Influence of Adjuvants on the Efficacy of Tolpyralate plus Atrazine for the Control of Annual Grass and Broadleaf Weeds in Corn with and without Roundup WeatherMAX ®

Tolpyralate is a new HPPD-inhibiting herbicide that is efficacious on annual grass and broadleaf weed species in corn. For maximum herbicide performance of tolpyralate, it is recommended that atrazine is tank mixed with tolpyralate along with the adjuvants methylated seed oil concentrate (MSO) plus urea ammonia nitrate (UAN). A common use pattern of tolpyralate plus atrazine will be in a tank mix with Roundup WeatherMAX® due to the high pro-portion of corn acres that are seeded to Roundup Ready® hybrids in Eastern Canada. There is no information in the peer-reviewed literature if the adjuvant system in Roundup WeatherMAX® is adequate for optimal herbicide performance of tolpyralate plus atrazine, or if MSO and UAN are still required. Six field trials were conducted over two years near Ridgetown and Exeter, ON, Canada to determine if adjuvants are still required when tolpyralate plus atrazine is tank mixed with Roundup WeatherMAX® in corn. Tolpyralate plus atrazine plus MSO and Roundup WeatherMAX® plus tolpyralate plus atrazine provided excellent control of velvetleaf, pigweed spp, common ragweed, lambsquarters, ladysthumb, wild mustard, flower-of-an-hour, barnyardgrass and green foxtail in this study. Results of this study show that in the absence of Roundup WeatherMAX®, weed control with tolpyralate plus atrazine was improved substantially with the addition of MSO; however, there was little to no increase in weed control with the addition of UAN. When tolpyralate plus atrazine was co-applied with Roundup WeatherMAX®, there was no improvement in weed control with the addition of MSO and/or UAN.


Introduction
Weed interference during the early stages of corn development can cause physiological changes in the plant and lead to yield loss [1]. A study conducted across the corn producing regions of the United States and Canada found that in the absence of any weed management tactics, corn yield loss due to weed interference was 50% [2]. The critical weed free period (CWFP) in corn delineates the time period during which weed interference must be minimized to prevent corn yield loss. The CWFP is crop specific due to the differences in crop growth and development and can vary across location and years [3]. Hall et al. 1992 [4] determined that the CWFP in Ontario for corn is from the 3 to 14 leaf-tip stage.
Using the CWFP as a guideline to time POST herbicide applications can minimize corn yield and economic losses [3].
Tolpyralate is a new 4-hydroxyphenyl-pyruvate dioxygenase (HPPD)-inhibitor belonging to the benzoylpyrazole family. This is the second benzoylpyrazole herbicide registered for use in corn. Classified as a Group 27 herbicide, HPPD-inhibitors affect sensitive weeds by interrupting the biosynthesis of plastoquinone and tocopherol and stopping the synthesis of carotenoid pigments [5]. The inability of plants to quench singlet oxygen and other reactive oxygen species leads to light induced destruction of proteins, lipids and the photosynthetic complex, releasing free chlorophyll [5] [6]. Free chlorophyll generates additional singlet oxygen that causes destruction of leaf pigments and results in the characteristic white bleaching symptoms associated with HPPD-inhibiting herbicides [5] [6]. Tolpyralate (30 -40 g•ai•ha −1 ) is recommended to be co-applied with a photosystem II inhibiting herbicide, such as atrazine (560 -1120 g•ai•ha −1 ) [7]. Previous research by Metzger et al. 2018 [8] reported that the addition of atrazine to tolpyralate increases the speed of herbicide activity and expands the spectrum of weed species controlled. Among weed species, there is variability in sensitivity to tolpyralate. Weed species such as velvetleaf (Abutilon theophrasti Medik.), common ragweed (Ambrosia artemisiifolia L.), lambsquarters (Chenopodium album L.), redroot pigweed (Amaranthus retroflexus L.) and green foxtail (Setaria viridis (L.) P. Beauv.) are very sensitive to tolpyralate and were controlled > 90% with lower than label rates of tolpyralate (15.5 g•ai•ha −1 ); in con- The efficacy of tolypyrate on weeds may be improved with the addition of adjuvants, such as methylated seed oil (MSO) concentrate (1% v/v) and a nitrogen source such as urea ammonium nitrate (UAN) or ammonium sulfate (AMS) (2.5% v/v) [11]. Adjuvants are additives to a spray solution, or present in an herbicide formulation, that can enhance herbicidal activity or application characteristics [12]. There are various types of adjuvants that are grouped based on function; special purpose adjuvants indirectly influence spray solution characteristics, while activator adjuvants modify physical and chemical properties of an herbicide [13]. Activator adjuvants can decrease surface tension and contact angle of the spray droplet (angle between the leaf surface and the spray droplet), as well as influence spreading of the herbicide on the leaf surface [14]. MSO and UAN are activator adjuvants that are added to a spray mixture. Zhang et al. 2013 [15] reported that MSO increased herbicide retention, absorption and translocation in plant leaves. Gronwald et al. 1993 [16] reported an increase in herbicide absorption into the plant with the addition of a nitrogen fertilizer due to the influence of ammonium ions, further increasing herbicidal activity. Commonly, herbicides require the addition of an adjuvant for optimal performance. For example, topramezone absorption and translocation into the plant is enhanced with the addition of MSO [15]. Wozinac et al. 2003 [17] reported enhanced quinclorac efficacy when it was co-applied with MSO and a nitrogen fertilizer. In some cases, increased corn injury may occur with the addition of adjuvants such as MSO and UAN to tolpyralate, however plants rapidly recovered [9] [18].
Herbicide formulations may be sold preformulated with adjuvants. All glyphosate products sold in Canada are formulated with adjuvants; for example, Roundup WeatherMAX® contains proprietary surfactants for effective retention and absorption of the herbicide in the plant tissue [19]. Previous research has found that tank mixes of glyphosate formulations with additional herbicides may influence crop injury [20], weed control efficacy [21] [22] or herbicidal activity of the tank mix partners [23].
Adjuvants may have some benefits; however, they are an additional weed management cost. Across Ontario, adjuvants can cost up to $10.00 ha −1 (Agris Co-operative, personal communication), in addition, they require the handling of multiple product containers thereby increasing time to fill sprayers in the field. In the literature, it is evident that there is an interaction between glyphosate and various herbicides, potentially a result of the aggressive adjuvant system in glyphosate products. Ninety-six percent of corn acres in Eastern Canada are seeded to Roundup Ready® hybrids (M. Reidy, Stratus, personal communication); therefore, tolpyralate plus atrazine will commonly be used in a tank mixture with glyphosate. The proprietary blend of surfactants in Roundup Wea-therMAX® enhances the ability of the herbicide to move into the plant [19]; however, it has not been reported if the adjuvant system in Roundup Weather-MAX® is adequate for optimal tolpyralate plus atrazine performance, or if additional adjuvants are still necessary. Therefore, the objective of this study was to

Materials and Methods
Over

Statistical Analysis
Data were analyzed using SAS v 9.4 (SAS Institute, Cary, NC) and the GLIMMIX procedure. Data were pooled across years and location for analysis; normality assumptions were evaluated using PROC UNIVARIATE. Each location-year represented a site for a total of six sites in this study. When weed species were not present at a site, that site was excluded from analyses. Fixed effects were Factors One and Two and their interactions; significance was determined using F-tests. Site (location-year), replication within site and the interaction of the site with each fixed effect were designated as random effects and tested using Z-tests. For all analyses, significance was set to α = 0.05. The Shapiro-Wilk test of normality in addition to normality and scatter plots of studentized residuals were used to determine if residuals met the assumptions (homogeneous, had a mean of zero and were normally distributed). The appropriate distribution and link for each parameter that best met assumptions was used. Weed control data was transformed using arcsine square root, except wild mustard control at 2 WAA and flower-of-an-hour (Hibiscus trionum L.) control at 4 WAA that was assigned a normal distribution. Weed density and biomass were analyzed using a lognormal distribution. Yield was analyzed using a normal distribution. Treatment comparisons for main effects were performed only when the interaction was not significant. When the interaction was significant, simple effects were analyzed. 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. Least-square means of each parameter were back-transformed to the original scale for presentation of results.

Results and Discussion
There were nine naturally-occurring weed species evaluated in this study. The

Velvetleaf
Analysis of variance determined that the interaction between Roundup Wea-therMAX® and tolpyralate + atrazine was not significant for velvetleaf density (P = 0.3347) ( 2013 [15] reported that MSO enhanced the efficacy of topramezone, a Group 27 herbicide, on velvetleaf by 1.0-fold due to increased absorption and translocation. Other research reported that bentazon absorption in velvetleaf was increased with the addition of a crop oil concentrate (COC) compared to UAN;  WeatherMAX® by tolpyralate + atrazine interaction for pigweed control 2, 4, and 8 WAA (P < 0.001), density (P = 0. 0001), and biomass (P = 0. 0003) ( Table 4).

Common Ragweed
Variance analysis of common ragweed data indicated a Roundup WeatherMAX® by tolpyralate + atrazine plus adjuvant treatment interaction for weed control at 2, 4 and 8 WAA (P < 0.0001) and common ragweed biomass (P = 0.0007) ( Table   6). There was not a significant interaction between Roundup WeatherMAX® and tolpyralate + atrazine for common ragweed density (P = 0.1035); however, for the tolpyralate + atrazine plus adjuvant treatment there was a significant main effect (P < 0.0001). Roundup WeatherMAX®, averaged across all tolpyralate plus adjuvant treatments, did not decrease common ragweed density (Table 6).
However, averaged across glyphosate at 0 and 900 g•ae•ha −1 , tolpyralate + atrazine reduced common ragweed density 81% compared to the control; there was no further decrease in density with the addition of MSO, UAN or MSO + UAN to tolpyralate + atrazine. Tolpyralate + atrazine controlled common ragweed ≥ 77% at 2, 4, and 8 WAA; there was no increase in common ragweed control with the addition of UAN ( Table 7). The addition of MSO or MSO + UAN to tolpyralate + atrazine improved common ragweed control to ≥ 94%. Tolpyralate + atrazine reduced common ragweed density 91%, there was no further decrease in common ragweed density when MSO, UAN or MSO + UAN were added to tolpyralate + atrazine. Results of this study are similar to Metzger et al. 2019 [11] who reported 99% control of common ragweed 2 WAA when tolpyralate + atrazine (30 + 1000 g•ai•ha −1 ) plus recommended adjuvants was applied early POST. Similar to this study, Bunting et al. 2004 [28] found that the addition of MSO to foramsulfuron improved control of common ragweed. However, the addition of MSO, UAN or MSO + UAN to tolpyralate + atrazine did not increase common ragweed control 8 WAA or reduce common ragweed biomass. Roundup Wea-therMAX® controlled common ragweed 88% -91% at 2, 4 and 8 WAA; control was not improved when tolpyralate + atrazine was added alone or in combination with MSO, UAN or MSO + UAN. Roundup WeatherMAX® reduced common ragweed biomass 99%; biomass was not reduced further with the addition of tolpyralate + atrazine to Roundup WeatherMAX®. The addition of Roundup WeatherMAX® to tolpyralate + atrazine improved control of common ragweed when applied alone and with UAN at 2 WAA and with tolpyralate + atrazine at 4 WAA. By 8 WAA, there was no increase in common ragweed control when Roundup WeatherMAX® was added to tolpyralate + atrazine.

Ladysthumb
Data analysis indicated that there was no interaction between Roundup Wea-therMAX® and tolpyralate + atrazine plus adjuvant treatment for ladysthumb density (P = 0.2773) or biomass (P = 0.0902) (

Wild Mustard
Data analysis concluded that the Roundup WeatherMAX® by tolpyralate + atrazine interaction was not significant for wild mustard density (P = 0.5125) or biomass (P = 0.1336). Averaged across all levels of each factor, Roundup Wea-therMAX® or tolpyralate + atrazine plus adjuvant treatment was there not a significant main effect for density or biomass (Table 12). There was a significant Roundup WeatherMAX® by tolpyralate + atrazine interaction for wild mustard control at 2 (P = 0.0011), 4 (P = 0.0080) and 8 (P = 0.0006) WAA (Table 12). Tolpyralate + atrazine controlled wild mustard 74, 76 and 97% at 2, 4 and 8 WAA, respectively (Table 13). There was no improvement in wild mustard

Barnyardgrass
Analysis of variance in barnyardgrass data concluded that the Roundup Wea-therMAX® by tolpyralate + atrazine interaction was not significant for barnyardgrass density (P = 0.3385) (   [28] showed that barnyardgrass control with foramsulfuron was greatest when MSO was the adjuvant and there was no benefit to the addition of a nitrogen source, similar to results of this study in N. M. Langdon et al.

Green Foxtail
Green foxtail control and biomass varied with Roundup WeatherMAX® and tolpyralate + atrazine plus adjuvant treatment, producing a significant Roundup WeatherMAX® by tolpyralate plus adjuvant interaction for control at 2, 4 and 8 WAA (P < 0.0001) and biomass (P < 0.0001) (

Crop Injury
Crop injury was less than 10% with all the treatments evaluated (data not presented). In contrast, Soltani et al. 2018 [20] reported increased corn injury from 2,4-D when it was co-applied with Roundup WeatherMAX®. Metzger et al. 2019 [9] reported similar results to this study where tolpyralate + atrazine caused minimal corn injury.

Crop Yield
The Roundup WeatherMAX® by tolpyralate + atrazine interaction was significant for corn grain yield (P < 0.0001) (  with all the herbicide treatments evaluated resulted in higher grain yield than the no tank mix partner (

Conclusions
Results from this study indicate that the addition of MSO to tolpyralate + atrazine improved the control of velvetleaf, pigweed, common ragweed, lambsquarters, ladysthumb, barnyardgrass and green foxtail with the exception of flower-of-an-hour at 2 WAA, common ragweed at 8 WAA and wild mustard and ladysthumb at all evaluations. The addition of UAN to tolpyralate + atrazine did not improve the control of velvetleaf, pigweed, common ragweed, lambsquarters, ladysthumb, wild mustard, flower-of-an-hour, barnyardgrass and green foxtail. Generally, there was no benefit of adding UAN to tolpyralate + atrazine + MSO in respect to weed control or corn yield. The current tolpyralate label recommends the use of both MSO and UAN for optimal weed control. The addition of MSO and UAN to tolpyralate + atrazine would require the farmer to spend an additional $10.00 ha −1 (Agris Co-operative Ltd., personal communication). Based on this research, it is not necessary to add UAN to the spray tank, potentially allowing farmers to save up to $2.50 ha −1 ; this cost may appear small but may be substantial over many hectares. Roundup WeatherMAX® controlled velvetleaf (96%), pigweed (97%), common ragweed (88%), lambsquarters (91%), ladysthumb (94%), wild mustard (97%), flower-of-an-hour (84%), barnyard grass (93%) and green foxtail (96%) at 2 WAA. There was no benefit of adding tolpyralate + atrazine to Roundup WeatherMAX® in respect to weed control with exception of lambsquarters control at 2 WAA and reduction in lambsquarters density at 8 WAA.
Although accentuated crop injury was not observed in this study with the addition of Roundup WeatherMAX® to tolpyralate + atrazine, there was an influence on weed control. The addition of Roundup WeatherMAX® to tolpyralate + atrazine improved the control of velvetleaf, pigweed, common ragweed, lambsquarters, ladysthumb, wild mustard, flower-of-an-hour, barnyard grass and green foxtail; however, there was no increase in weed control when Roundup WeatherMAX® was added to tolpyralate + atrazine + MSO with the exception of flower-of-an-hour at 2 WAA.
There was an increase in corn grain yield when Roundup WeatherMAX® was added to tolpyralate + atrazine, but there was no increase in yield when Roundup WeatherMAX® was added to tolpyralate + atrazine + MSO. Over 96% of corn acres in Eastern Canada are seeded to Roundup Ready hybrids and the vast majority of those acres will have glyphosate applied POST, so the most common use of tolpyralate + atrazine will be in a tank mix with glyphosate. Based on this research, there is no need to add additional adjuvants to the tank when tolpyralate + atrazine is co-applied with Roundup WeatherMAX®, potentially allowing farmers to save up to $10.00 ha −1 on adjuvants. The use of multiple effective modes of action is a good resistance management practice to preserve the American Journal of Plant Sciences long-term effectiveness of herbicides.
This study concludes that in the absence of Roundup WeatherMAX®, optimal weed control is achieved with tolpyralate + atrazine + MSO. When tolpyralate + atrazine is co-applied with Roundup WeatherMAX®, no additional adjuvants are required.