Early Postemergence Herbicide Tank-Mixtures for Control of Waterhemp Resistant to Four Herbicide Modes of Action in Corn

Multiple-herbicide-resistant (MHR) waterhemp has been confirmed and is difficult to control for growers in Ontario, Canada and in the Midwestern United States. The objective of this study was to evaluate early post-emergence (EPOST) herbicides for control of MHR waterhemp in field corn. Five field trials were conducted over a two-year period (2019, 2020) at sites on Walpole Island, ON and near Cottam, ON, Canada. Thirteen herbicide tank-mixtures containing multiple modes-of-action (MOA) were applied EPOST to 5 cm MHR waterhemp in field corn. Control of MHR waterhemp varied by site due to variable plant density, plant biomass, and number of herbicide-resistant individuals across research sites and years. Control of MHR waterhemp ranged from 90% to 100% with glyphosate + S-metolachlor/mesotrione/ bicyclopyrone/atrazine, glyphosate/2,4-D choline + rimsulfuron + mesotrione + atrazine, glyphosate + S-metolachlor/atrazine/mesotrione, glyphosate + mesotrione + atrazine, glyphosate/S-metolachlor/mesotrione + atrazine, glyphosate + S-metolachlor/mesotrione/bicyclopyrone, glyphosate/2,4-D choline + rimsulfuron + mesotrione, and glyphosate + pyroxasulfone + dicamba/atrazine at 4, 8, and 12 WAA. Control of MHR waterhemp ranged from 70% to 100% with glyphosate + topramezone/dimethenamid-P + dicamba/atrazine, glyphosate + isoxaflutole + atrazine, and glyphosate + tolpyralate + atrazine at 4, 8, and 12 WAA. Control of MHR waterhemp was similar for all herbicide programs, except glyphosate + dicamba/atrazine and glyphosate + S-metolachlor/atrazine which resulted in the lowest control at three of five sites that ranged from 63% to 89% and 61% to 76%, respectively. Crop injury was ≤10% for herbicide programs tested, except 28% to 31% corn injury with glyphosate/2,4-D choline + rimsulfuron + mesotrione + atrazine; however, How to cite this paper: Willemse, C., Soltani, N., Benoit, L., Jhala, A.J., Hooker, D.C., Robinson, D.E. and Sikkema, P.H. (2021) Early Postemergence Herbicide Tank-Mixtures for Control of Waterhemp Resistant to Four Herbicide Modes of Action in Corn. Agricultural Sciences, 12, 354-369. https://doi.org/10.4236/as.2021.124023 Received: February 13, 2021 Accepted: April 6, 2021 Published: April 9, 2021 Copyright © 2021 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Waterhemp [Amaranthus tuberculatus (Moq.) J. D. Sauer] has become one of the most problematic weed species in midwestern United States crop production. Reductions in tillage, greater reliance on herbicides for weed management, and the evolution of resistance to multiple herbicide modes of action (MOA) have contributed to the rapid increase of waterhemp in agricultural cropping systems [1] [2]. Waterhemp has been reported in 19 states of the USA and three provinces in Canada where it interferes with corn and soybean production [3] [4]. The rapid movement of waterhemp and evolution of herbicide resistance among individuals and populations is facilitated by its dioecism, rapid growth rate, high reproductive rate, delayed emergence, and extended emergence pattern [5] [6]. Resistance to photosystem II (PS II)-, acetolactate synthase (ALS)-, and protoporphyrinogen oxidase (PPO)-inhibiting herbicides was identified in waterhemp in 1990, 1993, and 2001, respectively [4] [7] [8] [9]. Waterhemp resistant to 5-enolpyruyl shikimate-3-phosphate synthase (EPSPS)-inhibitors was first reported in the USA in 2005 and Ontario, Canada in 2014 [4]. More recent reports from Ontario have identified multiple-herbicide-resistant (MHR) waterhemp populations resistant to ALS-, PS II-, EPSPS-, and PPO-inhibiting herbicides. Waterhemp continues to evolve resistance to currently used MOA and is the first weed species to develop resistance to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides [4] [7] [8] [9]. The first MHR waterhemp population with six-way resistance to synthetic auxins and ALS-, PS II-, EPSPS-, PPO-, and HPPD-inhibiting herbicides was identified in Missouri in 2015 [10]. Resistance to very-long-chain fatty-acid (VLCFA)-inhibiting herbicides has since been detected within a MHR waterhemp population resistant to 2,4-D and ALS-, PS II-, PPO-, and HPPD-inhibiting herbicides [11]. The ability of MHR waterhemp to rapidly evolve and accumulate traits that confer resistance to multiple MOA makes it difficult to manage in agricultural cropping systems [12].
Weed interference must be prevented during the early stages of corn (Zea mays L.) growth and development to prevent yield loss [13]. The relative time of crop and weed emergence has a greater effect on corn yield than weed density and biomass [13]. Weeds that emerge with the crop have the greatest impact on corn yield [13]. Steckel and Sprague [2] reported MHR waterhemp emerging at VE corn growth stage reduced grain yield 74% compared to only 2% yield loss when waterhemp emerged at V8 and was left uncontrolled for the remainder of the growing season. In contrast, Cordes et al. [14] reported corn yield loss was dependent on waterhemp density. When competing with corn, waterhemp can be placed at a disadvantage due to its characteristic late emergence; however, corn yield losses of up to 17% have been reported when densities of 369 to 445 plants m −2 emerge and compete up to V7 corn growth stage [14] [15]. Steckel and Sprague [2] reported corn yield reductions when waterhemp emerged before the V8 corn growth stage. In Ontario, corn yield losses of up to 48% have been reported when waterhemp populations are left uncontrolled [16]. The critical period of weed control in corn to prevent yield loss varies with the relative time of weed and crop emergence, weed density, species, and environment [17] [18]. It is recommended that corn remain waterhemp-free from emergence to V6 to maximize grain yield [2]. Early-season control of MHR waterhemp is imperative to reduce early-season weed interference, prevent corn yield loss, and reduce weed escapes. Current herbicide-based MHR waterhemp management strategies include preemergence (PRE), postemergence (POST), and PRE followed-by (fb) POST herbicide applications that utilize multiple effective MOA [16] [19] [20] [21]. The HPPD-inhibitors isoxaflutole, mesotrione, and tolpyralate are often applied in combination with a PS II-inhibitor such as atrazine and result in excellent control of MHR waterhemp [22] [23] [24] [25] [26]. Complementary activity between HPPD-inhibitors and atrazine has been reported for the control of triazine-susceptible and triazine-resistant redroot pigweed (Amaranthus retroflexus L.), waterhemp, and Palmer amaranth (Amaranthus palmeri S. Watson) [25] [26] [27]. HPPD-inhibitors inhibit the production of carotenoids, α-tocopherols, and plastoquinone, and atrazine increases the production of reactive oxygen species [28] [29]. The enhanced weed control efficacy when a HPPD-inhibitor is co-applied with a PS II-inhibitor is due to 1) increased binding efficiency of atrazine to the D1 protein of PS II-inhibitor caused by the shortage of plastoquinone, and 2) enhanced reactive oxygen species (ROS) levels due to the lack of quenching carotenoids, tocopherols, and plastoquinone. Synthetic auxin herbicides are another effective MOA for MHR waterhemp control; however, current literature reports variable responses [16] [30] [31]. Synthetic auxin herbicides provide control of broadleaf weeds by mimicking plant growth hormones which causes unregulated plant growth and death in some plants [32]. Superior MHR waterhemp control with dicamba/atrazine compared to other POST tank-mixtures has been reported [33]. Anderson et al. [30], Soltani et al. [16] and Vyn et al. [31] reported dicamba/atrazine provided ≥86% control of herbicide-resistant waterhemp. Benoit et al. [33] and Schryver et al. [34] found that POST applications of dicamba are more effective than PRE applications. The application of a new glyphosate/2,4-D choline formulation registered for application to ENLIST TM (Corteva Agriscience, Wilmington, DE) corn hybrids allows for a second synthetic auxin herbicide for MHR waterhemp control in corn [34] [35] [36]. ENLIST TM corn hybrids contain transgenes that confer resistance to glyphosate and glufosinate plus the aryloxyalkanoate dioxygenase-1 (AAD-1) transgene which enables them to exhibit resistance to glyphosate, glufosinate, and greater tolerance to 2,4-D and the arlyloxyphenoxy propionates than traditional glyphosate (RoundupReady ® ) (Bayer CropScience Inc., 160 Quarry Park Boulevard SE, Calgary, AB) and glufosinate (LibertyLink ® ) (BASF Canada Inc., 100 Milverton Drive, Mississauga, ON) resistant hybrids [35] [37] [38]. Robinson et al. [36] reported up to 94% control of common waterhemp (Amaranthus rudis Sauer) with 2,4-D (1120 g ae) and 99% control with 2,4-D + glyphosate (280 + 840 g ae); however, it is important to note glyphosate (840 g ae) alone provided 100% control 4 WAA in that study. Similarly, Miller and Norsworthy [39] [44] reported 8% to 9% greater control of ALS-resistant-waterhemp 1, 2, and 3 WAA when herbicides were applied to 5 cm waterhemp early POST (EPOST) compared to 10 cm waterhemp (POST) in soybean. Similarly, Hedges et al. [43] observed a 20% reduction in waterhemp control as POST applications were delayed from 5 to 25 cm tall waterhemp. These studies suggest differences between EPOST and POST can be attributed to slower herbicide activity on larger waterhemp plants and reduced interception due to shading of younger plants caused by the extended emergence pattern [33] [42] [43]. Corn producers should eliminate MHR waterhemp interference from VE to V6 corn growth stage and control it before it exceeds 10 cm in height [2] [44] [45].
Delayed POST herbicide applications can result in reduced control due to larger weed size at application and decreased corn yield due to early-season waterhemp interference. To achieve season-long control of MHR waterhemp, it is imperative that herbicide applications include effective MOA, provide soil residual and target small weed size (≤10 cm). We hypothesized that EPOST herbicide tank mixtures made to 5 cm MHR waterhemp will provide season-long control of MHR waterhemp in corn. The objective of this research was to identify effective EPOST herbicide tank-mixtures that provide control of emerged MHR waterhemp and season-long residual control in corn while stewarding currently available herbicide MOA.

Experimental Methods
Five field trials were conducted over a two-year period (2019, 2020) at sites on  (Table 1). Sites were disked or cultivated in the spring to prepare the seedbed for planting. Glyphosate-and glufosinate-resistant corn hybrid DKC45-65RIB (Monsanto, St. Louis, MO) was seeded in rows spaced 0.75 m apart at approximately 83,000 seeds ha −1 to a depth of 4 cm. Plots were 8 m long and 2.25 m (3 corn rows) wide. Fifteen herbicide treatments (Table 2) were arranged in a randomized complete block design with four replications. Replications included nontreated and weed-free controls and were separated by a 2 m alley. The weed-free control was maintained weed-free with a pre-emergence (PRE) application of atrazine/bicyclopyrone/mesotrione/S-metolachlor (2022 g·ha −1 ) followed by either atrazine/dicamba (1800 g·ha −1 ) applied postemergence (POST) up to V3-stage (5-leaf stage) of corn development, or glufosinate (500 g·ha −1 ) between V3 and V6; hand-weeding was performed throughout the remainder of the growing season as needed. Glyphosate (450 g ae ha −1 ) was applied POST to the entire experimental area, including the nontreated control, to remove susceptible waterhemp biotypes and other weed species.
Herbicide treatments were applied EPOST using a CO 2 -powered backpack sprayer equipped with four, 120-02 ultra low drift (ULD) nozzles (Pentair, New Brighton, MN) spaced 50 cm apart and calibrated to deliver 200 L·ha −1 at 240 kPa. All herbicide treatments were applied when MHR waterhemp reached an average 5 cm in height. Site 1 (S1) and S3 was separated temporally by applying herbicide treatments 5 days apart.
Data were collected on MHR waterhemp control estimates, density, biomass, visible corn injury, grain corn moisture content, and grain corn yield. Waterhemp control was evaluated visually on a 0% to 100% scale compared to the nontreated control at 4, 8, and 12 WAA. MHR waterhemp density and biomass were determined at 4 WAA by counting and harvesting the plants within two randomly placed 0.25 m 2 quadrats in each plot. The aboveground biomass of the plants within each quadrat was determined by cutting the MHR waterhemp at  the soil surface, the plants placed inside a paper bag, kiln-dried for three weeks to a consistent moisture, then weighed using an analytical balance to calculate MHR waterhemp biomass per unit area (g·m −2 ). Visible corn injury was assessed on a 0% to 100% scale at 1 and 4 weeks after herbicide application (WAA); 0% represented no visible injury and 100% represented complete plant death. Grain corn yield (t·ha −1 ) and moisture (%) were collected by harvesting two rows of each plot at maturity using a small-plot combine. Grain yields were adjusted to 15.5% moisture prior to statistical analysis.

Statistical Analysis
Data were subjected to variance analysis using the PROC GLIMMIX procedure in SAS v. 9.4 (SAS Institute Inc., Car, NC). An initial mixed model analysis was conducted to evaluate site-by-treatment interactions. Site, site-by-treatment, and replication within site were considered the random effect and the fixed effect was treatment. Site-by-treatment interaction was significant for all parameters with no difference between S1 and S3, and S2 and S5; therefore, data were combined for S1 and S3, and S2 and S5, and are presented separately for S4. A second mixed model analysis was conducted to analyze herbicide treatment ef-

Results and Discussion
Most EPOST herbicide tank-mixtures provided greater than 90% control of MHR waterhemp. The density, biomass, and population resistance profile are reflected in the differences in control between sites. At 4, 8, and 12 WAA, control of MHR waterhemp ranged from 61% to 100% across sites and was lower at S1, S3, and S4 due to greater density and biomass compared to S2 and S5 (Table   1, Tables 3-5). Density and biomass of MHR waterhemp in the nontreated control at S1, S3, and S4 averaged 263 to 962 plants m −2 and 70.2 to 259.4 g·m −2 , respectively, compared to 60 plants m −2 and 72.2 g·m −2 at S2 and S5. Vyn et al. [31] reported similar site differences in POST MHR waterhemp control which they attributed to plant density and site-specific MHR waterhemp resistance profiles.
In that study, one waterhemp population exhibited resistance to ALS-inhibiting herbicides and the other to both ALS-and PS II-inhibiting herbicides [31]. All sites contained waterhemp resistant to ALS-, PS II-, EPSPS-and PPO-inhibitors; however, the proportion of individuals resistant to each MOA varied by site. The MHR waterhemp population at S1, S3, and S4 contained a greater number of individuals exhibiting resistance to ALS-, PS II-, and PPO-inhibitors than that of S2 and S5.     [19] and [49]; in contrast, late emerging cohorts have been reported to reduce end-of-season control as well [2]. Corn injury was ≤10% for all herbicide treatments 1, 2, and 4 WAA at all sites except S2 (Table 5). Glyphosate/2,4-D choline + rimsulfuron + mesotrione + atrazine caused 28%, 31%, and 31% corn injury 1, 2, and 4 WAA , respectively at S2; symptoms included brace root malformation and lodging which resulted in reduced corn stand. Applications of glyphosate/2,4-D choline to non-ENLIST TM hybrids can cause stalk brittleness, leaning, malformed brace roots, and leaf rolling in the whorl [50] [51]. Ruen et al. [38] reported similar leaf necrosis and leaning of ENLIST TM corn hybrids treated with single applications of glyphosate/2,4-D choline at V4 and V7 corn growth stages and sequential applications at V4 fb V7. Interestingly, the addition of atrazine to glyphosate/2,4-D choline + rimsulfuron + mesotrione increased corn injury 25%, 25%, and 26% at 1, 2, and 4 WAA, respectively. We do not have an explanation for this observation; the response should be evaluated in future studies to determine if this is a real response. Tolerance of conventional corn hybrids to 2,4-D varies with hybrid, corn growth stage at application, soil characteristics, and weather conditions [38]. It is recommended that glyphosate/2,4-D choline (ENLIST DUO) only be applied to ENLIST TM field corn hybrids that contain the AAD-1 transgene [35]. Glyphosate/2,4-D choline applications can also be made up to the V8 corn growth stage; in this study, herbicides were applied to V4 corn (data not shown). Corn injury caused by glyphosate/2,4-D choline + rimsulfuron + mesotrione + atrazine resulted in lower corn yield than glyphosate + S-metolachlor/atrazine; however, yield was similar to the weed-free control. When waterhemp was left uncontrolled, corn yield was reduced 39% at S4 and was similar to another Ontario study that reported a corn yield reduction of 48% [16]. Relative to the weed-free control, corn yield was not reduced at S1, S2, S3, and S5 which could again be the result of comparatively lower MHR waterhemp density and biomass. Cordes et al. [14] reported corn yield reductions due to the late removal of waterhemp when plants reached 15 cm. This result supports previous research that suggests EPOST herbicide applications reduce early season weed interference and minimize corn yield loss [13].