazine, tolpyralate + atrazine, and topramezone + atrazine applied POST for MHR A. palmeri control.
At S2, S3, and S5, the HPPD inhibitors mesotrione, tolpyralate, and topramezone controlled MHR A. tuberculatus ≥95%, ≥97%, and ≥90%, respectively, across 4, 8, and 12 WAA; weed control was not improved by the addition of PS II inhibitors to the HPPD inhibitors. In contrast, the PS II inhibitors atrazine, bromoxynil, and bentazon controlled MHR A. tuberculatus ≥79%, ≥83%, and ≥71%, respectively, across 4, 8, and 12 WAA; the addition of HPPD inhibitors to atrazine, bromoxynil, and bentazon improved MHR A. tuberculatus control up to 20%, 17%, and 28%, respectively. Similarly, Benoit et al. (Reference Benoit, Soltani, Hooker, Robinson and Sikkema2019b) reported greater MHR A. tuberculatus control with mesotrione + atrazine, tembotrione + atrazine, topramezone + atrazine, and tolpyralate + atrazine compared with atrazine alone. Additionally, Chahal and Jhala (Reference Chahal and Jhala2018) reported greater control of HPPD- and PS II-resistant A. palmeri with POST-applied mesotrione + atrazine, tembotrione + atrazine, or topramezone + atrazine compared with each product alone.
Analysis of observed and calculated Colby’s expected values indicated the improvement in MHR A. tuberculatus control with the co-application of a HPPD inhibitors + PS II inhibitors was additive, except topramezone + bentazon at 4 WAA and topramezone + atrazine at 8 and 12 WAA at S2, S3, and S5, which were antagonistic. Kohrt and Sprague (Reference Kohrt and Sprague2017) reported a similar antagonistic response between tolpyralate and atrazine; however, they attributed this response to reduced absorption of tolpyralate due to the high rate of atrazine (35,900 g ha−1). Antagonistic interactions between topramezone and ALS inhibitors have been reported to reduce control of grass weed species; in contrast, this response was not observed in some species when topramezone was co-applied with atrazine (Kaastra et al. Reference Kaastra, Swanton, Tardif and Sikkema2008). We do not have an explanation for the antagonistic response between topramezone and atrazine or topramezone and bentazon; this response should be evaluated in future studies to determine whether this is a true effect.
MHR Amaranthus tuberculatus Density and Biomass
There was no interaction between the HPPD inhibitors and PS II inhibitors for MHR A. tuberculatus density and biomass at 4 WAA (Table 7); therefore, the main effects are presented. At S1 and S4, the HPPD inhibitors, when averaged across the PS II inhibitors, did not reduce MHR A. tuberculatus density; the PS II inhibitors, when averaged across the HPPD inhibitors, reduced MHR A. tuberculatus density 25% to 40%. At S2, S3, and S5, mesotrione, tolpyralate, and topramezone, when averaged across the PS II inhibitors, reduced MHR A. tuberculatus density and biomass 78% to 97%. Atrazine and bromoxynil reduced MHR A. tuberculatus density 79% and 84%, respectively, when averaged across HPPD inhibitors.
At S1 and S4, mesotrione reduced MHR A. tuberculatus biomass 88% when averaged across PS II inhibitors. When averaged across HPPD inhibitors, atrazine, bromoxynil, and bentazon reduced MHR A. tuberculatus biomass 16% to 43%. At S2, S3 and S5, the HPPD inhibitors reduced MHR A. tuberculatus biomass 93% to 99% when averaged across PS II inhibitors. When averaged across HPPD inhibitors, atrazine, bromoxynil, and bentazon reduced MHR A. tuberculatus biomass 79%, 86%, and 66%, respectively. These findings are consistent with those of Kohrt and Sprague (Reference Kohrt and Sprague2017), who reported greater reductions in ALS-, PS II-, and EPSPS-resistant A. palmeri with HPPD inhibitors compared with atrazine.
Corn Injury and Grain Yield
There was an interaction between HPPD inhibitors and PS II inhibitors for corn injury at 1 WAA at S1, S2, and S5 (Table 7); therefore, the simple effects are presented. There was no corn injury at S3 and S4 (data not shown). Atrazine did not cause corn injury; however, bromoxynil and bentazon caused 7% and 2% corn injury, respectively (Table 9). Similar corn leaf necrosis was observed at 1 WAA when bromoxynil and bentazon were co-applied with mesotrione, tolpyralate, and tembotrione. Carey and Kells (Reference Carey and Kells1995) and Vyn et al. (Reference Vyn, Swanton, Weaver and Sikkema2006) reported similar early-season corn leaf necrosis with bromoxynil applied POST. Compared with HPPD inhibitors alone, the addition of bromoxynil to mesotrione, tolpyralate, or topramezone increased corn injury 8% to 16%. Similarly, the addition of mesotrione, tolpyralate, or topramezone to bromoxynil increased corn injury 1% to 9%. Observed corn injury at 1 WAA was 5% to 9% greater than the calculated Colby’s expected value, indicating a synergistic increase in corn injury with the co-application of tolpyralate and bromoxynil, tolpyralate and bentazon, and topramezone and bromoxynil. Corn injury was transient; less corn injury was observed at 2 and 4 WAA. Transient leaf necrosis caused by bromoxynil is consistent with Cary and Kells (Reference Carey and Kells1995) and Vyn et al. (Reference Vyn, Swanton, Weaver and Sikkema2006), who reported no corn injury at 4 WAA.
Table 9. Corn injury due to 4-hydroxyphenylpyruvate dioxygenase (HPPD), photosystem II (PS II), and HPPD + PS II inhibitors applied POST across five field sites in 2019 and 2020 in Ontario, Canada.
a Within site groupings, means within column followed by the same letter (a–c) or means within row followed by the same letter (X–Z) are not significantly different according to Tukey-Kramer multiple range test (P < 0.05). WAA, weeks after POST application.
b Herbicide treatments with mesotrione included Agral® 90 (Syngenta Canada Inc., 140 Research Lane, Research Park, Guelph, ON, Canada) (0.2% v/v); with tolpyralate included methylated seed oil (MSO Concentrate®) (Loveland Products, 3005 Rocky Mountain Avenue, Loveland, CO, USA) (0.5% v/v) and urea ammonium nitrate (UAN 28-0-0) (Sylvite, 3221 North Service Road, Burlington, ON, Canada) (2.5% v/v); and with topramezone included Merge® (BASF Canada Inc., 100 Milverton Drive, Mississauga, ON, Canada) (0.5% v/v).
c Standard error of the mean.
d Values in parentheses represent expected values calculated from Colby’s analysis.
e Interaction was negligible; therefore, only treatment means and results from Colby’s analysis are shown.
* Significant difference of P < 0.05 between observed and expected values based on a two-sided t-test.
** Significant difference of P < 0.01 between observed and expected values based on a two-sided t-test.
There was an interaction between HPPD inhibitors and PS II inhibitors for corn grain yield at S1 and S4 (Table 7); therefore, the simple effects are presented. Greater MHR A. tuberculatus density and biomass at S1 and S4 and a greater number of herbicide-resistant individuals resulted in unacceptable control with HPPD inhibitors and PS II inhibitors (Table 8). At S1 and S4, MHR A. tuberculatus interference with the HPPD inhibitors and PS II inhibitors applied alone resulted in corn yield that was similar to the weedy control. The addition of atrazine, bromoxynil, or bentazon to mesotrione increased corn grain yield 2,100 to 2,400 kg ha−1. Similarly, the addition of mesotrione to atrazine, bromoxynil, or bentazon increased grain yield 3,600 to 4,300 kg ha−1. These results complement those of Vyn et al. (Reference Vyn, Swanton, Weaver and Sikkema2006), who reported 3,600 kg ha−1 greater corn yield with mesotrione + atrazine compared with atrazine alone.
In summary, these studies identify effective PRE and POST HPPD-inhibitor + PS II-inhibitor tank mixtures that result in season-long control of MHR A. tuberculatus. Within MHR A. tuberculatus populations, differences in plant density, biomass, and the number of individuals resistant to ALS, PS II, EPSPS, and PPO inhibitors resulted in differences in control. Results from the PRE study indicate linuron is an effective PRE herbicide for control of MHR A. tuberculatus in corn. Control of MHR A. tuberculatus was greater with PRE applications of isoxaflutole + atrazine, isoxaflutole + metribuzin, and isoxaflutole + linuron compared with each herbicide alone. Isoxaflutole + atrazine, isoxaflutole + metribuzin, and isoxaflutole + linuron are effective PRE tank mixtures that resulted in comparable corn grain yield. Interactions between isoxaflutole and all PS II inhibitors were additive for MHR A. tuberculatus control, biomass, and density; however, it is recommended that isoxaflutole be tank mixed with a PS II inhibitor for greater and more consistent MHR A. tuberculatus control. Results from the POST study indicate atrazine, bromoxynil, and bentazon controlled MHR A. tuberculatus 16% to 87%, while mesotrione, tolpyralate, and topramezone resulted in 45% to 97% control at 4, 8, and 12 WAA. The co-application of HPPD inhibitors and PS II inhibitors applied POST resulted in greater control of MHR A. tuberculatus when compared with these products applied alone. Interactions between HPPD inhibitors and PS II inhibitors were mostly additive; however, synergistic responses were observed with mesotrione + bromoxynil, mesotrione + bentazon, and tolpyralate + bromoxynil. It is recommended that POST-applied HPPD inhibitors be tank mixed with a PS II inhibitor for greater control of MHR A. tuberculatus. The application of bromoxynil and bentazon resulted in corn injury and was observed as foliar necrosis; however, injury was transient. The co-application of mesotrione + atrazine, mesotrione + bromoxynil, and mesotrione + bentazon resulted in greater corn yield than atrazine alone. We conclude from these studies that the atrazine alternatives metribuzin or linuron can be co-applied with isoxaflutole applied PRE and bromoxynil or bentazon can be co-applied with mesotrione or tolpyralate applied POST for control of MHR A. tuberculatus; these tank mixtures exhibit complementary activity and provide excellent season-long control of MHR A. tuberculatus in corn. Weed management programs often incorporate HPPD inhibitors and PS II inhibitors; future research should focus on the determination of other alternative HPPD-inhibitor tank-mixture partners, complementary herbicide tank mixtures, and integrated weed management strategies to reduce selection for HPPD inhibitors and PS II inhibitor–resistant biotypes. The ability of A. tuberculatus to rapidly evolve resistance to multiple effective herbicide MOAs, including the HPPD inhibitors, warrants the use of diverse weed management strategies.
Acknowledgments
We thank Chris Kramer for his technical support, Michelle Edwards for her statistical support, the University of Guelph, Ridgetown Campus summer staff for their field support, and Grain Farmers of Ontario and the Ontario Agri-Food Innovation Alliance for the funding to conduct this research. No conflicts of interest have been declared.
