Zea Mays L.) a Thesis Presented to the Faculty of Graduate

PREEMERGENCE AND POSTEMERGENCE INTERACTIONS OF SAFLUFENACIL IN CORN {Zea mays L.)

A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph

by MEGHAN ELIZABETH MORAN

In partial fulfillment of requirements for the degree of Master of Science April, 2010

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1+1 Canada ABSTRACT

PREEMERGENCE AND POSTEMERGENCE INTERACTIONS OF SAFLUFENACIL IN CORN (Zea mays L.)

Meghan Moran Advisors: University of Guelph, 2010 Clarence J. Swanton Peter H. Sikkema

Studies were conducted under field and growth room conditions to explore the tolerance of corn to postemergence treatments of saflufenacil and BAS 781 (saflufenacil plus dimethenamid-p), and to determine the safening effect of sodium compounds on saflufenacil applied postemergence. Saflufenacil and BAS 781 applied at the 3-to-4 leaf corn stage caused up to 28 and 65% injury, respectively, resulting in yield loss. The addition of Na-bentazon and baking soda to saflufenacil reduced this injury by decreasing foliar herbicide uptake. Field studies were conducted with BAS 781 applied preemergence to evaluate overall and species specific weed control, as well as the dose required for weed control when followed with glyphosate. The GR95 across locations ranged from 126 to 675 g/ha. Common ragweed was the most difficult weed to control; the GR95 was 933 g/ha. When followed by glyphosate, 46 to 1470 g/ha of BAS 781 resulted in optimum yields. ACKNOWLEDGEMENTS

Funding for this project came from BASF Canada, as well as the Ontario Ministry of Agriculture, Food, and Rural Affairs. Thank you for your financial contributions in support of this research.

There are a number of people to thank for their guidance, support, and assistance with this research project. I would like to thank my advisors, Clarence J. Swanton and Peter H. Sikkema, for their ideas and suggestions, and assistance during the writing process. Thank you to Nader Soltani and J. Christopher Hall of the University of Guelph, and Trevor Kraus of BASF Canada, for serving on my advisory committee and contributing in various ways to the research project. You have all been very kind and supportive, and have made this a great experience in which I have learned more than I expected to.

I would also like to thank those that helped manage my field experiments; Kevin Chandler, Todd Cowan, and Christy Shropshire. All of your help and advice was greatly appreciated. Additionally, I would like to thank all of the members of the Weeds Lab for their friendship and advice, and for lending a hand in the field. You have been an important source of support and entertainment over the past two years, and I will miss spending time working with you.

I would also like to thank my family, specifically my mother and grandfather, for taking good care of me and encouraging me along the way. I wouldn't have made it this far without you, and attribute much of my success to you. Thank you to my friends and partner, Chris, for encouraging me when I was overwhelmed, and for keeping me humble.

1 TABLE OF CONTENTS

Acknowledgements i Table of Contents ii List of Tables iv List of Figures vi 1.0 Literature Review: Protoporphyrinogen Oxidase Inhibiting Herbicides, and the Use of Herbicide Safeners 1.1 Introduction 1.2 Protoporphyrinogen Oxidase (PPO) and PPO Inhibiting Herbicides 1.3 A New PPO Inhibitor: Saflufenacil 1.4 Herbicide Safeners 1.5 Bentazon as an Herbicide Safener 1.6 The Role of Sodium Ions in the Safening Effect 2.0 Na-Bentazon Safens Saflufenacil Applied Postemergence to Corn 2.1 Abstract 2.2 Introduction 2.3 Materials and Methods 2.3.1 Tolerance of Corn to Saflufenacil and BAS 781 2.3.2 Growth Room 2.3.3 Dose Response of Saflufenacil 2.3.4 Safening Effect of Na-bentazon Under Growth Room Conditions 2.3.5 Safening Effect of Na-bentazon Under Field Conditions 2.3.6 Uptake of 14C-Saflufenacil With and Without Na-bentazon 2.3.7 Safening Effect of Baking Soda Under Growth Room Conditions 2.3.8 Statistical Analysis 2.4 Results and Discussion 2.5 Conclusion 2.6 Sources of Material 3.0 The Role of BAS 781 for Weed Control in Corn 3.1 Abstract 3.2 Introduction

n 3.3 Materials and Methods 3.4 Results and Discussion 3.5 Conclusion 3.6 Sources of Material 4.0 General Discussion 4.1 Contributions 4.2 Limitations 4.3 Future Research 5.0 Literature Cited 6.0 Appendix 1: Monthly Rainfall Averages for 2008 and 2009 at Each Field Location

in List of Tables

Table 2-1. Glyphosate tolerant corn hybrids used in each year at each location 50

Table 2-2. Soil characteristics for each location, including soil type, pH, percent composition of sand, silt and clay, and organic matter content 51

Table 2-3. Nozzle type, spray volume, operating pressure and plot size at each location 52

Table 2-4. Herbicide treatments and application timings for the study of corn tolerance to saflufenacil, dimethenamid-p and BAS 781 53

Table 2-5. Dates of seeding, corn seedling emergence, herbicide application relative to corn growth stage, and corn grain harvest in each year at each location 54

Table 2-6. Dates of seeding, corn seedling emergence, herbicide treatment, and corn grain harvest in 2009, at each location used in the study of the safening effect of bentazon on saflufenacil .55

Table 2-7. Herbicide treatments applied to 4 leaf corn in order to evaluate the safening effect of bentazon applied postemergence with saflufenacil under field conditions near Elora, Exeter, and Woodstock in 2009 56

Table 2-8. Average percent corn injury caused by saflufenacil, dimethenamid-p and BAS 781 (consistent with the order of your treatments) at 3, 7, 14, 21 28 and 56 days after application (DAT) at the preemergence, spike-to-2 leaf and 3-to-4 leaf stages of corn growth at Elora, Exeter, Woodstock and Ridgetown in 2008 and 2009 57

Table 2-9. Variance analysis of the dry weight of corn plants harvested 28 days after final herbicide treatment as a measure of crop injury at Elora, Exeter, Woodstock and Ridgetown in 2008 and 2009 59

Table 2-10. Variance analysis of the corn grain yield from experiments near Elora, Exeter, Woodstock and Ridgetown, ON in 2008 and 2009 60

Table 2-11. Mean corn yield from plots treated with saflufenacil, dimethenamid-p and BAS 781 at the preemergence, spike-to-2 leaf and 3-to-4 leaf stages of corn growth at the lx and 2x doses in Elora, Exeter, Woodstock and Ridgetown, ON in 2008 and 2009 61

Table 2-12. Variance analysis of percent corn collar height and dry weight reduction as compared to the untreated control 28 days after herbicide treatment and corn grain yield at Elora, Woodstock and Exeter, Ontario in 2009. All herbicides were applied at the 4th leaf stage of corn growth 62

IV Table 2-13. Distribution of C-safiufenacil in the leaf wash and parts of the treated leaf as a percent of the total amount recovered at 6 and 24 hours after treatment, with and without bentazon, in a controlled growth room experiment 63

Table 2-14. Variance analysis of plant dry weight following treatment with increasing doses of saflufenacil and baking soda in a controlled growth room, and the linear and quadratic regression coefficients at each dose of saflufenacil 64

Table 3-1. Glyphosate tolerant corn hybrids used in each year at each location 75

Table 3-2. Soil characteristics for each location, including soil type, pH, percent composition of sand, silt and clay, and organic matter content 76

Table 3-3. Nozzle type, spray volume, operating pressure and plot size at each location 77

Table 3-4. Dates of seeding, corn seedling emergence, herbicide application and corn grain harvest in each year at each location for the study of weed control with BAS 781 alone and as a part of a glyphosate program 78

Table 3-5. Dose response parameters by location for weed control with BAS 781 applied preemergence 79

Table 3-6. Dose response parameters for control of lamb's quarter, common ragweed, pigweed spp. and wild mustard with BAS 781 applied preemergence 81

Table 3-7. Corn yield for each treatment included BAS 781 applied alone and followed by glyphosate 83

v List of Figures

Figure 1-1. The structures of the reactant, protoporphyrinogen IX, and product, protoporphyrin IX, of the enzyme catalyst PPO, the last common step in chlorophyll and haem synthesis in plants.... 26

Figure 1-2. The biosynthetic pathway of chlorophyll and haem production in plants showing intermediates and enzyme catalysts, where PPO (protogen oxidase) catalyses the last step before chlorophyll and haem pathways diverge (Moulin and Smith 2005) 27

Figure 1-3. Chemical structure of acifluorfen 28

Figure 1-4. Chemical structure of oxyfluorfen. 29

Figure 1-5. Chemical structure of flumioxazin 30

Figure 1-6. Chemical structure of sulfentrazone 31

Figure 1-7. Chemical structure of carfentrazone 32

Figure 1-8. Chemical structure of saflufenacil 33

Figure 2-1. Relationship between dose of safluenacil and dry weight of corn, where data points represent the least square means of plant dry weight (% of the control). Experiments were conducted in a controlled growth room. The dose response equation defining the relationship is y = C + (D-C)/ (1 + exp[B(log(dose+10) - log I50))]) where C=18.9±2.01, D=100±0, B=9.9±1.4, I50=11.9±0.1771 65

Figure 2-2. The predicted dry weight of corn plants treated with increasing doses of saflufenacil and bentazon at the 4 leaf over stage of corn growth under growth room conditions, where weight is expressed as a percent of the control. Data points represent the least square means (standard error= ± 5.4456) of dry weight (% of control). The equation defining the relationship is y=81.642733 + (-6.154107*k) + (0.093985*b) + (0.141573*k*k) + (0.002243 *b*k) + (- 0.000073408*b*b) where b is the dose of bentazon applied, and k is the dose of saflufenacil set equal to 2.34, 4.69, 9.38, and 18.75 66

Figure 2-3. A 3-dimensional representation of predicted dry weight of corn plants treated with increasing doses of saflufenacil and bentazon at the 4 leaf stage of corn growth under growth room conditions, where weight is expressed as a percent of the control. The equation defining the relationship is y=81.642733 + (-6.154107*k) + (0.093985*b) + (0.141573*k*k) + (0.002243*b*k) + (-0.000073408*b*b) where b is the dose of bentazon applied, and k is the dose of saflufenacil applied. The data point is the saddle point at b=867.24 and k=14.86 67

vi 1.0 Literature Review: Protoporphyrinogen Oxidase Inhibiting Herbicides, and the Use of Herbicide Safeners

1.1 Introduction The development of efficacious herbicides causing minimal crop phytotoxicity, and low toxicity to non-target species, is a dynamic and progressive area of study. BASF is currently developing the herbicides BAS 800 (Kixor®, containing saflufenacil) and BAS 781 (Integrity®, containing saflufenacil and dimethenamid-p), which show promise in delivering broad spectrum weed control in corn and other crops. Saflufenacil is a pyrimidinedione compound that inhibits protoporphyrinogen-IX-oxidase (PPO). Herbicide research requires cross disciplinary studies to examine each facet of herbicide production and use, and it can take many years of research before the product is registered for use. Of particular interest in characterizing an herbicide, specifically one that inhibits enzyme function, are the metabolic pathway affected, enzyme structure and function, and the chemical properties of enzyme inhibition. The processes of cell and plant death are outlined, as well as the mammalian toxicity, environmental effects, and crop tolerance of an herbicide. Herbicidal compounds are also extensively analyzed for their weed control efficacy and appropriate use patterns. Another avenue of herbicide research is that of herbicide safeners, which increase herbicide selectivity between crop and weed plants. Bentazon is an herbicide which shows potential for use a safener for saflufenacil. The function and structure of the PPO enzyme will be discussed, as well as the development of PPO inhibiting herbicides and their mechanism of binding and subsequent plant injury. Saflufenacil will be introduced and studies of its crop tolerance and efficacy reviewed. Studies of bentazon and its potential as an herbicide safener will also be addressed, as well as the probable safening effect of sodium ions on saflufenacil treatments.

1.2 Protoporphyrinogen Oxidase (PPO) and PPO Inhibiting Herbicides Protoporphyrinogen oxidase (PPO) is an important enzyme in the porphyrin pathway from which chlorophyll and haem are derived. The porphyrin pathway is common to both plants and animals, and the PPO family of enzymes catalyzes oxidation reactions in mammals, yeast, bacteria and plants (Jacobs and Jacobs 1987). In plants, chlorophyll is an essential pigment for harvesting light in the photosynthetic process, while haem is a cofactor for a number of plant

1 proteins such as cytochromes and oxygenases (Li and Nicholl 2005). Within the chloroplast of plants, PPO catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX with the transfer of 6 electrons by a single FAD molecule (flavin adenine dinucleotide) (Corradi et al. 2006; Dailey and Dailey 1998; Jacobs and Jacobs 1987) (see Figure 1). This is the last common step in the biosynthesis of chlorophyll and haem, in a multistep process beginning with 5- aminolevulinate (ALA) (Jacobs and Jacobs 1987) (see Figure 2). Both protoporphyrinogen IX and protoporphyrin IX are heterocyclic macromolecules. Enzymatic oxidation of the colourless substrate yields a highly conjugated product, a property that gives rise to dark colour such as the common green and red associated with chlorophyll and haem, respectively (Arnould and Camadro 1998). Chlorophyll and haem biosyntheses diverge following protoporphyrin IX production (Jacobs and Jacobs 1987). Subsequently, the centre of the porphyrin ring structure of protoporphyrin IX is bound by a metal; chlorophyll molecules contain magnesium and haem molecules contain iron (Jacobs and Jacobs 1987; Matringe et al. 1989). PPO is critical for synthesis of both chlorophyll and haem.

The PPO family of enzymes has a highly conserved structure (Arnould and Camadro 1998), and in plant species the enzymes can be found in both chloroplasts and mitochondria (Koch et al. 2004). The crystal structure of mitochondrial PPO in tobacco has been revealed by Corradi et al. (2006). The heterodimeric protein has three lobes (Corradi et al. 2006). One lobe is an FAD-binding domain, where the redox cofactor carries out a six-electron exchange with the substrate (Corradi et al. 2006; Koch et al. 2004). A six-electron oxidation is somewhat atypical with just one FAD cofactor present; however, the exchange is facilitated by its proximity to the binding site and substrate in the nearby lobe (Koch et al. 2004). The lobe containing the substrate binding site is a narrow cavity just beneath the bound FAD (Corradi et al. 2006; Koch et al. 2004). The third lobe of the PPO protein is an a-helical domain which associates with the membrane (Corradi et al. 2006; Koch et al. 2004). Membrane-bound proteins commonly express the a-helix secondary structure for anchoring into a membrane. It is a coil shape with hydrophilic amino acid side chains facing the interior of the a-helical structure, and hydrophobic amino acid side chains facing the outside of the helix allowing insertion into the hydrophobic membrane (Branden and Tooze 1998). In plant cells PPO is anchored in the inner mitochondrial membrane,

2 the chloroplast envelope, and the thylakoid membranes (Matringe et al. 1992). It is within the chloroplast that herbicidal inhibition of PPO occurs.

Upon inhibition of PPO, levels of protoporphyrinogen IX within the chloroplast increase. Accumulation is rapid and causes export of the lipophilic protoporphyrinogen IX to the cytosol (Jacobs et al. 1996; Lehnen et al. 1990). Extraplastidic protoporphyrinogen IX is converted to protoporphyrin IX by an unidentified mechanism. The production of protoporphyrin IX may occur through uncontrolled oxidation by toxic oxygen species present in the cytosol (Nandihalli et al. 1992), or by another oxidizing mechanism located outside of the chloroplast (Jacobs et al. 1991; Lee et al. 1993; Lee and Duke 1994). Jacobs et al. (1991) outlined a model scheme where oxidation is mediated by a separate PPO enzyme associated with the plasma membrane. Lee et al. (1993) investigated the theory and found that this mechanism is apparently not inhibited by diphenyl ether herbicides, and has non-specific substrate activity (Jacobs et al. 1991, Lee et al. 1993). Low specificity could be the result of a non-specific enzyme or the presence of many enzymes specific to various compounds (Lee et al. 1993). There could, in fact, be PPO enzymes in cell fractions outside the chloroplast that are not sensitive to herbicides (Jacobs et al. 1991; Lee et al. 1993; Lee and Duke 1994). It was not confirmed that the mechanism is enzymatic, but the activity is heat labile (Jacobs et al. 1991). It is important to understand how protoporphyrinogen IX is converted to protoporphyrin IX outside the chloroplast because protoporphyrin IX is shown to be the only porphyrin to correlate well with herbicidal injury, and other porphyrin molecules have not shown this correlation (Becerril and Duke 1989). Following inhibition of PPO, export of accumulating protoporphyrinogen IX from the chloroplast, and extraplastidic oxidation of this substrate to protoporphyrin IX, light is required to cause plant damage.

PPO inhibiting herbicides only induce plant damage in the presence of light. The highly conjugated protoporphyrin IX molecules in the cytosol absorb light photons, creating chemical energy by exciting electrons and generating singlet oxygen i.e. free radicals (Becerril and Duke 1989; Matringe et al. 1989; Matringe and Scalla 1988). If treated plants are left in the dark they accumulate protoporphyrin IX which is quickly oxidized after exposure to light (Jacobs et al. 1996). Symptoms appear within 20 minutes after light exposure in growth room studies, or 2 hours in field studies (Rebeiz et al. 1990). The symptoms observed are caused by photooxidation of polyunsaturated fats of the cell and organelle membranes, via peroxidation chain reactions initiated by protoporphyrin carrying a free radical (Becerril and Duke 1989; Matringe et al. 1989; Matringe and Scalla 1988). A radical can remove a hydrogen atom from unsaturated lipids in the membranes, producing a lipid carrying a radical. Lipid peroxidation is propagated by the continued generation of radicals. Exposure to high intensity light, ozone, air pollutants and high or low temperatures can increase the production of reactive oxygen species (Jung and Back 2005). As a result of lipid peroxidation, the membrane integrity is compromised. Membrane structure and function are lost, and membrane proteins cannot operate, leading to cell lysis and death. Measurements of ion and water loss, decay of S-labelled thylakoid bound sulfolipids, and accumulation of short chain hydrocarbons are used to indicate the level of peroxidation that has occurred (Nicolaus et al. 1989). Tonoplasts and plasmalemma are the first to lyse, followed by the chloroplast membrane (Lehnen et al. 1990). This sequence confirms that the PPO substrate is transported out of the chloroplast, to the cytosol where radical-scavenging carotenoids and tocopherals are not plentiful (Becerril and Duke 1989; Nandihalli et al. 1992). It is agreed that lipid peroxidation is the main cause of cell damage and plant death.

There is another well documented circumstance that contributes to the toxic effect of PPO inhibition. The haem molecule acts as a feedback inhibition mechanism for the porphyrin synthesis pathway; haem production regulates ALA biosynthesis in healthy tissues (Nandihalli et al. 1992; Rebeiz et al. 1990). This down-regulation of ALA production by haem is likely a method of regulating haem production, but in turn controls the accumulation of the highly reactive intermediate molecules (Ferreira et al. 1988; Rebeiz et al. 1990). Inhibition of PPO channels protoporphyrinogen IX away from haem synthesis when it is exported from the chloroplast, because all enzymes of haem biosynthesis are contained in this organelle (Matringe et al. 1992). This deregulates the pathway, as lack of haem stimulates ALA production resulting in uncontrolled flow of carbon into the porphyrin synthesis pathway (Becerril and Duke 1989; Masuda et al. 1990; Nandihalli et al. 1992). In this way herbicidal PPO inhibition causes enhanced protoporphyrinogen IX synthesis leading to increased photooxidative damage (Becerril and Duke 1989; Masuda et al. 1990).

4 ' Injury caused by PPO inhibitors is often rapid. Symptoms can appear within hours of application (Falk et al. 2006; Li and Nicholl 2005). The injury symptoms first appear as a water- soaked lesion that subsequently turns yellow, brown or black as wilting gives way to chlorosis and necrosis (Falk et al. 2006; Johnson et al. 1978). The chemical cause of PPO inhibition and resultant injury and death has been characterized extensively. Herbicide molecules bind PPO at the same site as the natural substrate, protoporphyrinogen IX (Arnould and Camadro 1998; Camadro et al. 1991). The herbicide does not bind covalently or permanently, so binding at this site is classified as competitive (Camadro et al. 1991; Nandihalli et al. 1992). In effect, the competition reduces the affinity of the natural substrate for the enzyme. The inhibitor must have a structure complementary to the substrate, protoporphyrinogen IX, to bind at this site (Koch et al. 2004; Nandihalli et al. 1992). A bicyclic structure corresponds to one half of the substrate structure, and is of sufficient chemical similarity for bonding (Nandihalli et al. 1992). Rings joined by a hydrocarbon chain or other type of flexible bridging structure are good inhibiting compounds as they are non-planar and pliant like protoporphyrinogen IX (Nandihalli et al. 1992). The shape of compounds, such as acifluorfen, and the location of substituents and delocalized electron orbitals permit electrostatic interactions with conserved amino acid residues of the protein, forming a transient bond structure called aromatic stacking (Corradi et al. 2006; Koch et al. 2004). By stacking an aromatic ring (containing conjugated bonds, such as a phenyl group) of the herbicide molecule between aromatic residues of phenylalanine or tyrosine on the PPO enzyme, the rings are held together like a stack of coins via non-covalent Van der Waal forces (Koch et al. 2004). PPO has a broad specificity for binding, and many molecules with a variety of chemical structures can bind effectively (Nandihalli et al. 1992). There are a number of different herbicide compounds that can bind and inhibit PPO.

Photobleaching herbicides that inhibit PPO were in use before the active site and mode of action had been elucidated. Nitrofen, for example, was one of the first diphenyl ether herbicides used and was removed from the market in 1980, at least 8 years before it was linked to the porphyrin pathway (Report on Carcinogens 2008). Many researchers attempted to uncover the mechanism of action of diphenyl ethers. Hailing and Peters (1987) found that acifluorfen damage was greatest in tissues that synthesize chlorophyll, and that the herbicide interacted specifically with developing plastids. Matringe and Scalla (1988) were the first to report protoporphyrin IX

5 as the molecule accumulating following herbicide treatment, and that plant injury was caused by its photooxidative dynamics. They also saw reduced herbicidal activity when acifluorfen treatment was combined with a known inhibitor of protoporphyrin IX synthesis. Lydon and Duke (1988) found that acifluorfen prevented the production of protochlorophyll, an intermediate of protoporphyrin IX and chlorophyll. They also stated that gabaculine, an inhibitor of ALA biosynthesis, protected cucumber plants from damage by the diphenyl ether herbicides acifluorfen, nitrofen, fluorodifen, and oxyfluorfen, but not from the photosynthetic disruptor paraquat. Their findings were similar to those reported by Matringe and Scalla (1988). While some reports postulated that diphenyl ether herbicides inhibit other enzymes, such as Mg- chelatase in chlorophyll synthesis (Witowski and Hailing 1988), it has since been established that diphenyl ether herbicides bind and inhibit PPO in the porphyrin pathway (Matringe et al. 1989; Witowski and Hailing 1989). Today there are many herbicides known to cause photobleaching by disrupting the porphyrin pathway. The two main chemical structures of these commercial herbicides are diphenyl ethers and phenyl heterocycles containing nitrogen (Meazza et al. 2004).

Diphenyl ether compounds were the first PPO inhibiting herbicides used. Diphenyl ether herbicides continue to be important in crop protection after 40 years of use. Reports on acifluorfen were first published in the late 1970's, in which the high efficacy on hard to control broadleaf weeds in soybean (Glycine max) was outlined (Johnson et al. 1978). Acifluorfen causes some injury to soybean, however the plants recover quickly. Preemergence treatments require a higher dose for activity than postemergence, and herbicidal activity is at its highest 3-7 days after application. Lee and Oliver (1982) reported that efficacy is variable under different environmental conditions and is specific to the size of annual weeds. Acifluorfen is an historically important PPO inhibitor because it was used in initial studies which elucidated the site of action of diphenyl ethers (Matringe et al. 1989; Matringe and Scalla 1988). Oxyfluorfen is another diphenyl ether herbicide, and its herbicidal activity was first studied in the late 1970's and early 1980's. Oxyfluorfen is active on broadleaf weeds and grasses, with greater efficacy when applied preemergence (Biroli et al. 1981). Biroli et al. (1981) reported oxyfluorfen safety applied preemergence in cotton (Gossyypium hirsutum), soybean, corn, wheat (Triticum aestivum L.), tomatoes (Solarium lycopersicum), and transplanted onions (Allium spp.), and postemergence in vineyards, orchards and coffee plantations. Oxyfluorfen exhibits good initial and

6 residual control, and good synergy in combination with paraquat or dalapon. Research continues on the appropriate doses of oxyfluorfen and effective tank mixes for various crops, because growers of some crops, such as broccoli (Brassica oleracea) and cauliflower, have limited herbicide options (Sikkema et al. 2007). Diphenyl ethers remain in use today, however other PPO inhibiting compounds have been discovered.

Many of the articles published in the last 15 years on the topic of PPO inhibitors concern the organic synthesis of new compounds. Since the decline of diphenyl ether research in the 1990's, studies on phenyl heterocycles containing nitrogen have been pursued (Luo et al. 2008; Meazza et al. 2004). Flumioxazin was patented in 1987 and established as a potent herbicide for the control of many troublesome broadleaf weeds in soybean, such as common purselane {Portulaca oleracea), lamb's quarters (Chenopodium album), and ivy leaf and tall morningglories {Ipomoea spp.), to name a few (Nagano et al. 1987). It has since been studied for its weed control efficacy in potato {Solarium tuberosum), peanut {Arachis hypogaea), grain sorghum {Sorghum bicolor), and cotton crops. In the late 1990's phenyl triazolinone derivatives became commercially available, which are a sub-group of phenyl heterocycles containing nitrogen (Dayan et al. 1996; Dayan et al. 1997). These newer molecules are of particular importance to agriculture today as they are active at very low doses; just a few grams per hectare (Wakabayashi and Boger 2002; Zhang et al. 2004). Sulfentrazone was one of the first phenyl triazolinone herbicides commercially available (Luo et al. 2008). Sulfentrazone is applied to soybeans preplant incorporated and postemergence for broadleaf weed control (Meazza et al. 2004; Vidrine et al. 1996). However, when applied postemergence in a study by Vidrine et al. (1996), soybean injury was observed for up to four weeks. In spite of injury, soybean yields were similar for pre and post-emergence application. In a foliar applied trial by Hancock (1994), doses as low as 18 g/ha provided weed control. Carfentrazone, another phenyl traizolinone, is a compound similar in structure to sulfentrazone but more active (Dayan et al. 1997). Carfentrazone, developed in 1997, is fast acting and has little to no residual activity (Dayan et al. 1997; Thompson and Nissen 2002). It can cause crop injury symptoms, but row crops recover quickly (Thompson and Nissen 2002). The herbicide is registered as a burn down product in a variety of crops such as cereal grains, oilseeds, legumes, roots and tubers, cole crops and other fruits and vegetables (Anonymous 2007). Research and development of phenyl heterocycles

7 containing nitrogen continues, with many new and highly active PPO inhibitors reported in the literature (Luo et al. 2008; Zhang et al. 2004).

PPO inhibiting herbicides have been in development for over 40 years, where motivation of studies often lies in establishing the most effective use patterns. Acifluorfen was first studied in the late 1970's for use in soybeans, as a result of broadleaf weed escapes seen with the common practice of preplant and preemergence spraying (Johnson et al. 1978). Today PPO herbicides are sprayed at both pre and postemergence stages, and they are absorbed by foliage, shoots and roots (Johnson et al. 1978; Meazza et al. 2004). Activity is generally greater on dicotyledonous plants (Li and Nicholl 2005). Reports indicate that PPO inhibiting herbicides are effective on many hard to control weeds, such as pitted morning glory, cocklebur (Xanthium strumarium), and velvetleaf (Abutilon theophrasti) (Johnson et al. 1978; Meazza et al. 2004). The key benefit to the use of PPO inhibiting herbicides is that they control weeds at low doses (Johnson et al. 1978; Meazza et al. 2004; Witkoski and Hailing 1989). Damage is greatest in developing tissue where chlorophyll biosynthesis is occurring at an increased rate during greening (Hailing and Peters 1987; Johnson et al. 1978). For this reason, younger plants are more effectively controlled with PPO inhibitors (Hailing and Peters 1987). Reports are continually published outlining the specific use patterns in various crops that will offer the best weed control and lowest crop injury.

Herbicides that cause plant damage are also potentially harmful to other non-target organisms and the environment. The PPO enzyme is present in both plants and mammals. In mammals, PPO is crucial for the biosynthesis of haem and is present in mitochondria. Herbicide molecules are not selective and can bind both the plant and mammalian enzymes (Birchfield et al. 1998; Shaner 2003). However, mammalian toxicity is low. In rats, embryo growth retardation, teratogenicity, and lethality caused by loss of haem have been observed but similar results have not been obtained for other mammals (Kawamura et al. 1996). In a study of oxyfluorfen effect on human erythroblast precursors, results showed toxicity only at very high herbicide concentrations, and it was reported that while oxyfluorfen did inhibit PPO there was no effect on cell proliferation (Rio et al. 1997). The toxicity is species dependant, conferring harm to rats but not other mammals (Kawamura et al. 1996). The PPO inhibiting herbicides are, in most cases,

8 rapidly metabolized by mammals at the doses used in agriculture (Shaner 2003). They do not affect biosynthesis of compounds as they do in plants, because the herbicide does not accumulate in tissues and is not exposed to light which causes generation of reactive oxygen species. Information on the environmental implications of these herbicides must be researched individually, however it is frequently stated that negative effects are kept to a minimum because they are applied at low doses, and they have low residual activity (Meazza et al. 2004; Zhang et al. 2004). The Environmental Protection Agency in the USA reported that flumioxazin is highly susceptible to hydrolysis and photolysis in soil and water and does not leach below 3 inches of soil depth (United States Environmental Protection Agency 2001). Conversely, sulfentrazone is highly mobile and persistent in soils, with a strong potential to leach into groundwater (United States Environmental Protection Agency 1997). It is stable to hydrolysis and biodegradation. Each herbicide will have a different profile for environmental persistence and safety or tolerance in non-target organisms.

The discovery of the genes causing tolerance to PPO inhibiting herbicides would provide an opportunity for the development of transformed crop plants carrying this trait, similar to glyphosate and glufosinate tolerant crops. In 2006, 70% of all genetically modified crops planted worldwide were glyphosate-tolerant or glufosinate-tolerant (GMO Compass 2007). The use of glyphosate-tolerant corn in eastern Canada has increased each year since its introduction in 2001. In 2005, 21% of all corn was planted to glyphosate-tolerant varieties (Sikkema and Soltani 2007). There are some clear benefits associated with the use of herbicide-tolerant crops. By preventing crop injury, a non-selective weed control product can be used and may result in improved levels of broad-spectrum weed control. For example, it can be difficult to adequately control grassy weeds as well as broadleaf weeds in a corn crop without injuring the corn. The use of non-selective glyphosate in glyphosate-tolerant corn overcomes this problem (Anonymous 2010; Sikkema and Soltani 2007). Additionally, the use of herbicide-tolerant crops often widens the window of herbicide application, increasing flexibility in a grower's weed control program. Many PPO inhibiting herbicides cause rapid damage to foliage, but a tolerant crop would allow for postemergence application. There are also some concerns with the use of herbicide tolerant crops, one of which is the development of resistant weed species (Sikkema and Soltani 2007; Tardiff 2007). With an increase in the amount of land planted to an herbicide-tolerant crop, there

9 will be an increased frequency of application of that herbicide or group of herbicides and thus increased selection pressure for herbicide-resistant weeds as has occurred with glyphosate (Sikkema and Soltani 2007; Tardiff 2007). One of the most important resistance management strategies is to rotate herbicide modes of action. The option of planting a crop tolerant to PPO inhibitors would provide growers with the benefits associated with herbicide-tolerant crops, as well as increasing their ability to rotate herbicide modes of action.

Some researchers have directed their studies toward the discovery of a gene that would provide tolerance to PPO inhibiting herbicides. Randolf-Anderson et al. (1998) isolated a chlorophyll producing algae which is normally susceptible to PPO inhibiting herbicides that has a single amino acid substitution which prevents herbicide binding to the enzyme and confers herbicidal tolerance. A naturally occurring mutation like this may be useful in crop plants if screening and breeding, or transformation, leads to development of an herbicide tolerant crop. Lermontova and Grimm (2000) induced herbicidal tolerance in tobacco (Nicotiana tabacum) plants by a transformation to over-express PPO in plastids. Indeed, the transformed tobacco plants showed an increase in PPO activity of 5 to 6x, which prevented protoporphyrinogen IX export from the chloroplast and allowed the completion of chlorophyll synthesis. The increased availability of PPO enzymes neutralized acifluorfen injury. There have also been studies on the presence of protoporphyrinogen IX degradation outside of the chloroplast which can prevent herbicide injury. Jacobs et al. (1996) reported that plants which express some herbicide tolerance, such as mustard, have high levels of protoporphyrinogen IX degrading factor in the soluble protein fraction of leaves which prevents accumulation of porphyrins that can lead to plant injury (Jacobs et al. 1994; Jacobs et al. 1996). They saw species differences in the level of effect of this degrading factor and noted that older plants, which have always been known to have increased tolerance to PPO inhibiting herbicides, have increased porphyrin degradation outside of chloroplasts. It is also reported that crop plants are inherently less susceptible to herbicide injury. Soybean recovers from PPO inhibitor injury without yield penalty, and Falk et al. (2006) found soybean to sustain less injury than wild mustard {Sinapsis arvense), susceptible common waterhemp (Amaranthus tuberculatus var. rudis), and herbicide tolerant common waterhemp with identical treatments of acifluorfen, fomesafen and sulfentrazone (Vidrine et al.

10 1996). Studies on herbicide tolerance can uncover why the herbicides do not control certain weeds, as well as potentially lead to the development of crop plants with herbicide resistance.

1.3 A New PPO Inhibitor: Saflufenacil BASF Crop Protection is developing a new herbicide, BAS 800 (Kixor®), with the active ingredient saflufenacil (Kixor® Worldwide Technical Brochure 2008). Saflufenacil is a pyrimidinedione that inhibits PPO in susceptible plants (Figure 3) (Grossman et al. 2010). Saflufenacil was proposed for full registration by Health Canada's Pest Management Regulatory Agency in 2009, for the control of a number of major broadleaf weeds (Health Canada 2009). The general proposed use pattern for saflufenacil is preplant, preplant incorporated, to postemergence in small grains and legumes, corn, sorghum, cotton, soybean, tree fruits, nut crops, and fallow. Its use in corn is proposed to be at a dose of 50 to 125 g ai/ha preemergence, depending on soil type. It is translocated primarily in the xylem of plants, and is a non-volatile herbicide. Saflufenacil can be applied as a water-based spray on its own, or in combination with other herbicides. Saflufenacil can also be tankmixed with glyphosate and applied preplant for effective burndown of emerged weeds in reduced or no-till corn (Liebl et al. 2008). An adjuvant is required for foliar application, and a fertilizer containing ammonium, such as ammonium sulphate, should be included in the spray mixture. Saflufenacil is absorbed by roots, shoots and leaves, offering rapid burndown as well as soil residual activity.

There are many broadleaf weeds controlled by saflufenacil, including velvetleaf {Abutilon theophrasti), common ragweed {Ambrosia artemisiifolia), giant ragweed {Ambrosia trifidd), common cocklebur, ladysthumb {Polygonum persicaria), redroot pigweed {Amaranthus retroflexus), common waterhemp and common lamb's quarter including triazine and acetolactate synthase resistant biotypes (Kixor® Worldwide Technical Brochure 2008; Liebl et al. 2008). Saflufenacil is also compatible with burndown and residual graminicides (Kixor® Worldwide Technical Brochure 2008). BASF is developing an herbicide mixture for use in corn. BAS 781 (Integrity®) includes saflufenacil and dimethenamid-p. Dimethenamid-p is a chloroacetamide herbicide used for the control of many annual grass weed species including giant foxtail {Setaria faberii), green foxtail {Setaria viridis), yellow foxtail {Setaria glauca), large crabgrass {Digitaria sanguinalis ), smooth crabgrass {Digitaria ischaemum), barnyardgrass {Echinochloa crusgalli),

11 fall panicum (Panicum dichotomiflorum), and witchgrass (Panicum capillare), as well many small-seeded annual broadleaf weeds such as redroot pigweed, American black nightshade {Solarium americanum), and eastern black nightshade (Solarium ptycanthum) (Anonymous 2010; Senseman 2007). Dimethenamid-p can be applied preemergence and early postemergence in corn for season-long weed control. Dimethenamid-p inhibits very long chain fatty acid synthesis in weed species, but rarely injures corn at the registered application doses (Bernards et al. 2006; Matthes et al. 1998). Partnering saflufenacil and dimethenamid-p offers an effective herbicide application for season-long broad spectrum weed control in field corn.

As a PPO inhibitor, saflufenacil offers a novel mode of action compared to the most widely used herbicides in Ontario today. Atrazine, dicamba, isoxaflutole and mesotrione are, respectively, a photosystem I inhibitor, a synthetic auxin, and HPPD inhibitors (Senseman 2007). Incorporating saflufenacil into a pesticide program for field corn will reduce the selection pressure caused by repeated use of one mode of action, which results in resistant weeds (Soltani et al. 2009). Atrazine is commonly used in many pesticide programs and triazine resistant weeds are widespread as a result. Saflufenacil does not require tank-mixing with atrazine for good broadleaf weed control (Kixor® Worldwide Technical Brochure 2008). Saflufenacil provides better common ragweed control than mesotrione, and better lamb's quarter control than isoxaflutole (Anonymous 2010; Kixor® Worldwide Technical Brochure 2008). The velvetleaf control in field corn is greater than that of atrazine, and it offers better wild mustard control and longer residual control of broadleaf weeds than dicamba (Anonymous 2010; Kixor® Worldwide Technical Brochure 2008). Also, unlike isoxaflutole and dicamba, saflufenacil does not have soil type restrictions (Anonymous 2010; Kixor® Worldwide Technical Brochure 2008).

There have been a few studies published on saflufenacil weed control efficacy. Geier et al. (2009) conducted a study to determine the dose response of saflufenacil applied preemergence and postemergence in doses of 6, 12, 18, 24 and 30 g ai/ha to blue mustard (Chorispora tenella), flixweed (Descurainia sophia) Palmer amaranth (Amaranthus palmer7), redroot pigweed, and tumble pigweed (Amaranthus albus), under growth room conditions. In the preemergence treatments there were no interactions between weed species and herbicide dose, and control of all five weeds was 82 to 98% by 6 to 30 g ai/ha of saflufenacil based on dry weight as a percent of

12 the untreated controls (Geier et al. 2009). Doses of 9 g ai/ha caused an average of 90% biomass reduction across all species (i.e. GR.9o=9 g ai/ha). Similarly, the DR90 for population density was 9 g ai/ha averaged across all weed species. When saflufenacil was applied postemergence, the GR90 was 6 g ai/ha averaged across all weed species. However, based on reduction in population density, the weed control was not as good. The regression analysis showed a density reduction of 88% for saflufenacil doses up to 30 g ai/ha. Greater than 30 g ai/ha would be required to reduce the population density by more than 90% in postemergence applications. The individual species had some differences in reduction of population density in postemergence applications. Blue mustard was the most sensitive to saflufenacil applied postemergence, followed by the three Amaranthus species and then flixweed (Geier et al. 2009). In another growth room study, Frihauf (2009) looked at saflufenacil control of flixweed and henbit at doses of 13, 25 and 50 g ai/ha with a non-ionic surfactant (NIS) applied when plants measured 1 to 2.5 cm in diameter. Flixweed was more sensitive than henbit, and was controlled 95 to 99% at 14 days after treatment. Henbit control reached a maximum of 70% at 7 days after treatment with 50 g ai/ha, and by 21 days after treatment the dry weight was only reduced to 27% of the control. Growth room studies give an indication of relative saflufenacil efficacy, but field studies are required to determine its true performance.

Field studies have been conducted to determine the weed control efficacy of saflufenacil on selected weeds. Knezevic et al. (2009a) reported on weed control in Nebraska with five doses of saflufenacil (6.25, 12.5, 25, 50, and 100 g ai/ha) and four adjuvant types (no adjuvant; NIS, 0.25% v/v; crop oil concentrate [COC], 1% v/v; methylated seed oil [MSO], 1% v/v). The treatments were applied after the emergence of dandelion {Taraxacum officinale), field bindweed {Convolvulus arvensis), field pennycress {Thlaspi arvense), henbit {Lamium amplexicaule), prickly lettuce {Lactuca serriola), and shepherd's-purse {Capsella bursa-pastoris), over two years without a crop present. Knezevic et al. (2009a) stated that saflufenacil was highly effective on most'weeds tested, and that MSO was the best adjuvant for providing enhancement of herbicide activity, followed closely by COC; NIS provided the lowest level of enhancement. Based on dose response curves generated from visual control ratings, the GR90 values at 28 days after treatment with saflufenacil alone were 93, 71, 103, 98, 110 and 128 g ai/ha, respectively, for the above mentioned weeds. The addition of MSO to the treatments improved the GR90

13 values to 2, 7, 34, 35, 11 and 19 g ai/ha, respectively (Knezevic et al. 2009a). Knezevic et al. (2009b) conducted another similar field study in Nebraska over two years that included the same list of weeds mentioned above, as well as kochia {Kochia scoparia), wild buckwheat {Polygonum convolvulus) and horseweed (Conyza canadensis). The GR90 values at 28 days after treatment were, in general, higher in this study, which may be a result of using visual ratings. With saflufenacil alone, GR90 values ranged from 82 to 217 g ai/ha, where the most difficult to control were kochia and horseweed. The GR90 values for saflufenacil plus an MSO ranged from 21 to 78 g ai/ha. In both studies it was concluded that by following the proposed label recommendations for saflufenacil, as well as using an appropriate surfactant, effective weed control and burn down can be achieved (Knezevic et al. 2009b).

Frihauf (2009) studied flixweed, blue mustard and henbit control with saflufenacil at doses of 13, 25 and 50 g ai/ha with COC. The experiment was conducted at two Kansas locations over two years where weed seeds were broadcast and treatments were applied after weed emergence. Saflufenacil provided over 95% control of blue mustard at all doses and locations. Flixweed control was only 73 and 84% at the two locations when saflufenacil was applied at 13 g ai/ha, but was greater than 94% at both locations with doses of 25 g ai/ha or more. Control of henbit was very low at one location, from 30 to 45%, and somewhat higher at the other location, from 75 to 88%. Overall, field studies on the weed control efficacy of saflufenacil indicate that it provides very good broadleaf weed control and will be an asset to growers. Weed control studies with saflufenacil have not yet been published for weeds common to southern Ontario. An excellent level of weed control is not the only important factor in herbicide use; an herbicide must also be safe for application in the crop.

Studies on the tolerance of spring barley (Hordeum vulgare), oats (Avena sativa), and wheat have been published. Sikkema et al. (2008) conducted a two year study in Ontario on the injury caused to spring-planted barley, oats and wheat by saflufenacil applied preemergence and postemergence in doses of 50 and 100 g ai/ha. No more than 1% injury was seen in preemergence treatments, but postemergence applications included a surfactant (Merge®) and caused between 35 and 76% visual injury. Yields were reduced in barley and wheat by 24 and 13% (Sikkema et al. 2008). Saflufenacil is to be registered as a preemergence product in spring

14 cereals, so these results are consistent with the label recommendations. Knezevic et al. (2010) have also explored saflufenacil injury to wheat in preemergence and postemergence applications, in northeast Nebraska over two years. Preemergence doses ranged from 25 to 400 g ai/ha, and postemergence doses of 6.25 to 200 g ai/ha were applied to wheat in both the fall (5 cm height) and spring (40 cm height), alone and with NIS and COC. Preemergence treatments did not cause any visual injury or yield loss, even at the very high dose of 400 g ai/ha. Visual injury and yield loss were greatest in fall and spring postemergence treatments that included COC, and a 5% yield loss was seen with 10 g ai/ha of saflufenacil applied with COC in the fall, and 4 g ai/ha in the spring. Injury and yield reduction were lowest where saflufenacil was applied alone. Saflufenacil applied alone caused 5% injury in the fall and spring at doses of 82 and 51 g ai/ha, respectively, and yield losses of 5% in the fall and spring at doses of 54 and 24 g ai/ha, respectively.

Tolerance studies have also been conducted on saflufenacil applied to corn (Zea mays). Soltani et al. (2009) conducted field trials over one year in two Ontario locations to evaluate corn tolerance to saflufenacil in doses of 50, 100 and 200 g ai/ha applied at the preemergence, spike and 2-to-3 leaf stages of corn growth with and without an adjuvant (Merge®). The preemergence treatments did not show visual injury above 1%. Plants treated at the spike stage without an adjuvant had up to 2% injury at 21 days after treatment, and those with treatments including an adjuvant sustained up to 4% injury. At one location, plants treated with saflufenacil alone at the 2-to-3 leaf stage had 4% injury with 50 g ai/ha, and 21 to 25% injury at higher rates. The inclusion of an adjuvant increased injury to 42 to 65%. At the second location the results were quite different. There was no visual injury for treatments that did not include an adjuvant, but those with an adjuvant caused an average of 99% injury. The yield results were interesting in that at one location there was no yield loss caused by any of the treatments, but at the other location saflufenacil applied with an adjuvant to 2-to-3 leaf corn at all doses caused yield losses from 49 to 59%o. Soltani et al. (2009) suggest that postemergent applications of saflufenacil at 50 to 100 g ai/ha without an adjuvant are safe up to the 2-to-3 leaf stage of corn growth, and that preemergence applications are safe up to 200 g ai/ha. This is currently the only field study published on corn tolerance to saflufenacil, but laboratory studies also indicate that corn shows tolerance to the herbicide.

15 There is an indication that corn has some tolerance to saflufenacil. Grossman et al. (2010) have shown that corn has an inplanta tolerance to saflufenacil. When treated with saflufenacil, crude plastid preparations from velvetleaf and black nightshade shoots had an 8-fold and 25-fold greater production in protoporphyrin IX, respectively, than controls. Protoporphyrin IX levels in plastid preparations from corn coleoptiles were only increased 2-fold. Also, velvetleaf and black nightshade leaf discs were treated with luM of saflufenacil and left in the dark for 22 hours, and the levels of reactive oxygen species increased 134 and 142%, respectively, after 4 hours of light exposure. Corn leafs discs did not show an increase in levels of reactive oxygen species when treated with luM of saflufenacil. These results indicate that upon treatment with saflufenacil, corn plants do not generate as many harmful reactive oxygen species as some broadleaf weed species and therefore may not be injured to the same degree as weedy plants when sprayed in the field. Because corn shows some tolerance to saflufenacil, the use of an herbicide safener with saflufenacil may increase its selectivity between crop and weed plants and allow for postemergence applications.

1.4 Herbicide Safeners The use patterns of some herbicides are expanded with the addition of herbicide safeners. Historically called herbicide antidotes, antagonists, and protectants, safeners were first discovered by Otto Hoffman approximately sixty years ago (Abu-Qare and Duncan 2002; Davies 2001). Safeners are synthetic compounds that reduce the phytotoxicity of herbicides to the desired crop (DeRidder and Goldsbrough 2006). They protect crop plants without reducing the efficacy of the herbicide in controlling weeds; safeners allow for selective killing of weeds (Davies 2001). They are chemically diverse, but the structure of a safener is often similar to that of the herbicide it safens (Davies 2001). The compounds are applied with herbicides in dose ratios that often range between 1:6 to 1:30 of safenenherbicide, indicating that safeners induce their effect at relatively small doses (Hatzios and Burgos 2004). The level of protection provided is affected by interactions occurring between the safener, herbicide, environmental conditions and the specific plant to which the mixture is applied (Bernards et al. 2006). The objective of safener use is to prevent damage to a crop while allowing the herbicide to kill undesirable weeds, so the practicality of a safener hinges on the selectivity they achieve (DeRidder and Goldsbrough

16 2006). A successful safener-herbicide partnership can improve crop safety as well as weed control.

With crop protection through the use of herbicide safeners, increased doses of herbicide can be used to allow for increased weed control (Davies and Caseley 1999; Matola and Jablonkai 2007). Ultimately an increased herbicide dose can prevent the survival of tolerant or resistant weeds (Nelson and Penner 2006). Alternately, safener use can reduce costs and permit the use of less selective chemicals at lower application doses (Davies and Caseley 1999). With a high degree of selectivity between plant species, safeners allow for control of weeds botanically related to the crop, such as wild rice growing within rice (Hatzios and Burgos 2004). The options of weed control for growers of minor crops are expanded with safeners as well (Davies and Caseley 1999). Herbicide development within these smaller markets is limited because the potential revenue gains there are less than gains in field crops grown on a greater number of acres, such as corn. There are fewer herbicide options for minor use crops, so safeners can be useful (Davies and Caseley 1999). In fact, safeners expand the use of herbicides in a number of ways. Their inclusion in tank mixes can enable a grower to spray a particular herbicide under adverse conditions that might normally result in crop damage (Davies and Caseley 1999; Nelson and Penner 2006). For example, some herbicides are not to be sprayed in poor weather conditions, such as cool or wet conditions, where the chance of crop injury is greater (Nelson and Penner 2006). The inclusion of a safener can overcome these limitations. Many herbicides cause crop damage to more sensitive cultivars, or at certain growth stages of the crop. Again, safeners can extend the use patterns of these herbicides (Nelson and Penner 2006). This ability to increase selectivity and expand the use of older herbicides, along with the push for reduced pesticide use, can stimulate development of compounds that are less specific but have reduced environmental impact (Davies and Caseley 1999; Hatzios and Burgos 2004).

Compounds can operate as herbicide safeners through a few different mechanisms. They may reduce uptake or translocation of the herbicide, compete for binding at the herbicide target site or influence other physiological mechanisms, but possibly the most dynamic and predominant impact is that on the metabolic degradation pathways (Abu-Qare and Duncan 2002; Siminsky 2006). A safener can guard the plant from injury by increasing breakdown of the

17 herbicide molecules, thus preventing or reducing a toxic effect (Riechers et al. 2003). Proteins and enzymes from endogenous secondary metabolism are enlisted, and increasing the activity of these detoxifying enzymes speeds metabolism of the herbicide (Cummins et al. 2006; Riechers et al. 2003). The complete plant response to safeners is unclear, but it seems they can induce the transcription of genes for a variety of proteins, including metabolism-based enzymes, directly or indirectly (Hatzios and Burgos 2004; Matola and Jablonkai 2007). There is evidence indicating that the entire detoxification system can be regulated and coordinated by the activity of safeners (Riechers et al. 2003). In vitro studies have ruled out the possibility that safeners act on metabolic enzymes directly (Davies and Caseley 1999). One current example of an herbicide safener is cyprosulfamide, which was developed to extend the application window of isoxaflutole (Nelson and Penner, 2006; Philbrook and Santel, 2008). Isoxaflutole is a preemergence herbicide used in corn, but the addition of cyprosulfamide permits application postemergence up to the 3 leaf stage of corn growth. Cyprosulfamide increases isoxaflutole detoxification by monooxygenase enzymes (Pesticide Properties DataBase 2009). There is opportunity for the development of safeners for a number of current herbicides, which may expand their use patterns in various crops.

1.5 Bentazon as an Herbicide Safener Bentazon is being studied for use as a safener for saflufenacil applied to field corn. It is believed that the addition of Na-bentazon will reduce crop injury in preemergence and early postemergence applications. Bentazon is a broadleaf weed control product registered in Ontario for postemergence use on small, actively growing weeds in field corn at doses up to 1080 g/ha (Anonymous 2010). A number of studies have indicated that mixing Na-bentazon with various types of herbicides results in reduced injury caused by reduced herbicide uptake. In soybean leaves, Na-bentazon at a dose of 560 g ai/ha has been shown to reduce thifensulfuron (a sulfonylurea) injury from 30% to 16% (Ateh et al. 1995). Weinberg et al. (2007) reported antagonism of uptake of tritosulfuron in white bean by bentazon added at a dose of 442.5 g ai/ha, which reduced injury caused this sulfonylurea. In pinto bean (Phaseolus vulgaris), injury by an ALS inhibitor, imazethapyr, was also reduced by the addition of bentazon (Bauer et al. 1995). Paraquat injury to peanut was reduced with bentazon (Wehtje et al. 1992). However, the safening effect is largely cosmetic because it has been shown that while foliar paraquat injury can be

18 significant, it does not reduce yield (Wehtje et al. 1992). There are a number of examples in the literature indicating that bentazon has the potential to safen a variety of herbicide compounds, reducing injury in various crops.

The mechanism of safening by Na-bentazon has been evaluated in a number of reports. Both uptake and translocation can be reduced with the addition of Na-bentazon. Phloem translocation can be affected by Na-bentazon, a photosystem II inhibitor, as it directly acts upon photosynthesis and may have a lasting negative impact on membrane ATPases, reducing active herbicide loading into the phloem (Weinberg et al. 2007). However, most reports state that the mechanism of safening is a reduction in herbicide uptake. Weinberg et al. (2007) reported tritosulfuron safening by Na-bentazon, but safening was not seen with the H-bentazon or NH4- bentazon forms. Antagonism of sethoxydim was also seen with only the Na-bentazon form of the herbicide (Wanamarta et al. 1989). Wanamarta et al. (1989) concluded that it was Na-bentazon specifically that caused reduced herbicide uptake because the Na+ complexes with sethoxydim, decreasing its penetration into the leaf cuticle. Weinberg et al. (2007) later came to the same conclusion; they saw a crystalline deposit form on the leaf surface where Na-bentazon was added to tritosulfuron, and predicted that the safening effect was caused by reduced leaf penetration. Wanamarta et al. (1989) conducted uptake studies using 14C-sethoxydim and saw reduced absorption into quackgrass leaves when the herbicide was mixed with bentazon. Similarly, Frihauf (2009) analyzed winter wheat uptake of 14C-saflufenacil in the presence of bentazon. Frihauf reported a 13% uptake of saflufenacil at 7 days after treatment, but in the presence of bentazon only 6±2% moved into the leaf. It has been established that bentazon can decrease herbicide uptake.

This specific interaction was further studied through proton nuclear magnetic resonance (NMR) spectrometry to determine if Na+ from Na-bentazon interacts chemically with the sethoxydim compound (Thelen et al. 1995). The results showed that the interaction causing antagonism is indeed chemical. The NMR output established that Na+ associates with the hydroxyl oxygen of the sethoxydim ring, converting sethoxydim from a weak acid to a Na+ salt that is somewhat more polar (Thelen et al. 1995; Wanamarta et al. 1989). The extent to which an element attracts bonding electrons is called its electronegativity, and when two atoms have

19 different electronegativities the bond between them is polar, where a greater difference corresponds to a more polar bond (Olmsted and Williams 2002). The electronegative difference between oxygen and Na+ is greater than that between oxygen and H+, thus the electron distribution is altered and the newly formed Na-sethoxydim salt is more polar (Olmsted and Williams 2002; Wanamarta et al. 1989). The larger molar volume of the Na+ salt may also cause some reduced permeance, but more importantly a more polar compound can express reduced penetration because of the overall lipophilic character of the cuticle (Wanamarta and Penner 1989). Increasing the polarity of a compound will increase its hydrophilic nature and thus solubility in water. The cuticle has an overall negative charge as well as a waxy, hydrophobic epicuticular layer that represents a significant barrier to herbicides, especially those that are . water soluble (Hess and Foy 2000; Wanamarta and Penner 1989). Hydrophilic herbicides diffuse passively into and through the cuticle, as do lipophilic herbicides, but the hydrophilic exhibit relatively lower partitioning into the cuticle and through to the cell wall (Hess and Foy 2000). A reduction in herbicide uptake in the crop plant can reduce the injury caused to the crop, and may or may not cause decreased weed control.

There are reports on bentazon mixtures with herbicides where the antagonism of herbicide uptake caused reduced weed control. Wanamarta et al. (1989) reported that addition of Na-bentazon, equivalent to 840 g/ha, to sethoxydim resulted in 40% of the sethoxydim being absorbed into quackgrass leaves compared to 70% absorption with sethoxydim alone. This decreased absorption led to reduced quackgrass control. Similarly, it has been reported that weed control has been reduced by adding bentazon to the graminicide haloxyfop, and in ALS inhibiting broadleaf herbicides halosulfuron and prosulfuron (Hart 1997). However, weed control is not compromised in all cases. Lycan and Hart (1999) reported on a growth room study in which thifensulfuron reduced velvetleaf and lamb's quarters dry weight to 9% and 16% of the control, respectively (Lycan and Hart 1999). Addition of bentazon did not increase the dry weights of velvetleaf and lamb's quarter, but did safen thifensulfuron applied to soybean (Lycan and Hart 1999). In a similar study, Hager and Renner (1994) stated that thifensulfuron with bentazon also provided equal or greater control of common ragweed than either herbicide applied alone. Generally speaking, weed control is species dependant, and this must be considered for any tank mix. The addition of 280, 560 and 840 g ai/ha of bentazon to paraquat

20 (70 and 140 g ai/ha) reduced visible peanut injury, reduced Florida beggarweed (Desmodium tortuosum) control in all dose combinations, and reduced sicklepod (Cassia obtusifolia) control when the bentazon dose was above 560 g ai/ha (Wehtje et al. 1992). However, control of smallflower morningglory (Jacquemontia tamnifolia) was increased with the addition of bentazon, because it is not highly susceptible to paraquat (Wehtje et al. 1992). Bentazon may safen herbicide applications without reducing injury to weeds.

Depending on the herbicide considered, the dose absorbed by weed plants after some loss of activity by bentazon antagonism may be adequate for weed .control. The basis of this selectively is unclear, but is likely to be in part derived from the hybrid vigor of the crop species growers use (Edwards et al. 2000). In general, hybrid crop plants have a greater ability to metabolize xenobiotic compounds than weeds do and often express greater quantities of the detoxifying enzymes and conjugation substrates, such as the conjugating enzyme glutathione-S- transferase (GST) and its substrate y-glutamylcysteinylglycine (Edwards et al. 2000; Ekler et al. 1993). GST plays a major role in xenobiotic metabolism, and GST quantities can be up to 20- fold greater in crops than in weed plants (Edwards et al. 2000). Hybrid crop plants have a better general stress response than inbred plants or annual weed plants and thus often have a greater tolerance to herbicides in general (Edwards et al. 2000; Stephenson et al. 1993). Indeed, safeners are much less efficacious when applied to inbred crop lines (Bernards et al. 2006). Saflufenacil is believed to be detoxified through metabolism, as is the structurally similar compound carfentrazone-ethyl (Elmarakby et al. 2001; Thompson and Nissen 2000). The rate of metabolism of carfentrazone-ethyl is negatively correlated with plant sensitivity to the herbicide (Thompson and Nissen 2000). Plants that can quickly metabolize the herbicide compound to non-toxic products prevent translocation of the toxin and show reduced injury. It is in this way that selectivity is conferred to increased activity on weedy plants rather than crop plants. In a study by Thompson and Nissen (2000), carfentrazone-ethyl was quickly absorbed in corn, soybean and velvetleaf, but was metabolized much more rapidly in corn, followed by soybean and then velvetleaf. Dayan et al. (1996) reported that the selectivity of aryl triazolinones is also based on rates of metabolic detoxification. With this evidence it is predicted that bentazon will reduce saflufenacil uptake and thus reduce injury to corn, but it is expected that there will still be high activity on susceptible weeds.

21 Bentazon fits the model for a potential herbicide safener, and may safen saflufenacil applied to corn postemergence. Frihauf (2009) has conducted studies on saflufenacil and bentazon applied to winter wheat. In a growth room study, saflufenacil was applied to 15 to 25 cm tall wheat in doses of 13, 25 and 50 g ai/ha with NIS (0.25% v/v), alone and in combination with 560 g ai/ha of bentazon and 533 g ae/ha of 2,4-D amine. Saflufenacil applied alone caused between 19 and 38% necrosis and reduced plant dry weight to between 99 and 68% of the control. The addition of bentazon had a significant safening effect, and only 1 to 9% necrosis was observed. Plant dry weight was greater than the control when bentazon was included in the tank mix; dry weights were between 113 and 123% of the control. Adding 2,4-D amine to saflufenacil caused a high degree of injury to wheat, ranging from 24 to 40% and reduced dry weight to between 70 and 53% of the control. Frihauf (2009) also conducted a field study exploring winter wheat injury caused by saflufenacil at rates of 13 and 25 g ai/ha (plus COC), mixed with dicamba, MCPA ester, 2,4-D amine, 2,4-D ester and bentazon. The experiment was conducted at two locations in Kansas over two years. Treatments including bentazon (560 g ai/ha) did not have necrosis above 9% at any location or time. Treatments including dicamba (140 g ai/ha) had necrosis up to 11% at 3 to 5 days after treatment, and 2,4-D (533 g ae/ha) between 5 and 13%. All other herbicides combined with saflufenacil caused necrosis between 10 and 33%. In Frihauf s studies, saflufenacil treatments that included bentazon caused the least amount of injury to winter wheat (Frihauf 2009). It is expected that similar results would be seen with saflufenacil and bentazon applied to corn. Frihauf s weed control studies on saflufenacil mixed with various herbicides, including bentazon, also provide a good evaluation of how these products will perform in a corn field.

In the growth room study by Frihauf (2009) mentioned above, the treatments including saflufenacil, bentazon and 2,4-D amine were applied to henbit and flixweed with NIS. Henbit necrosis caused by saflufenacil reached 70% at 7 days after treatment with 50 g ai/ha, and dry weight was reduced by as much as 27% of the control. The addition of bentazon resulted in levels of henbit necrosis and final dry weights not significantly different than saflufenacil alone. The addition of 2,4-D amine increased weed control. Up to 90% necrosis was achieved at 7 days after treatment, and henbit dry weight was reduced to 3% of the control. The control of flixweed

22 was better than henbit control overall. Saflufenacil at all three doses caused between 92 and 96% necrosis of flixweed by 7 days after treatment, however this level of necrosis was only achieved by bentazon plus saflufenacil at doses of 25 g ai/ha and above at 7 days after treatment. Saflufenacil plus 2,4-D amine provided faster and more complete control of both weeds. In the growth room studies, bentazon provided an exceptional safening effect and generally did not antagonize saflufenacil control of henbit or flixweed. Saflufenacil alone or in combination with 2,4-D amine provided similar or better weed control, but crop injury was significantly higher with these treatments. Again, as mentioned above, Frihauf s field study on winter wheat tolerance included a study of weed control with saflufenacil (13 and 25 g ai/ha) combined with dicamba, MCPA ester, 2,4-D amine, 2,4-D ester and bentazon. Control of blue mustard and flixweed with saflufenacil plus COC was 93% or greater at 18 to 20 days after treatment, and control of henbit was 25%. Weed control with saflufenacil at 13 and 25 g ai/ha plus bentazon at 560 g ai/ha was variable. Control of blue mustard was 81 and 91% at one location, increasing with increasing dose of saflufenacil, and was 55 and 100% at the second location. With a similar pattern, flixweed control was 90 and 100% at one location, increasing with increasing dose of saflufenacil, and was 32 and 53% at the second location. Henbit control with saflufenacil plus bentazon was between 10 and 23%. Generally, the other herbicide combinations provided significantly greater weed control than saflufenacil plus bentazon. The dose of saflufenacil used in this experiments is lower than for corn; 25 vs 75 g ai/ha, respectively. When bentazon is combined with the higher dose of saflufenacil, there may be a safening effect as well as adequate weed control.

1.6 The Role of Sodium Ions in the Safening Effect With evidence indicating that the safening effect of Na-bentazon is caused by the Na+ component, it is possible that other monovalent cations can induce a safening effect. Just as Ca2+ and Mg2+ can reduce glyphosate phytotoxicity, monovalent cations such as Li+ and K+ have been shown to reduce sethoxydim absorption (Wanamarta et al. 1989). Also, it is possible that Na+ derived from sources other than Na-bentazon would decrease herbicide uptake. Wanamarta et al. (1989) showed a decrease in 14C-sethoxydim absorption from 60% when applied alone to 31% when applied with Na-bentazon, 31 % when applied with sodium acetate, and 40% when applied with sodium bicarbonate (baking soda). Thelen et al. (1995) concluded with NMR that Na+ from

23 sodium bicarbonate and sodium chloride (table salt) associated with sethoxydim the same as it does when derived from Na-bentazon. It is also possible, however, that the addition of sodium bicarbonate increases the pH of the solution and causes reduced uptake due to the weak acid herbicide being in the ionized state (McMullan 1994). Once in the ionized state, the herbicide may bind sodium ions in the solution, giving the NMR result stated above. If safening of saflufenacil is seen with Na-bentazon when applied to corn, it would be interesting to test whether the same safening effect is seen when sodium bicarbonate or sodium chloride is applied with saflufenacil.

The use of safeners can expand the application patterns of herbicides, and increase their selectivity. Na-bentazon has been shown to possess safening qualities when applied with weak acid herbicides. The mechanism of the safening effect induced by mixing certain herbicides with Na-bentazon, is a reduced uptake of the herbicide. This is caused by Na+ binding the herbicide molecule to form a salt with increased polarity and decreased ability to penetrate the leaf cuticle. It is predicted that combining Na-bentazon with saflufenacil will have the same effect in corn leaves and will reduce saflufenacil injury in postemergence applications. Antagonism of herbicide uptake by Na-bentazon has caused reduced weed control is some cases, but not all. It is anticipated that because of the rapid action of saflufenacil and high degree of susceptibility in weed plants, the weed control will not be compromised. Establishing Na-bentazon as an adequate safener for saflufenacil applied to field corn would provide some flexibility in application timing for corn growers.

Publications on saflufenacil at this time are limited, as it is a new product. All of the reports currently available have been cited in this review. It is evident that saflufenacil provides good weed control with preemergence application; however, the published studies on weed control were conducted in Kansas and Nebraska. A report on weed control with saflufenacil in eastern Canada would benefit Canadian corn growers and researchers. Also, reports have not been published on weed control with BAS 781, the combination of saflufenacil and dimethenamid-p to be registered for preemergence weed control in corn. Similarly, there have not been corn tolerance studies that include BAS 781, or tolerance studies conducted on the safening effect of bentazon on saflufenacil applied in corn. Following complete registration of

24 saflufenacil products in Canada, the number of publications on efficacy and crop tolerance will likely increase. The compound is proving to be highly efficacious and will be of great benefit to growers of a variety of crops. It is clear the future holds many opportunities to extend the use pattern of this product following exploration of its full potential.

25 HjC

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Figure 1-1. The structures of the reactant, protoporphyrinogen IX, and product, protoporphyrin IX, of the enzyme catalyst PPO, the last common step in chlorophyll and haem synthesis in plants.

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27 Figure 1-3. Chemical structure of acifluorfen.

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Figure 1-4. Chemical structure of oxyfluorfen.

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Figure 1-5. Chemical structure of flumioxazin.

30 HF2C

Figure 1-6. Chemical structure of sulfentrazone.

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Figure 1-7. Chemical structure of carfentrazone.

32 Figure 1-8. Chemical structure of saflufenacil.

33 2.0 Na-Bentazon Safens Saflufenacil Applied Postemergence to Corn

2.1 Abstract Saflufenacil is a preemergence herbicide for the control of broadleaf weeds. Field and growth room studies were conducted to explore the tolerance of corn to postemergence treatments of saflufenacil and BAS 781 (saflufenacil plus dimethenamid-p). Additionally, the potential use of sodium as a safener for saflufenacil was evaluated. Crop injury caused by saflufenacil or BAS 781 was 8 and 38%, respectively, when applied at the 2X dose at the spike- to-2 crop leaf stage. This injury increased to 28 and 65%, respectively, when applied at the 3-to- 4 leaf stage. This level of crop injury resulted yield loss, particularly when applied at 3-to-4 leaf. The addition of Na-bentazon to saflufenacil reduced this injury and increased crop dry weight under both field and laboratory conditions. In the field, Na-bentazon also increased corn collar height and yield compared to saflufenacil applied alone. Na-bentazon reduced injury through a reduction in foliar uptake of saflufenacil. Sodium derived from baking soda also provided a safening effect, only at the lowest dose of saflufenacil tested.

2.2 Introduction Saflufenacil is a new herbicide registered for the control of broadleaf weeds in a number of crops, including field corn. The compound inhibits protoporphyrinogen oxidase (PPO) and has a pyrimidinedione structure (Grossman et al. 2010). Saflufenacil will control weeds such as velvetleaf (Abutilon theophrasti), common ragweed {Ambrosia artemisiifolia), giant ragweed (Ambrosia trifida), common cocklebur (Xanthium strumarium), lady's thumb (Polygonum persicaria), redroot pigweed (Amaranthus retroflexus), common waterhemp (Amaranthus tuberculatus var. rudis) and common lamb's-quarter (Chenopodium album) including triazine and acetolactate synthase resistant biotypes (Kixor® Worldwide Technical Brochure, 2008; Liebl et al. 2008). It provides both contact and soil residual control of many of these weeds. In Canada, the proposed dose of saflufenacil in corn is 75 g ai/ha applied preplant, preplant incorporated or preemergence. It can be tank mixed with other herbicides such as glyphosate, dimethenamid-p or atrazine. The preformulated mixture of saflufenacil plus dimethenamid-p (BAS 781), applied preplant, preplant incorporated or preemergence is being developed in

34 Canada for grass and broadleaf weed control in corn. The proposed dose of BAS 781 is 735 g ai/ha.

Corn tolerance to saflufenacil is affected by the corn growth stage at the time of herbicide application. Corn is known to have an inplanta tolerance to saflufenacil (Grossmann et al. 2010). Saflufenacil applied prior to corn seedling emergence has resulted in very limited crop injury. For example, Soltani et al. (2009) reported that saflufenacil applied alone at doses as high as 200 g ai/ha caused crop injury of 1% or less, 21 days after treatment. Within the same study saflufenacil applied postemergence without the addition of an adjuvant, however, has been shown to cause injury ranging from 4 to 25% at doses of 50, 100 and 200 g ai/ha when applied at the 2-to-3 leaf stage of corn growth. Despite this level of injury no yield loss was reported.

The in planta tolerance of corn to saflufenacil may provide an opportunity to enhance crop tolerance to saflufenacil applied postemergence. Herbicide safeners have been used to reduce crop injury and expand the window of application for several herbicides. For example, metolochlor applied preplant or preemergence in corn can cause crop injury under cool, wet soil conditions (Foy and Witt 1991). The addition of the safener, benoxacor, to metolachlor was found to reduce corn injury caused by metolachlor under unfavourable soil conditions (Foy and Witt 1991; Viger et al. 1991). Also, the addition of the safener, cyprosulfamide, to isoxaflutole in corn has extended the use pattern of this herbicide from preemergence to postemergent use up to the 2 leaf-collar stage of corn growth (Nelson and Penner 2006; Philbrook and Santel 2008). In addition, bentazon as a sodium salt has been reported to have herbicide safening characteristics (Bauer et al. 1995; Weinberg et al. 2007).

Na-bentazon has been shown to reduce crop injury when included in solution with a wide variety of herbicides, such as tritosulfuron, thifensulfuron, and imazethapyr (Bauer et al. 1995; Lycan and Hart 1999; Weinberg et al. 2007). The safening effect of Na-bentazon has been reported to be the result of reduced foliar uptake of the herbicide (Wanamarta et al. 1989; Weinberg et al. 2007). This reduction in foliar uptake has been attributed to antagonism of ATPase function by bentazon (Couderchet and Retzlaff 1991). However, formulations of bentazon other than Na-bentazon, such as H-bentazon and NH3-bentazon, have not provided this

35 safening effect (Wanamarta et al. 1989). Consequently, a second hypothesis emerged; that sodium ions in the herbicide solution are the cause of reduced herbicide uptake (Thelan et al. 1995; Wanamarta et al. 1989). In experiments studying sethoxydim absorption, both Na- bentazon and baking soda (NaHCC^) reduced foliar uptake when they were included in the herbicide solution (Wanamarta et al. 1989). Furthermore, Thelan et al. (1995) have shown that Na+ from both Na-bentazon and baking soda chemically associated with sethoxydim in solution. The linkage of sodium to the sethoxydim molecule was suggested to increase the polarity which in turn would decrease cuticle penetration and subsequent leaf uptake (Thelan et al. 1995; Wanamarta et al. 1989; Wanamarta and Penner 1989).

The goal of this study was to explore the role of sodium as a potential safener for saflufenacil. Sodium ions were derived from bentazon as a sodium salt or from commercial baking soda. Field studies were conducted initially to determine the tolerance of corn to saflufenacil applied alone and in combination with dimethenamid-p, as a preemergence or postemergence treatment. Once the degree of crop tolerance was determined from these field studies, we then tested the hypothesis under laboratory and field conditions that sodium in solution with saflufenacil will decrease foliar uptake of saflufenacil thereby reducing crop injury.

2.3 Materials and Methods 2.3.1 Tolerance of corn to saflufenacil and BAS 781 Field experiments were conducted in 2008 and 2009 at the Elora, Huron, Ridgetown and Woodstock Research Stations located in southern Ontario. At the Elora and Woodstock Research Stations, seedbed preparation consisted of conservation tillage practices which included either a chisel plow or an offset disk followed by a cultipacker. At both Exeter and Ridgetown, a moldboard plow was employed as the primary tillage followed by cultivation to ensure a level seedbed. All sites were fertilized according to soil test results; corn was planted in rows 75 cm apart at seeding densities ranging from 75000 to 79100 per hectare. Locally adapted glyphosate tolerant corn hybrids were selected for each location based on their maturity ranking and yield potential (Table 1). Soil characteristics and spray parameters are listed in Tables 2 and 3, respectively. Plots were organized in a randomized complete block design with four replications at each location.

36 The herbicide treatments were designed to test the effect of saflufenacil and dimethenamid-p applied alone and the preformulated mixture (BAS 781). Saflufenacil (75 and 150 g/ha), dimethenamid-p (660 and 1320 g/ha) and BAS 781 (735 and 1470 g/ha) were each applied to corn at the recommended label dose and at the 2X dose. Each dose was applied preemergence to the corn, at the spike-to-2 leaf tip (approximately VE to VI), and at the 3-to-4 leaf tip stage (approximately VI to V2) of corn growth (Table 4). Dates of seeding, corn seedling emergence, herbicide application relative to corn growth stage, and final grain harvest are listed in Table 5. In order to facilitate observations of crop injury, all plots were kept weed free with glyphosate applied at approximately the 2 leaf stage and again as needed to control weeds throughout the season. Corn plants were assessed for visual injury at 3, 7, 14, 21, 28 and 56 days after application. A single injury rated accounted for leaf desiccation, growth restriction (buggy whipping), and visible height reduction when compared to the control, on a scale of 0 (no injury) to 100% (plant death). At 28 days after the last herbicide treatment, 5 consecutive plants from each of the middle two rows (10 plants per plot, N=40 per treatment) were cut at the soil surface and dried to a constant weight at 80°C. Corn yields were harvested from the remaining plants within the centre two rows and adjusted to 15.5% moisture.

2.3.2 Growth Room Trials Experiments evaluating the safening effect of Na-bentazon were conducted in a controlled environment in the growth room at the University of Guelph, Guelph ON. The temperature of the growth room was 25°C during the day and 20°C at night, and the relative humidity was 75%. There was a 16 hour photoperiod, using both incandescent and florescent lights. Plants were watered by hand with a fertilizer solution of 20-20-20. The corn hybrid used in growth room trials was Pioneer 39K37. For uniformity of germination, seeds were pre- germinated within wet paper towels, in a clear plastic covered tray to increase humidity, for 48 hours. Germinated seeds (i.e., emerged root radicle) were then planted in 10.3 cm pots, filled with Pro-Mix PGX1 growth medium, and placed in trays in the growth room.

Plants were treated at the 4 leaf stage, with the 5th leaf tip visible in the whorl. Plants were selected that were uniform in height and leaf number. The potting medium was covered

37 with vermiculite before spraying to prevent any soil activity of the herbicide, and to isolate foliar contact activity. The vermiculite was removed 24 hours after application, prior to watering. The sprayer nozzle in the spray chamber was a stainless steel 8002E flat fan Teejet2, calibrated to deliver 210 L ha"1 at 276 kPa. The nozzle height was set at 45 cm above the middle of the corn plants. Plants were returned to the growth room once the applied herbicide had dried, and arranged in a randomized complete block design in trays, where plants were aligned with their pots touching. To homogenize and randomize the effect of light interception due to the size of neighbouring plants and the pattern of the light bulbs, plants were rearranged every 2 to 3 days within the randomized complete block design.

2.3.3 Dose Response ofSaflufenacil. In order to select the appropriate herbicide doses for this study, it was first necessary to characterize the dose response of saflufenacil applied to corn under growth room conditions. Four leaf corn was treated with saflufenacil at doses of 0, 0.58, 1.17, 2.34, 4.69, 9.38, 18.75, 37.50, and 75.00 g/ha. All treatments, with the exception of the zero dose, included the surfactant monosodium methylarsonate (Merge®), at 0.5% volume/volume. Preliminary experiments confirmed limited uptake of saflufenacil without the addition of the surfactant (data not shown). Visual injury was recorded every 2 to 3 days for 9 days after treatment, where ratings of percent injury accounted for leaf desiccation, growth restriction due to buggy whipping, and height reduction compared to the control on a scale of 0% (no injury) to 100% (plant death) (data not shown). Preliminary experiments confirmed that by 9 days after treatment the plant response to the herbicide was no longer changing, and the untreated controls were growing too large to remain in the 10.3 cm pots. Therefore, at 9 days after treatment, plants were cut at the soil surface and dried at 80° C to a constant weight. The experiment was run 3 times with 4 replicates, and a fourth time with 10 replicates. Based on this experiment the doses of 0, 2.34, 4.69, 9.38, and 18.75 g /ha were selected for the following experiment, based on a range of visual injury ratings from approximately 0 to 90% and a range of plant dry weight from 20 to 60% (Figure 1).

38 2.3.4 Safening Effect of Na-bentazon Under Growth Room Conditions To characterize the potential safening effect of Na-bentazon on saflufenacil applied postemergence on corn, a range of doses of Na-bentazon were mixed with a range of doses of saflufenacil. Each of five doses of saflufenacil (0, 2.34, 4.69, 9.38, and 18.75 g/ha) were combined with each of five doses of Na-bentazon (0, 250, 500, 750 and 1000 g/ha), for a total of 25 different treatments. The dose of 1000 g ai/ha is within the registered field dose range of Na- bentazon in Ontario. All treatments, with the exception of the control, included the surfactant monosodium methylarsonate, at 0.5% volume/volume. In order to reduce experimental variability, a piece of cotton ball was inserted into the whorl before spraying. Cotton balls were removed at the time of vermiculite removal, at 24 hours after spraying. Visual injury was recorded every 2 days after treatment for 8 days, where ratings of percent injury leaf desiccation, growth restriction due to buggy whipping, and visible height reduction when compared to the control on a scale of 0 (no injury) to 100% (plant death) (data not shown). At 8 days after treatment, plants were cut at the soil surface and dried at 80° C to a constant weight. The experiment was run 2 times with 4 replicates, and a third time with 5 replicates.

2.3.5 Safening Effect of Na-bentazon Under Field Conditions Field experiments were conducted in 2009 at the Elora, Exeter and Woodstock Research Stations. Seedbed preparations, fertilization and corn hybrid selection at these locations were as described previously. Soil characteristics and herbicide application information are described in. Tables 2 and 3, respectively. Dates of seeding, corn seedling emergence, herbicide treatment, and corn harvest at each location are listed in Table 6. Plots were organized in a randomized complete block design with four replications at each location.

The 12 herbicide treatments, listed in Table 7, included Na-bentazon applied at 0, 600, 1200 and 2400 g/ha, saflufenacil applied at 0, 75 and 150 g/ha, and all combinations of Na- bentazon and saflufenacil. S-metolachlor/benoxacor/atrazine (Primextra II Magnum®) at 2160 g/ha was applied preemergence to the control plots for annual grass and broadleaf weed control. All plots were maintained weed free with glyphosate applied at the label dose at the 2-leaf stage of corn growth, and again when necessary depending on weed emergence. To assess the safening effect caused by Na-bentazon, visual injury was recorded at 3, 7, 14, 21, 28 and 56 days after

39 application on a scale of 0% (no injury) to 100% (plant death). A single injury rated accounted for leaf desiccation, growth restriction due to buggy whipping, and visible height reduction. Collar height was measured from 10 random plants per plot (N=40) at 28 days after treatment. At this time 5 consecutive plants from each of the middle two rows (10 plants per plot, N=40 per treatment) were cut at the soil surface and dried to a constant weight at 80°C. Corn yields were harvested from the remaining plants within the centre two rows and adjusted to 15.5% moisture content.

2.3.6 Uptake of C-saflufenacil With and Without Na-bentazon To determine the potential effect of Na-bentazon on the uptake of saflufenacil into corn leaves, a study was conducted using radio-labeled saflufenacil3. Based on the dose values obtained from the previous study, there were two treatments in the uptake study; saflufenacil applied alone at 15 g/ha, and saflufenacil at 15 g /ha plus Na-bentazon at 870 g/ha. Plants were first sprayed with the technical grade of each herbicide in the spray chamber, followed by 14C-saflufenacil application. The specific activity of l4C-saflufenacil was 5.54 MBq/mg. 14C-saflufenacil was dissolved in water, and 10 uL of solution containing 100 000 dpm was applied in 1 uL droplets to the mid-upper surface of the third leaf using a 10 uL Wiretrol4 micropipette, avoiding the mid- vein. The droplet solution also contained Na-bentazon at 870 g /ha when applied to plants sprayed with saflufenacil plus Na-bentazon. Plants were harvested at 6 and 24 h after treatment. At each harvest time, the third leaf was cut from the plant and dissected into the treated area of the leaf, and untreated portions distal and proximal to the treated area, respectively. The rest of the plant was harvested by cutting at the soil surface. To determine the amount of 14C- saflufenacil remaining on the leaf surface, the treated area was washed in two 22 ml scintillation vials each containing 10 mL of a foliar rinse treatment. The rinse contained 10% v/v ethanol and 0.5% v/v Tween 20, and vials were then filled with Ecolite3 scintillation cocktail. A Beckman LS6K-SC6 scintillation counter was used to quantify the radioactivity of the rinse. Each of the three portions of the third leaf were wrapped in tissue paper and dried at 80° C to a constant weight. Samples were combusted to 14CC>2 and trapped in a carbon-14 scintillation cocktail using a model OX300 oxidiser7, to quantify the radioactivity present within the leaf. The l4C02 recovery was 90% based on combustion of known quantities of D- C-glucose .

40 2.3.7 Safening Effect of Baking Soda Under Growth Room Conditions To evaluate the role of sodium as a safener, a study was conducted using saflufenacil and commercial grade baking soda (NaHCOa). Each of 4 doses of saflufenacil (0, 4.69, 9.38, and 18.75 g ai/ha) were combined with baking soda. The number of moles of sodium in the baking soda was equivalent to the number of moles of saflufenacil at each dose. In addition, based on each dose of saflufenacil, treatments of 10, 100 and 1000 times that number of moles in baking soda were added to the experimental protocol for a total of 20 treatments. Cotton balls were placed in the whorl of the plants at the time of spraying. At 8 days after treatment, plants were cut at the soil surface and dried at 80° C to a constant weight. The experiment was repeated twice with 5 replicates in each run.

2.3.8 Statistical Analysis Tolerance of corn to saflufenacil and BAS 781. Dry weight data was expressed as a percent of the untreated control within each block. Variances were partitioned into the random effects of location, year, location by year, and replicate by location within year and fixed effects of herbicide, dose nested within herbicide, time of application, time of application by herbicide, and time by dose within herbicide. Least square means, standard errors and pre-planned estimates were computed using the PROC MIXED procedure of SAS . Estimate statements were used instead of contrasts to show the magnitude of the difference between means. The control treatment was removed from the ANOVA (analysis of variance). Visual injury data was subjected to an arcsine square root transformation to correct for non-constancy of error variance, and converted back to the original scale for presentation of results. Transformation did not improve the distribution of dry weight residuals, therefore untransformed data was used. Yield data met the assumptions of the model (random, homogeneous, and normal distribution of error) therefore no transformation was needed.

Dose response of saflufenacil. Dry weight data was expressed as a percent of the untreated control within each block. Variances were partitioned into the random effects of run and replicate nested within run, and the

41 fixed effect of herbicide dose. Least square means and standard errors were computed using the PROC MIXED procedure, and a dose response equation was generated from dry weight data using PROC NLIN. Least square means of dry weight and the dose response curve of predicted dry weights were graphically displayed using Sigma Plot10. Herbicide doses were enabled to carry their own error variance; therefore standard assumptions of the analysis of variance were not tested.

Safening effect ofNa-bentazon under growth room conditions. Dry weight data was expressed as a percent of the untreated control within each block. Variances were partitioned into the random effects of run and replicate within run, and fixed effects of saflufenacil dose, Na-bentazon dose, and their interaction. Doses were further sub partitioned into linear and lack of fit parameters. Least squared means and standard errors were computed using PROC MIXED, and a response surface analysis was conducted using the PROC RSREG procedures to create a 3-dimensional curved hill modelling predicted dry weights. Predicted dry weight values for the 4 doses of saflufenacil were then expressed 2-dimensionally over increasing doses of Na-bentazon, using Sigma Plot. Error assumptions of the variance analysis including a random, homogenous and normal distribution of error were confirmed.

Safening effect of Na-bentazon under field conditions. Dry weight data was expressed as a percent of the untreated control within each block. Variances were partitioned into the random effects of location and replicate within location, and fixed effects of saflufenacil dose, Na-bentazon dose, and saflufenacil dose by Na-bentazon dose. Least square means, standard error, and planned contrasts were computed using PROC MIXED. Field locations were analyzed separately for each parameter, and data for locations were pooled only when there were significant effects shown in the contrasts statements. Collar height data was pooled across Elora, Exeter and Woodstock locations, biomass data was analyzed for Exeter alone, and yield data was pooled for Elora and Exeter. Error assumptions of the variance analysis including a random, homogenous and normal distribution of error were confirmed. For the data to meet the .assumption of normality, one outlying data point was removed from the dry weight data and one from the height data, based on their non-alignment shown in a plot of residuals against ranked residuals and the box plot of the PROC UNIVARIATE procedure.

42 Uptake of C-saflufenacil with and without Na-bentazon. Variances were partitioned into the random effects of run and replicate within run, and fixed effects of treatment, time and treatment by time. Least squared means, standard errors and contrasts were computed using PROC MIXED. Error assumptions of the variance analysis including a random, homogenous and normal distribution of error were confirmed. The amount of' C-saflufenacil in the leaf rinse and each part of the treated leaf was calculated by dividing the amount present in the sample by the total amount recovered in the rinse plus all leaf parts, and converting to a percentage. The total amount translocated was calculated by adding the amount above and below the treated area and dividing by the total amount recovered, and converting to a percentage.

Safening effect of baking soda under growth room conditions. Dry weight data was expressed as a percent of the untreated control within each block. Variances were partitioned into the random effects of run, replicate within run, BAS800 dose by run, baking soda dose by run, and saflufenacil dose by baking soda dose within run and the fixed effects of saflufenacil dose, baking soda dose, and saflufenacil dose by baking soda dose. Doses were further sub-partitioned into linear, quadratic and lack of fit parameters. Least square means, standard error and coefficients of regression equations for each dose of saflufenacil were computed using PROC MIXED. Assumptions of random and homogenous error variance were confirmed. The assumption of normality of residuals was not met, however it has been shown that F-tests have minimal sensitivity to small deviations from normality (Box, 1953; Box and Watson 1962). A histogram of residuals indicted a near-normal pattern (data not shown).

2.4 Results and Discussion Saflufenacil and BAS 781 cause injury and yield loss when applied postemergence. Saflufenacil or BAS 781 applied at the spike-to-2 leaf or 3-to-4 leaf stage of corn resulted in visible crop injury and a decrease in plant dry weight. In contrast, no injury was observed when saflufenacil or BAS 781 was applied preemergence (Table 8). At the spike-to-2 leaf stage saflufenacil and dimethenamid-p applied at the 2X dose resulted in crop injury up to 8 and 2%, respectively. BAS 781 applied at the same stage of growth resulted in injury as high as 38% at

43 the 2X dose. Saflufenacil and BAS 781 applied at the 3-to-4 leaf stage resulted in crop injury of up to 28% and 65%, respectively, at the 2X dose. This injury was transient and decreased with time after treatment. Percent dry weight of corn seedlings 28 days after treatment varied with time of herbicide application (Table 9). Corn seedlings sprayed at the spike-to-2 leaf stage had a dry weight 4.5% lower than the preemergence treatment. In addition, plants sprayed at the 3-to-4 leaf stage had a dry weight 18.7% less than plants treated preemergence.

The ability of corn plants to recover from herbicide injury was reflected in yield. No yield differences were detected between the preemergence and early postemergence treatments (Table 10). Saflufenacil, dimethenamid-p or BAS 781 applied preemergence or early postemergence had yields greater than 11.90 T/ha (Table 11). BAS 781 (2X dose) applied at the 3-to-4 leaf stage reduced corn yield. Generally, corn yield was reduced more when saflufenacil or BAS 781 was applied at the 3-to-4 leaf stage. No corn yield loss was observed with the application of dimethenamid-p at the three application timings included in this experiment.

The degree of injury and yield loss caused by saflufenacil and BAS 781 in corn was dependent on corn growth stage at the time of application. Yield losses caused by BAS 781 treatments at the 3-to-4 leaf stage were greater than that of saflufenacil. Similarly, Soltani et al. (2009) reported up to 25% injury with 200 g ai/ha of saflufenacil applied at the 2-to-3 leaf stage of corn growth, although no yield loss occurred. Other PPO inhibiting herbicides are known to cause injury to corn when applied postemergence. For example, carfentrazone-ethyl applied to corn at 35 g ai/ha at the 4th leaf stage resulted in injury of 10-to-20% at 7 days after treatment (Thompson and Nissen 2002). PPO injury to corn caused by carfentrazone-ethyl was attributed to warm, moist environmental conditions at the time of herbicide application, however, it is suggested that corn tends to recover quickly with little to no yield penalty (Thompson and Nissen 2002).

Growth room studies to determine the safening effect of Na-bentazon. Growth room studies were conducted to determine the role of Na-bentazon and sodium ions as a potential safener for saflufenacil. For these studies the dose response of saflufenacil under controlled conditions was determined and doses were selected causing an appropriate level

44 of injury for safener experiments. Secondly, Na-bentazon and common baking soda (NaHCOs) were applied with saflufenacil to plants grown in the growth room to determine their safening effect.

Increasing doses of saflufenacil caused increased injury. Corn seedlings grown under controlled environmental conditions were very sensitive to saflufenacil applied postemergence. The reduction in plant dry weight was rapid; approximately 1 g of dry matter for each 0.25 g /ha increase in dose of saflufenacil ranging from 0 to 18.75 g /ha (Figure 1). The relationship of dry weight and herbicide dose was defined by the equation y =

C + (D-C)/ (1 + exp[B(log(dose+10) - log I50))]) where C=18.9±2.01, D=100±0, B=9.9±1.4, 150=11.9±0.1771. From the results of this experiment, herbicide doses that caused a mid-range level of corn injury (i.e. 20 to 60% dry weight reduction compared to the control) were selected for the safener experiments. The selected doses were 2.34, 4.69, 9.38, and 18.75 g/ha.

Na-bentazon safened saflufenacil applied to 4 leaf corn under growth room conditions. The addition of Na-bentazon to the saflufenacil solution reduced injury and increased corn seedling dry weight. The safening effect was described using a response surface. The equation defining this relationship was described as y=81.642733 + (-6.154107*k) + (0.093985*b) + (0.141573*k*k) + (0.002243*b*k) + (-0.000073408*b*b) where k is the dose of saflufenacil applied, and b is the dose of Na-bentazon applied. To estimate the predicted dry weights of corn seedlings when sprayed with saflufenacil, the variable k was set to doses of 2.34, 4.69, 9.38, and 18.75, to generate a 2-dimensional quadratic graph of predicted dry weight versus dose of Na-bentazon (Figure 2). The greatest degree of safening was observed with the highest doses of saflufenacil. For example, when 18.75 g/ha of saflufenacil was combined with Na- bentazon doses from 0 to 750 g /ha, the change in corn seedling dry weight was approximately 1% of the control for each 12 g /ha increase in dose of Na-bentazon. In addition, from the equation described above a 3-dimensional graph was generated to determine the saddle point, i.e. the point of inflection (Figure 3). This point was chosen to represent an optimum combination dose of 15 g/ha of saflufenacil and 870 g /ha of Na-bentazon. This dose combination for both herbicides was selected for the uptake analysis study.

45 Na-bentazon safened saflufenacil applied to 4 leaf corn under field conditions. Under field conditions, saflufenacil (75 or 150 g/ha) applied alone to corn at the fourth leaf tip stage reduced collar height more than when combined with Na-bentazon (Table 12). Saflufenacil at 75 or 150 g/ha reduced corn collar height by 12 and 26%, respectively 28 days after treatment. In contrast, the addition of Na-bentazon to saflufenacil at 75 or 150 g/ha reduced corn collar height only 3 to 12%, respectively, indicating a safening effect of the Na-bentazon. In addition, all doses of Na-bentazon in solution with 150 g ai/ha of saflufenacil resulted in less reduction in plant dry weight and an increase in yield when compared to treatments of saflufenacil alone. For example, final grain yield was 8.8 T/ha when saflufenacil was applied at 150 g/ha. The addition of Na-bentazon, at doses ranging from 600 to 2400 g ai/ha, increased yield to 9.8 to 10.2 T/ha. This same trend was observed at the 75 g /ha dose of saflufenacil with the addition of 600 g /ha of Na-bentazon in solution.

The addition of increasing doses of Na-bentazon to saflufenacil had a concomitant safening effect under growth room conditions, lessening the reduction in corn plant dry weight caused by herbicide injury. In the field, a tankmix of Na-bentazon plus saflufenacil resulted in less corn injury as measured by corn plant collar height, dry weight and yield compared to saflufenacil applied alone. There is little published information on the use of safeners with PPO inhibiting herbicides; however, there are a number of studies on the safening effect of Na- bentazon when combined with other herbicides. For example, under growth room conditions, thifensulfuron reduced soybean dry weight by 58% when applied postemergence (Lycan and Hart 1999). With the addition of Na-bentazon to the solution, however, the dry weight reduction was only 36%. Similarly, Na-bentazon safened imazethapyr applied to pinto bean (Bauer et al. 1995). Visual injury decreased and pinto bean trifoliate dry weight increased with the addition 420 to 1680 g ai/ha of Na-bentazon to a solution of 53 g ai/ha of imazethapyr applied under greenhouse conditions. When imazethapyr was applied at the second trifoliate stage of pinto bean under field conditions, the addition of Na-bentazon to the solution reduced injury by up to 10% and prevented a delay in physiological maturity, but did not increase seed yield (Bauer et al. 1995).

Na-bentazon reduced foliar uptake of C-saflufenacil in corn.

46 The addition of Na-bentazon to a solution of safiufenacil reduced the uptake of radio labeled saflufenacil by corn leaf tissue. The amount of 14C-safiufenacil washed off of the leaf surface at both 6 and 24 hours after application when Na-bentazon was included in the treatment was 94% of the amount applied (Table 13). Without Na-bentazon, 76% and 63% of the applied 14C-saflufenacil washed off the leaf at 6 and 24 hours after treatment, respectively. The amount of C-saflufenacil in the leaf tissue after treatment was lower in treatments containing Na- bentazon. The total amount of saflufenacil in the leaf 6 and 24 hours after treatment was 12 and 18.6% when saflufenacil was applied alone, but was only 3 to 3.2%, respectively when Na- bentazon was included in the solution. The amount of 14C-saflufenacil translocated to the proximal portion of the leaf was 0.2 to 0.4% 6 and 24 hours after treatment, respectively, but decreased to 0.1 % at both evaluation timings when Na-bentazon was added to the saflufenacil solution. The amount of 14C-saflufenacil translocated to the distal portion of the leaf did not differ 6 hours after treatment; however, after 24 hours the amount translocated was different between treatments. As a result, the total amount translocated did not differ between treatments after 6 hours but was different 24 hours after treatment. The amount of 14C-saflufenacil in the wash and all other parts of the treated leaf could be pooled across harvest times. The amount of l4C-saflufenacil translocated to the tip of the leaf was greater than the amount translocated towards the base of the leaf. This evidence suggests that saflufenacil was translocated primarily in the xylem.

Na-bentazon has been reported in several studies to reduce herbicide uptake into plant tissue. The addition of bentazon to sethoxydim, haloxyfop, fenoxaprop-ethyl, tritosulfuron, acifluorfen, and paraquat has been shown to reduce foliar uptake in several plant species (Brommer et al. 2000; Gerwick 1988; Sorensen et al. 1987; Wanamarta et al. 1989; Wehtje et al. 1992; Weinberg et al. 2007). In each case bentazon was formulated as a sodium salt. Wanamarta et al. (1989) showed a reduction in absorption of sethoxydim of 21-to-29% in quackgrass leaves at 10 hours after treatment when combined with Na-bentazon. Similarly, in a more recent paper, Weinberg et al. (2007) reported a reduction in absorption of 60% at 7 days after treatment when tritosulfuron was combined with Na-bentazon and sprayed onto the surface of white bean leaves. The results of this study provide further evidence of the safening effect of Na-bentazon.

47 Baking soda safened saflufenacil appliedpostemergence in corn. The addition of baking soda to a solution of saflufenacil reduced injury when applied to corn at the 4th leaf tip stage of growth. This reduction in injury was evident only at the lowest dose of saflufenacil (i.e. 4.69 g/ha) (Table 14). At this dose of saflufenacil, there was a quadratic relationship between dry weight and dose of baking soda. As the dose of saflufenacil increased, however, a safening effect was not observed.

Reductions in herbicide uptake caused by bentazon have only been observed with the sodium salt of bentazon. The baking soda experiment supports the hypothesis that sodium ions in solution chemically associate with the herbicide molecule. In a similar study, Wanamarta et al. (1989) observed reduced sethoxydim absorption into quackgrass leaves when the herbicide was in solution with Na-bentazon, sodium acetate ^I-bNaCh) and baking soda. Thelan et al. (1995) used nuclear magnetic resonance spectroscopy to confirm that Na+ from Na-bentazon, salt (NaCl) and baking soda chemically associates with sethoxydim in solution. When Na+ binds with a weak acid herbicide, the compound becomes more polar and less lipophilic. This chemical change results in reduced leaf cuticle penetration (Thelan et al. 1995; Wanamarta et al. 1989; Wanamarta and Penner 1989). While the results of this study are consistent with the literature, baking soda may also have increased the pH of the solution to cause a change in herbicide polarity which would reduce leaf cuticle penetration (McMullan 1994).

2.6 Conclusion In summary, this study concludes that saflufenacil and BAS 781 cause injury and yield loss when applied postemergence in corn, particularly at the 3-to-4 leaf stage of corn growth. The addition of Na-bentazon safens saflufenacil applied postemergence under both growth room and field conditions. Additionally, Na-bentazon in solution with saflufenacil reduced foliar uptake of saflufenacil in corn leaves. Baking soda in solution with saflufenacil also provided a safening effect. This study suggests that sodium ions in solution with saflufenacil cause reduced foliar uptake of the herbicide caused by a change in polarity of the molecule. With further research it may be possible to develop a safener for postemergence applications of saflufenacil in corn that is derived from Na-bentazon or other sodium-containing compounds. This is the first report on the use of Na-bentazon as a safener for saflufenacil applied postemergence in corn under both growth room and field conditions.

48 2.6 Sources of Materials 1 Pro-mix PGX®, Premier Horticulture Inc., 127 South Fifth Street, #300 Quackertown, PA 18951.

2 8002 flat-fan nozzle, Teejet®, Spraying Systems Co., North Avenue, Wheaton, IL 60189-7900.

J 14C-saflufenacil, specific activity 5.54 MBq/mg, BASF, Landwehrweg, Germany

4 Wiretrol micropipette, Drummond Scientific Company, 500 Parkway, Box 700, Broomall, PA 19008.

5 Ecolite, ICN Biomedicals Inc., 15 Morgan, Irvine, CA 92618.

6 Scintillation counter, Beckman Instruments Inc., 2500 Harbor Blvd., Fullerton, CA 92634.

7 OX300, RJ Harvey Instrument Co., Hillsdale, NJ, USA

8 D-14C-glucose MP Biomedicals, OH, USA

9 SAS®, Version 9.1.3, SAS Institute Inc., 100 SAS Campus Drive, Cary, NC 27513.

10 Sigma Plot®, Version 10.0, Systat Software Inc., 1735 Technology Drive, Ste 430, San Jose, CA95110.

49 Table 2-1. Glyphosate tolerant corn hybrids used in each year at each location.

Location Year Variety Elora 2008 Pioneer 38N37 2009 Pioneer 39B23 Exeter 2008 Pioneer 38M58 2009 Pioneer 38M58 Ridgetown 2008 Pioneer 35F44 2009 Pioneer 35F44 Woodstock 2008 Pioneer 38N37 2009 Pioneer 39B23

50 Table 2-2. Soil characteristics for each location, including soil type, pH, percent composition of sand, silt and clay, and organic matter content.

Location Soil type pH Sand Silt Clay Organic matter % Elora Tavistock silt loam 7.4 31 50 19 4.2 Exeter Brookston clay loam 7.9 34 33 33 3.0 Ridgetown sandy clay loam 6.6 48 28 24 6.7 Woodstock Guelph loam 7.7 40 43 17 4.8

51 Table 2-3. Nozzle type, spray volume, operating pressure and plot size at each location.

Location Nozzle Spray volume Pressure Plot size L/ha kPa m x m Elora flat fan XR80 02 200 170 2x9 Exeter ultra low drift 120 02 200 241 2x10 Ridgetown ultra low drift 120 02 200 241 2x8 Woodstock flat fan XR80 02 200 170 2x9

52 Table 2-4. Herbicide treatments and application timings for the study of corn tolerance to saflufenacil, dimethenamid-p and BAS 781.

Herbicide Dose Time of Application g ai/ha None saflufenacil 75 preemergence saflufenacil 150 preemergence dimethenamid-p 660 preemergence dimethenamid-p 1320 preemergence BAS 781 735 preemergence BAS 781 1470 preemergence saflufenacil 75 spike-2 leaf saflufenacil 150 spike-2 leaf dimethenamid-p 660 spike-2 leaf dimethenamid-p 1320 spike-2 leaf BAS 781 735 spike-2 leaf BAS 781 1470 spike-2 leaf saflufenacil 75 3-4 leaf saflufenacil 150 3-4 leaf dimethenamid-p 660 3-4 leaf dimethenamid-p 1320 3-4 leaf BAS 781 735 3-4 leaf BAS 781 1470 3-4 leaf

53 Table 2-5. Dates of seeding, corn seedling emergence, herbicide application relative to corn growth stage, and corn grain harvest in each year at each location.

Location Year Seeding Emergence Preemergence Spike-2 Leaf 3-4 Leaf Harvest Elora 2008 1-May 24-May 5-May 27-May 7-Jun 5-Dec 2009 8-May 24-May 12-May 26-May 10-Jun 1-Dec Exeter 2008 22-Apr 9-May 29-Apr 13-May 29-May 17-Oct 2009 6-May 21-May 6-May 22-May 30-May 19-Oct Ridgetown 2008 6-May 20-May 13-May 23-May 2-Jun 17-Oct 2009 5-May 16-May 5-May 18-May 25-May 2-Nov Woodstock 2008 16-May 29-May 22-May 2-Jun 11-Jun 22-Oct 2009 13-May 24-May 19-May 30-May 10-Jun 17-Nov

54 Table 2-6. Dates of seeding, corn seedling emergence, herbicide treatment, and corn grain harvest in 2009, at each location used in the study of the safening effect of bentazon on saflufenacil.

Location Planting Emergence Treatment Harvest Elora 8-May 24-May 10-Jun 1-Dec Exeter 6-May 21-May 2-Jun 19-Oct Woodstock 13-May 24-May 10-Jun 17-Nov

55 Table 2-7. Herbicide treatments applied to 4 leaf corn in order to evaluate the safening effect of bentazon applied postemergence with saflufenacil under field conditions near Elora, Exeter, and Woodstock in 2009.

Herbicide Dose gai/ha control bentazon 600 bentazon 1200 bentazon 2400 saflufenacil 75 saflufenacil 150 saflufenacil + bentazon 75 + 600 saflufenacil + bentazon 75 + 1200 saflufenacil + bentazon 75 + 2400 saflufenacil + bentazon 150 + 600 saflufenacil + bentazon 150+1200 saflufenacil + bentazon 150 + 2400

56 Table 2-8. Average percent corn injury caused by saflufenacil, dimethenamid-p and BAS 781 (consistent with the order of your treatments) at 3, 7, 14, 21 28 and 56 days after application (DAT) at the preemergence, spike-to-2 leaf and 3-to-4 leaf stages of corn growth at Elora, Exeter, Woodstock and Ridgetown in 2008 and 2009.

Time of Application Herbicide Dose 3 DAT 7 DAT 14 DAT 21 DAT 28 DAT 56 DAT g ai/ha %- preemergence saflufenacil 75 0 0 0 0 0 0 saflufenacil 150 0 0 0 0 0 0

dimethenamid-•P 660 0 0 0 0 0 0 dimethenamid- P 1320 0 0 0 0 0 0 BAS 781 735 0 0 0 0 0 0 BAS 781 1470 0 0 0 0 0 0 spike-to-2 leaf saflufenacil 75 4 6 4 1 0 0 saflufenacil 150 6 8 3 1 0 0 dimethenamid-•P 660 1 2 1 0 0 0 dimethenamid-•P 1320 0 1 2 0 0 0 BAS 781 735 24 12 5 2 1 1 BAS 781 1470 38 25 14 4 4 3 3-to-4 leaf saflufenacil 75 13 18 9 2 2 0 saflufenacil 150 19 28 19 5 5 1 dimethenamid-•P 660 2 1 0 1 0 0 dimethenamid-•P 1320 10 6 2 1 0 0 BAS 781 735 40 43 34 14 12 2 BAS 781 1470 64 65 60 38 30 7 LSD (0.05) 12 16 12 4 4 1

57 Table 2-9. Variance analysis of the dry weight of corn plants harvested 28 days after final herbicide treatment as a measure of crop injury at Elora, Exeter, Woodstock and Ridgetown in 2008 and 2009.

Covariance Parameters Estimate Standard Error Z value Pr>Z location 0 - - - year 101.5 209.49 0.48 0.31 location*year 0 - - - rep*location(year) l 726.9 192.03 3.79 <0.0001 residual 302.3 18.62 16.23 <0.0001 Effect Num DF Den DF F value Pr>F herbicide 2 527 115.25 <0.0001 dose(herbicide) 3 527 12.03 <0.0001 time 2 527 60.35 <0.0001 herbicide*time 4 527 30.10 <0.0001 dose*time(herbicide) 6 527 1.86 0.08 Estimated Contrast Difference* Standard Error t value Pr>F preemergence vs spike-to-2 leaf -4.5 1.78 2.54 0.01 preemergence vs 3-to-4 leaf -18.7 1.78 -10.53 <0.0001 spike-to-2 leaf vs 3-to-4 leaf -14.2 1.78 63.82 <0.0001 *Estimated difference in plant dry weights expressed as a percent of the control as influenced by herbicide timing. Plants were treated with saflufenacil, dimethenamid-p and BAS 781 at the preemergence, spike-to-2 leaf and 3-to-4 leaf stages of corn growth at the IX and 2X doses

58 Table 2-10. Variance analysis of the corn grain yield from experiments near Elora, Exeter, Woodstock and Ridgetown, ON in 2008 and 2009.

Covariance Parameters Estimate Standard Error Z value Pr>Z Location 1.80 2.012 0.90 0.185 Year 0.96 1.784 0.54 0.296 location*year 1.12 0.960 1.16 0.122 rep*location(year) 0.18 0.067 2.71 0.003 Residual 0.89 0.055 16.05 <0.0001 Effect Num DF Den DF F value Pr>F herbicide 2 515 34.77 <0.0001 dose(herbicide) 3 515 15.01 O.0001 time 2 515 14.35 <0.0001 herbicide*time 4 515 14.71 <0.0001 dose*time(herbicide) 6 515 2.84 0.010 Estimated Contrast Difference* Standard Error t value Pr>F preemergence vs spike-to-2 leaf -0.04 0.097 0.44 0.660 preemergence vs 3-to-4leaf -0.47 0.097 -4.84 O.0001 spike-to-2 leaf vs 3-to-4 leaf -0.43 0.097 -4.40 <0.0001

*Estimated difference in grain yield as influenced by herbicide timing. Plants were treated with saflufenacil, BAS 781 and dimethenamid-p at the preemergence, spike-to-2 leaf and 3-to-4 leaf stages of corn growth at the lXx and 2Xx doses.

59 Table 2-11. Mean corn yield from plots treated with saflufenacil, dimethenamid-p and BAS 781 at the preemergence, spike-to-2 leaf and 3-to-4 leaf stages of corn growth at the lx and 2x doses in Elora, Exeter, Woodstock and Ridgetown, ON in 2008 and 2009.

Time of Application Herbicide Dose Yield g ai/ha T/ha Control 12.63 preemergence saflufenacil 75 12.28 saflufenacil 150 12.20 dimethenamid-•P 660 13.01 dimethenamid-•P 1320 12.62 BAS 781 735 12.85 BAS 781 1470 12.43 spike-2 leaf saflufenacil 75 12.75 saflufenacil 150 12.46 dimethenamid-•P 660 12.74 dimethenamid-•P 1320 12.79 BAS 781 735 12.50 BAS 781 1470 11.90 3-4 leaf saflufenacil 75 12.53 saflufenacil 150 11.99 dimethenamid-•P 660 12.93 dimethenamid-•P 1320 12.79 BAS 781 735 11.94 BAS 781 1470 10.38

60 Table 2-12. Variance analysis of percent corn collar height and dry weight reduction as compared to the untreated control 28 days after herbicide treatment and corn grain yield th at Elora, Woodstock and Exeter, Ontario in 2009. All herbicides were applied at the 4 leaf stage of corn growth.

Collar height Dry weight Treatment Dose reduction3 reduction13 Yield0 g ai/ha % % T/ha Saflufenacil 75 12 33 9.4 150 26 61 8.8 saflufenacil+ bentazon 75 + 600 5 18 10.6 75 + 1200 5 23 10.0 75 + 2400 3 24 10.1 150+600 12 37 9.8 150 + 1200 12 33 10.2 150 + 2400 11 33 9.8 Contrasts saflufenacil vs saflufenacil + bentazon 75 vs 75 + 600 0.0179* 0.0046** 0.0024** 75vs75 + 1200 0.0231* 0.0596 0.1361 75 vs 75 +2400 0.0031** 0.0795 0.0628 150 vs 150+ 600 <0.0001** <0.0001* 0.0077* 150 vs 150+ 1200 <0.0001** <0.0001* 0.0007** 150 vs 150+ 2400 O.0001** <0.0001* 0.0116* * PO.05 **P<0.005 a Data analyzed for all locations b Data analyzed for Exeter location only. No dry weight reduction detected at Elora and Woodstock at the time of sampling c Data analyzed for Exeter and Elora locations. No differences detected in yield at Woodstock. Yield was adjusted to 15.5% moisture.

61 Table 2-13. Distribution of 14C-saflufenaci, l in the leaf wash and parts of the treated leaf as a percent of the total amount recovered at 6 and 24 hours after treatment, with and without bentazon, in a controlled growth room experiment.

Distribution of 14C as % of recovered Harvest time Part of treated leaf Treatment 6h 24h Leaf rinse (% not absorbed) saflufenacil 75.9 (4.99)* 62.8 (4.99)* saflufenacil + bentazon 94.0 (4.99)* 93.6 (4.99)* Total in leaf saflufenacil 12.0 (2.49)* 18.6(2.49)* saflufenacil + bentazon 3.0 (2.49)* 3.2 (2.49)* Treated portion saflufenacil 10.5(2.13)* 14.9(2.13)* saflufenacil + bentazon 2.4(2.13)* 2.4(2.13)* Total translocated saflufenacil 1.5(0.47) 3.7 (0.47)* saflufenacil + bentazon 0.6 (0.47) 0.8 (0.47)* Distal portion saflufenacil 1.3(0.45) 3.3 (0.45)* saflufenacil + bentazon 0.5 (0.45) 0.7 (0.45)* Proximal portion saflufenacil 0.2 (0.04)* 0.4 (0.04)* saflufenacil + bentazon 0.1 (0.04)* 0.1 (0.04)* * Indicates a significant difference at P<0.05 between the two treatments within a plant part at a harvest time

62 Table 2-14. Variance analysis of plant dry weight following treatment with increasing doses of saflufenacil and baking soda in a controlled growth room, and the linear and quadratic regression coefficients at each dose of saflufenacil.

Covariance Parameter Estimate Standard Error Z value Pr>Z Run 27.20 58.370 0.47 0.3206 rep(run) 56.93 31.138 1.83 0.0338 run*saflufenacil 0 - - - run*soda 0 - - - Run*saflufenacil*soda 27.56 16.046 1.72 0.0429 Residual 106.70 12.239 8.72 <0.0001 Effect Num DF Den DF F value Pr>F Saflufenacil 3 3 .234.03 0.0005 Soda 4 4 6.06 0.0546 soda linear 1 4 13.93 0.0203 soda quadratic 1 4 7.29 0.0541 soda lack of fit 2 4 1.50 0.3259 saflufenacil*soda 12 12 2.04 0.1153 saflufenacil*soda linear 3 12 2.96 0.0752 saflufenacil*soda quadratic 3 12 3.67 0.0438 saflufenacil*soda lack of fit 6 12 0.77 0.6073 Regression Coefficients Saflufenacil dose Linear Quadratic g ai/ha Estimate P value Estimate P value 0 -0.0240 0.7309 0 0.7503 4.69 -0.2872 0.0117 0.0002 0.0017* 9.38 -0.0997 0.2015 0.0001 0.1878 18.75 0.0160 0.8188 0 0.7438 indicates a significant quadratic relationship at P<0.05 between dry weight and the dose of baking soda in solution with saflufenacil.

63 100

c uo o

.c 53

Dose of saflufenacil (g ai/ha)

64 * saflufenacil 2.34 g ai/ha • saflufenacil 4.69 g ai/ha A saflufenacil 9.38 g ai/ha • saflufenacil 18.75 g ai/ha

200 400 600 800 1000 Rate of bentazon (g ai/ha)

Figure 2-2. The predicted dry weight of corn plants treated with increasing doses of saflufenacil and bentazon at the 4 leaf over stage of corn growth under growth room conditions, where weight is expressed as a percent of the control. Data points represent the least square means (standard error= ± 5.4456) of dry weight (% of control). The equation defining the relationship is y=81.642733 + (-6.154107*k) + (0.093985*b) + (0.141573*k*k) + (0.002243 *b*k) + (-0.000073408*b*b) where b is the dose of bentazon applied, and k is the dose of saflufenacil set equal to 2.34, 4.69, 9.38, and 18.75.

65 — 100

66 3.0 The Role of BAS 781 for Weed Control^ Corn

3.1 Abstract BAS 781 (saflufenacil plus dimethenamid-p) is a new herbicide for preemergence control of grass and broadleaf weeds in corn. Field experiments were conducted in 2008 and 2009 at four locations in southern Ontario. The objective of this study was to determine the dose of BAS 781 required for overall weed control and species specific weed control, as well as the dose required for early season weed control when followed with glyphosate at the 6-to-8 leaf stage. Based on weed dry weight, the GR95 across locations ranged from

126 to 675 g/ha. The GR95 for common ragweed, lamb's-quarter, pigweed spp. and wild mustard were 933, 325, 186 and 115 g/ha, respectively. Optimal yields were achieved with BAS 781 applied alone at doses ranging from 368 to 1470 g/ha. When followed by glyphosate, the dose range of BAS 781 required for optimum corn yield was 46 to 1470 g/ha. A minimum dose of 184 g/ha was required to out-yield the single treatment of glyphosate applied alone.

3.2 Introduction Saflufenacil is a new herbicide applied preplant, preplant incorporated or preemergence for use in a number of crops, including field corn (Zea mays L.). Saflufenacil is a protoporphyrinogen oxidase (PPO) inhibitor that provides both contact and soil residual control of broadleaf weeds. It can be tank mixed with other herbicides such as glyphosate or atrazine. Crop tolerance to saflufenacil alone has been reported in the literature. Knezevic et al. (2010) stated that up to 400 g/ha can be applied safely as a preemergence treatment in winter wheat; however, postemergence treatments resulted in unacceptable crop injury and yield loss. Similarly, spring planted barley (Hordeum vulgar-e L.), oats (Avena sativa L.) and wheat {Triticum aestivum L.) do not tolerate saflufenacil applied postemergence (Sikkema et al. 2008). Corn, however, has an in planta tolerance to postemergence treatments of saflufenacil (Grossmann et al. 2010). Corn has been shown to tolerate 100 g/ha with no yield loss when saflufenacil was applied preemergence, at the spike stage or the 2-to-3 leaf stage of corn growth, provided an adjuvant was not added to the spray solution (Soltani et al. 2009).

67 Geier et al. (2009) tested the effectiveness of preemergence and postemergence treatments of saflufenacil on various weed species under growth room conditions. They did not find a differential species response to preemergence treatments; however, control was more variable with postemergence treatments. In this study, doses of saflufenacil required to reduce weed biomass by 90% were 9 and 6 g/ha for preemergence and postemergence treatments. Similarly, the doses which reduced weed population density by 90% were 9 g/ha for preemergence treatments and greater than 30 g/ha for postemergence treatments. Knezevic et al. (2009) conducted a field study with saflufenacil applied in the fall for postemergence burn-down, and noted that the doses which reduced dry weight by 90% depended on the weed species. The doses required to achieve a 90% reduction in dry weight of field bindweed {Convolvulus arvensis), dandelion (Taraxacum officinale), henbit (Lamium amplexicaule), field pennycress (Thlaspi arvense), prickly lettuce (Lactuca serriola), and shepherd's-purse (Capsella bursa-pastoris) and were 71, 93, 98, 103, 110, and 128 g/ha, respectively. The inclusion of an adjuvant, however, lowered the doses required for 90% dry weight reduction.

Today, many growers use glyphosate tolerant corn hybrids. Increased use of glyphosate, however, has led to an increase in glyphosate resistant weed species (Tardif 2007). Repeated application of glyphosate in a field, without rotating herbicide modes of action, increases the selection pressure which can cause resistant weed species (Tardif 2007). In glyphosate-tolerant crops it is important to include the use of herbicides with different modes of action. This can be accomplished through the use of preemergence residual herbicides.

BASF has developed a new preformulated mixture of saflufenacil and dimethenamid-p (BAS 781) for preemergence control of grass and broadleaf weeds in corn. The registered dose of BAS 781 is 735 g/ha. The low rate is for use in planned two- pass program with BAS 781 applied preemergence followed by glyphosate applied postemergence in glyphosate-tolerant corn. The high rate is for full season residual control of both grass and broadleaf weeds in conventional corn. There are no studies

68 published on the field performance of BAS 781. The goal of this study was to determine the biologically effective dose for weed control with BAS 781 applied preemergence in corn, as well as the doses required for species-specific weed control. Studies were also conducted to determine the dose of BAS 781 which provided adequate early season weed control when followed by glyphosate applied at the 6-to-8 leaf stage of corn growth.

3.3 Materials and Methods Field experiments were conducted in 2008 at research stations near Elora, Exeter, and Ridgetown in southern Ontario. In 2009, these same locations were utilized with the addition of the Woodstock Research Station. At the Elora and Woodstock Research Stations, seedbed preparation consisted of conservation tillage practices which included either a chisel plow or an offset disk followed by a cultipacker. At both Exeter and Ridgetown, a moldboard plow was used as the primary tillage followed by cultivation to ensure a level seedbed. All sites were fertilized according to soil test results, and corn was seeded in rows spaced 75 cm apart at seeding densities ranging from 75 000 to 79 000 per hectare. Glyphosate tolerant corn hybrids were selected for each location based on their maturity ranking (Table 1). Soil characteristics and spray parameters are listed in Tables 2 and 3, respectively.

Seven of the 17 herbicide treatments included BAS 781 applied preemergence at doses of 46, 92, 184, 368, 551, 735 and 1470 g ai/ha. The remaining treatments included the same doses of BAS 781 applied preemergence, followed by glyphosate applied at the 6-8 leaf stage (V4 toV6) at 900 g ae/ha. There was also a treatment of glyphosate only, applied at the 6-8 leaf stage. Each experiment included a weedy and weed-free control. The weed-free control was maintained weed-free with s-metolachlor/benoxacor/atrazine (Primextra II Magnum®) at 2160 g ai/ha applied preemergence. Dates of seeding, corn emergence, herbicide application, and corn grain harvest are listed in Table 4. Weeds were harvested 28 days after crop emergence from a 1 m long section between the centre two corn rows of plots treated with BAS 781 alone. Weeds were cut at the soil surface, separated by species, counted and bagged. Weeds were then dried at 80° C to a constant weight. Corn grain was machine harvested at maturity from the centre two rows of each

69 plot and the seed moisture content was adjusted to 15.5%.

Statistical Analysis Plots were organized in a randomized complete block design with four replications. Total weed dry weight per plot (weight of all species combined) was expressed as a percent of the control within each replicate. Weed dry weight by species was expressed as a percent of the average control weight within each location. For all weed dry weight, data variances were partitioned into the random effect of replicate nested within environment, and fixed effects of environment, dose of BAS 781, and environment by dose of BAS 781, where environment represents the location and year. For yield data, variances were partitioned into the random effects of location and replicate within location, and the fixed effects of treatment, year and treatment by year. Least square means, standard errors and pre-planned contrasts were computed using the PROC MIXED procedure of SAS1. For total weed dry weight per plot data, the environment by dose effect was significant so each environment was analyzed separately. For yield data, the assumption of normality of residuals was not met. A histogram of residuals, however, showed a near-normal pattern (data not shown). It has been shown that F-tests have minimal sensitivity to small deviations from normality (Box, 1952; Box and Watson, 1962).

A dose response equation was generated from total weed dry weight data for each environment (location year), using PROC NLIN and the log-logistic model Y= C + (D- C)/ {1 + exp[B(log(X+10) - loglso)]}; Y is percent weed control, C is the lower limit, D is the upper limit, B is the slope of the line, X is the herbicide dose, and I50 minus 10 equals the dose at which control is 50%. A dose response equation was not generated for total dry weight data from Exeter in 2008, and Ridgetown in 2009, because the response was flat-lined across all treatments. A dose response equation was generated for weed dry weight by species for lamb's quarter (Chenopodium album), pigweed (Amaranthus spp.), common ragweed (Ambrosia artemisiifolia) and wild mustard (Sinapsis arvensis) using the same log-logistic model given above. Weed species were selected based on uniformity across all replicates of the control plots. If one replicate within an

70 environment had a low dry weight, the environment was removed from the analysis because all plots are assumed to be equal barring the herbicide treatment applied. As a result, only four species were selected for analysis and the number of environments analyzed for each of the species varied. Pigweed spp. and wild mustard each met this requirement at only one site year. The most frequently occurring species were lamb's- quarters and common ragweed. For these species, the dry weight data was pooled across environments, because there was no significant environment by BAS 781 dose interaction. The doses at which weed dry weight were reduced by 80 and 95% (GR80 and GR95) were calculated for total weed dry weight and dry weight by species, using the dose response equations generated with PROC NLIN.

3.4 Results and Discussion Weed control with BAS 781 measured as total weed dry weight 28 days after treatment was dependant upon herbicide dose, location and weed species. The estimated GR95 value for total weed dry weight ranged from 675 g/ha in 2009 at the Woodstock Research Station to 126 g/ha at Exeter in 2009 (Table 5). A similar trend was observed for GR50 and GRso values. At each location rainfall patterns were sufficient in both years to provide activation of all preemergence treatments (data not shown). As a result, this wide range in the dose of BAS 781 required to achieve a dry weight reduction of 95% was influenced mainly by the weed species present. Common ragweed was the most difficult to control of the four species analyzed, followed by lamb's-quarter, pigweed, and finally wild mustard (Table 6). At 28 days after treatment, the GR95 value for common ragweed was estimated to be 933 g/ha of BAS 781, compared to 325, 186, and 115 g/ha for lamb's-quarter, pigweed spp., and wild mustard, respectively.

No previous research has been published on the dose range and weed species response to BAS 781. Research conducted with saflufenacil, however, has shown that weed species do not respond differently to saflufenacil applied preemergence under growth room conditions (Geier et al. 2009). This study included blue mustard {Chorispora tenella), flixweed (Descurainia sophia), Palmer amaranth {Amaranthns palmeri), redroot pigweed {Amaranthns retroflexus) and tumble pigweed (Amaranthus

71 albus). In another study, Knezevic et al. (2009) applied saflufenacil as a fall burn-down treatment for emerged weeds. In contrast to Geier et al. (2009), the estimated GR90 values at 28 days after treatment ranged from 71 to 128 g/ha depending on the species.

The yield response of corn to BAS 781 was influenced by herbicide dose when applied alone as a preemergence treatment, or in combination with glyphosate. When BAS 781 was applied alone as a preemergence treatment, optimal yields were achieved at a dose of 368 g/ha or greater (Table 7). For example, in this dose range, yields were 10.95 to 11.86 T/ha. The registered dose in Ontario is 735 g/ha, which is within the dose range achieving optimum yield. When glyphosate was applied as a follow-up postemergence treatment at the 6-to-8 leaf tip stage of corn, the dose range of BAS 781 required for optimum corn yield was 46 g/ha or greater. Yields achieved with these doses ranged from 11.70 to 12.37 T/ha. A minimum dose of 184 g/ha of BAS 781 followed by glyphosate was required to out-yield the single treatment of glyphosate applied alone at the 6-to-8 leaf stage.

3.5 Conclusion In summary, BAS 781 is an effective preemergence herbicide for the control of weeds in field corn. Weed species varied in their response to BAS 781, with common ragweed being the most difficult to control. This data suggests that common ragweed may be the first weed to escape treatment with BAS 781, as the preemergence dose required for 95% control was greater than the recommended field dose of 735 g/ha. Yield data suggested that the registered dose of 735 g/ha of BAS 781 applied preemergence will provide sufficient season long weed control for optimal corn yield. There was, however, greater flexibility, in the effective dose providing early season weed control when glyphosate was applied no later than the 8 leaf tip stage of corn growth. Applying a preemergence product can prevent yield loss caused by early season weed competition. The ability to use a reduced dose will also decrease the amount of active ingredient introduced into the environment. Additionally, the use of a preemergence herbicide is an important aspect of glyphosate stewardship; applying herbicides with a different mode of action in combination with glyphosate will lessen selection pressure

72 3.6 Sources of Material 'SAS ®, Version 9.1.3, SAS Institute Inc., 100 SAS Campus Drive, Cary, NC 27513.

73 Table 3-1. Glyphosate tolerant corn hybrids used in each year at each location.

Location Year Variety Elora 2008 Pioneer 38N37 2009 Pioneer 39B23 Exeter 2008 Pioneer 38M58 2009 Pioneer 38M58 Ridgetown 2008 Pioneer 35F44 2009 Pioneer 35F44 Woodstock 2009 Pioneer 39B23

74 Table 3-2. Soil characteristics for each location, including soil type, pH, percent composition of sand, silt and clay, and organic matter content.

75 Table 3-3. Nozzle type, spray volume, operating pressure and plot size at each location.

76 ' Table 3-4. Dates of seeding, corn seedling emergence, herbicide application and corn grain harvest in each year at each location for the study of weed control with BAS 781 alone and as a part of a glyphosate program.

Bas 781 Glyphosate Location Year Seeding Emergence application application Harvest Elora 2008 1-May 24-May 5-May 18-Jun 5-Dec 2009 8-May 24-May 12-May 22-Jun 1-Dec Exeter 2008 27-Apr 8-May 29-Apr 11-Jun 10-Oct 2009 12-May 25-May 12-May 22-Jun 19-Oct Ridgetown 2008 6-May 20-May 8-May 17-Jun 14-Oct 2009 5-May 17-May 6-May 16-Jun 3-Nov Woodstock 2009 13-May 24-May 19-May 22-Jun 17-Nov

77 Table 3-5. Dose response parameters by location for weed control with BAS 781 applied preemergence.

Dose response parameters (±SE) BAS 781 dose

Location Year D C b I50 *GR50i GRso GR95 g ai/ha Elora 2008 100(0) 0(0) 1.9(0.42) 54 (6.6) 44 103 250 Elora 2009 100(0) 0(0) 2.0 (0.46) 40 (5.2) 30 68 157 Exeter 2009 100(0) 0.4(1.67) 2.4 (0.48) 39(4.1) 29 60 126 Ridgetown 2008 100(0) 0(0) 1.7(0.18) 90 (5.6) 80 191 483 Woodstock 2009 100(0) 0(0) 1.3(0.25) 74(11.5) 64 201 675 ''GRso, GRso, and GR95 are the dose required to reduce weed dry weight by 50, 80 and 95%, respectively.

78 Table 3-6. Dose response parameters for control of lamb's quarter, common ragweed, pigweed spp. and wild mustard with BAS 781 applied preemergence.

Regression parameters (±SE) BAS 781 dose No. Location Weed Species Years D C B 150 GR50 GRso GR95 g ai/ha aCHEAL 5 100(0) 0.8 (2.86) 1.7(0.32) 54 (5.3) 44 114 325 AMBEL 4 99(11.8) 0(0) 1.4(0.39) 116(31.7) 106 299 933 AMASS 1 100(0) 1.1 (4.20) 2.8(1.02) 63 (6.9) 53 95 186 SINAR 1 100 (0) 2.3 (4.20) 2.6(1.17) 32 (9.9) 22 47 115 Abbreviations: CHEAL, common lamb's quarter; AMBEL, common ragweed; AMASS, pigweed spp.; SINAR, wild mustard. b GR50, GRso, and GR95 are the dose required to reduced weed dry weight by 50, 80 and 95%, respectively.

79 Table 3-7. Corn yield for each treatment included BAS 781 applied alone and followed by glyphosate.

Yield

i /na Weed free control 12.30 Weedy control 4.34 Dose of BAS 781 BAS 781 alone BAS 781 + glyphosate g ai/ha •11.2BC 46 6.1 E 11.7 ABC 92 7.0 E 11.9AB 184 9.1 D 12.4 A 368 11.0C 12.2 A 551 11.0 BC 12.2 A 735 11.9 ABC 12.3A 1470 11.6 ABC 11.6 ABC

All doses of BAS 781 were applied preemergence and glyphosate was applied at the 6-8 leaf stage in a dose of 900 g ae/ha. Means followed by the same letter are not significantly different at P=0.05 by a Tukey's multiple means comparison test. *Glyphosate applied alone, without a preemergence treatment.

80 4.0 General Discussion

4.1 Contributions Few studies have been published on the crop tolerance and weed control efficacy of saflufenacil and BAS 781, as they are new herbicide products that have not yet been registered in Canada. The studies reported here on the use of an herbicide safener in postemergence applications of saflufenacil have shown that it is possible to reduce crop injury with the addition of Na-bentazon or other Na+ containing compounds to the herbicide solution. There are many published studies which show that Na-bentazon can reduce crop injury caused by various herbicides through a reduction in herbicide uptake. This research shows that Na-bentazon reduced the uptake of saflufenacil into corn leaves, and reduced the injury caused to corn by saflufenacil applied postemergence. Additionally, a safening effect was seen with the addition of baking soda to the herbicide solution. This research contributes to the literature on the interactions between Na- bentazon and herbicide uptake, as well as the safening effect of other Na+ containing products. The research indicates that it may be possible to develop an effective herbicide safener with this type of chemistry, for postemergent applications of saflufenacil. The development of a safener would extend the window of application of saflufenacil, providing growers with some flexibility with the use of this efficacious herbicide.

The research also provides growers with information on saflufenacil and BAS 781 that will help them make decisions on the use of these products in their weed control programs. Through field studies we have shown that saflufenacil and BAS 781 cause injury to corn when applied postemergence, however yield losses were only observed with applications at the 3-to-4 leaf stage of corn growth. The studies also show that BAS 781 provides effective weed control as a stand-alone product or as part of a glyphosate stewardship program. BAS 781 alone reduces weed dry weight by 95% at doses ranging from 126 to 675 g/ha, which are below the recommended field dose of 735 g/ha. Common ragweed may be the first weed to escape treatment with BAS 781, as the dose required to provide 95% control is estimated to be 933 g/ha. As a preemergence product used in conjunction with post-applied glyphosate, BAS 781 provides early season weed

81 control at doses ranging from 184 to 735g/ha. The use of BAS 781 in a glyphosate program not only improved yields compared to glyphosate alone, but allows the grower to apply a second mode-of-action in their glyphosate program which will reduce the selection pressure causing glyphosate resistant weeds. This data allows growers to make informed decisions about which products to use in their specific fields, and what doses to apply.

4.2 Limitations The herbicide safener studies show that it may be possible to extend the window of application of saflufenacil, however, further research is required for the development of an effective safener that will prevent crop injury in postemergent applications. One limitation to this study is the lack of data on weed control with saflufenacil plus Na- bentazon. It is possible that the inclusion of Na-bentazon in the saflufenacil solution will compromise weed control. An effective safener will reduce crop injury without reducing weed control, so without further research we cannot state that Na-bentazon is an appropriate compound to use as a safener for saflufenacil. Additionally, the safener study was only conducted in the field over one year at three locations. If the experiment were conducted in a second year we may have more confidence in the results because there would be a greater number of replications, and more diverse conditions under which the experiment was conducted. In the data analysis we were not able to pool the results of the various observations, and with a greater number of replications we may have had more replicates within each of these analyzed parameters and thus a greater confidence in the results of the analyses. These limitations were noted concerning the field studies, and additional limitations were recognized regarding the growth room and laboratory studies.

Within the herbicide safener studies conducted in the growth room and laboratory there were some factors that were not addressed as to the mechanism of reduced uptake. It was concluded from these experiments that sodium ions in solution with saflufenacil bind the herbicide molecule and cause reduced uptake because of an increase in polarity. This mechanism was suggested in the literature and is continuous with our results however it is also possible that adding Na-bentazon or baking soda to the herbicide

82 causes a change in the pH of the solution (McMullan 1994). A change in the pH of an herbicide solution can also cause a change in the polarity of the herbicide molecule, causing reduced herbicide uptake. It is therefore possible that the sodium ions are not directly affecting the uptake of the compound, but that the potential safeners have merely changed the pH of the spray solution to the same effect.

The study of species-specific weed control with BAS 781 was conducted according to methods that are generally accepted in published literature. In the data analysis, however, a strict guideline was followed for selecting by-species data generated from the experiment. The guidelines were that all four replicates of the randomly distributed control plots must contain a similar amount of the weed, so it can then be assumed that all plots within the experiment contain the weed and differ only in the herbicide treatment applied. Weed species within a location year that did not meet these criteria were not analyzed. If the experiment were conducted with a postemergence herbicide, the weeds present prior to application could be used as a covariate against which to compare the level of control. With a preemergence herbicide there is not a covariate to compare to, and it cannot be determined whether weeds are absent in a particular plot because of the herbicide treatment or because of the random pattern of weeds within the field. It would be difficult to design a field experiment for a preemergence herbicide that has a covariate for weed control. One solution would be to increase the number of control plots present in the experiment, and to potentially have a control plot beside each treated plot to act as a covariate.

4.3 Future Research These studies outline some important information for growers on the tolerance of corn to saflufenacil and BAS 781, and the level of weed control achieved with BAS 781 as a stand-alone product or as part of a glyphosate program. Additionally, the role of an herbicide safener in postemergence applications of saflufenacil in field corn has been explored. In terms of weed control, there have not been any reports published on BAS 781 and it would be beneficial to conduct a species-specific weed control study under growth room conditions, in which many weeds common to Ontario are treated pre- and

83 postemergence. It would benefit local growers using the product to know which weeds may escape treatment with BAS 781.

Regarding the herbicide safener study, these experiments are a preliminary effort in determining how to extend the window of application of saflufenacil. Further experiments on the crop safety of saflufenacil plus Na-bentazon would need to be conducted, and derivatives of Na-bentazon and other sodium containing compound should also be tested. Future research should also focus on application at postemergent stages earlier than the 4 leaf stage of corn growth. The postemergent stage of application at which growers apply saflufenacil following this future research would likely be closer to the time of emergence, particularly because the herbicide already shows greater crop safety at earlier stages. Additionally, Na-bentazon or other potential safeners combined with saflufenacil must be tested for their weed control efficacy in comparison to saflufenacil applied alone; an effective safener should not reduce weed control. Future research could go so far as to develop a safener for BAS 781, and this would require even more experimental research because the composition of saflufenacil alone and the formulated mixture of saflufenacil and dimethenamid-p may interact differently with the safener. Any other compounds added to the safener plus herbicide, such as UAN or a surfactant must also be tested for crop safety and weed control efficacy before the safener can be used in combination with these other inputs.

Further research could also be conducted to confirm the mechanism by which Na- bentazon safens saflufenacil applied to corn. It has been concluded in these studies that sodium ions in solution increase the polarity of the herbicide molecule, however, the Na- bentazon and baking soda may in fact be changing the pH of the spray solution which would also cause a change in polarity and reduce herbicide uptake. Future experiments could include testing the pH of the spray solutions used in the Na-bentazon and baking soda studies, as well as manipulating the pH of saflufenacil alone in solution. It would be important to determine the change in uptake of the herbicide in these two studies to attempt to correlate uptake with pH.

84 5.0 Literature Cited

Abu-Qare, A., and H. Duncan. 2002. Herbicide safeners: uses, limitations, metabolism, and mechanisms of action. Chemosphere. 48: 965-974.

Anonymous. 2007. Aim EC herbicide label. FMC Corporation publication, EPA Reg. No. 279-3241. Philadelphia, PA: FMC Corporation. 4 p.

Anonymous. 2010. Guide to Weed Control, Publication 75. Toronto, ON: Ontario Ministry of Agriculture, Food and Rural Affairs Canada.

Arnould, S., and J. Camadro. 1998. The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. Proc. Nat. Acad. Sci. 95:10553- 10558.

Ateh, C. M., R. G. Harvey, J. W. Albright, and T. M Anthon. 1995. Yield penalties associated with soybean injury from postemergence herbicides. Res. Rep. N. Cent. Weed Sci. Soc. 52:374-375.

Bauer, T. A., K. A. Renner, and D. Penner. 1995. 'Olathe' pinto bean (Phaseolus vulgaris) response to postemergence imazethapyr and bentazon. Weed Sci. 43: 276-282.

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97 6.0 Appendix 1: Monthly rainfall averages for 2008 and 2009 at each field location.

Location Year Monthly rainfall average Apr. May June July Aug. Sept. -mm- Elora 2008 68 71 101 141 121 119 2009 106 109 79 86 92 55 Exeter 2008 42 85 161 121 70 144 2009 138 64 91 59 47 72 Ridgetown 2008 38 74 92 91 54 112 2009 108 31 61 32 91 34 Woodstock 2008 56 95 109 126 90 108 2009 133 130 67 131 117 45