Influence of Adjuvants on the Control of Glyphosate-Resistant Canada Fleabane and Waterhemp in Corn with Tolpyralate

Tolpyralate 
is a new benzoylpyrazole herbicide for weed management in corn. It is recommended to be co-applied 
with atrazine along with the adjuvants methylated seed oil concentrate (MSO) 
plus an ammonium nitrogen fertilizer, such as urea ammonium nitrate (UAN). Two studies were conducted on glyphosate-resistant 
(GR) Canada fleabane and GR waterhemp to determine if an additional adjuvant is 
still required when tolpyralate plus atrazine are tankmixed with a commercial 
glyphosate formulation (Roundup WeatherMAX®) in corn. Trials were conducted over a two-year 
period (2018-19) on farms in south western Ontario with 
confirmed GR populations. When co-applied with Roundup WeatherMAX®, the addition of MSO to tolpyralate + atrazine increased 
control of GR waterhemp 9%; however, there was no increase in GR Canada 
fleabane control from the addition of additional adjuvants. At 8 WAA, all 
treatments provided > 91% and > 84% control of GR waterhemp and GR Canada fleabane, 
respectively. This study concludes that the addition of Roundup WeatherMAX® 
to tolpyralate plus atrazine improves the control of GR waterhemp and GR Canada 
fleabane in corn.


Introduction
Glyphosate-resistant (GR) waterhemp (Amaranthus tuberculatus) and GR Canada fleabane (Conyza canadensis L. Cronq.) are prevalent across parts of south-plant [12] [15]. Canada fleabane seeds have a small attached pappus that aids in wind dispersal; seeds can travel long distances of up to 500 km [16]. Canada fleabane seeds germinate on or near the soil surface, at depths up to 0.5 cm, making this species well-adapted to no-till crop production systems [17]. Multiple applications of glyphosate are often used for weed control in no-till GR cropping systems; this may have contributed to the evolution of GR Canada fleabane biotypes [17]. The first confirmed population of GR Canada fleabane was discovered in 2001 in a GR soybean field near Delaware, USA, that received glyphosate applications for three consecutive years [18]. In 2010, the first population of GR Canada fleabane was discovered in Ontario in Essex County, and by 2015, GR populations were found in 30 counties across southern Ontario from the Michigan to the Quebec border [19]. Resistance in GR Canada fleabane is primarily caused by non-target site resistance mechanisms: vacuolar sequestration and enhanced metabolism where the herbicide does not reach the target site at a lethal dose [20] [21]. More recently, a proline to serine amino acid substitution at position 106 was identified that confers resistance to glyphosate by altering the target site for herbicide binding [22]. Resistance to herbicides adds complexity to the management of Canada fleabane.
Tolpyralate plus atrazine, applied POST, provides control of annual grass and broadleaf weed species including Canada fleabane and Amaranthus spp. [24] [27]. On the Canadian and US labels, tolpyralate plus atrazine provides "control" of both common (A. rudis) and tall (A. tuberculatus) waterhemp; however, only the US label lists "partial control" of Canada fleabane [25] [28].
The recommended adjuvants with tolpyralate are MSO Concentrate (1% v/v) and a nitrogen source such as UAN or ammonium sulfate (AMS) (2.5% v/v) to improve herbicide efficacy [28]. Defined by the Weed Science Society of America, an adjuvant is any substance added to the spray tank, or present in an herbicide formulation that enhances herbicidal activity or application characteristics.
Selection of an appropriate adjuvant at the proper concentration can improve herbicide coverage on various leaf surfaces [29]. Adjuvants can decrease surface tension and contact angle (angle between the leaf surface and the spray droplet), as well as influence wetted area and evaporation time, and play a role in the deposition patterns on leaf surfaces [30]. Many herbicides require the addition of an adjuvant for optimal performance. Topramezone, another Group 27 herbicide, has enhanced efficacy with the addition of MSO due to increased absorption and translocation of the herbicide in the plant [31]. Quinclorac efficacy is enhanced with the addition of MSO and a nitrogen fertilizer that combats antagonistic salts present in the carrier water [32]. Adjuvants can also be already present in the herbicide formulation. For example, most commercial glyphosate formulations in Canada are formulated with an adjuvant. For example, Roundup WeatherMAX® contains proprietary surfactants to improve the penetration of the herbicide into the plant tissue [33]. In a tankmix, glyphosate formulations have been found to influence crop injury [34], weed control [35] [36] or absorption and translocation of the tank mix partners. Adjuvants add an additional cost to the complete weed control program. In Ontario, adjuvants can cost up to $10.00 ha −1 (Agris Co-operative, personal communication) and require the handling of multiple product containers.
Based on previous research, it is evident that there is an interaction between commercial glyphosate formulations and various herbicides that may be the re-

Materials and Methods
This manuscript summarizes two distinct studies: Study 1-Impact of adjuvants on tolpyralate + atrazine efficacy for the control of GR waterhemp, and Study 2-Impact of adjuvants on tolpyralate + atrazine efficacy for the control of GR Canada fleabane.

Study 1
Six field trials (as listed in Table 1 Corn injury at 1, 2 and 4 WAA, and weed control at 2, 4, 8 and 12 WAA was assessed visually on a percent scale where 0 indicates no corn injury/weed control and 100 complete plant death. Waterhemp density and dry biomass were determined by counting and cutting waterhemp at the soil surface from two 0.25 m 2 quadrats per plot, placing them in paper bag, drying the biomass to a constant mass at 60 C, and then weighing. Corn was harvested from two rows in each plot at maturity with a small plot combine; grain weight and moisture content were recorded. Corn grain yield was expressed as tonnes ha −1 and adjusted to 15.5% moisture before statistical analysis.

Study 2
Materials and methods were similar to Study 1, with the exception that the weed species evaluated was GR Canada fleabane and present at different sites located near Dresden, Thamseville, Harrow, Ridegtown and Zone Centre; all sites were managed no-till. Table 2 lists GR Canada fleabane sites, planting and herbicide application dates.

Statistical Analysis
For both studies, data were analyzed as a 2 by 5 factorial, variance analysis was performed using PROC GLIMMIX in SAS v. 9.4 (SAS Institute, Cary, NC). Data were pooled across years and locations for analysis. Each location-year indicates an environment for a total of 6 environments in study 1 and 5 environments in study 2. Variances were partitioned into random and fixed effects. Random effects were environment, replication within environment, environment and its interaction with each fixed effect. Fixed effects were Factor One, Factor Two and the interaction between Factor One and Two. The significance of fixed effects was tested using F-tests and random effects were tested using Z-tests with α N. M. Langdon et al. assigned as 0.05. The assumptions of variance analyses (residuals have mean of zero, are homogeneous, and normally distributed) were tested using a Shapiro-Wilk test of normality and scatterplot of studentized residuals. An appropriate distribution and link were assigned to each parameter to meet those assumptions. Weed control data were transformed using arcsine square root; weed density and biomass data were log-transformed. Treatment comparisons for main effects were performed only when the interaction was not significant. Simple effects were analyzed when the interaction was significant. Treatment comparisons were made based on least-square means using Tukey-Kramer's multiple range test and letter codes were assigned to illustrate statistically significant differences. The means of transformed data were converted back to the original scale for presentation of results.

Study 1
Data analysis indicated that the Roundup Weather MAX® by tolpyralate + atrazine interaction was not significant for GR waterhemp density (P = 0.6348) and biomass (P = 0.9906) and corn yield (P = 0.3225) ( Table 3). Averaged across all tolpyralate + atrazine treatments, the addition of Roundup WeatherMAX® was not significant for GR waterhemp density and biomass and corn yield ( Table 3). Averaged across the 0 and 900 g•ae•ha −1 of Roundup WeatherMAX®, tolpyralate + atrazine reduced GR waterhemp density and biomass, the decrease in density and biomass were greater with the addition of MSO or MSO + UAN compared to UAN. There was no effect of Roundup WeatherMAX® or tolpyralate + atrazine treatment on corn yield (Table 3). There was a significant glyphosate by tolpyralate + atrazine interaction for weed control at 2, 4, 8 and 12 WAA (P < 0.0001) ( Table 4).

Crop Injury
Corn injury was less than 10% in all treatments (data not presented).

Study 2
Based on data analysis, the Roundup WeatherMAX® by tolpyralate + atrazine interaction was not significant for GR Canada fleabane density (P = 0.7426) or biomass (P = 0.4563) ( Table 5). Averaged over all tolpyralate + atrazine treatments, the addition of Roundup WeatherMAX® had no effect on GR Canada fleabane biomass or density (

Crop Injury
Crop injury was less than 10% in all treatments (data not presented).

Weed Control
Tolpyralate + atrazine controlled GR Canada fleabane 58% at 2 WAA; there was no improvement in control with the addition of UAN ( Table 6). The addition of  (Table 6). Roundup WeatherMAX® and Roundup WeatherMAX® + tolpyralate + atrazine controlled GR Canada fleabane 25% and 91%, respectively, there was no increase in control with the addition of MSO, UAN or MSO + UAN.

Yield
GR Canada fleabane interference reduced corn yield up to 50% (highest yielding treatment compared to the weedy control). Reduced GR Canada fleabane interference with tolpyralate + atrazine, without and with adjuvants, increased corn yield 84% to 101% relative to the weedy control.

Discussion
POST-applied herbicides must contact the leaf surface of the target species and cross the cuticle and enter the living portion of the plant to be effective. Adjuvants improve herbicide performance by altering application characteristics, physical and/or chemical properties of herbicides [37]. The tolpyralate label recommends the addition of MSO and UAN as adjuvants for enhanced herbicide efficacy [25] [28]. Glyphosate (Roundup WeatherMAX®) has a proprietary blend of surfactants included in the formulation [26]. The addition of Roundup WeatherMAX® to tolpyralate + atrazine improved the control of GR waterhemp and Canada fleabane at 2 and 4 WAA, in the absence of MSO, UAN or MSO + UAN. At 2 and 4 WAA, the addition of Roundup WeatherMAX® to tolpyralate + atrazine increased GR waterhemp control by 6% and 10%, and increased GR Canada fleabane control by 20% and 18%, respectively. Armel et al. [52] also reported improved control of common ragweed and giant foxtail with mesotrione (105 or 140 g•ai•ha −1 ) when in a tankmix with glyphosate compared to mesotrione alone. Soltani et al. [34] reported an interaction between glyphosate and 2,4-D where corn injury is enhanced with the addition of glyphosate, speculated to be caused by the aggressive adjuvant system in a commercial glyphosate formulation. In contrast to results from Soltani et al. [34], crop injury was not accentuated with the addition of glyphosate to tolpyralate + atrazine, however there was an influence on weed control. Glyphosate tank mixtures are reported to be complementary for herbicides like saflufenacil, chlorimuron and imazethapyr, exhibiting an additive effect [35] [38]. In contrast, addition of glyphosate to fomesafen and sulfentrazone was antagonistic and reduced absorption and translocation within various weed species [38]. Research by Deen et al. [36] found that quizalofop-p-ethyl (high rate) plus glyphosate and quizalofop-p-ethyl plus the recommended adjuvant provided similar control of GR volunteer corn, suggesting that the adjuvant in the glyphosate formulation could replace the recommended adjuvant on the quizalofop-p-ethyl label. At lower rates of quizalofop-p-ethyl, greatest control was achieved when the recommended adjuvant was used [36].
Removing unnecessary adjuvants from herbicide applications allows farmers to spend less money on their weed control program while still achieving a high level of weed control. In Ontario, adjuvants can cost between $1.00 to 10.00 ha −1 .
In this specific example, using both UAN and MSO with tolpyralate would require the farmer to spend over $10.00 ha −1 on the adjuvant system (Agris In the absence of Roundup WeatherMAX®, control of GR waterhemp and GR Canada fleabane was affected by adjuvant selection. Without the addition of an adjuvant, tolpyralate + atrazine provided less than 70% control of Canada fleabane (Table 6) and less than 80% control of waterhemp (Table 3) at 2 and 4 WAA. Previous research by Dayan et al. [39] found that in the absence of adjuvants, sulfentrazone absorption and phototoxic effects on weeds was reduced, although when included, adjuvants overcame barriers to herbicide absorption in plant leaves. As an activator adjuvant, MSO is an oil adjuvant made from soybean and influences various properties of an herbicide spray droplet leading to N. M. Langdon et al.
improved efficacy [40]. Tolpyralate + atrazine + MSO provided the greatest control of GR Canada fleabane at 2 and 4 WAA, and waterhemp at 4 WAA. The increased herbicidal activity observed with the addition of MSO may be attributed to the greater absorption and retention of the herbicide on plant leaf as a result of various physical and chemical changes to the herbicide solution [31] [41]. Reduced surface tension and contact angle of spray droplets promote the penetration of herbicides through the leaf cuticle [31]. Herbicidal activity of topramezone and isoxaflutole, two other Group 27 herbicides, is also improved with the addition of MSO [31] [41]. Hutchinson et al. [42] found that MSO enhanced activity of metribuzin, resulting in greater control of lambsquarters, hairy nightshade, redroot pigweed and kochia when compared to nonionic surfactants. The addition of UAN to tolpyralate + atrazine with MSO did not increase the control of GR waterhemp and GR Canada fleabane (Table 3 and Table 6). This study concludes that the addition of MSO to tolpyralate + atrazine improves the control of GR waterhemp and GR Canada fleabane, but there is no improvement in weed control when UAN is added to tolpyralate + atrazine. This is consistent with research conducted by Idziak et al. [43] and Young and Hart [41] where no increase in weed control was observed with the addition of UAN to MSO. In contrast, Gronwald et al. 1993 [44] reported an increase in herbicidal activity with the addition of a nitrogen fertilizer due to the influence of ammonium ions on herbicide absorption into the plant. There was no improvement in GR waterhemp and GR Canada fleabane with the addition of an adjuvant at 8 and 12 WAA. This study found that the addition of MSO to tolpyralate + atrazine increases the speed of GR waterhemp and GR Canada fleabane control, but end-of-season weed control is similar. The current tolpyralate label in the US claims partial control of GR Canada fleabane; GR Canada fleabane does not appear on the Canadian label. Results from this study conclude that tolpyralate + atrazine controls GR Canada fleabane [25] [28]. Metzger et al. [27] also found GR Canada fleabane control with tolpyralate + atrazine equivalent to bromoxynil + atrazine and dicamba/atrazine, the current industry standards for control of GR Canada fleabane in corn in Canada. In addition, results of this study are similar to Benoit et al. [8] who reported that POST-applied tolpyralate + atrazine controlled GR waterhemp in corn. Osipitan et al. [45] found that tolpyralate + atrazine controlled waterhemp at lower than the label rate at 60 days after application.
Corn is susceptible to herbicide injury when plants are young and rapidly growing, possessing a thin cuticle that allows for rapid uptake of the herbicide [46]. White bleaching is a characteristic corn injury symptom from HPPD-inhibiting herbicides. Corn injury across all experiments was less than 10% (data not shown); however, injury symptoms appeared as gray, water-soaked lesions, likely a result of the adjuvants used [47]. The addition of MSO to mesotrione increased herbicide uptake in sorghum resulting in increased levels of crop injury, sorghum outgrew the injury and yield was not impacted [43]. Metzger et al. [47] found tolpyralate + atrazine + MSO + UAN to  [34] that reported increased crop injury when glyphosate was added to 2,4-D, the addition of glyphosate to tolpyralate + atrazine did not enhance crop injury. Any visible crop injury symptoms in these studies did not affect corn yield. Left uncontrolled, GR Canada fleabane reduced corn grain yields by up to 50% in this study, which is similar to research by Ford et al. [48] that observed a 69% yield loss due GR Canada fleabane interference in corn.

Conclusion
Results indicate that the addition of Roundup WeatherMAX® enhances herbicidal activity of tolpyralate + atrazine; however, the addition of MSO is still required to enhance the speed of activity. The inclusion of a commercial glyphosate formulation in the tankmix will increase the spectrum of weeds controlled and an additional mode of action will reduce the selection intensity for  [52]. Incorporating multiple effective modes of action, in addition to a diverse crop and herbicide rotation will enhance the long-term effectiveness of this herbicide mode-of-action.