Improving the Consistency of Glyphosate-resistant Canada fleabane (Conyza
canadensis) Control with Saflufenacil: Distribution and Control in Soybean
(Glycine max)
By Christopher Murray Budd
A Thesis Presented to The University of Guelph
In partial fulfillment of requirements For the degree of Master of Science In Plant Agriculture
Guelph, Ontario, Canada © Christopher M. Budd, April, 2016 ABSTRACT Improving the Consistency of Glyphosate-resistant Canada fleabane (Conyza canadensis) Control with Saflufenacil: Distribution and Control in Soybean (Glycine max) Christopher M. Budd Advisors: University of Guelph, 2016 Peter H. Sikkema Darren E. Robinson
In 2010, glyphosate-resistant (GR) biotypes of Canada fleabane were first confirmed in
Essex County, Ontario. From surveys conducted in 2010 to 2015, inclusive, GR Canada fleabane has now been confirmed in 30 counties and multiple-resistant biotypes (glyphosate and cloransulam-methyl) in 22 counties. Saflufenacil was recommended for GR Canada fleabane control in soybean, however variable control had been reported. The biologically effective rate
(BER) of saflufenacil is 25 g a.i. ha-1 for 90% control eight weeks after application (WAA). At 8
WAA, the BER of metribuzin when tankmixed with glyphosate (900 g a.i. ha-1) plus saflufenacil
(25 g a.i. ha-1) was 61, 261, and 572 g a.i. ha-1 for 90, 95 and 98% control, respectively. Tankmix partners with glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) were investigated, and the best three-way tankmixes were with either 2,4-D or metribuzin for full season control of multiple-resistant Canada fleabane. The optimal time of day (TOD) for controlling GR Canada fleabane with glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) was 09:00-21:00 h. GR
Canada fleabane height and density were found to have a minimal effect on control with glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1).
ACKNOWLEDGEMENTS
This journey would never have happened without the help and support of many people and groups. Saying thank you only begins to reflect my appreciation for my advisors Dr. Peter Sikkema and Dr. Darren Robinson. The amount of support and opportunity each of you provided me with was fantastic and how you make your graduate students and their work a priority with your busy schedules was amazing. I have taken so much away from this experience because of your efforts and constant encouragement. A simple conversation with you often acted as a shot of espresso for motivating others and myself. Thank you to Dr. Dave Hooker and Rob Miller from BASF Canada for being a part of my advisory committee, for providing lots of feedback, and challenging me to explain why I thought something was interesting. Thank you to Dr. Nader Soltani for your continual support through the manuscript publication process. The field and greenhouse work was no small feat and would not have been possible if not for Chris Kramer. A very big thank you to Chris for your expertise and for all the time you put towards helping out those on the Kramer Crew. Special thanks to my fellow graduate students Mike Schryver, Jordan Eyamie, Matt Underwood, Holly Byker, Annemarie Van Wely and Scott Ditschun for your assistance in the field and/or greenhouse. Thank you to the summer students Rebecca Sterling, Shelby Johnston, Rebecca Jennen and April Stead for your hours of help in the field, and for putting up with my ‘Dad Quotes’. I would like to thank Hilary Gbinije for your help with the greenhouse work and keeping the place so clean and organized. Thank you, Christy Shropshire, for your expertise with the statistical analysis. Thank you to Kris McNaughton, Dave Bilyea, and Lynnette Brown for your help with some greenhouse watering and my many random questions. Thank you to BASF Canada, Grain Farmers of Ontario and the Agricultural Adaptation Council for providing the funding and supporting this project. Thank you to the farmer co-operators for allowing this research to be conducted on their land. Thank you to the many members of the agricultural industry who contacted me for the survey. Finally I would like to thank my family and friends for their support, especially after I
accepted a Field Biologist position and became overwhelmed to complete my studies.
iii Table of Contents
ACKNOWLEDGEMENTS ...... iii!
List of Tables ...... viii!
List of Figures ...... x!
Chapter 1: Literature Review: Improving the Consistency of Glyphosate-resistant
Canada fleabane (Conyza canadensis) Control with Saflufenacil: Distribution and
Control in Soybean (Glycine max)...... 1!
1.1 Introduction ...... 1!
1.2 Biology of Conyza canadensis (L.) Cronq...... 3!
1.2.1 Description and Distribution ...... 3!
1.2.2 Germination and Development ...... 5!
1.2.3 Reproduction ...... 6!
1.2.4 Control ...... 8!
1.2.5 Herbicide Resistance ...... 10!
1.3 Saflufenacil ...... 11!
1.3.1 Chemistry ...... 11!
1.3.2 Weed Control ...... 13!
1.3.3 Canada Fleabane Control ...... 15!
1.3.4 Factors Affecting Control ...... 16!
1.3.5 Crop Safety ...... 18!
1.4 Glyphosate ...... 19!
1.4.1 Mode of Action ...... 19!
1.4.2 Behaviour in Plants ...... 20!
1.4.3 Behaviour in Soil ...... 22!
1.4.4 Environmental Interactions ...... 23!
1.4.5 Glyphosate-Resistant Crops ...... 25!
iv 1.5 Glyphosate-Resistant Weeds ...... 27!
1.5.1 Background and Fitness Costs ...... 27!
1.5.2 Species ...... 28!
1.5.3 Mechanisms of Resistance ...... 30!
1.5.4 Non-Target Based Resistance ...... 31!
1.5.5 Target Based Resistance ...... 33!
1.6 Glyphosate-Resistant Canada fleabane ...... 34!
1.6.1 Inheritance and Hybridization ...... 34!
1.6.2 Mechanisms of Resistance ...... 36!
1.6.3 Spread of Resistance and Fitness Costs ...... 40!
1.6.4 Control of Resistant Biotypes ...... 42!
1.7 Hypothesis and Objectives ...... 45!
Chapter 2: Distribution of glyphosate and cloransulam-methyl resistant Canada fleabane
(Conyza Canadensis L. Cronq.) in Ontario ...... 46!
2.1 Abstract ...... 46!
2.2 Introduction ...... 47!
2.3 Materials and Methods ...... 49!
2.3.1 Seed Collection ...... 49!
2.3.2 Resistance Screening ...... 50!
2.4 Results and Discussion ...... 51!
2.5 Implications ...... 52!
Chapter 3: Glyphosate resistant Canada fleabane [Conyza canadensis (L.) Cronq.] dose- response to saflufenacil, saflufenacil plus glyphosate, and metribuzin plus saflufenacil plus glyphosate in soybean [Glycine max (L.) Merr.] in Ontario ...... 62!
3.1 Abstract ...... 62!
3.2 Introduction ...... 62!
v 3.3 Materials and Methods ...... 67!
3.4 Results and Discussion ...... 70!
3.4.1 Biologically Effective Rate of Saflufenacil Alone ...... 70!
3.4.2 Biologically Effective Rate of Saflufenacil plus Glyphosate Tankmix ...... 72!
3.4.3 Biologically Effective Rate of Metribuzin plus Saflufenacil plus Glyphosate Tankmix ...... 73!
3.5 Conclusions ...... 75!
Chapter 4: Control of glyphosate-resistant Canada fleabane with saflufenacil plus tankmix partners in soybean ...... 81!
4.1 Abstract ...... 81!
4.2 Introduction ...... 82!
4.3 Materials and Methods ...... 84!
4.4 Results and Discussion ...... 86!
4.5 Conclusions ...... 90!
Chapter 5: Efficacy of saflufenacil for control of glyphosate-resistant Canada fleabane
[Conyza canadensis] as affected by height, density and time of day ...... 93!
5.1 Abstract ...... 93!
5.2 Introduction ...... 93!
5.3 Materials and Methods ...... 97!
5.4 Results and Discussion ...... 101!
5.4.1 Study 1. Effect of Time of Day on Glyphosate plus Saflufenacil Efficacy ...... 101!
5.4.2 Study 2. Effects of GR Canada Fleabane Height on Glyphosate plus Saflufenacil Efficacy ...... 104!
5.4.3 Study 3. Effects of GR Canada Fleabane Density on Glyphosate plus Saflufenacil Efficacy ...... 106!
5.5 Conclusions ...... 107!
Chapter 6: General Discussion ...... 116!
6.1 Contributions ...... 116!
vi 6.2 Limitations ...... 117!
6.3 Future Research ...... 119!
Chapter 7: Literature Cited ...... 121!
Chapter 8: Appendixies ...... 130!
8.1 Chapter 5.4.1 Additional Figures ...... 130!
8.2 Chapter 5.4.2 Additional Figures ...... 137!
8.3 Chapter 5.4.3 Additional Figures ...... 143!
8.4 SAS Code for Analyzing Regressions in Chapter 3 ...... 149!
8.5 SAS Code for Analyzing Means Comparisons in Chapter 4 ...... 151!
8.6 SAS Code for Analyzing Regressions in Chapter 5 ...... 153!
vii List of Tables
Table 2.1- Number of sites per county with at least one plant surviving five weeks after application of glyphosate or cloransulam-methyl from greenhouse screening from a 2013-2015 survey of Canada fleabane in Ontario, Canada...... 54!
Table 3.1- Location, agronomic information and height and density of glyphosate-resistant Canada fleabane in biologically effective rate experiments in Ontario, Canada in 2014 and 2015 ...... 77!
Table 3.2- Regression parameters of exponential to a maximum and inverse exponential equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, dry weight, and density for saflufenacil alone field dose experiments conducted in 2014 and 2015 in Ontario, CanadaZ ...... 78!
Table 3.3- Regression parameters of exponential to a maximum and inverse exponential equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, dry weight, and density for saflufenacil plus glyphosate field dose experiments conducted in 2014 and 2015 in Ontario, Canada ...... 79!
Table 3.4- Regression parameters of exponential to a maximum and inverse exponential equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, dry weight, and density for metribuzin plus saflufenacil plus glyphosate field dose experiments conducted in 2014 and 2015 in Ontario, Canada Z ...... 80!
Table 4.1- Location, agronomic information, height and density of glyphosate-resistant Canada fleabane during field experiments conducted in Ontario, Canada in 2014 and 2015 .. 91!
Table 4.2- Percent control of GR Canada fleabane at 4 and 8 weeks after treatment application (WAA) and density and biomass at 8 WAA of glyphosate plus saflufenacil plus tankmix partners and soybean yield during field experiments conducted across six locations in Ontario, Canada in 2014 and 2015 ...... 92!
Table 5.1- Location and agronomic information for factors influencing glyphosate-resistant Canada fleabane control with glyphosate plus saflufenacil studies in Ontario, Canada in 2014 and 2015 ...... 110!
Table 5.2- Environmental measurements at each application for the effect from time of day of glyphosate plus saflufenacil application on glyphosate-resistant Canada fleabane control study from 2014 and 2015 in Ontario, Canada ...... 111!
Table 5.3- Regression parameters of parabolic curve equation for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, density, dry weight, and soybean yield for time of day of glyphosate plus saflufenacil application study conducted in 2014 and 2015 in Ontario, CanadaZ ...... 113!
viii Table 5.4- Regression parameters of linear equation for glyphosate-resistant Canada fleabane control 1, 2, 4, and 6 WAA, dry weight of treated plants, and percent dry weight of treated plants of untreated plants for height at application of glyphosate plus saflufenacil study conducted in 2014 and 2015 in Ontario, CanadaZ ...... 114!
Table 5.5- Regression parameters of exponential and linear equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 6 WAA, dry weight of treated plants, and percent dry weight of treated plants of untreated plants for density at application of glyphosate plus saflufenacil study conducted in 2014 and 2015 in Ontario, CanadaZ ...... 115!
ix List of Figures
Figure 2.1. Ontario counties with glyphosate-resistant Canada fleabane since 2010. Adapted from Byker et al. (2013c)...... 56!
Figure 2.2. Ontario counties with multiple-resistant Canada fleabane since 2011. Adapted from Byker et al. (2013c) ...... 57!
Figure 2.3. Ontario counties with multiple-resistant Canada fleabane since 2012. Adapted from Byker et al. (2013c)...... 58!
Figure 2.4. Ontario counties with multiple-resistant Canada fleabane since 2013...... 59!
Figure 2.5. Ontario counties with multiple-resistant Canada fleabane since 2014...... 60!
Figure 2.6. Ontario counties with multiple-resistant Canada fleabane since 2015...... 61!
Figure 5.4.1.1. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 1 week after application for six combined sites in ON across 2014 and 2015...... 130!
Figure 5.4.1.2. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 2 weeks after application for six combined sites in ON across 2014 and 2015...... 131!
Figure 5.4.1.3. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 2 weeks after application for six combined sites in ON across 2014 and 2015...... 132!
Figure 5.4.1.4. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 8 weeks after application for six combined sites in ON across 2014 and 2015...... 133!
Figure 5.4.1.5. Reduction in glyphosate-resistant Canada fleabane density with saflufenacil with different time of day applications, 8 weeks after application for six combined sites in ON across 2014 and 2015...... 134!
Figure 5.4.1.6. Reduction in glyphosate-resistant Canada fleabane dry weight with saflufenacil with different time of day applications, 8 weeks after application for six combined sites in ON across 2014 and 2015...... 135!
Figure 5.4.1.7. Reduction soybean yield from glyphosate-resistant Canada fleabane interference due to different time of day applications of saflufenacil, for six combined sites in ON across 2014 and 2015...... 136!
Figure 5.4.2.1. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 1 week after application for six combined sites in ON
x across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm...... 137!
Figure 5.4.2.2. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 2 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm...... 138!
Figure 5.4.2.3. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 4 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm...... 139!
Figure 5.4.2.4. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm...... 140!
Figure 5.4.2.5. Reduction in glyphosate-resistant Canada fleabane dry weight at different Canada fleabane heights at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm...... 141!
Figure 5.4.2.6. Percent dry weight of untreated control for glyphosate-resistant Canada fleabane at different Canada fleabane heights at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm...... 142!
Figure 5.4.3.1. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 1 week after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41- 100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2...... 143!
Figure 5.4.3.2. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 2 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41- 100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2...... 144!
Figure 5.4.3.3. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 4 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41- 100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2...... 145!
xi Figure 5.4.3.4. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41- 100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2...... 146!
Figure 5.4.3.5. Reduction in glyphosate-resistant Canada fleabane dry weight at different Canada fleabane densities at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m- 2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2...... 147!
Figure 5.4.3.6. Percent dry weight of untreated control for glyphosate-resistant Canada fleabane at different Canada fleabane densities at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1- 20 plants m-2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2...... 148!
xii Chapter 1: Literature Review: Improving the Consistency of Glyphosate- resistant Canada fleabane (Conyza canadensis) Control with Saflufenacil: Distribution and Control in Soybean (Glycine max).
1.1 Introduction
Glyphosate is a non-selective herbicide that controls grass and broadleaf weeds by targeting the shikimate biosynthetic pathway; glyphosate has very low animal and insect toxicity because these organisms do not have the shikimate pathway (Franz et al. 1997). The first glyphosate-resistant (GR) crops were released in 1996, being soybean and canola released in the
USA and Canada, respectively (Duke and Powles 2008; Dill 2005). Since then, more transgenic crops have been developed with resistance to glyphosate, which allowed for increased use of glyphosate (Duke and Powles 2008). The repeated use, and overreliance on glyphosate for weed management, created strong selection pressure for the selection of GR weeds (Nandula et al.
2005; Powles et al. 1998).
In 1996, rigid ryegrass ( Lolium rigidum Gaud.) in Australia was the first reported GR weed. Today, there are 32 weed species, globally, with GR biotypes (Heap 2016). Currently there are more herbicide resistance mechanisms known to glyphosate, than for any other herbicide (Sammons and Gaines 2014). There are two broad classes of herbicide resistance: non- target site and target-site based resistance (Nandula 2010, Powles and Preston 2006). Non-target site mechanisms reduce the amount of active herbicide that reaches the target site (Yuan et al.
2006). Target-site resistance mechanisms reduce the lethality of the herbicide at the target site. In
GR Canada fleabane (Conyza canadensis L. Cronq.), the resistance mechanisms are only non- target based, being herbicide sequestration and enhanced metabolism (Ge et al. 2010; González-
Torralva et al. 2012).
1 Canada fleabane was the first broadleaf weed with GR biotypes, which was in Delaware,
USA in 2001 (VanGessel 2001). In Canada, GR Canada fleabane was identified in 2010 as the second GR weed in the country (Heap 2016). Although Canada fleabane can be found in all
Canadian provinces except Newfoundland (Weaver 2001), GR biotypes have only been reported in Ontario (Heap 2016). GR Canada fleabane is expected to spread due to some of its morphological features and the high use of glyphosate for weed management in Ontario (Davis et al. 2009). Canada fleabane is self-pollinated (Smisek 1995) capable of producing over 200,000 seeds per plant that are wind dispersed (Weaver 2001). Seeds can be found predominately within
100-m of the mother plant (Dauer et al. 2007), however, viable seed has been collected from the planetary boundary layer where it could travel up to 500 km (Shields et al. 2006). GR Canada fleabane control in soybean is challenging since postemergent herbicides do not provide consistent control (Davis et al. 2009), therefore farmers must rely on preplant (PP) or preemergence (PRE) herbicides. Control of GR Canada fleabane in soybean is very important since interference with soybean can reduce yield up to 93% (Byker et al. 2013b).
Saflufenacil is a group 14 herbicide that provides control of some broadleaf weeds, including biotypes that are resistant to other modes of action such as GR Canada fleabane (Liebl et al. 2008; Soltani et al. 2010; Trolove et al. 2011). However, there have been reports from farmers and in published research of variable control of GR Canada fleabane with saflufenacil.
In a study by Ikley (2012), saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) provided
61, 67 and 57% control of GR Canada fleabane at 7, 14, and 28 days after application (DAA); the decrease in control over time was due to plant regrowth. Mellendorf et al. (2013) suggested that control of GR Canada fleabane with saflufenacil may be optimized when plants are relatively small and to include a third herbicide with a different mode of action as a tankmix;
2 these and other factors should be investigated to improve the consistency of GR Canada fleabane control with saflufenacil in soybean.
1.2 Biology of Conyza canadensis (L.) Cronq.
1.2.1 Description and Distribution
Canada fleabane is a member of the Asteraceae Family with specific morphological features that aid in its identification. The fruit (achenes) of Canada fleabane are small, transparent, 1 to 2 mm long (Frankton and Mulligan 1987), are flattened and oblong with an attached greyish white to tan pappus 3 to 5 mm long (Royer and Dickenson 1999). Canada fleabane has a short taproot with branched laterals (Frankton and Mulligan 1987). The cotyledons are 1 to 2-mm wide and 2- to 3.5-mm long, ovate shaped and hairless (Royer and
Dickenson 1999). The first leaves are spatula-shaped, and hairy on the upper surface and leaf margin (Royer and Dickenson 1999). Mature leaves are alternate, 2- to 10-cm long, and oblong to lance shaped (Royer and Dickenson 1999). Mature plants have leaves with no petioles and are smaller towards the top of the plant (Loux et al. 2006). The lower leaves are often bristly-haired particularly near the base (Royer and Dickenson 1999), and can have smooth or slightly toothed margins (Loux et al. 2006). The upper leaves are smaller and have smooth margins (Royer and
Dickenson 1999). The stem and leaves have a faint odour suggestive of carrots when crushed
(Royer and Dickenson 1999). The stem grows erect with short bristly hairs and is unbranched at the base unless damaged by mowing, animal or insect feeding or herbicides (Loux et al. 2006), and can grow up to 180-cm in height (Frankton and Mulligan 1987). Near the top of the plant are numerous, short, leafless flowering branches containing small flower heads 3- to 5-mm in diameter (Frankton and Mulligan 1987), arranged into branched terminal clusters (Royer and
Dickenson 1999). The flower heads are composed of white ray and yellow disc florets, with the ray florets often hidden by floral bracts, 2- to 4-mm long and arranged in 2 to 3 overlapping rows
3 (Royer and Dickenson 1999). The flowers contain 20 to 40 yellow, perfect disc florets (Loux et al. 2006), with 60 to 70 seeds per capitulum (Frankton and Mulligan 1987). The above morphological features help distinguish Canada fleabane from other Asteraceae species.
Canada fleabane can emerge in the fall or spring. Plants that germinate in the fall form a basal rosette of dark green hairy leaves on petioles, with coarsely-toothed margins, and are less than 1-cm wide (Frankton and Mulligan 1987). Canada fleabane plants that germinate in the spring do not form a rosette (Bhowmik and Bekech 1993). For fall-germinated Canada fleabane, the stem elongates as the rosette deteriorates in the spring (Frankton and Mulligan 1987). The time of germination and emergence influences the morphology of Canada fleabane.
Canada fleabane is a cosmopolitan weed. Canada fleabane is distributed worldwide, but is most common in the North Temperate Zone (Weaver 2001). It is native to North America and considered the most completely naturalized plant of American origin in Europe (Frankton and
Mulligan 1987) that was likely introduced into Europe within the past 350 years (Thebaud and
Abbott 1995). Canada fleabane can be broadly found between latitudes N 55 and S 45, suggesting that it has few specialized climatic requirements (Weaver 2001). In Canada, Canada fleabane can be found in all provinces except Newfoundland (Weaver 2001); relative occurrence throughout the country can be ranked as high, low and medium in eastern Canada, the prairie provinces and British Columbia, respectively (Cici and Van Acker 2009). Canada fleabane is widely adapted to the temperate, semi-arid and Mediterranean climates throughout the world.
Canada fleabane density is influenced by soil texture, drainage and tillage. Canada fleabane is most commonly found on coarse-textured, well-drained soils (Frankton and Mulligan
1987), because it prefers rough and stony, sandy or loam land (Weaver 2001). Canada fleabane endures drought well, but is intolerant of flooding (Weaver 2001). In regards to tillage systems,
Canada fleabane is more prevalent where rosettes remain undisturbed in minimum tillage
4 systems compared to those in conventionally tilled systems (Bhowmik and Bekech 1993).
Canada fleabane is commonly found in coarse-textured, well-drained, undisturbed soils.
1.2.2 Germination and Development
Canada fleabane can germinate throughout the year. The seed of Canada fleabane is non- dormant and given suitable conditions can readily germinate (Buhler and Owen 1997) after being released from a mature plant (Loux et al. 2006). Canada fleabane seed has different base temperatures for different populations [Ontario (8-9.5°C), Iran (9.5-11°C), Spain (12.5-14°C),
UK (11-12.5°C)] (Tozzi et al. 2013), but was previously documented as having only one at 13°C
(Steinmasus et al. 2000). Germination of Canada fleabane is optimal at 24/20C (day/night) temperatures and can germinate in complete darkness, but a 13-hour photoperiod is more favourable (Nandula et al. 2006). In Canada, most Canada fleabane emerges from late August through October and forms rosettes that overwinter, while only a small fraction act as spring annuals (Weaver 2001). In an area of large seed populations, emergence may occur for an extended period because of the continued availability of viable seed (Buhler and Owen 1997). In undisturbed systems, Canada fleabane may solely act as a winter annual due to reduced space for later germinating plants (Buhler and Owen 1997). Crop canopies can help protect Canada fleabane seeds on the soil surface from temperature extremes, desiccation and erosion events, while in the winter, crop residues protect living rosettes which can exploit environmental resources when competitor weeds are dormant (Reghr and Bazzaz 1979). In Ontario, Canada fleabane has a long emergence period that is modified by environmental factors.
Soil and residue cover can inhibit Canada fleabane emergence. When Canada fleabane seed is buried 1-cm below the soil surface compared to surface-sown seeds, emergence can be reduced by 90% (Weaver 2001). Seeds at a depth of 0.5 cm can overwinter and germination occurs in the spring (Tozzi et al. 2014). Emergence of Canada fleabane can be delayed and
5 reduced by 80% from crop residues on the soil surface (Bond et al. 2007).Germination can be reduced if residues of rye (Secale cereale L.) are present in the soil profile (Bond et al. 2007).
Seed burial depth and crop residues affect Canada fleabane emergence.
1.2.3 Reproduction
Canada fleabane is primarily self-pollinated (Smisek 1995) and self-compatible (Weaver
2001). Pollen is released before flower heads are completely opened. Smisek (1995) found that an average of 96% of florets was self-pollinated since the flower heads were closed at the time of pollination. However, insects have been observed visiting open flowers (Smisek 1995).
Outcrossing ranges from 1.2 to 14.5%, with 4% as the average in a population within Essex
County, Ontario (Smisek 1995). Self-pollination allows for the rapid increase of alleles within populations of Canada fleabane.
Winter warm spells can impact the flowering time of Canada fleabane. Warm spells are periods of successive temperatures in the 80th percentile of average temperature (Tozzi et al.
2014). The later the warming spell, the earlier the plants flower (Tozzi et al. 2014). With later warming spells, the air temperature is high enough afterwards to prevent the plants from returning to a dormant, flower-inducing state (Tozzi et al. 2014). A long cold period followed by a late warming spell creates conditions that allow for high concentrations of flowering-inducing hormones in Canada fleabane and coupled with high post warming spell temperatures, causes earlier flowering (Tozzi et al. 2014). Canada fleabane can flower earlier if exposed to a late winter warming spell.
Both seed production and their viability are variable depending on plant growth, density and environment. Canada fleabane reproduces by seeds which mature approximately three weeks after fertilization (Weaver 2001). The number of flower heads per plant, and therefore seed production, is proportional to stem height (Regehr and Bazzaz 1979; Smisek 1995). A Canada
6 fleabane plant that is 40-cm tall has been shown to produce approximately 2000 seeds, while a
1.5-m tall plant may produce 230,000 seeds (Weaver 2001). The number of seeds produced per plant depends on the relative plant population density. Highly dense populations of Canada fleabane can delay flowering and reduce seed production per unit area (Palmblad 1967). Time of emergence can affect seed production, with fall-emerging Canada fleabane usually producing more seed than spring emerging (Regehr and Bazzaz 1979). Data on seed longevity of Canada fleabane is varied. In laboratory conditions, seed longevity was found to be 2 to 3 years, while in field conditions, fall-emerged plants produced seed that was viable for one year (Weaver 2001).
In contrast, viable seeds have been found in a 20-year old abandoned pasture that was free of weed plants because of a prominent dwarf bamboo (Sasa senanensis Rehd.) population
(Tsuyuzaki and Kanda 1996). Canada fleabane produces a large number of seeds per plant that remain viable in the soil for a short period of time.
The morphology of Canada fleabane seed allows for long distance dispersal. The seeds of
Canada fleabane can be dispersed by wind and water (Weaver 2001). Canada fleabane and dandelion (Taraxacum officinale L.) have similar mechanisms of seed dispersal because they have a similar ratio of seed surface area to biomass, but Canada fleabane has a lower settlement velocity and therefore a greater dispersal distance (Weaver 2001). The attached pappus on
Canada fleabane seeds decreases the seed settlement velocity (Dauer et al. 2006). The mean settlement velocity of Canada fleabane seeds was documented by Andersen (1993) to be 0.2778 m per second using field collected seeds in a plexiglass tube. The small seed with an attached pappus facilitates long distance dispersal of Canada fleabane seed.
Canada fleabane seed can move varying distances from the parent plant. Air movement is complex in nature with gusts, updrafts and interactions with boundary layers that affect the dispersal of Canada fleabane seed (Dauer et al. 2006). Ninety-nine percent of Canada fleabane
7 seed lands within 100-m of the mother plant, with a relatively small amount moving further distances (Dauer et al. 2007). However, seeds have been found further than 1.5 km, easily entering 10s to 100s of neighbouring farms (Dauer et al. 2007). Wind speeds of 18 km per hour and greater can move Canada fleabane seeds 72 to 145 km in a single flight (Shields et al. 2006).
Viable Canada fleabane seed has been found in the Planetary Boundary Layer (PBL) which allows the seed to move hundreds of kilometers (Shields et al. 2006). It is estimated by Shields et al. (2006) that with winds in the PBL often exceeding 72 km hr-1 a 550 km seed movement in a single flight is likely. Canada fleabane can spread very far distances and can establish in previously uninfested areas.
1.2.4 Control
Canada fleabane can be managed through mechanical and biological techniques, but is less affected by cultural control methods. In crop production systems where tillage is used, fall or spring tillage generally controls Canada fleabane (Brown and Whitwell 1988). Shallow disking can provide control of Canada fleabane prior to seeding (Brown and Whitwell 1988), however only small plants at the time of tillage operation would be controlled (Shrestha et al. 2008).
Mowing however, is generally not a viable mechanical control method as it tends to stimulate branching and can harden off the plants making control with subsequent herbicide application more difficult (Shrestha et al. 2008). Having little diversity in crop rotations can reduce control.
For example, in a study throughout Indiana, Canada fleabane was similarly found in 63% of double-crop soybean fields, 51% in continuous soybean fields, and 47% in a corn-soybean rotation (Loux et al. 2006). Canada fleabane establishment may be reduced if emergence is delayed or growth is slowed in the fall from increased residue cover and competition; small, weak seedlings are more susceptible to mortality during winter (Buhler and Owen 1997). A possible biological control method may be with the use of a Phytoparasitic bacterium,
8 Pseudomonas syringae, which has been shown to cause severe disease symptoms that eliminated
Canada fleabane in field trials in corn (Johnson et al. 1996). However, cultural control can be variable in the field due to adverse environmental conditions and the short life of biocontrol agents (Ross and Lembi 1999). Canada fleabane can be controlled with biological and mechanical weed control techniques.
Canada fleabane in soybean can be controlled with herbicides. In no-tillage fields, herbicides must be used to control Canada fleabane (Bruce and Kells 1990). It is best to apply herbicides in the fall or early spring when rosettes are small and actively growing (Weaver
2001). Herbicides should be applied pre-emergence because post-emergence herbicides are limited in effectiveness (Loux et al. 2006). Canada fleabane can emerge following non-selective pre-plant herbicide applications (Buhler and Owen 1997), making residual herbicide application a more desirable option for early planted soybean (Loux et al. 2006). In Ontario, herbicides that provided acceptable levels of Canada fleabane control in soybean were chlorimuron-ethyl, cloransulam-methyl, amitrole and flumetsulam, with the most economic being a reduced rate of cloransulam-methyl (Tardif and Smith 2003). Other herbicides have shown variable control in no-till soybean, such as linuron, imazaquin, imazethapyr, metribuzin (Bruce and Kells 1990), clomozone, flumioxazin, and pyroxasulfone plus flumioxazin (Byker 2013a). In a study by
Byker (2013a), metribuzin at 1120 g a.i ha-1 provided over 97% control of spring-germinated
Canada fleabane in no-till soybean. In the same study, glyphosate burndown applications enhanced control when tankmixed with saflufenacil, saflufenacil/dimethenamid-p along with residual activity applications of cloransulam-methyl or flumetsulam. There are only a few herbicide options for the control of Canada fleabane in soybean.
9 1.2.5 Herbicide Resistance
Canada fleabane has developed resistance to a number of herbicides. To-date, 16 countries have Canada fleabane populations resistant to at least one herbicide (Heap 2016). The first report of herbicide resistant Canada fleabane was by Japanese researchers in 1980, who found a population resistant to paraquat (Loux et al. 2006). Since then, resistance to paraquat has been reported in Hungary, United States, and several orchards in Essex County, Ontario, where paraquat had been applied four to five times per year for at least 10 years (Smisek et al. 1998;
Weaver 2001). Populations resistant to triazine herbicides have been found in the United
Kingdom, France, Belgium, Switzerland, Poland, Czechoslovakia, and Spain (Weaver 2001). It has been reported that triazine-resistant Canada fleabane plants have a more limited capacity for acclimation to high and low temperatures than susceptible plants (Weaver 2001). There are also populations in Ohio and Poland that are resistant to acetolactate synthase (ALS) inhibitors, and in 2001, the first confirmed glyphosate resistant population was found in a no-till soybean field in Delaware (VanGessel 2001). There are biotypes of Canada fleabane with resistance to a number of herbicide modes-of-action.
There are Canada fleabane populations with resistance to multiple herbicides and some are resistant to herbicides with different modes of action (Weaver 2001). Canada fleabane has been found to be resistant to both paraquat and atrazine in Hungary (Weaver 2001), and to both atrazine and chlorsulfuron in Israel (Heap 2016). The greatest amount of multiple herbicide resistance has been found in the United States, with populations resistant to atrazine, diuron and simazine as well as chlorimuron-ethyl, cloransulam-methyl and glyphosate, and finally to both glyphosate and paraquat (Heap 2016). In Canada, there are populations of Canada fleabane resistant to both cloransulam-methyl and glyphosate (Heap 2016). Canada fleabane is resistant to
10 multiple herbicides in different areas of the world; a key area of research is the search for efficacious herbicides with alternate modes-of-action.
1.3 Saflufenacil
1.3.1 Chemistry
Saflufenacil is a protoporphyrinogen-oxidase (PPO) inhibiting herbicide that belongs to the pyrimidinedione class of chemicals and the N-phenylnitrogen heterocycle family of PPO inhibitors (Grossman et al. 2010). In general, saflufenacil affects plant growth by inhibiting the photosynthetic system (Grossmann et al. 2010), since PPO is a necessary enzyme for chlorophyll, heme and cytochrome synthesis in the chloroplast (Grossman et al. 2011). Inhibition of PPO by saflufenacil prevents the normal conversion of protoporphyrinogen IX to protoporphyrin IX in the chloroplast (Grossmann et al. 2010). As a result, protoporphyrinogen
IX accumulates in the chloroplast and then diffuses into the cytoplasm where it is oxidized to protoporphyrin IX at sites on plasma membranes (Jacobs et al. 1991), and/or enzymes in the cytoplasm (Frihauf et al. 2010). Protoporphyrin IX is a strong photosensitizer and in the presence of oxygen and light it produces singlet oxygen (Duke et al. 1991). The singlet oxygen peroxidises the unsaturated fatty acids of cell membranes resulting in rapid loss of membrane integrity and function (Grossmann et al. 2011). The loss in membrane integrity leads to ion leakage and water loss from the cell (Grossman et al. 2010), as well as bleaching of chloroplast pigments, further tissue necrosis and ultimately growth inhibition and plant death (Grossmann et al. 2011). The efficacy of PPO inhibitors is dependent on light and is closely correlated with the level of protoporphyrin IX that accumulates in the cytoplasm (Duke et al. 1991). Saflufenacil causes an increase in free radicals which results in loss of cell membrane integrity and plant death.
11 Saflufenacil has limited movement in plants, differing between plant species. An acidic proton from a nitrogen atom in the amide position on a side chain allows saflufenacil to have a weak acid property that is unique among commercial PPO inhibitors (Grossmann et al. 2011).
While saflufenacil is principally translocated in the xylem in corn (Liebl et al. 2008), in some plant species, it also has limited mobility in the phloem. Grossmann et al. (2011), found saflufenacil is retained in the sieve tube of susceptible weed species including black nightshade
(Solanum nigrum L.) and tall morningglory (Ipomoea purpurea L.), long enough to be transported to remote parts of the plant. Within 16 hours after a foliar application of saflufenacil,
Grossman et al. (2011) found 7% of absorbed 14C saflufenacil had been translocated to shoot cells in black nightshade. The integrity of a plant’s vascular tissue is necessary for systemic translocation of herbicides (Grossmann et al. 2011). A general expectation of PPO inhibitors is that they cause rapid necrosis to vascular tissues, lowering their potential for systemic translocation (Grossmann et al. 2011). Saflufenacil causes less inhibition of PPO compared to other PPO inhibitors, and it is thought that the delayed injury to vascular tissues allows for long- distance transport of the herbicide before vascular tissue is destroyed (Grossmann et al. 2011).
Saflufenacil has differing mobility between plant species.
Saflufenacil has low persistence in soil. Saflufenacil is applied at relatively low doses and has low environmental, toxicological and ecotoxicological impact (Knezevic et al. 2009; Soltani et al. 2012). In general, pesticides are degraded by biological, chemical and photochemical processes (Camargo et al. 2013). Camargo et al. (2013), observed that saflufenacil persisted two to three times longer in the environment under saturated field conditions compared to when soil moisture was at field capacity. The half-life of saflufenacil is 59 and 33 days for saturated and field capacity conditions respectively, indicating that faster dissipation of saflufenacil resulted from aerobic respiration then anaerobic metabolism (Camargo et al. 2013). The weak acid
12 property of saflufenacil with a pKa of 4.4 means that in most agricultural soils, pH >5, keeps it primarily in anionic form (Hixson 2008). As soil clay content increases there is a repulsion of the anionic saflufenacil molecules and negatively charged clay particles causing less saflufenacil sorption (Hixson 2008). Soil organic matter content also influences saflufenacil sorption with greater affinity resulting from high (>4%) organic matter content (Hixson 2008). Several factors influence the persistence of saflufenacil in soil overall, resulting in a low persistence compared to other herbicides.
1.3.2 Weed Control
Saflufenacil can be used as a PP burndown herbicide prior to seeding several crops.
Saflufenacil is registered for pre-emergence application in barley (Hordeum vulgare L.), wheat
(T.a) and corn (Zea mays L.) (field and sweet) and as a pre-plant only application in soybean
(Glycine max L. Merr.) in eastern Canada (Anonymous 2014a). In western Canada, saflufenacil is also registered for pre-emergence or pre-plant application on canary seed (Phalaris canariensis L.), chickpeas (Cicer arietinum L.), lentils (Lens culinaris M.), oats (Avena sativa
L.) and field peas (Pisum avense L.) (Anonymous 2014b). Saflufenacil has been approved as a harvest aid for desiccation of soybean, dry bean and triticale in eastern Canada (Anonymous
2014a); additionally it has been approved for canola (Brassica napus L.), flax (Lunum usitatissimum L.), lentils, mustard (Brassica spp.), field peas, and sunflower (Helianthus annuus
L.) in western Canada (Anonymous 2014b). A broad-spectrum of both grass and broadleaf weeds are controlled when glyphosate is tankmixed with saflufenacil (Mellendorf et al. 2013).
The tankmix of saflufenacil with glyphosate applied as a burndown results in improved control of emerged weeds in reduced or no-till cropping systems (Soltani et al. 2010). In addition, saflufenacil provides residual control of broadleaf weeds (Soltani et al. 2012). The length of broadleaf weed control is influenced by the rate applied (Soltani et al. 2012). Saflufenacil
13 provides control of broadleaf weeds applied pre-plant, pre-emergent or as a harvest aid for desiccation in several crops.
Saflufenacil has a range of application rates for burndown and residual control of some broadleaf weeds. Susceptible weed species show injury symptoms within a few hours and may die within one to three days (Owen et al. 2011). Saflufenacil applied as a PP burndown is registered in eastern Canada for the control of broadleaf plantain (Plantago major L.), Canada fleabane, common ragweed (Ambrosia artemisiifolia L.), dandelion (Taraxacum officinale
Weber ex Wigg.) (suppression), giant ragweed (Ambrosia trifida L.), lady’s thumb (Polygonum persicaria L.), common lamb’s-quarters (Chenopodium album L.), perennial sow thistle
(Sonchus arvensisL.) (top growth), prickly lettuce (Lactuca serriola L.) (top growth), redroot pigweed (Amaranthus retroflexus L.), shepherd’s-purse (Capsella bursa-pastoris L.), stinkweed
(Thlaspi arvense L.), wild buckwheat (Polygonum convolvulus L.), wild mustard (Sinapis arvensis L.) and velvetleaf (Abutilon theophrasti Medik.) (Anonymous 2014a). In addition, residual control has been documented for common cocklebur (Xanthium strumarium L.), velvetleaf, redroot pigweed, common waterhemp (Amaranthus rudis Sauer), common ragweed, giant ragweed, lady’s thumb and common lambsquarters (Knezevic et al. 2009). Liebl et al.
(2008) reported that 63 to 125 g ai ha-1 of saflufenacil provided complete control of large-seeded broadleaf species including velvetleaf, common cocklebur, giant ragweed, common ragweed, common sunflower (Helianthus annuus) and morningglory (Ipomoea spp.). At rates up to 100 g ai ha-1 complete control of emerged flixweed (Descurainia sophia L. Webb), kochia [Kochia scoparia (L.) Schrad.], and wild buckwheat was achieved (Frihauf et al. 2010). Soltani et al.
(2012) found that the dose required to provide 95% control of common ragweed, common lamb’s-quarters, wild buckwheat, green smartweed (Polygonum scabrum Moench.) and wild mustard to be 72 to >100, >100, 74, 58, and >100 g ai ha-1 respectively. Low rates of 6-30 g ai
14 ha-1 have activity on blue mustard (Chorispora tenella Willd.), flixweed, palmer amaranth
(Amaranthus palmeri S. Wats.), redroot pigweed and tumble pigweed (Amaranthus albus L.)
(Geier et al. 2009). Saflufenacil provides excellent control of selected broadleaf weeds depending on application rate.
Saflufenacil provides control of selected broadleaf weeds that are resistant to other modes of action. Saflufenacil provides control of Group 2, 4, 5 and 9 resistant Canada fleabane and prickly lettuce (Liebl et al. 2008; Soltani et al. 2010; Trolove et al. 2011). Saflufenacil has activity on weed species that are resistant to glyphosate, ALS inhibitors, triazine and dicamba herbicides.
1.3.3 Canada Fleabane Control
Saflufenacil provides control of glyphosate resistant Canada fleabane before and after crop emergence. Davis et al. (2010) reported that saflufenacil applied early in the spring at 50 and 100 g ai ha-1 provided greater than 90% residual control for 8 and 12 weeks, respectively.
Davis et al. (2010) found that saflufenacil, applied late in the spring, reduced Canada fleabane densities by greater than 90%, whereas glyphosate + 2,4-D was less. Mellendorf et al. (2013) found that control of Canada fleabane increased when the rate of saflufenacil increased from 25 to 50 g ai ha-1, but there was no further increase in control with rates above 50 g ai ha-1.
Saflufenacil, applied PP at 25 and 50 g ai ha-1, provided greater than 90% control of emerged
Canada fleabane with equivalent residual control 7 and 30 days after planting (Owen et al. 2011).
Saflufenacil applied prior to crop emergence can provide control of glyphosate resistant Canada fleabane making it an effective tool in conservation tillage systems.
The height of Canada fleabane at the time of saflufenacil application has little impact on control. In a study by Mellendorf et al. (2013), the height of Canada fleabane at the time of application influenced the level of control with saflufenacil (25 g ai ha-1) in one of a two year
15 study. For every 8 cm increase in height, the control of Canada fleabane decreased by 1%; however, even at a height of 45 cm, control was 94% (Mellendorf et al. 2013). Saflufenacil applied at 50 g ai ha-1 or greater, provided at least 98% control of Canada fleabane regardless of plant height (Mellendorf et al. 2013). With glyphosate plus saflufenacil (25 g ai ha-1), there was no influence of Canada fleabane height on efficacy.
Variable control of GR Canada fleabane with saflufenacil has been observed. Saflufenacil
(25 g ai ha-1) applied alone provided 35, 32 and 20% control at 7, 14, and 28 DAA, respectively
(Ikley 2012). When glyphosate was added to saflufenacil, efficacy increased to 61, 67 and 57% at 7, 14 and 28 DAA, respectively (Ikley 2012). At 7 and 14 DAA, saflufenacil + glyphosate provided greater control than glyphosate alone at 61 vs 11% and 67 vs 35% respectively; at 28
DAA there was not a significant difference between saflufenacil + glyphosate and glyphosate alone at 57 and 37% control respectively (Ikley 2012). The decrease in control as time after application increased was due to extensive regrowth from the Canada fleabane rosettes since the application of saflufenacil broke apical dominance (Ikley 2012). Mellendorf et al. (2013) suggested that glyphosate resistant Canada fleabane may be controlled the best when saflufenacil plus glyphosate are applied when plants are relatively small, and by including a third herbicide with another mode of action in the tankmix. There is variability in the control of glyphosate resistant Canada fleabane with saflufenacil.
1.3.4 Factors Affecting Control
The efficacy of saflufenacil can be enhanced with the addition of some adjuvants.
Adjuvants can increase the efficacy of saflufenacil by increased penetration into the leaf by improved wetting and dispersion of spray droplets on the leaf cuticle (Frihauf et al. 2010).
Examples of spray adjuvants include: non-ionic surfactants (NIS), petroleum derived oils, or plant derived oils (Frihauf et al. 2010). Methylated seed oil (MSO) has been found to be the most
16 effective spray adjuvant, with crop oil concentrate intermediate and NIS as having the least effect in improving the efficacy of saflufenacil on Canada fleabane (Knezevic et al. 2010a;
Eubank et al. 2013). Increasing the concentration of MSO from 1 to 2% v/v improved the efficacy of saflufenacil (Eubank et al. 2013). The addition of ammonium sulfate (AMS) does not improve control of Canada fleabane (Eubank et al. 2013). The burndown of saflufenacil is improved by the addition of some spray adjuvants.
The properties of the carrier water can affect the efficacy of saflufenacil. Weak acid herbicides can be affected by cations in hard water solutions as seen with glyphosate and 2,4-D; however, the addition of AMS can reduce these effects on the efficacy of saflufenacil by dissociating into ammonium and hydrogen sulfate ions which bind to the cations (Roskamp et al.
2013). The pH of the water can also impact the efficacy of saflufenacil (Roskamp et al. 2013).
For example, the control of lambsquarters with saflufenacil with water pH of 4.0, 5.2, 7.7 and 9.0 was 15, 17, 71 and 58%, respectively (Roskamp et al. 2013). Similarly, control of giant ragweed with saflufenacil was 44, 47, 84 and 80% with a carrier water pH of 4.0, 5.2, 7.7 and 9.0, respectively (Roskamp et al. 2013). The solubility of saflufenacil decreases as water pH decreases from >5,000 mg L-1 at pH 9.0 to 10.1 mg L-1 at pH 4.0, contributing to the decrease in efficacy seen with acidic water (Roskamp et al. 2013). If the pH of the water cannot be changed it is suggested by Roskamp et al. (2013), to increase water volume to allow for more saflufenacil to dissolve. Applying saflufenacil using water with a low pH can negatively affect efficacy.
The addition of glyphosate to saflufenacil can significantly increase efficacy. The control of GR Canada fleabane is greater with the tankmix of glyphosate and saflufenacil than when either product is applied alone (Mellendorf et al. 2013; Ikley 2012; Eubank et al. 2013). Control of Canada fleabane with saflufenacil (25 g ai ha-1) can be increased equivalently with the addition of glyphosate or by increasing the rate of saflufenacil to 50 g ai ha-1 (Mellendorf et al.
17 2013). The addition of glyphosate can improve saflufenacil absorption (Waggoner 2010) and conversely saflufenacil can improve glyphosate absorption in GR Canada fleabane. Interestingly, there is no increase in absorption on glyphosate-susceptible Canada fleabane which suggests negative cross-resistance (Eubank et al. 2013). The addition of glyphosate to saflufenacil improves efficacy which provides broad-spectrum weed control.
1.3.5 Crop Safety
Saflufenacil has a range in crop tolerance when applied prior to crop-emergence. Crop- tolerance to saflufenacil is influenced by its physical placement and rate of metabolism. Tolerant crops include soybean, corn, cereals and some legumes (Liebl et al. 2008). However, some crops are less tolerant, with cotton and some legumes being more susceptible to damage from saflufenacil than corn or cereal crops (Owen et al. 2011; Soltani et al. 2010; Soltani et al. 2012;
Knezevic et al. 2010b; Grossman et al. 2011). In cotton, saflufenacil applied pre-emergence at 50 g ai ha-1 or greater resulted in injury and stand loss (Owen et al. 2011). In dry beans, saflufenacil applied pre-emergence at 100 g ai ha -1 or greater caused unacceptable injury and yield loss, while processing pea were tolerant at same rates (Soltani et al. 2010). The tolerance of soybean to saflufenacil varies by cultivar. It is hypothesized that tolerant cultivars can rapidly metabolize saflufenacil, whereas susceptible cultivars metabolize the herbicide more slowly (Ikley 2012). It is recommended to use cultivars known to be tolerant to saflufenacil to reduce the risk of injury and yield loss (Miller 2012). Cereal crops including wheat, spring barley and oat (Avena sativa
L.) are very tolerant to higher doses of saflufenacil when applied pre-emergence (Knezevic et al.
2010b; Soltani et al. 2012). Corn has intermediate tolerance, with rates of up to 125 g ai ha-1 showing minimal to no injury, perhaps due to low root translocation and rapid metabolism of the herbicide (Grossman et al. 2011, Trolove et al. 2011). Saflufenacil is very safe when applied pre-
18 emergence to cereal crops and corn, but rates must be adjusted when used for weed control in cotton or certain legume crops.
Winter wheat tolerance to post-emergence applications of saflufenacil is influenced by surfactant use and herbicide tankmixes. In one study, saflufenacil applied post-emergence to wheat alone or with NIS or COC resulted in unacceptable injury and yield loss (Knezevic et al.
2010b). However, saflufenacil absorption has been shown to increase when applied with 2,4-D amine (Frihauf et al. 2010). In contrast, the tankmix of bentazon and saflufenacil reduced injury to wheat (Frihauf et al. 2010). There is not an adequate margin of crop safety for saflufenacil applied postemergence (POST) in wheat, but further research may identify tankmixes which provide acceptable weed control and crop tolerance.
1.4 Glyphosate
1.4.1 Mode of Action
Glyphosate is a broad-spectrum, systemic herbicide with a unique mode of action. The primary target of glyphosate is 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS), an enzyme in the shikimate acid pathway (Franz et al. 1997). EPSPS is located primarily in the chloroplast, but there is also a cytoplasmic form (Baylis 2000). The shikimate pathway, and
EPSPS, are found in plants and microorganisms because most living organisms rely on their diet for the products of this process (Franz et al. 1997). There are several effects of glyphosate, and inhibition of EPSPS, on plant development resulting in plant death. Glyphosate causes insufficient production of aromatic amino acids preventing protein synthesis; and increases carbon flow to the shikimate pathway which results in deficient carbon for other essential pathways (Duke and Powles 2008). When glyphosate inhibits the EPSPS reaction, chorismate production halts (Franz et al. 1997). Chorismate is required to produce many aromatic plant metabolites including the essential aromatic amino acids (phenylalanine, tyrosine, tryptophan),
19 ubiquinone, tetrahydrofolate and vitamin K (Franz et al. 1997). Without primary plant metabolite production, secondary plant products such as anthocyanins, lignin, growth promoters, growth inhibitors, flavonoids and other phenolics are not produced (Dill 2005; Franz et al. 1997;
Steinrucken and Amrhein 1980). Either directly from blockage of the shikimate pathway or due to feedback mechanisms, many physiochemical and physiological processes are affected; such as reductions in photosynthesis, degradation of chlorophyll, inhibition of auxin transport and greater auxin oxidation (Baylis 2000). The inhibition of EPSPS by glyphosate results in broad- spectrum weed control.
Glyphosate inhibits EPSPS by binding to the enzyme. Normally the substrates shikimate-
3-phosphate (S3P) and phosphoenolpyruvate (PEP) bind to EPSPS, producing chorismate (Duke and Powles 2008). All glyphosate-susceptible plants have glyphosate-sensitive EPSPS (Franz et al. 1997). When glyphosate is applied it competes with PEP for binding to EPSPS, but is uncompetitive with S3P, resulting in a very stable glyphosate-EPSPS-S3P complex (Dill 2005).
Intracellular concentrations of PEP are generally unaffected by glyphosate, however, S3P accumulates following glyphosate application, resulting in an increase in the concentration of the
EPSPS-S3P binary complex (Franz et al. 1997). EPSPS cannot function once the ternary complex with glyphosate and S3P is formed.
1.4.2 Behaviour in Plants
Glyphosate is rapidly absorbed by plants. The rate of glyphosate absorption varies between plant species, partially accounting for some differences in glyphosate susceptibility
(Duke and Powles 2008). Diffusion across the plant cuticle is the most likely method of glyphosate absorption since it is a highly-polar molecule with a low lipophilic property (Franz et al. 1997; Duke and Powles 2008). Established orchard and other woody plants are safe as glyphosate cannot penetrate woody stems or bark (Carlisle and Trevors 1987). When glyphosate
20 is applied, a strong concentration gradient of glyphosate is formed across the plant cuticle causing an initial phase of rapid uptake, followed by a slower phase of subsequent uptake (Franz et al. 1997). The speed of glyphosate absorption is influenced by plant species, plant morphology and environmental conditions.
Glyphosate is a systemic herbicide. After absorption, glyphosate is translocated via the symplast and apoplast (Franz et al. 1997), with limited movement in the apoplast (Jachetta et al.
1986). The distribution pattern of photoassimilates, such as sucrose, influences glyphosate movement as it translocates to active metabolic sinks including meristems, growing leaves and roots, storage tissues and any other actively growing tissues. The absorption and translocation of glyphosate is influenced by adjuvants, the distribution of photoassimilates, plant growth stage and environmental conditions (Duke and Powles 2008; Franz et al. 1997). Translocation of glyphosate is influenced by itself in sugar beets (Beta vulgaris L.) (Geiger et al. 1999). Since glyphosate affects carbon translocation and the movement of glyphosate and carbon coincides, the rapid action of glyphosate in sugar beets actually influences its own distribution (Geiger et al.
1999). The distribution of glyphosate explains why injury symptoms first appear in immature leaves and growing points (Shaner et al. 2014). The efficacy of glyphosate is aided by its slow but thorough distribution to actively growing plant tissues.
Surfactants and carrier water volumes affect the phytotoxicity of glyphosate. In general, surfactants increase the absorption of glyphosate but do not enhance translocation (Franz et al.
1997). In general, glyphosate activity increases as carrier water volume decreases. Buhler and
Burnside (1983) reported increased phytotoxicity when the carrier water volume was decreased from 190-to 24-L ha-1 (Buhler and Burnside 1983). At carrier water volumes of 48 L ha-1 and greater, phytotoxicity can be improved by the addition of a surfactant (Buhler and Burnside
1983). Even in cases of environmental stress, glyphosate efficacy can be increased by adding a
21 surfactant (McWhorter and Azlin 1978). Surfactants and carrier water volume can influence glyphosate absorption which ultimately can influence glyphosate efficacy.
1.4.3 Behaviour in Soil
Glyphosate is rapidly adsorbed to soil colloids (Sprankle et al. 1975). The amount of glyphosate that binds to soil is greatly determined by the level of phosphate in the soil and the amount of cations present, but is not influenced by soil pH (Sprankle et al. 1975). Less glyphosate is adsorbed as the level of inorganic phosphate increases (Sprankle et al. 1975), and the amount of Fe3+ and Al3+ cations decrease (Franz et al. 1997). The binding of glyphosate to soil may be due to the phosphonate moiety of glyphosate competing for phosphate-ion binding sites (Franz et al. 1997). In lighter textured soils glyphosate may not be bound tightly due to less unoccupied phosphate binding sites (Franz et al. 1997). Crops can be seeded immediately after glyphosate application, however, in rare situations injury has been observed on light textured soils (Carlisle and Trevors 1987). Light textured soils such as Arenosols have a low immobilization potential for glyphosate due to a low clay content and decreased cation capacity
(Tesfamariam et al. 2009). Crop injury can also occur from the remobilization of glyphosate by competitive desorption by phosphorous fertilizer, particularly on soils with fewer phosphate binding sites such as Arenosols; this is unlikely for Luvisols due to more phosphate binding sites and a high calcium carbonate content that forms insoluble salts with glyphosate and calcium cations (Bott et al. 2011). The rapid binding of glyphosate to soil prevents PRE herbicidal activity.
The persistence of glyphosate in soil is determined by how strong it is adsorbed and the speed of degradation (Carlisle and Trevors 1987). The half-life of glyphosate can range from less than a week to several months (Franz et al. 1997), but on average is 47 days (Shaner et al. 2014).
Due to glyphosate binding tightly to soil, it has little mobility in the soil and is not volatile (Franz
22 et al. 1997). The soil mobility of glyphosate increases at high pH levels and when there are high levels of inorganic phosphate (Sprankle et al. 1975). Once tightly bound to soil, glyphosate is immobile but can persist for a period of time, but is non-phytotoxic.
Glyphosate degradation is affected by many different factors. The primary method of glyphosate degradation is metabolism by soil microflora (Franz et al. 1997). The rate of degradation is correlated with the level of microbial activity that is influenced by many soil factors, in both aerobic and anaerobic conditions (Franz et al. 1997). The initial rate of degradation is rapid compared to a slower rate that is observed later which is suspected to be from the metabolism of free glyphosate, followed by soil-bound glyphosate (Carlisle and Trevors
1987). Factors that decrease glyphosate adsorption such as increased phosphate and decreased
Fe3+ and Al3+levels, can increase glyphosate degradation (Franz et al. 1997). Soil bacteria break glyphosate into aminomethylphosphonic acid (AMPA) or sarcosine, and then eventually to inorganic phosphate, ammonia and carbon dioxide (Baylis 2000). The rate of degradation for glyphosate varies due to soil characteristics and factors that influence soil microbial activity.
1.4.4 Environmental Interactions
Glyphosate is rapidly inactivated in water. Soil colloids, bottom silt and soil particles in suspension bind to glyphosate in water causing inactivation (Franz et al. 1997). Not only is adsorption rapid in water but degradation is as well with the first-order half-life of glyphosate ranging from 1.5 to 3.5 days (Franz et al. 1997). Glyphosate can control aquatic weeds because of its rapid absorption and translocation to submerged roots (Carlisle and Trevors 1987).
Glyphosate is safe to use for weed control near water due to its rapid inactivation.
Glyphosate has low toxicity to insects and animals. The main reason for the safety of glyphosate to insects, birds, fish, mammals and man is that these organisms do not have the shikimate biosynthetic pathway (Franz et al. 1997). A common chemical such as sodium
23 -1 chloride has an acute oral LD50 for rats of 2.69 ± 0.12-g kg (Boyd et al. 1966), which is far
-1 more toxic then glyphosate which has an oral LD50 of greater than 5 g kg (Franz et al. 1997).
Glyphosate has not been found to be a carcinogen, reproductive toxin, or have any subacute chronic toxicity (Duke and Powles 2008). Glyphosate has been found to have little indirect effect on animal communities, although some bird species populations may show decreased densities due to reduced habitat from glyphosate use (Carlisle and Trevors 1987). Fish and invertebrates are more sensitive to glyphosate especially under increased water temperatures and a pH increase from 6.5 to 7.5 but not higher; however commercial field rates are not expected to have any negative effects on aquatic life (Carlise and Trevors 1987). The safety of glyphosate to non- target species is an important feature of this herbicide.
Surfactant use with glyphosate can influence the toxicological properties. A study with the original Roundup formulation showed that surfactants used with glyphosate may be the primary toxic agent in the herbicide, especially to fish and aquatic invertebrates (Folmar et al.
1979). Earlier life stages of fish are more susceptible to the surfactant and glyphosate mixture, therefore caution needs to be used when young fish are in nearby waters (Folmar et al. 1979).
Like glyphosate itself, glyphosate with surfactant becomes more toxic with increased temperature, with toxicity to rainbow trout doubling at 17° compared to 7°C; it is also more toxic to bluegills at 27° than at 17°C (Folmar et al. 1979). The toxicity of glyphosate plus surfactant to fish is influenced by pH. An increase in pH from 6.5 to 7.5, but not higher, resulted in increased fish toxicity (Folmar et al. 1979). The characteristics that allow for glyphosate inactivation in water aid in reducing toxicological effects to aquatic life (Folmar et al. 1979). A more recent
Roundup formulation contains a surfactant, polyoxyethylene amine, which is the primary toxic component to aquatic organisms (Tsui and Chu, 2004). This surfactant is very absorptive to water sediment and toxicity decreases as sediment organic carbon increases (Tsui and Chu,
24 2004). The newer Roundup formulation has little acute effects expected to occur in the environment (Tsui and Chu 2004). Regardless care should still be taken when applying glyphosate to prevent adverse effects to non-target organisms such as fish and aquatic invertebrates.
1.4.5 Glyphosate-Resistant Crops
The greatest factor that has contributed to the increased use of glyphosate has been the adoption of GR crops. In 1996, transgenic GR soybean and canola were introduced in the USA and Canada, respectively under the Roundup Ready brand (Duke and Powles 2008; Dill 2005).
In 2008, there were five GR crops grown on 74 million hectares in 13 countries (Dill et al. 2008).
Globally, the production of GR crops is expected to continue to increase. Of all transgenic crops grown globally, 90% are GR (Duke and Powles 2008). Of the genetically-modified corn hectares planted globally, GR corn accounts for 61%; of all soybean planted globally, 64% are GR (Dill et al. 2008). The rapid adoption of GR crops is attributed to crop safety, excellent broad- spectrum weed control, convenience and economics (Dill 2005). Due to the wide spread and repeated use of glyphosate, the future use of glyphosate is at risk due to the increasing presence of GR weeds both in terms of number of species resistant to glyphosate and the number of hectares affected (Duke and Powles 2008). The introduction of GR crops has revolutionized weed management.
Several techniques were attempted to incorporate glyphosate-resistance in crops. These techniques included: overproduction of EPSPS, introduction of an EPSPS with decreased affinity for glyphosate and the introduction of a glyphosate degradation gene (Padgette et al. 1996).
Although the overproduction of EPSPS did increase glyphosate resistance in plants, it was insufficient to commercial rates of glyphosate (Padgette et al. 1996). An altered EPSPS (CP4
EPSPS) with resistance to glyphosate was found in an Agrobacterium sp. (Duke and Powles
25 2008). When the CP4 EPSPS gene plus a promoter was inserted into the genome of alfalfa, corn, cotton and soybean, resistance to commercial glyphosate rates was achieved (Duke and Powles
2008). This is successful because of changes to the amino acid sequence outside the glyphosate/PEP binding region, and an increased affinity for PEP to CP4 EPSPS that allows the shikimate pathway to function normally after glyphosate application (Dill 2005). In some plant species such as soybean, glyphosate is degraded slowly to AMPA and glyoxylate by glyphosate oxidoreductase (GOX), so with CP4 EPSPS allows for normal shikimate pathway function, and the plant is still able to breakdown and remove glyphosate (Duke and Powles 2008). In canola, a gene from Ochrobactrum anthropic was used to encode GOX and was introduced, along with
CP4 EPSPS because GOX alone did not provide sufficient resistance (Duke and Powles 2008,
Dill 2005). In corn, a single multiple missense mutation to corn EPSPS generated by using site directed mutagenesis of a corn cell line, was the method used to create glyphosate-resistance
(Dill 2005). Glyphosate-resistance in crops was achieved by the insertion of an altered EPSPS enzyme and enhanced glyphosate metabolism by glyphosate oxidoreductase.
GR crop weed management systems have lower environmental risks than previous technology. The only known environmental risk of GR crops is transgene flow to wild or weedy related species (Duke and Powles 2008). There are three known cases including transgene flow in GR canola, transgenic creeping bentgrass (Agrostis stonlonifera L.) to non-transgenic bentgrass and GR alfalfa (Medicago sativa L.) to organic alfalfa (Duke and Powles 2008).
Introgression is seen as the largest potential risk of transgenic crops because the genes cannot be recalled, however hybrids between crop species and weedy relatives are generally unfit and the required backcrosses to cause full movement into the genome is unlikely (Duke and Powles
2008). Compared to previous weed management techniques, GR crops pose low environmental risks.
26 1.5 Glyphosate-Resistant Weeds
1.5.1 Background and Fitness Costs
Glyphosate resistance in weed species was once thought unlikely to occur. Resistance to glyphosate did not seem likely due to 25 years of use with no reports of resistance from 1972-
1997, little plant metabolism of the molecule and no knowledge of active transporters for glyphosate (Bradshaw et al. 1997). Since glyphosate has no residual activity in the soil, one application of glyphosate creates only a short, selection event to emerged plants (Powles 2008).
Non-emerged weeds remain unaffected by a glyphosate application, and along with tillage or other weed management options, the overall weed population remains largely unselected (Powles
2008). Since the adoption of glyphosate resistant (GR) crops, the use of glyphosate has increased greatly due to the flexibility in applying the herbicide either PP or POST (Nandula et al. 2005).
Concomitantly, there has been a decrease in the use of other weed management tactics (Powles
2008). As a result, the selection pressure for glyphosate resistance has increased and resistance has evolved from repeated use of glyphosate (Nandula et al. 2005; Powles et al. 1998). The extensive and repeated use of glyphosate has resulted in evolved weed resistance that originally was thought to be unlikely.
The inheritance of glyphosate resistance traits and dominance varies by species and resistance mechanism. The unselected frequency of the first mutations to confer glyphosate resistance in weed species is difficult to estimate since little is known about all mutation possibilities that can generate resistance (Nandula 2010). Single gene, nuclear traits were thought to be the only means of inheritance of glyphosate resistance (Powles and Preston 2006); however, some species such as rigid ryegrass (Lolium rigidum Gaud.) inherit a two-gene trait for resistance, conferring a target-site mutation mechanism along with a reduced translocation mechanism (Nandula 2010; Preston et al. 2009). Resistance traits can vary from incompletely
27 dominant to dominant in rigid ryegrass exhibiting reduced herbicide translocation of glyphosate
(Nandula 2010). However, incomplete dominance is common in biotypes with reduced herbicide translocation and altered target site resistance mechanisms (Nandula 2010), whereas Powles and
Preston (2006) found dominance to range from high to moderate for reduced translocation mechanisms in Lolium spp. Interestingly, among populations of rigid ryegrass, all with the same glyphosate resistance mechanism, there is variation in the degree of dominance (Preston and
Wakelin 2008). The knowledge of inheritance and dominance of glyphosate resistance traits will continue to change as more mechanisms of resistance and populations with more than one resistance mechanism are identified.
There may be fitness costs to weed species carrying herbicide resistant alleles. In populations without herbicide selection, the low frequency of herbicide resistant alleles is considered due to fitness costs from the allele (Nandula 2010; Preston and Wakelin 2008;
Preston et al. 2009). This has been reported by Preston et al. (2009), when the frequency of GR rigid ryegrass plants decreased over three years when no glyphosate selection pressure was present. However, the fitness cost may depend on the mechanism of resistance. In the rigid ryegrass population studied by Preston et al. (2009), which had reduced herbicide translocation, there was a significant fitness penalty associated with glyphosate resistance (Nandula et al.
2013). Vila-Aiub et al. (2014), observed fitness penalty in terms of height and biomass allocation to vegetative and reproductive parts in Palmer amaranth (Amaranthus palmeri S. Wats.) with target-site-amplification. Fitness costs appear to depend on the weed species and mechanism of glyphosate resistance.
1.5.2 Species
There are now many weed species that have evolved resistance to glyphosate. The first
GR weed species documented was rigid ryegrass, found in Australia in 1996 with a 7-11-fold
28 resistance factor (Powles et al. 1998). The first documented GR broadleaf weed was Canada fleabane in Delaware USA in 2000 with an 8-13-fold resistance factor (VanGessel 2001). In
2016, there were 32 GR weed species on six continents (Heap 2016). In 2016 in Canada, there were five GR weed species that include giant ragweed (Ambrosia trifida L.) [Ontario 2008],
Canada fleabane [Ontario 2010], common ragweed (Ambrosia artemisiifolia L.) [Ontario 2011], kochia (Kochia scoparia L.) [Alberta, Saskatchewan 2012], and waterhemp (Amaranthus tuberculatus (Moq.) J. D. Sauer) [Ontario 2014] (Heap 2016). The number of weed species with glyphosate resistance and the number of hectares affected will continue to increase globally.
Few plant species have natural tolerance to glyphosate. Natural tolerance to glyphosate is when plants are not controlled by glyphosate in the absence of selection pressure (Rojano-
Delgado et al. 2012). Some naturally tolerant plant biotypes/species include field bindweed
(Convolvus arvensis L.), birdsfoot trefoil (Lotus corniculatus L.), tropical spiderwort
(Commelina benghalensis L.) and Asiatic dayflower (Commelina communis L.) (Nandula et al.
2005). Tropical spiderwort and Asiatic dayflower have been reported as difficult to control with glyphosate since they have natural tolerance; however, these species were not a problem until the repeated use of glyphosate in GR crops; the buildup of these weed species is a process termed a weed shift (Nandula et al. 2005). The velvet bean (Mucuna pruriens L. DC.) has reduced uptake and translocation of glyphosate along with two glyphosate detoxifying pathways, which have not been reported in other plant species (Rojano-Delgado et al. 2012). Between both detoxifying pathways, glyphosate is broken down in velvet bean to aminomethylphosphonic acid (AMPA), glyoxylate, sarcosine and formaldehyde (Rojano-Delgado et al. 2012). Natural tolerance to glyphosate, a very efficacious, broad-spectrum herbicide, has been found in some plant species.
29 1.5.3 Mechanisms of Resistance
There are more known mechanisms of resistance to glyphosate than for any other herbicide (Sammons and Gaines 2014). These mechanisms can be placed in one of two classes: non-target site or target site based resistance (Nandula 2010; Powles and Preston 2006). Non- target site involves mechanisms that decrease the amount of active herbicide that reaches the target site (Yuan et al. 2006). Target-site-based resistance occurs when the herbicide reaches the target site at a lethal dose, but there are changes at the target site that limit the impact of the herbicide (Yuan et al. 2006). Three mechanisms are currently known within the non-target based resistance class: i) reduced absorption and translocation, ii) herbicide sequestration and iii) enhanced metabolism (González-Torralva et al. 2012; Nandula 2010). Within the target based resistance class there are two mechanisms: i) target-site mutation and ii) overexpression of the target-site (González-Torralva et al. 2012; Nandula 2010). Interestingly, the target-site based and metabolism mechanisms are less common for GR weeds than for acetolactate synthase (ALS) or acetyl-CoA carboxylase (ACCase) herbicide-resistant weeds (Sammons and Gaines 2014). As knowledge of glyphosate resistant mechanisms continues, it is expected that more mechanisms may be found.
Greater resistance to glyphosate is likely in weed species that have more than one resistance mechanism (Preston et al. 2009; Nandula et al. 2013). Under selection, it is more favourable to have multiple resistance mechanisms and so it is common to see combinations in weed populations and individuals, especially in cross-pollinated species (Sammons and Gaines
2014). A single weed population may have individuals coexisting with different resistance mechanisms (Alarcón-Reverte et al. 2014). Weed species that involve combinations of resistance mechanisms include rigid ryegrass with target-site mutations and reduced translocation, and
Italian ryegrass (Lolium multiflorum Lam.) with reduced absorption and translocation of
30 glyphosate (Preston et al. 2009). In some cases, target-site mutations or gene duplication were found, but did not explain the high levels of resistance, perhaps indicating unknown mechanisms
(Bell et al. 2013; Collavo and Sattin 2012). As selection of glyphosate continues, more weed species may be selected with multiple resistance mechanisms.
Cold air temperatures can have an impact the efficacy of glyphosate on GR weeds.
Populations of Johnsongrass (Sorghum halepense L. Pers.) and Lolium spp. with reduced translocation mechanisms have been made sensitive to glyphosate at suboptimal temperatures
(Vila-Aiub et al. 2013). GR Canada fleabane growing at 8°C was sensitive to glyphosate because the herbicide sequestration mechanism was inhibited (Ge et al. 2011). Membrane dependent resistance mechanisms may be inhibited by the cold; however, plants with multiple mechanisms can remain resistant to glyphosate (Sammons and Gaines 2014). GR weeds can be sensitive to glyphosate under suboptimal conditions if they have a membrane dependant resistance mechanism.
1.5.4 Non-Target Based Resistance
1.5.4.1 Reduced Absorption and Translocation
Reduced absorption acts as a first step in glyphosate resistance. In GR sourgrass
(Digitaria insularis L. Mez ex Ekman), De Carvalho et al. (2012) found at least 12% less glyphosate was absorbed 48 hours after treatment. GR Italian ryegrass had reduced herbicide interception and retention due to a differing leaf angle then susceptible species (Michitte et al.
2007). In association with other mechanisms of resistance, reduced absorption can confer resistance to glyphosate.
Since glyphosate needs to be translocated to meristematic tissue for optimal herbicidal activity, reduced translocation can result in glyphosate resistance. In a rigid ryegrass population, glyphosate is accumulated in the tips of leaves rather than shoot and root meristems, preventing
31 inhibition of the shikimate biosynthetic pathway (Preston et al. 2009). This is very visual in GR giant ragweed populations, where the glyphosate accumulates in older leaf tissue that senesces and falls off, and has been termed, rapid necrosis response (Robertson 2010). Reduced translocation confers resistance to glyphosate in some weed species.
1.5.4.2 Herbicide Sequestration
Sequestration of glyphosate prevents movement to root and shoot meristems. This mechanism has been found in GR Canada fleabane where glyphosate was sequestered in vacuoles (Ge et al. 2010). Net uptake of glyphosate was similar between susceptible and resistant
Canada fleabane plants; however, glyphosate was rapidly sequestered into vacuoles and about one-half of glyphosate was translocated from the source to sink leaves in resistant plants compared to susceptible ones (Ge et al. 2010). Glyphosate sequestration occurs throughout the plant including the meristematic tissues, further reducing the amount available to inhibit the shikimate pathway (Ge et al. 2010). Glyphosate entering the vacuole by crossing the tonoplast appears to be unidirectional in all species studied since 2013 (Ge et al. 2013), and a tonoplast transporter may be responsible (Ge et al. 2010). The transporter may be specific to resistant biotypes or present in higher quantities than in the susceptible biotypes (Ge et al. 2010).
Glyphosate sequestered in the vacuole appears to remain there indefinitely as long as the tissue is intact (Ge et al. 2010). Glyphosate sequestration is a highly effective resistance mechanism, possibly aided by an active transporter.
1.5.4.3 Enhanced Metabolism
Metabolism of glyphosate has only been found in a few GR plant species. Two glyphosate degradation pathways exist in plants: i) degradation by glyphosate dehydrogenase, to
AMPA and glyoxylate, and ii) degradation by C-P lyase to sarcosine (De Carvalho et al. 2012).
32 In GR sourgrass, greater than 90% of glyphosate was degraded by resistant plants compared to
11% in the susceptible at 168 hours after treatment (De Carvalho et al. 2012). A GR Canada fleabane biotype from Spain showed rapid glyphosate metabolism compared to a susceptible biotype (González-Torralva et al. 2012). Rapid metabolism of glyphosate decreases the amount that reaches the chloroplast where it inhibits EPSPS in the shikimate biosynthetic pathway.
1.5.5 Target Based Resistance
1.5.5.1 Overexpression of the Target Site
Target site overexpression is another way of avoiding the toxic effects of herbicides in plants. Target site overexpression begins with replication of a DNA segment that results in more than one gene copy within the genome (Innan and Kondrashov 2010). Gene duplication is possible by unequal crossing-over events, chromosome duplication, or transposable element events (Zhang 2003). The duplication of the gene itself does not provide resistance until EPSPS mRNA and protein expression is increased (Sammons and Gaines 2014). The first case of a weed species exhibiting an overexpression resistance mechanism was a population of GR Palmer amaranth in Georgia, USA (Culpepper et al. 2006). In several cases, the level of glyphosate resistance was found to be positively correlated with higher EPSPS genomic copy number
(Gaines et al. 2011; Ribeiro et al. 2014; Salas et al. 2012). Such is the case for Palmer amaranth, where a higher EPSPS copy number (53-fold) resulted in greater glyphosate-resistance than plants with a lower (21-fold) copy number (Vila-Aiub et al. 2014). Similarly, GR kochia (Kochia scoparia L.) uses many EPSPS copies (3.1-8.5-fold) to provide resistance (Wiersma et al. 2014).
Glyphosate is essentially unable to inhibit all of the EPSPS enzymes when they are present at high frequencies, making this an effective mechanism of resistance.
1.5.5.2 Target-Site Mutation
33 A change in the target enzyme for a herbicide can reduce binding affinity or change the shape of the active site, thereby reducing molecule binding ability. The first case of a target-site mutation conferring glyphosate resistance in a plant species was in a goosegrass (Eleusine indica
L. Gaertn.) population from Malaysia (Nandula et al. 2005). This mutation was due to an amino acid substitution at position 106 of the EPSPS protein sequence of proline to serine (Pro106)
(Nandula et al. 2005). Interestingly, Pro106 is not directly involved with the interactions between glyphosate or PEP, but it causes a change in the structure of the active site, thereby reducing space for glyphosate binding (Healy-Fried et al. 2007). The first case of this resistance mechanism in a broadleaf weed species was reported in a Mississippi population of common waterhemp, also with a Pro106Ser mutation (Nandula et al. 2013). Common waterhemp is a dioecious weed that allows for a higher level of polymorphism than monoecious amaranthus species (Costea et al. 2005). Currently, six weed species have been reported to have EPSPS target site mutations, commonly among Lolium spp. and have included changes of Pro106 to alanine, leucine, serine or threonine (Sammons and Gaines 2014). Interestingly, a threonine to isoleucine substitution at position 102 of the EPSPS sequence has been reported in a goosegrass population along with the Pro106Ser mutation, the first case of a double mutation in an evolved weed species (Yu et al. 2015). The level of glyphosate-resistance from a single-target-site- resistance mechanism is generally modest (2-15-fold) (Preston et al. 2009), however when two mutations are present, the second can increase the overall level of resistance (Yu et al. 2015). By preventing glyphosate from effectively binding to EPSPS, the herbicidal effects can be reduced.
1.6 Glyphosate-Resistant Canada fleabane
1.6.1 Inheritance and Hybridization
Different models have been proposed to classify the inheritance of glyphosate resistance in Canada fleabane. In studies by Zelaya et al. (2004), glyphosate resistance was found to be
34 governed by a single-locus gene that is incompletely dominant. This was confirmed using a cross of resistant and susceptible biotypes of parent plants and observing an intermediate resistance response from glyphosate application on the heterozygous F1 hybrid (Zelaya et al. 2004). In another study, glyphosate resistance in Canada fleabane was conferred by a single, dominant nuclear gene (Feng et al. 2004). The mechanism of resistance to glyphosate in the study by Feng et al. (2004) was reduced glyphosate translocation. Due to evidence for different resistance inheritance models, this may support having multiple mechanisms of glyphosate resistance among Canada fleabane biotypes (Zelaya et al. 2004).
It is probable the glyphosate resistance in Canada fleabane has developed from many founding populations. If biotypes of GR Canada fleabane were from the same ancestry, then it would be expected that there would be only one mechanism of resistance (Yuan et al. 2010).
This would simplify the management of GR Canada fleabane, because strategies could be more effective and specific to control sources of seed or pollen (Yuan et al. 2010). However, even though independent evolution has occurred in many locals, glyphosate resistance may still be similar due to only a few mechanisms of resistance (Yuan et al. 2010). The independent selection of resistant biotypes increases the complexity in control, compared to if they originated from one source with the same mechanism of resistance.
GR Canada fleabane can cross with other Conyza species forming new hybrids (Zelaya et al. 2007). Canada fleabane was successfully crossed with Conyza ramosissima Cronq. (Dwarf horseweed) to form a Conyza hybrid with the two parents being GR and glyphosate susceptible
(GS) respectively (Zelaya et al. 2007). The success of the hybridization is due to both parents being diploid (2n=18) allowing for successful chromosome pairing during meiosis (Zelaya et al.
2007). Phenotypically the hybrid was intermediate between the parents with a larger rosette than the susceptible dwarf horseweed with more and denser leaves than the GR Canada fleabane
35 (Zelaya et al. 2007). Interestingly, the level of resistance to glyphosate was not intermediate of the parents but followed a hybrid resistance model that predicts greater resistance in hybrid populations compared to their parents (Zelaya et al. 2007). This interspecific hybrid has not been found in the field, howeve,r successful occurrence is possible but unlikely due to the highly autogamous ability of Conyza (Zelaya et al. 2007). A vigorous and GR Conyza hybrid would be well adapted to survive and multiply in current high-glyphosate use crop production systems.
1.6.2 Mechanisms of Resistance
1.6.2.1 Target Site-Based Resistance
Currently there are few biotypes of Canada fleabane where resistance to glyphosate is due to target site based resistance mechanisms. At two days after glyphosate application, shikimate accumulated in both GR and GS Canada fleabane biotypes (Mueller et al. 2003).
Shikimate levels were found to decrease in the resistant population and increase in the susceptible, from two-to-four days after treatment; however, there were no significant differences between the two biotypes at each timing (Mueller et al. 2003). Since there was not significantly less shikimate accumulated in the GR compared to the GS biotype, Mueller et al.
(2003) concluded that resistance in this biotype was not solely due to an insensitive EPSPS.
Shikimate accumulation was also noted in a GR Canada fleabane biotype which indicated that a glyphosate-tolerant EPSPS could not be present (Dinelli et al. 2006). However, at four-to-eight
DAA, shikimate concentration decreased and the plants survived which Dinelli et al. (2006) suggested was due to the ability of the GR biotypes to metabolize the shikimate, possibly from an overexpression of glyphosate-sensitive EPSPS. In the GR biotype EPSPS mRNA levels before glyphosate application were two-to-three fold higher, although the synthesis of EPSPS transcripts was not induced in response to glyphosate application (Dinelli et al. 2006). When the
EPSPS gene was sequenced from a GR biotype, it did not reveal a point mutation at codon 106,
36 suggesting that the mechanism of resistance for this biotype was not a target-site mutation (Tani et al. 2015). The GR biotype studied by Tani et al. (2015) involved a synchronization of overexpression of EPSPS and ABC-transporter genes which will be covered more in more detail later. The EPSPS gene was overexpressed positively as glyphosate rate was increased and the response was within one day after treatment; this overexpression had a prolonged effect compared to the expressed transporter genes (Tani et al. 2015). Target-site based glyphosate resistance is common in other weed species, and this mechanism also confers resistance in some
Canada fleabane biotypes.
1.6.2.2 Reduced Absorption and Translocation
Reduced translocation confers resistance to glyphosate in some Canada fleabane biotypes while there are no documented cases of reduced absorption. In several cases, absorption of glyphosate between the GR and GS Canada fleabane biotypes was similar and found unlikely to provide resistance to glyphosate (Feng et al. 2004; González-Torralva et al. 2012). Studies examining reduced glyphosate translocation have found decreased leaf to root translocation, indicating reduced phloem transport (Dinelli et al. 2006; Feng et al. 2004; Nandula et al. 2005;
Powles and Preston 2006). Glyphosate translocation out of a treated leaf has been found to range from 28- 48% less in the GR compared to the GS biotypes from different states (Feng et al. 2004,
Nandula et al. 2005). Not only does reduced glyphosate translocation to the root occur, but increased translocation of glyphosate in the xylem has been reported in a GR compared to a GS biotype (Dinelli et al. 2006). The reduced translocation method does not completely prevent glyphosate from reaching the meristematic tissues, indicating another mechanism of resistance may be active as well (Dinelli et al. 2006). In combination with other resistance mechanisms, reduced translocation contributes to glyphosate resistance in Canada fleabane.
1.6.2.3 Enhanced Metabolism and Herbicide Sequestration
37 Enhanced metabolism of glyphosate has been rarely found in GR Canada fleabane. In studies conducted by Dinelli et al. (2006) and Feng et al. (2004) they reported no difference in glyphosate metabolism in GR and GS Canada fleabane biotypes. In contrast, a GR Canada fleabane biotype in Spain has been reported to express enhanced glyphosate metabolism which along with vacuole sequestration confers resistance to glyphosate (González-Torralva et al.
2012). To date, only one case of enhanced metabolism in GR Canada fleabane has been reported.
Vacuolar sequestration of glyphosate confers resistance to glyphosate in many GR
Canada fleabane biotypes. Essentially, the sequestration of glyphosate into cell vacuoles removes it from the cytoplasm at a rate faster than cellular uptake can occur, thereby creating a concentration limit and reducing the amount that can translocate via the phloem (Ge et al. 2014).
Several studies have concluded that the mechanism of resistance in some GR Canada fleabane biotypes is due to vacuolar sequestration (Dinelli et al. 2006; Ge et al. 2014; González-Torralva et al. 2012; Tani et al. 2015). The difference in glyphosate sequestration rates between GR and
GS biotypes can differ by ten times (Ge et al. 2013). Interestingly, temperature has been found to affect vacuole sequestration of glyphosate in Canada fleabane (Ge et al. 2011). When glyphosate was applied at 8 °C, vacuole sequestration of glyphosate was inhibited and the resistant biotypes were susceptible to that treatment, suggesting temperature can affect the process of vacuole sequestration (Ge et al. 2011). More work needs to be done to investigate the process of vacuole sequestration as a mechanism of glyphosate-resistance in Canada fleabane.
The sequestration of glyphosate into cell vacuoles as a mechanism of resistance in
Canada fleabane appears to be due to non-passive transport. As stated previously, translocation of glyphosate in GR Canada fleabane biotypes is predominantly in the xylem, not the phloem, suggesting herbicide sequestration occurs in the apoplast environment (Dinelli et al. 2006). The
38 transport of ionic compounds such as glyphosate in the phloem is driven by the ion trap effect which occurs due to pH differences between the apoplast and the symplast (Dinelli et al. 2006).
Alteration of the membrane ionic pumps in resistant biotypes could acidify the apoplast and inhibit transport of glyphosate across the plasmalemma, while simultaneously reducing phloem translocation and increasing apoplast transport (Dinelli et al. 2006). The transport of glyphosate cannot be due to the tonoplast phosphate portal since the plant cells are able to maintain normal pH levels, after being adjusted with ammonia additions, and after the pH adjustment, glyphosate sequestration continues, not supporting acidic or ionic trapping of glyphosate as a method of sequestration (Ge et al. 2014). It appears that glyphosate and alternative substrates aminomethylphosphonate (AMPA) and N-methyl glyphosate (NMG), compete for sequestration into the vacuole; this coupled with no observed efflux of glyphosate from the vacuole tissues while intact, provides further evidence that glyphosate vacuolar sequestration is transporter- mediated and not non-passive (Ge et al. 2014). Glyphosate is sequestered into vacuoles through active transport.
The sequestration of glyphosate into vacuoles in resistant Canada fleabane biotypes may be dependent on adenosine triphosphate (ATP). As ATP levels are decreased, the amount of vacuolar sequestration of glyphosate decreases (Ge et al. 2014). In a study using vandate, a compound with very high pH (pH>13), glyphosate sequestration was reduced as vandate interfered with the dephosphorylation of ATP, inhibiting a transmembrane pump dependent on
ATP (Ge et al. 2014). Where temperature was found to impact the resistance of Canada fleabane to glyphosate, the hypothesis was that low temperatures (8°C) reduce the activity of the transmembrane pump resulting in reduced glyphosate transport (Ge et al. 2011). The function of transmembrane pumps for sequestering glyphosate appears to be dependent on ATP.
39 The transmembrane pumps responsible for vacuolar sequestration of glyphosate in
Canada fleabane is believed to be regulated by ABC transporter genes. ABC transporter genes code for active transport proteins that are dependent on ATP (Yuan et al. 2010). It was observed by Yuan et al. (2010) that ABC transporter genes were highly up-regulated after glyphosate application to resistant Canada fleabane. An induction period from glyphosate application to vacuole sequestration may be involved, as the cytosolic content of glyphosate was similar for six hours after application in both GS and GR biotypes of Canada fleabane (Ge et al. 2014). The
ABC transporter genes involved in glyphosate sequestration are M10, M11, M7 and P3 (Tani et al. 2015). Shortly after glyphosate application (1 day), all of the ABC transporter genes and the gene for EPSPS were overexpressed in a resistant compared to a susceptible biotype at a high and low rate, with the exception of P3; the overexpression of P3 was correlated positively with glyphosate rate (Tani et al. 2015). The length of overexpression differed between genes, with M7 and P3 only overexpressed shortly after application, while EPSPS and M10 had prolonged expression; this difference implies that each gene may have a different contribution to the resistance mechanism, which requires further investigation (Tani et al. 2015). The overexpression of ABC transporter genes appears to depend on the rate of glyphosate applied to resistant Canada fleabane biotypes and each have their own role in this resistance mechanism.
1.6.3 Spread of Resistance and Fitness Costs
There has been rapid spread of GR Canada fleabane in Ontario. The first case of GR
Canada fleabane was confirmed in Delaware USA in 2001 (VanGessel 2001). GR Canada fleabane was first confirmed in Canada 2010 at eight sites in Essex county (Byker et al. 2013c).
By 2012, 155 sites were confirmed with GR Canada fleabane in Canada, all within the Ontario counties of Elgin, Essex, Haldimand, Huron, Kent, Lambton, Middlesex and Niagara (Byker et
40 al. 2013c). Not only are there biotypes that are resistant to glyphosate, but there are multiple resistant biotypes, to both glyphosate and chloransulam-methyl. Multiple resistant Canada fleabane has been confirmed in Elgin, Essex, Kent, Lambton and Middlesex counties (Byker et al. 2013c). GR Canada fleabane has spread rapidly across Ontario.
Agronomic and herbicide application practices can impact the spread of GR Canada fleabane. In a study by Davis et al. (2009) management systems that involve crop rotation, cover crops and the use of residual pre-plant followed by non-glyphosate POST herbicides the ratio of resistant: susceptible biotypes was shifted from 3:1 to 1:6 after four years. Sometimes herbicides with different modes of action are rotated each year to prevent the spread of glyphosate resistance, however, this only reduces exposure of weeds to each compound individually and depletion of resistance will only occur if there is a high fitness cost for resistant biotypes relative to the rotation interval (Evans et al. 2015). A practice commonly suggested to reduce GR weeds is to employ mixing of herbicides with different modes of action (Evans et al. 2015). The mixing of herbicides with different modes of action depletes resistance alleles by decreasing the likelihood of weed survival but only works if each mixed component individually suppresses the weed (Evans et al. 2015). The potential for selection of multiple resistance to multiple herbicides with different modes of action increases as herbicide mixing occurs and the cost of mixing can be more expensive for farmers making them reluctant to employ this strategy (Evans et al. 2015).
Long-term strategies to reduce the spread of GR Canada fleabane and other weed species will require diversified crop production and weed management strategies.
The morphology of GR Canada fleabane aids in the dispersion of resistance. The ability of Canada fleabane to establish in undisturbed soils in both crop and non-crop land provides it with a large amount of area for establishment especially with the popularity of no-tillage
41 practices (Nandula et al. 2005). The self-compatibility of Canada fleabane and its ability to produce over 200,000 wind-borne seeds per plant allows it to easily spread to nearby areas
(Weaver 2001, Zelaya et al. 2004). The sole involvement of wind-dispersal of seeds for spreading resistance is unlikely given the distance between new resistant populations within a short period of time (Dinelli et al. 2006). The production of a large number of small, air-borne seed aids in the dispersal of GR Canada fleabane.
A fitness penalty for GR Canada fleabane would impact the density and spread of resistant biotypes. However, no fitness penalty has been observed in GR compared to GS biotypes (Zelaya et al. 2004). Accelerated growth has been reported in GR compared to GS
Canada fleabane biotypes, such as earlier stem elongation and flowering, seed formation, and overall plant height was greater (Shrestha et al. 2010). Fewer growing degree days were required for GR Canada fleabane biotypes to reach development stages compared to GS (Shrestha et al.
2010). Due to the more rapid growth of the GR biotypes, it has been suggested that herbicide applications be made early to target the small rosette stage of Canada fleabane when it is more susceptible (González-Torralva et al. 2012; Shrestha et al. 2010). There is no fitness penalty between GR and GS Canada fleabane biotypes so that in the absence of glyphosate both biotypes would continue to persist.
1.6.4 Control of Resistant Biotypes
Herbicides, applied PP in soybean, can provide effective control of GR Canada fleabane.
In an area where Canada fleabane grows predominantly as a summer annual, spring-applied PP herbicides provide greater and more consistent control than if applied in the previous fall (Davis et al. 2007; Davis et al. 2009). The use of a herbicide mixture that provides POST control of emerged Canada fleabane plus residual control of Canada fleabane is important because
42 additional plants emerge after non-residual herbicide treatments (Davis et al. 2007). Control eight weeks after application of 85-95% has been reported using 2,4-D ester (1120 g a.i ha-1) + glyphosate and 73-95% control with 560 g a.i ha-1 2,4-D (Byker et al. 2013b).
Saflufenacil/dimethenamid-p (490 g a.i ha-1) applied PP provided 98-100% control of GR
Canada fleabane at five of six sites, but poor control of only 49% control was reported at one site at eight weeks after application (Ford et al. 2014b). The most consistent PP herbicide found by
Ford et al. (2014b) was s-metolachlor + flumetsulam + clopyralid, providing 95-99% control eight weeks after application possibly due to the combination of the two residual herbicides.
Where variable control with PP herbicides was reported, it has been suggested that fall- germinated Canada fleabane which is bigger at the time of application then spring germinated plants, are tougher to kill and are responsible for this variation (Buhler and Owen 1997; Ford et al. 2014b). Using PP herbicides to control GR Canada fleabane is important since current POST soybean herbicides are not effective (Davis et al. 2009).
New herbicide resistant traits in field crops will increase options for controlling GR
Canada fleabane. In 2,4-D resistant crops (Enlist) a single versus sequential application of 2,4-D choline/glyphosate DMA applied PP provided 86 and 99% control, respectively (Ford et al.
2014a). The efficacy of 2,4-D choline/glyphosate DMA was not influenced by GR Canada fleabane size (10 to 30 cm in height) at the time of application (Ford et al. 2014c). The use of an effective residual herbicide applied PP followed by 2,4-D choline/glyphosate DMA POST provided excellent full season control of GR Canada fleabane and incorporates the use of multiple herbicide modes of action (Ford et al. 2014b). In dicamba resistant crops (Roundup
Ready Xtend soybean) a single POST application does not provide acceptable control (Byker et al. 2013b). A PP application of glyphosate + dicamba can provide excellent control (Byker et al.
43 2013b). The use of new herbicide resistant traits will allow for effective POST herbicides for the control of GR Canada fleabane escapes (Byker et al. 2013b).
Crop rotation can be an effective agronomic practice to reduce GR Canada fleabane persistence. Even a simple rotation of soybean-corn compared to a continuous soybean rotation can decrease the density of GR Canada fleabane by two-fold in four years (Davis et al. 2009). By using crop-rotation, different herbicides can be applied for weed control, use of cover crops can be employed and the crops grown can have differences in their competition with GR Canada fleabane, all influencing the overall effect on Canada fleabane density (Davis et al. 2009).
Tillage is also a very effective control option however this is not desired for growers who use no- till systems for reduced soil erosion potential (Brown and Whitwell 1988; Davis et al. 2009).
Agronomic practices other than herbicide use can contribute to the control of GR Canada fleabane.
GR Canada fleabane is a very competitive weed that can dramatically impact soybean yields. In GR crop systems a GR weed can significantly reduce crop yields (Steckel and
Gwathmey 2009). If a PP herbicide is not used and a POST application of dicamba is applied in dicamba-resistant soybean, the yield loss can be up to 31% due to GR Canada fleabane interference (Byker et al. 2013b). If glyphosate alone is applied to GR Canada fleabane, soybean yield can be reduced from 35 to 42% (Byker et al. 2013b). GR Canada fleabane interference can reduce soybean yields up to 93% when no weed management tactics are employed (Byker et al.
2013b). Clearly, GR Canada fleabane should be controlled early in the season to minimize soybean yield losses (VanGessel et al. 2009).
44 1.7 Hypothesis and Objectives
GR Canada fleabane has been confirmed in eight counties in Ontario from 2010 to 2012.
Due to the limited effectiveness of POST herbicides on GR Canada fleabane in soybean, the PP herbicide saflufenacil is an important herbicide for the control of GR Canada fleabane, to minimize soybean yield losses and to prevent spread of resistance. Variable control of GR
Canada fleabane with saflufenacil in no-till soybean has been reported. Research is needed to document distribution of GR Canada fleabane in Ontario and to identify factors that influence the control of GR Canada fleabane with saflufenacil in no-till soybean.
Our hypotheses include the following: 1. GR Canada fleabane will be found in additional counties in Ontario, and 2. GR Canada fleabane can be controlled in soybean with saflufenacil by optimizing the application rate, timing and tankmix partner. The objectives of this research are: 1. To document the distribution of GR Canada fleabane in Ontario through surveys in 2014 and 2015. 2. To determine the biologically effective rate of saflufenacil, saflufenacil tankmixed with glyphosate, and saflufenacil tankmixed with glyphosate and metribuzin for the control of GR Canada fleabane. 3. To determine the level of GR Canada fleabane control with saflufenacil tankmixes applied PP. 4. To determine the effect of time of day at application on the control of GR Canada fleabane. 5. To ascertain the effect of GR Canada fleabane height and density on saflufenacil efficacy.
45 Chapter 2: Distribution of glyphosate and cloransulam-methyl resistant Canada fleabane (Conyza Canadensis L. Cronq.) in Ontario
2.1 Abstract
Glyphosate-resistant (GR) Canada fleabane was first documented in Essex County
Ontario, Canada in 2010. An initial survey was conducted in 2011-2012 to identify the occurrence of GR Canada fleabane in southern Ontario. From 2013-2015 an expanded survey was conducted to record the distribution of GR and multiple-resistant Canada fleabane with resistance to glyphosate and cloransulam-methyl across Ontario. Seed was collected from fields with Canada fleabane escapes and plants were grown in a greenhouse for resistance screening.
Glyphosate (900 g a.i. ha-1) or cloransulam-methyl (17.5 g a.i. ha-1) was applied to Canada fleabane plants at the 10 cm rosette stage. 27, 47, and 82 additional sites were found with GR
Canada fleabane in 2013, 2014 and 2015 respectively; of those 4, 9, and 32 were found to be multiple-resistant. By 2015, 22 additional counties were identified with GR Canada fleabane since 2012 for a total of 30 counties in Ontario. This expanded survey shows that GR Canada fleabane is throughout Ontario and will be useful for Ontario farmers when planning their weed management program.
46 2.2 Introduction
Canada fleabane is a member of the Asteraceae family (Shrestha et al. 2008), and can germinate in the fall or spring (Weaver 2001). The cotyledons of Canada fleabane are small (2-
3.5-mm long and 1-2-mm wide), ovate shaped and hairless (Royer and Dickenson 1999). Fall- germinated Canada fleabane plants form a basal rosette (Frankton and Mulligan 1987), while spring-germinated plants do not (Bhowmik and Bekech 1993). With fall-germinated Canada fleabane the basal rosette deteriorates in the spring as the stem elongates (Frankton and Mulligan
1987). The first leaves of Canada fleabane are spatula-shaped and have many hairs on the upper leaf surface; mature leaves are also hairy but have an oblong to lance shape (Royer and
Dickenson 1999), with no petioles and have an alternate arrangement on the stem (Loux et al.
2006). Lower leaves can have slightly toothed margins (Loux et al. 2006), while the upper leaves are relatively smaller and have smooth margins (Royer and Dickenson 1999). The stem of
Canada fleabane is unbranched at the base, covered in short bristly hairs (Loux et al. 2006), and can grow up to 180-cm in height (Frankton and Mulligan 1987). Towards the top of the stem, several short leafless branches form that contain small flower heads (3-to-5-mm in diameter)
(Frankton and Mulligan 1987). The flowers consist of 20 to 40 yellow, perfect disc florets (Loux et al. 2006), that are primarily self-pollinated (Smisek 1995) and self-compatible (Weaver 2001).
GR Canada fleabane was first reported in Delaware USA in 2001 (VanGessel 2001). In
Canada, it was first reported in Essex county Ontario in 2010 (Byker et al. 2013c). Two years later, GR Canada fleabane was reported in Elgin, Essex, Haldimand, Huron, Kent, Lambton,
Middlesex and Niagara counties (Byker et al. 2013c). Multiple-resistant biotypes of Canada fleabane have been reported in Elgin, Essex, Kent, Lambton and Middlesex counties, with biotypes that are resistant to glyphosate and cloransulam-methyl (Byker et al. 2013c).
47 Tillage can be an effective option for control of GR Canada fleabane (Brown and
Whitwell 1988; Kapusta 1979), however plants must be small to ensure control (Shrestha et al.
2008). For no-tillage crop production systems, herbicides are used to control GR Canada fleabane (Bruce and Kells 1990). Control of GR Canada fleabane with herbicides must be PP in soybean since POST herbicides are not effective (Davis et al. 2009), or have limited control
(Loux et al. 2006). It is desirable to use a PP herbicide with residual activity on GR Canada fleabane in soybean (Loux et al. 2006), as Canada fleabane can later emerge after non-residual herbicide applications (Buhler and Owen 1997). The most consistent control of multiple-resistant
Canada fleabane in glyphosate-resistant soybean in Ontario is with a combination of glyphosate
(900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1), with the addition of metribuzin (400 g a.i. ha-1) or 2,4-D (500 g a.i. ha-1) that provide 96 and 95% control 8 weeks after application (WAA) respectively (Budd et al. 2016).
Interference from GR Canada fleabane can reduce crop yields. Cotton lint yield can be reduced by 46% due to GR Canada fleabane interference at a density of 25 plants m-2 (Steckel and Gwathmey 2009). Where no weed control strategies are implemented, grain corn yield can be reduced up to 69% from GR Canada fleabane interference (Ford et al. 2014a). Soybean yield is most sensitive to GR Canada fleabane interference where yield loss can be up to 93% (Byker et al. 2013b).
Several morphological features of GR Canada fleabane contribute to its rapid spread. A
GR Canada fleabane plant can produce up to 230,000 seeds (Weaver 2001). Each of these seeds has a pappus, which is a structure that aids in wind-dispersal (Royer and Dickenson 1999). Air movement is complex and can affect the movement of GR Canada fleabane seed with gusts and updrafts (Dauer et al. 2006). Most seeds (99%) are found within 100-m of the mother plant
(Dauer et al. 2007). In a study by Shields et al. (2006), viable Canada fleabane seed was found in
48 the Planetary Boundary Layer (PBL). In the PBL Shields et al. (2006), estimates that a Canada fleabane seed can travel 550 km.
Undisturbed soils in cropped and non-cropped areas are ideal for Canada fleabane establishment; the increased use of no-tillage crop production, especially for soybean, provides a large area for the spread of resistant Canada fleabane biotypes (Nandula et al. 2005). Since the first report of the occurrence of GR Canada fleabane in Ontario (Byker et al. 2013c), now four years ago, Ontario farmers need to know the geographical spread of resistant biotypes in the province. The objective of this survey was to provide an update on the distribution of glyphosate- and cloransulam-methyl-resistant Canada fleabane in Ontario.
2.3 Materials and Methods
2.3.1 Seed Collection
Canada fleabane seed was collected from 27 sites in 2013, 54 sites in 2014, and 129 sites in 2015. This survey investigated counties where GR Canada fleabane was not confirmed from a survey in southwestern Ontario in 2011-2012 (Byker et al. 2013c). The seed collection sites were identified by roadside surveying of fields with Canada fleabane escapes, or reports from agronomists and farmers concerned with poor Canada fleabane control; several surveys of resistant weeds have been conducted using this methodology (Byker et al. 2013c; Falk et al.
2005; Follings et al. 2013; VanWely et al. 2015; Vink et al. 2012).
Seed collection occurred in September to October of each year. The seed collection sites were primarily soybean fields, however some seed was collected from dry-bean, and harvested winter wheat and canola fields. The sites contained single to dense patches of Canada fleabane plants. Seed was collected as a composite sample from approximately 20 plants (where possible) per site throughout the field. The field locations were recorded using a global positioning system
(Garmin GPSMAP 76CSx).
49 2.3.2 Resistance Screening
Canada fleabane seed can readily germinate upon maturity, as there is no dormancy requirement (Buhler and Owen 1997). Germination trays were filled with a soilless mixture
(Sunshine Professional Growing Mix) and then watered. Canada fleabane seed was spread over the soil then covered with 0.5 mm of the soilless mixture to improve seed to soil contact but allow light for germination (Nandula et al. 2006). The greenhouse was set to provide a 16 hour photoperiod and day/night temperature of 25 and 18°C, respectively and the trays were watered daily as needed.
Canada fleabane seedlings were transplanted at the three to four leaf stage into 10 cm diameter pots, one seedling per pot, filled with soilless potting mixture. For each sample, 35 plants were transplanted of which the 28 most uniform were selected and 12 were sprayed with glyphosate, 12 were sprayed with cloransulam-methyl and four were left untreated. Resistance screening examined if the Canada fleabane seedlings were resistant to glyphosate, or cloransulam; if the sample had at least one plant survive the glyphosate and one plant survive the cloransulam-methyl application, it was classified as multiple-resistant. Herbicide applications were made when the Canada fleabane plants were at the rosette stage, 10 cm in diameter (Byker et al. 2013c). Glyphosate was sprayed at 900 g a.e. ha-1 and cloransulam-methyl was sprayed at
17.5 g a.i. ha-1 + Agral 90 + UAN 28% in a spray chamber with a flat fan even nozzle calibrated to deliver 200 L ha-1 at 2.54 km hr-1 and 276 kPa. Herbicide dose was selected based on previous resistance screening work that was based on previous dose response work (Byker et al. 2013c).
Previously determined susceptible populations of Canada fleabane from Middlesex county and a non-cropped area at the University of Guelph Ridgetown Campus were used as susceptible checks. Control ratings were conducted on a scale of 0 (no herbicide injury) to 100
(complete necrosis) at 4 and 5 weeks after application (WAA), plants were classified as dead
50 (growing point was necrotic) or alive (Byker et al. 2013c; VanGessel 2001). If at least one plant in the sample survived, the site was identified as resistant (Byker et al. 2013c; VanWely et al.
2015)
2.4 Results and Discussion
Additional counties were confirmed with GR Canada fleabane from the 2013-2015 survey. After the application of glyphosate, the total number of sites with at least one plant surviving at 5 WAA was 27, 47, and 82 in 2013, 2014, and 2015 respectively (Table 2.1). In
2013, four additional counties confirmed with GR Canada fleabane, Brant, Durham, Norfolk and
Oxford (Figure 2.4). In 2014, 16 additional counties were confirmed with GR Canada fleabane:
Hamilton, Perth, Waterloo, Wellington, Bruce, Duffering, Peel, Halton, York, Peterborough,
Northumberland, Hastings, Lennox Addington, Prince Edward, Ottawa, Stormont Dundas &
Glengarry. (Figure 2.5). In 2015, two additional counties were identified with GR Canada fleabane, Simcoe and Frontenac (Figure 2.6). At the end of the 2015 growing season there were
30 counties in Ontario with GR Canada fleabane (Figure 2.6).
In 2013, 2014, and 2015 there were 4, 9, and 32 additional sites confirmed with multiple- resistant Canada fleabane with resistance to glyphosate and cloransulam-methyl (Table 2.1).
Huron County was confirmed in 2013 with multiple-resistant Canada fleabane (Figure 2.4). In
2014, multiple-resistant Canada fleabane was confirmed in Northumberland, Perth,
Peterborough, Waterloo and Wellington (Figure 2.5). In 2015, 11 counties were identified with multiple-resistant Canada fleabane including: Oxford, Norfolk, Brant, Haldimand, Niagra,
Hamilton, Halton, Peel, York, Lennox Addington, Stormont Dundas & Glengarry. There are now
22 counties in Ontario with multiple-resistant Canada fleabane (Figure 2.6).
The geographic spread of GR Canada fleabane in Ontario since 2010-2012 (Byker et al.
2013c) has been very rapid. Several factors such as the morphology of Canada fleabane, crop
51 rotation, and weed control tactics contribute to the dispersal of GR Canada fleabane (Loux et al.
2006; Shields et al. 2006; Shrestha et al. 2008; Weaver 2001). Some morphological features include Canada fleabane being primarily self-pollinated and its ability to produce over 200,000 seeds (Weaver 2001), which can travel by wind due to an attached pappus on the seed and spread up to 500 km (Shields et al. 2006). A diverse crop rotation can reduce populations of Canada fleabane as heavy tillage can provide some control (Shrestha et al. 2008), heavy crop residues can reduce germination, and crops such as corn (Zea mays L.) or wheat (Triticum spp.) are more competitive than soybean (Loux et al. 2006). Weed control tactics have become more simplified with the repeated use of glyphosate for weed control, which has created a strong selection pressure and a welcoming habitat for resistant weed biotypes (Nandula et al. 2005; Powles et al.
1998).
2.5 Implications
This survey provides updated information on the distribution of GR Canada fleabane throughout Ontario since 2012. This survey does not provide information on the frequency of
GR Canada fleabane in Ontario since the methodology involved non-random site selection procedures (Beckie et al. 2000). With sites confirmed with GR Canada fleabane in Stormont,
Dundas & Glengarry county, this means GR Canada fleabane is present up to 750 km away from where it was originally confirmed in Essex county 2010 (Byker et al. 2013c). Knowledge of GR
Canada fleabane being present across the province of Ontario is important to farmers so that they can implement weed control tactics to manage resistant populations. The identification of
Ontario counties with multiple-resistant Canada fleabane is also important for farmers as use of cloransulam-methyl post-emergence in soybean was common, but now cannot be relied on and other control tactics should be implemented. Diversifying crop rotations or mixing herbicides
52 with alternative modes of action is encouraged to manage resistant populations of Canada fleabane.
53 Table 2.1- Number of sites per county with at least one plant surviving five weeks after application of glyphosate or cloransulam-methyl from greenhouse screening from a 2013-2015 survey of Canada fleabane in Ontario, Canada.
County 2013 2014 2015 G Res.Z G + C Res. G Res. G + C Res. G Res. G + C Res. Brant 4 0 -Y - 8 3 Bruce - - 1 0 2 0 Dufferin - - 1 0 0 0 Durham 1 0 2 0 1 0 Elgin 6 1 - - - - Frontenac - - - - 1 0 Grey - - 0 0 0 0 Haldimand - - - - 10 3 Halton - - 1 0 4 1 Hamilton - - 5 0 5 2 Hastings - - 2 0 2 0 Huron 1 1 - - 7 4 Kawartha Lakes - - - - 0 0 Leeds & Grenville - - - - 0 0 Lennox & Addington - - 2 0 4 0 Middlesex 4 2 - - - - Niagra - - - - 3 1 Norfolk 7 0 - - 5 2 Northumberland - - 6 1 4 3 Ottawa - - 2 0 1 0 Oxford 4 0 1 0 5 4 Prince Edward - - 2 0 1 0 Peel - - 2 0 3 1 Perth - - 4 2 3 2 Peterborough - - 1 1 2 0 Prescott & Russell - - - - 0 0 Simcoe - - 0 0 2 0 Stormont, Dundas & - - 1 0 1 1 Glengarry
54 Waterloo - - 4 3 3 3 Wellington - - 9 2 4 0 York - - 1 0 1 1 Total 27 4 47 9 82 32 Z G= Glyphosate (900 g a.i. ha-1), C= Cloransulam-methyl (17.5 g a.i. ha-1) applied with Agral 90 and UAN 28% Y A dash indicates that no samples were taken from that county in that year
55 County Legend:
A. Essex
Figure 2.1. Ontario counties with glyphosate-resistant Canada fleabane since 2010. Adapted from Byker et al. (2013c).
56 County Legend: A. Essex B. Lambton C. Kent D. Middlesex E. Elgin F. Niagra
Figure 2.2. Ontario counties with multiple-resistant Canada fleabane since 2011. Adapted from Byker et al. (2013c)
57 County Legend: A. Essex B. Lambton C. Kent D. Middlesex E. Elgin F. Niagra G. Huron H. Haldimand
Figure 2.3. Ontario counties with multiple-resistant Canada fleabane since 2012. Adapted from Byker et al. (2013c).
58 County Legend: A. Essex B. Lambton C. Kent D. Middlesex E. Elgin F. Niagra G. Huron H. Haldimand I. Oxford J. Norfolk K. Brant L. Durham
Figure 2.4. Ontario counties with multiple-resistant Canada fleabane since 2013.
59 County Legend: A. Essex B. Lambton C. Kent D. Middlesex E. Elgin F. Niagra G. Huron H. Haldimand I. Oxford J. Norfolk K. Brant L. Durham M. Bruce N. Perth O. Wellington P. Waterloo Q. Hamilton R. Dufferin S. Peel T. Halton U. York V. Peterborough W. Northumberland X. Hastings Y. Prince Edward Z. Lennox & Addington AA. Ottawa BB. Stormont Dundas & Glengarry
Figure 2.5. Ontario counties with multiple-resistant Canada fleabane since 2014.
60 County Legend: A. Essex B. Lambton C. Kent D. Middlesex E. Elgin F. Niagra G. Huron H. Haldimand I. Oxford J. Norfolk K. Brant L. Durham M. Bruce N. Perth O. Wellington P. Waterloo Q. Hamilton R. Dufferin S. Peel T. Halton U. York V. Peterborough W. Northumberland X. Hastings Y. Prince Edward Z. Lennox & Addington AA. Ottawa BB. Stormont Dundas & Glengarry CC. Simcoe DD. Frontenac
Figure 2.6. Ontario counties with multiple-resistant Canada fleabane since 2015.
61 Chapter 3: Glyphosate resistant Canada fleabane [Conyza canadensis (L.) Cronq.] dose-response to saflufenacil, saflufenacil plus glyphosate, and metribuzin plus saflufenacil plus glyphosate in soybean [Glycine max (L.) Merr.] in Ontario
3.1 Abstract
The control of glyphosate-resistant (GR) Canada fleabane in soybean (Glycine max) has been variable with glyphosate plus saflufenacil. The objective of this research was to determine the biologically effective rate of saflufenacil, saflufenacil tankmixed with glyphosate (900 g a.i. ha-1), and metribuzin tankmixed with saflufenacil (25 g a.i. ha-1) and glyphosate (900 g a.i. ha-1) applied preplant (PP). For each study, seven field trials were completed during a two-year period
(2014, 2015) in fields previously confirmed with GR Canada fleabane. The addition of glyphosate to saflufenacil reduced the rate of saflufenacil required to 11, 15, and 25 g a.i. ha-1 for
90, 95, and 98% control of GR Canada fleabane 4 weeks after application (WAA), respectively; at 8 WAA, 25, 34, and 47 g a.i. ha-1 of saflufenacil was required to provide 90, 95, and 98% control respectively compared to the label rate of saflufenacil of 25 g a.i. ha-1 in Ontario. The rate of metribuzin applied in a tankmix with glyphosate (900 g a.i. ha-1) and saflufenacil (25 g a.i. ha-
1) for 90, 95, and 98% control GR Canada fleabane 8 WAA was 61, 261, and 572 g a.i. ha-1, respectively. The addition of metribuzin as a tankmix partner with saflufenacil plus glyphosate, improved control and provided a second effective herbicide mode of action for the control of GR
Canada fleabane. The use of a three-way herbicide tankmix is an effective tactic in a robust herbicide resistance management strategy.
3.2 Introduction
Glyphosate is a nonselective aromatic amino acid synthesis inhibitor that provides broad- spectrum weed control (Franz et al. 1997). Glyphosate targets the 5-enolpyruvoylshikimate-3- phosphate synthase (EPSPS) enzyme in the shikimate acid pathway (Franz et al. 1997). A
62 reduction in photosynthesis, inhibition of auxin transportation, greater auxin oxidation and degradation of chlorophyll are some of the physiochemical and physiological processes that are also affected due to EPSPS inhibition (Baylis 2000). Glyphosate is symplastically, and to a limited amount, apoplastically translocated (Jachetta et al. 1986). Movement to actively growing meristematic cell regions explains why injury symptoms (chlorosis) appear in immature leaves and growing points first (Shaner et al. 2014). Glyphosate is rapidly bound to soil and the amount is primarily determined by the concentrations of phosphate and number of cations in the soil, but is not influenced by soil pH (Sprankle et al. 1975). On light-textured soils, glyphosate may not be adsorbed as tightly due to fewer unoccupied phosphate binding sites (Franz et al. 1997).
Evidence of glyphosate adsorption to the soil includes its limited mobility in the soil and low volatility (Franz et al. 1997). Since the adoption of GR crops in 1996, the use of glyphosate has increased rapidly (Duke and Powles 2008). Excellent broad-spectrum weed control, convenience, crop safety and economics have contributed to the increase in GR crops (Dill
2005), along with the ability to apply the herbicide PP or POST (Nandula et al. 2005).
The repeated use of glyphosate has resulted in the evolution of GR weeds (Duke and
Powles 2008). The first reported GR weed species was rigid ryegrass (Lolium rigidum Gaud.), found in Australia in 1996 (Powles et al. 1998). A population of Canada fleabane in Delaware
USA in 2000 was reported as the first GR broadleaf weed species and had a resistance factor of
8-13-fold (VanGessel 2001), similar to the 7-11-fold resistance factor found in the first reported
GR weed species (Powles et al. 1998). There were 32 GR weed species in 2015, with five in
Canada (Heap 2016). The five GR weed species in Canada were giant ragweed (Ambrosia trifida
L.) [Ontario 2008], Canada fleabane [Ontario 2010], common ragweed (Ambrosia artemisiifolia
L.) [Ontario 2011], kochia (Kochia scoparia L.) [Alberta, Saskatchewan 2012], and waterhemp
(Amaranthus tuberculatus (Moq.) J. D. Sauer) [Ontario 2014].
63 The mechanisms of herbicide resistance are both target site and non-target site based, both of which confer resistance to glyphosate. There are more known mechanisms of resistance to glyphosate than any other herbicide (Sammons and Gaines 2014). Currently two mechanisms are known to confer target-site based resistance: i) altered target-site, and ii) overexpression of the target site (González-Torralva et al. 2012; Nandula 2010). Non-target site based resistance is due to: i) reduced absorption and translocation, ii) herbicide sequestration, and iii) enhanced metabolism (González-Torralva et al. 2012; Nandula 2010). Populations of weed species may have multiple resistance mechanisms that can provide greater resistance to glyphosate (Preston et al. 2009; Nandula et al. 2013).
GR Canada fleabane was first confirmed in Ontario in 2010, and by 2012 it was reported in eight counties within Ontario (Byker et al. 2013a). Five of those counties had multiple- resistant Canada fleabane populations to glyphosate and cloransulam-methyl (Byker et al.
2013c). The rapid spread of GR Canada fleabane can be attributed to the plant’s ability to produce a large number (up to 230,000) of small seeds per plant (Weaver 2001), with an attached pappus that allows for wind-dispersal (Royer and Dickenson 1999). Viable Canada fleabane seed has been collected from the Planetary Boundary Layer, that could move a seed 550 km from the parent plant (Shields et al. 2006); however, ninety-percent of the seed lands within 100-m of the parent plant (Dauer et al. 2007). Canada fleabane can act as a spring or winter annual; in Canada most emergence occurs between late August and October, during which rosettes are formed that overwinter and continue growth early the following spring (Weaver 2001).
Mechanical and chemical control of Canada fleabane has been shown to be variable.
Newly emerged Canada fleabane can be mechanically controlled (Brown and Whitwell 1988); however, larger plants such as established winter annual rosettes may escape tillage (Shrestha et al. 2008). Herbicides must be used to control Canada fleabane in no-tillage crop production
64 systems (Bruce and Kells 1990). Control of GR Canada fleabane should focus on the use of PP or PRE herbicides because POST herbicides are limited in effectiveness (Loux et al. 2006), and there are multiple-resistant biotypes to POST herbicides in Ontario (Byker et al. 2013c). A PP herbicide application with residual activity is a desirable option due to the long emergence pattern of Canada fleabane (Loux et al. 2006). Effective herbicide options for the control of GR
Canada fleabane in Ontario have included glyphosate tankmixes with amitrole (2000 g a.i. ha-1), saflufenacil (25 g a.i. ha-1), flumetsulam (70 g a.i. ha-1), and metribuzin (1120 g a.i. ha-1) (Byker et al. 2013a).
Saflufenacil is a protoporphyrinogen-oxidase inhibiting herbicide (Grossman et al. 2010), that when tankmixed with glyphosate, provides broad-spectrum grass and broadleaf weed control
(Mellendorf et al. 2013). The application timing for saflufenacil in soybean is PP and can be used as a desiccant prior to harvest (Anonymous 2014a). Saflufenacil provides activity on several weed species resistant to glyphosate, ALS inhibitors, triazine, and dicamba herbicides, including
Canada fleabane (Liebl et al. 2008; Soltani et al. 2010; Trolove et al. 2011). Control of GR
Canada fleabane increased as the rate of saflufenacil increased from 25 to 50 g a.i. ha-1 where control was then maximized (Mellendorf et al. 2013). In comparison, Owen et al. (2011) reported equivalent residual control of GR Canada fleabane of greater than 90%, 30 days after planting, with saflufenacil applied at either 25 or 50 g a.i. ha-1. Saflufenacil applied PP in soybean can cause injury due to cultivar sensitivity, depending on environmental conditions soon after application (Miller 2012). The most sensitive cultivar tested by Miller (2012) was cv. ‘OAC
Hanover’ that had up to a 10% reduction in yield from 22 g a.i. ha-1 vs 46 g a.i. ha-1 under cool and wet, versus warm and dry conditions respectively. Variable control of GR Canada fleabane with saflufenacil has been reported by Ikley (2012) in a greenhouse study where 25 g a.i. ha-1 of saflufenacil provided 35, 32, and 20% control at 7, 14, and 28 DAA respectively. The addition of
65 glyphosate to saflufenacil (25 g a.i. ha-1) increased GR Canada fleabane control to 61 and 67% at
7 and 14 DAA, respectively with no significant increase at 28 DAA (Ikley 2012).
Metribuzin alone does not provide acceptable control of Canada fleabane at current application rates in Ontario. Tardif and Smith (2003) reported 73% control of Canada fleabane with 1120 g a.i. ha-1 of metribuzin. The addition of glyphosate to a lower rate of metribuzin (420 g a.i. ha-1) has provided 58% control 4 WAA. A high rate of metribuzin with glyphosate can improve GR Canada fleabane control as reported by Byker et al. (2013a), where 1120 g a.i. ha-1 of metribuzin provided greater than 97% control 8 WAA, however high rates of metribuzin can cause soybean injury, especially on coarse-textured, high pH soils.
GR Canada fleabane is widely distributed in Ontario; its distribution is expected to increase (Byker et al. 2013c) due to the large number of windblown seeds. The widespread use of no-tillage crop production practices creates a large area for GR Canada fleabane to establish as it readily establishes in undisturbed soils (Nandula et al. 2005). The increasing prevalence of
GR Canada fleabane coupled with its ability to reduce soybean yield up to 93% where no control strategies were applied (Byker 2013b), illustrates the need for a reliable control strategy. A common method of controlling GR weeds is to tankmix herbicides with different modes of action (Evans et al. 2015). Tankmixing herbicides will only reduce resistant weed populations if each herbicide has activity, and tankmixing can be expensive for farmers (Evans et al. 2015). To produce consistent control of GR Canada fleabane with saflufenacil, Mellendorf et al. (2013) suggested growers apply glyphosate plus saflufenacil when the plants are relatively small and to include a third tankmix partner with another mode of action. Three-way herbicide tankmixes with glyphosate plus saflufenacil (25 g a.i. ha-1) were investigated by Budd et al. (2016) to determine options for controlling GR Canada fleabane in soybean. Metribuzin (400 g a.i. ha-1) and 2,4-D ester (500 g a.i. ha-1) were determined to be the best tankmix partners for saflufenacil
66 plus glyphosate in GR soybean (Budd et al. 2016). It has also been suggested by Loux (2014) that glyphosate plus saflufenacil plus metribuzin is very effective for the control of GR Canada fleabane. The objective of this study was to determine the dose response of saflufenacil, saflufenacil tankmixed with glyphosate, and metribuzin plus saflufenacil plus glyphosate in a tankmix for the control of GR Canada fleabane in soybean. It is hypothesised that GR Canada fleabane can be controlled in soybean by optimizing the rate of a third tankmix partner.
3.3 Materials and Methods
Three distinct studies were conducted to evaluate the dose-response of saflufenacil alone, saflufenacil plus glyphosate (900 g a.i. ha-1), and metribuzin plus saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) as a tankmix for the control of GR Canada fleabane applied PP in soybean. Each of these studies had seven location-years over a two-year period (2014, 2015), totaling 21 field trials with previously confirmed GR Canada fleabane across southwestern
Ontario. A randomized complete block design with four replications was used for each trial. The plot dimensions were 2.25 m wide by 8 m in length with 3 soybean rows spaced 0.75 m apart. A backpack sprayer was used to apply all herbicide treatments which was calibrated to deliver 200
-1 L ha of spray mixture at 240 kPa using pressurized CO2 as the propellant. The sprayer boom was 1.5 m wide with four ULD120-02 nozzles (Hypro, New Brighton, MN) spaced 50 cm apart.
All treatments included Merge© surfactant at 1 L ha-1 and the saflufenacil formulation was 342 g
L-1 SC. Untreated (weedy) and weed-free controls were included in each replicate of all experiments. Weed-free controls were established with a PP tankmix of glyphosate (1800 g a.i. ha-1), saflufenacil (25 g a.i. ha-1), and metribuzin (400 g a.i. ha-1), followed by hand hoeing as required. Herbicide treatments in the biologically effective rate of saflufenacil trial consisted of saflufenacil applied PP at 3.125, 6.25, 12.5, 25, 50, 100, and 200 g a.i. ha-1. Treatments in the biologically effective rate of saflufenacil plus glyphosate trial were the same as the previous trial,
67 but with the addition of glyphosate (900 g a.i. ha-1) to all treatments. The biologically effective rate trial for metribuzin plus saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) consisted of saflufenacil and glyphosate in all treatments and metribuzin at 12.5, 25, 50, 100, 200, 400,
800, and 1600 g a.i. ha-1. Quizalofop-p-ethyl (36 g a.i. ha-1) and glyphosate (900 g a.i. ha-1) were applied as maintenance sprays in 2014 and 2015, respectively, to remove potentially confounding effects of other weed species. Soil characteristics, seeding and herbicide application dates, and Canada fleabane height and density at application for all trials are listed in Table 3.1.
Canada fleabane control was visually assessed 4 and 8 weeks after application (WAA) using a scale of 0 to 100% where 0 was no control and 100 was plant death. At 8 WAA, GR
Canada fleabane density and dry weight were measured by counting the plants in two, 0.25 m2 quadrants per plot; these plants were cut at the soil surface, placed in paper bags, and dried to a constant moisture at 60°C and then weighed. At soybean maturity, seed yield was determined by harvesting a 2-m length of the center row and threshing it in a stationary machine. Moisture content and weight of the harvested grain was recorded for each plot. Soybean grain yield is presented in tonnes ha-1 at 13% dry grain moisture. At 2 and 4 weeks after soybean emergence, soybean injury was assessed visually on a scale of 0 (no injury) to 100% (plant death).
The PROC NLIN procedure in SAS (Ver. 9.4, SAS Institute Inc., Cary, NC) was used to analyze the biologically effective rate in all studies. All environments were combined for analysis. The weedy and weed-free controls were not included in regression analysis. Weed dry weight and density were converted to a percent of the glyphosate alone treatment prior to analysis for the biologically effective rate of saflufenacil alone, and saflufenacil plus glyphosate studies (Byker et al. 2013a). Weed dry weight and density were kept in original form for the biologically effective rate of metribuzin plus saflufenacil plus glyphosate study because the percent data was flawed due to low values in the zero metribuzin plots.
68 Regression equations (models) used
All parameters in the biologically effective rate of saflufenacil and the biologically effective rate of saflufenacil plus glyphosate studies were regressed against saflufenacil dose, represented by DOSE in the equations. The parameters in the biologically effective rate of metribuzin plus saflufenacil plus glyphosate study were regressed against metribuzin dose, also represented by
DOSE in the equations. The exponential to a maximum curve was used to analyze the GR
Canada fleabane control data and was obtained from one of three equations, depending on study:
Exponential to a maximum Y = a + b (1 - e-c DOSE) Equation [1]
Where a is the intercept, b is the magnitude, and c is the slope.
Exponential to a maximum Y = a – b (e –c DOSE) Equation [2]
Where a is the upper asymptote, b is the magnitude, and c is the slope.
Exponential to a maximum Y = a – c (b DOSE) Equation [3]
Where a is the upper asymptote, b is the slope, and c is the magnitude.
The inverse exponential curve was used for the dry weight and density data for all studies and was obtained from the equation [4]:
Inverse exponential Y = a + be –c DOSE Equation [4]
Where a is the lower asymptote, b is the reduction in y from intercept to a, and c is the slope.
The biologically effective rate of saflufenacil study used the exponential to a maximum
Equation [2] for all control data; however, Equation [3] provided similar fit and predicted values.
The biologically effective rate of saflufenacil plus glyphosate study used the exponential to a maximum in Equation [3] for all control data because it had the best fit to the data. The biologically effective rate of metribuzin plus saflufenacil plus glyphosate study used the exponential to a maximum in Equation [1] for 1 and 2 WAA control data and Equation [2] for 4 and 8 WAA control data. Saflufenacil provided short residual control that meant little metribuzin
69 was required for control at 1 and 2 WAA. At 4 and 8 WAA, the residual control from saflufenacil decreased and more metribuzin was required to provide control. The use of both equations is appropriate to describe the control in these results based on the short residual that saflufenacil provides for controlling GR Canada fleabane.
Predicted values
Regression equations were used to calculate predicted saflufenacil or metribuzin doses (g a.i. ha-1) that resulted in 90, 95, or 98% weed control, or reduction in GR Canada fleabane dry weight or density (ED90, ED95, and ED98). Where any dose was predicted to be greater than the range of doses evaluated in these studies, it was expressed as ‘-‘ because it would be improper to extrapolate outside of the range. Also in the biologically effective rate of metribuzin plus saflufenacil plus glyphosate study, if the equation predicted no metribuzin was required, it was expressed as ‘-‘ as well.
3.4 Results and Discussion
Soybean injury was minimal (<10%) in both saflufenacil alone and saflufenacil plus glyphosate biologically effective rate experiments. The injury consisted of leaf puckering and distortion, but it was observed only at high doses and where there was high soil moisture during crop emergence. Soybean injury in the metribuzin plus saflufenacil plus glyphosate biologically effective rate experiment was up to 40 and 20% at 2 and 4 weeks after emergence (WAE), respectively. This consisted of leaf burn of the lower leaves for treatments with high metribuzin rates (800 and 1600 g a.i. ha-1), and when soil moisture was high after crop emergence.
3.4.1 Biologically Effective Rate of Saflufenacil Alone
At 1 week after application (WAA), the saflufenacil dose required for 90 and 95% control of GR Canada fleabane was 11 and 18 g a.i. ha-1, respectively (Table 3.2). At 2 WAA,
70 the rate of saflufenacil increased to 18 and 27 g a.i. ha-1 for 90 and 95% control, respectively.
The ED98 for the control of GR Canada fleabane could not be calculated at 1 and 2 WAA. The
-1 ED90 and ED95 values were less than the label rate of saflufenacil (<25 g a.i. ha ) with the exception of ED95 at 2 WAA that required 1.1X the label rate. Similarly, Mellendorf et al. (2013) found 25 g a.i. ha-1 of saflufenacil provided 92% control 2 WAA and that 50 g a.i. ha-1 provided significantly higher control at 98%. To obtain 90, 95, and 98% control of GR Canada fleabane at
4 WAA 13, 18, and 30 g a.i. ha-1 of saflufenacil was required. In contrast, Knezevic et al. (2009) reported that 78 g a.i. ha-1 of saflufenacil were required for 90% control of Canada fleabane 4
WAA with methylated seed oil instead of Merge©. At 4 WAA, less that the label rate of saflufenacil (<25 g a.i. ha-1), was required for 90 and 95% control, while a 1.2X rate was required to achieve 98% control. At 8 WAA, higher rates (≥25 g a.i. ha-1) of saflufenacil were required to provide 90 and 95% control, than at 1, 2, and 4 WAA (ED98 could not be calculated).
Saflufenacil at 25 and 36 g a.i. ha-1 provided 90 and 95% control 8 WAA respectively, or 1.4X the label rate for 95% control. This is in contrast to Soltani et al. (2012) who reported greater than 58 g a.i. ha-1 of saflufenacil was required for 95% control of five different annual broadleaf weeds (Ambrosia artemisifolia L., Chenopodium album L., Polygonum convolvulus L.,
Polygonum scabrum Moench., and Sinapis arvensis L.) 8 weeks after oat (Avena sativa L.) emergence.
The dose of saflufenacil required to reduce GR Canada fleabane dry weight by 90 and
95% were similar to the dose for weed control at 8 WAA. Saflufenacil at 26, 36, and 61 g a.i. ha-
1 reduced GR Canada fleabane dry weight by 90, 95, and 98% respectively, or 1.0, 1.4, and 2.4X the label rate of saflufenacil was required (Table 3.2). A lower dose of saflufenacil of 16 and 22 g a.i. ha-1 reduced GR Canada fleabane density by 90 and 95%, respectively which is less than the label rate for saflufenacil (<25 g a.i. ha-1). This is in contrast to Mellendorf et al. (2013)
71 where 25 and 50 g a.i. ha-1 of saflufenacil reduced GR Canada fleabane density 76 and 97% respectively, 4 WAA. The ED98 could not be calculated for GR Canada fleabane density reduction.
3.4.2 Biologically Effective Rate of Saflufenacil plus Glyphosate Tankmix
The addition of glyphosate (900 g a.i. ha-1) reduced the dose of saflufenacil required for the control of GR Canada fleabane compared to the saflufenacil alone. At 1 WAA 8, 11, and 20 g a.i. ha-1 of saflufenacil was required for 90, 95, and 98% control, respectively (Table 3.3). This is similar to Byker et al. (2013a) who reported 98% GR Canada fleabane control with glyphosate
(900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1). Waggoner (2010) stated that the addition of glyphosate to saflufenacil did not improve control 1 WAA due to the short rainfastness of saflufenacil observed in a rainfast study. At 2 WAA, a slightly higher dose of saflufenacil of 10
-1 and 16 g a.i. ha was required for 90 and 95% control, respectively. At 2 WAA, the ED98 of saflufenacil for the control of GR Canada fleabane could not be calculated. Similarly, Byker et al. (2013a) reported that glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) provided greater than 90% control at 2 WAA. At 4WAA, a saflufenacil dose of 11, 15, and 25 g a.i. ha-1 was required for 90, 95 and 98% GR Canada fleabane control, respectively. In contrast, 25 g a.i. ha-1 of saflufenacil provided only 91% control, which was significantly less than 75 g a.i. ha-1 of saflufenacil that provided 98% control 4 WAA with glyphosate (900 g a.i. ha-1) (Mellendorf et al. 2013). Similar to the results from this study, Byker et al. (2013a) reported that glyphosate
(900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) provided greater than 95% control 4 WAA and regrowth in plant escapes was reported. Ford et al. (2014b) found that glyphosate (900 g a.i. ha-1) plus saflufenacil (50 g a.i. ha-1) provided greater than 93% control at four sites, but less than 44% control at a fifth site at 4 WAA. The dose of saflufenacil for ED90, ED95, and ED98 for GR
Canada fleabane control 1, 2, and 4 WAA was less than or equal to the label rate of saflufenacil
72 (≤ 25 g a.i. ha-1) when tankmixed with glyphosate. At 8 WAA, saflufenacil at 25, 34, and 47 g a.i. ha-1 provided 90, 95, and 98% control, respectively. To provide 95 and 98% control 8 WAA,
1.4 and 1.9X the label rate of saflufenacil was required. Similarly, four of five sites in a study by
Ford et al. (2014b) showed greater than 98% control with 50 g a.i. ha-1 of saflufenacil 8 WAA, however in contrast, one site had only 49% control.
The dose of saflufenacil required to reduce GR Canada fleabane dry weight by 90, 95,
-1 and 98% was greater than required for control at 8 WAA. Saflufenacil at 31, 42 and 59 g a.i. ha reduced GR Canada fleabane dry weight by 90, 95, and 98% or 1.2, 1.7, and 2.4X the label rate, respectively (Table 3.3). Ford et al. (2014b) reported similar results at four of five sites where 50 g a.i. ha-1 of saflufenacil plus glyphosate reduced GR Canada fleabane dry weight greater than
95% while in contrast one site showed a 58% reduction. The dose of saflufenacil required to provide 90, 95 and 98% reduction in GR Canada fleabane density was less than required for dry weight. Saflufenacil at 16, 21, and 28 g a.i. ha-1 reduced GR Canada density 90, 95, and 98%, respectively. ED90 and ED95 for density required less than the label rate of saflufenacil (<25 g a.i.
-1 ha ), while ED98 required 1.1X the label rate. In contrast, Mellendorf et al. (2013) reported 25 g a.i. ha-1 and 50 g a.i. ha-1 of saflufenacil provided 91 and 97% reduction in density respectively.
In contrast to our result, Ford et al. (2014b) reported 50 g a.i. ha-1 of saflufenacil provided an
85% reduction in density at one site; however, greater than 97% density reduction was reported at four other sites.
3.4.3 Biologically Effective Rate of Metribuzin plus Saflufenacil plus
Glyphosate Tankmix
At 1 WAA 5, 6, and 9 g a.i. ha-1 of metribuzin was required for 90, 95 and 98% control of
GR Canada fleabane, respectively (Table 3.4). At 2 WAA, 6 and 11 g a.i. ha-1 of metribuzin was required for 90 and 95% control, respectively, while the ED98 could not be calculated. The
73 exponential to a maximum in Equation [1] was used for the 1 and 2 WAA weed control regression due to the high level of control observed where the metribuzin dose was 0 g a.i. ha-1.
The label rate of saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) provided short residual control of GR Canada fleabane, requiring low doses of metribuzin to achieve 90, 95 and
98% control. In a study by Budd et al. (2016), 25 g a.i. ha-1 of saflufenacil provided 99% control
4 WAA which explains the low requirement for metribuzin at 4 WAA in this study. At 4 WAA,
ED90 and ED95 could not be calculated using the exponential to a maximum in Equation [2] because the saflufenacil provided residual control; however, at 4 WAA 217 g a.i. ha-1 of metribuzin was required for 98% control of GR Canada fleabane. Interestingly, Tardif and Smith
(2003) reported 73% control of glyphosate-susceptible Canada fleabane with metribuzin (1120 g a.i. ha-1) 4 WAA; over 5X the amount needed to provide 98% control with saflufenacil (25 g a.i. ha-1) in this study. Eubank et al. (2008) reported glyphosate plus metribuzin (420 g a.i. ha-1) provided 58% control of GR Canada fleabane 4 WAA. At 8 WAA, 61, 261, and 572 g a.i. ha-1 of metribuzin was required for 90, 95, and 98% control, respectively. Similarly, Budd et al. (2016) reported that saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) plus metribuzin (400 g a.i. ha-1) provided 96% control of GR Canada fleabane 8 WAA.
A higher dose of metribuzin was required to reduce GR Canada fleabane dry weight compared at 8 WAA. Metribuzin at 523, 820 and 1251 g a.i. ha-1 was required for 90, 95, and
98% reduction in GR Canada fleabane dry weight, respectively (Table 3.4). Budd et al. (2016) reported a 92% reduction in GR Canada fleabane dry weight with glyphosate plus saflufenacil plus metribuzin (400 g a.i. ha-1), which is slightly less than the rate required in this study. Also in contrast, Byker et al. (2013a) found that glyphosate (900 g a.i. ha-1) plus metribuzin (1120 g a.i. ha-1) reduced GR Canada fleabane dry weight by 100% at 8 WAA. Metribuzin at 283 and 557 g
-1 a.i. ha reduced GR Canada fleabane density 90 and 95%, respectively, while the ED98 for GR
74 Canada fleabane density could not be calculated. Interestingly, glyphosate (900 g a.i. ha-1) plus metribuzin (420 g a.i. ha-1) reduced GR Canada fleabane density 66% (Eubank et al. 2008). In contrast to these results, Budd et al. (2016) reported glyphosate (900 g a.i. ha-1) plus saflufenacil
(25 g a.i. ha-1) plus metribuzin (400 g a.i. ha-1) reduced GR Canada fleabane density by 98%.
3.5 Conclusions
In this study, the label rate of saflufenacil (25 g a.i. ha-1) provided 90% control of GR
Canada fleabane up to 8 WAA. The control of GR Canada fleabane with saflufenacil was improved with the addition of glyphosate (900 g a.i. ha-1). Mellendorf et al. (2013) reported that the addition of glyphosate to saflufenacil reduced the frequency of GR Canada fleabane regrowth compared to saflufenacil applied alone. Where regrowth was observed, it began more than 4
WAA, an observation also reported by Byker et al. (2013a).
The addition of metribuzin to glyphosate plus saflufenacil reduces GR Canada fleabane escapes and provides residual control of late emerging fleabane (Ikley 2012). The addition of metribuzin (572 g a.i. ha-1) as a tankmix partner provided 98% control of GR Canada fleabane with no crop injury. In this study, soybean injury was observed when metribuzin was applied at
800 g a.i. ha-1 and greater, however, metribuzin has been reported to cause soybean injury at 400 g a.i. ha-1 on light-textured soils that have high soil pH (Peter Sikkema, pers. comm.).
Saflufenacil compliments glyphosate for the control of weeds that are resistant to several herbicide groups (2, 4, 5, and 9) (Liebl et al. 2008; Soltani et al. 2010; Trolove et al. 2011). For the control of GR Canada fleabane, an application of glyphosate plus saflufenacil would mean there is only one effective herbicide mode of action. If a third herbicide with a different mode of action was included in the tankmix, then there would be two effective modes of action on GR
Canada fleabane, which is why the inclusion of metribuzin in the tankmix is very desirable. The use of a three-way tankmix for GR Canada fleabane control is a robust weed management
75 strategy that provides excellent control of GR Canada fleabane and reduces the potential for the selection of herbicide resistant biotypes (Mellendorf et al. 2013).
76 Table 3.1- Location, agronomic information and height and density of glyphosate-resistant Canada fleabane in biologically effective rate experiments in Ontario, Canada in 2014 and 2015
Soil Characteristics (0-15cm) Seeding Spray Canada fleabaneZ Location Year Closest Town Texture OM (%) pH Date Date Size Density Year (cm) (# m-2) Q1Y 2014 Mull Loam 3.1 6.6 June-10 June-6 up to 5 5648 Q2 2014 Blenheim Sandy Loam 2.9 6.5 June-20 June-4 up to 10 1232 Q3 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-28 up to 10 321 Q4 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-28 up to 7 674 Q5 2015 Mull Loam 2.6 6.0 June-12 June-4 up to 9 1048 Q6 2015 Blenheim Sandy Loam 4.2 6.2 June-6 June-1 up to 8 340 Q7 2015 Harrow Sandy Loam 2.5 6.1 May-29 May-21 up to 7 349 R1 2014 Mull Loam 3.1 6.6 June-10 June-6 up to 6 2781 R2 2014 Blenheim Sandy Loam 2.9 6.5 June-20 June-4 up to 11 724 R3 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-28 up to 11 145 R4 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-28 up to 11 796 R5 2015 Mull Loam 2.6 6.0 June-12 June-4 up to 7 826 R6 2015 Blenheim Sandy Loam 4.2 6.2 June-6 June-1 up to 8 505 R7 2015 Harrow Sandy Loam 2.5 6.1 May-29 May-21 up to 11 501 S1 2014 Mull Loam 3.1 6.6 June-10 June-6 up to 5 1379 S2 2014 Blenheim Sandy Loam 2.9 6.5 June-20 June-4 up to 10 973 S3 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-28 up to 7 146 S4 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-28 up to 8 299 S5 2015 Mull Loam 2.6 6.0 June-12 June-4 up to 8 604 S6 2015 Blenheim Sandy Loam 4.2 6.2 June-6 June-1 up to 11 783 S7 2015 Harrow Sandy Loam 2.5 6.1 May-29 May-21 up to 6 121 Z Canada fleabane size and density at time of treatment application from untreated control plots Y Q 1-7, Locations for biologically effective rate of saflufenacil alone experiments; R 1-7, Locations for biologically effective rate of saflufenacil plus glyphosate experiments; S 1-7, Locations for the biologically effective rate of metribuzin plus saflufenacil plus glyphosate experiments
77 Table 3.2- Regression parameters of exponential to a maximum and inverse exponential equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, dry weight, and density for saflufenacil alone field dose experiments conducted in 2014 and 2015 in Ontario, CanadaZ
Regression Parameters y (se) Saflufenacil dose (g a.i. ha-1) Exponential to maximum a b c ED90 ED95 ED98 Weed Control 1 WAA 95.7 (1.5) 94.5 (3.4) 0.3 (0.02) 11 18 - 2 WAA 97.0 (1.3) 95.9 (2.6) 0.1 (0.009) 18 27 - 4 WAA 98.4 (1.4) 100.1 (2.9) 0.2 (0.01) 13 18 30 8 WAA 97.5 (1.9) 95.4 (3.4) 0.1 (0.009) 25 36 - Inverse exponential Dry weight 1.7 (6.3) 102.5 (11.1) 0.1 (0.03) 26 36 61 Density 2.4 (3.0) 98.8 (6.0) 0.2 (0.02) 16 22 - z Abbreviation: WAA, weeks after application y Parameters: a, upper asymptote (Exponential to maximum) or lower asymptote (Inverse exponential); b, magnitude; c, slope; ED, the effective dose for 90, 95, and 98% control or reduction in dry weight or reduction in density compared to the control
78 Table 3.3- Regression parameters of exponential to a maximum and inverse exponential equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, dry weight, and density for saflufenacil plus glyphosate field dose experiments conducted in 2014 and 2015 in Ontario, Canada
Regression Parameters y (se) Saflufenacil dose (g a.i. ha-1) Exponential to maximum a b c ED90 ED95 ED98 Weed Control 1 WAA 98.1 (1.0) 0.7 (0.02) 85.3 (2.3) 8 11 20 2 WAA 96.8 (1.0) 0.8 (0.01) 71.7 (2.2) 10 16 - 4 WAA 98.6 (1.3) 0.8 (0.02) 64.8 (2.8) 11 15 25 8 WAA 99.4 (2.3) 0.9 (0.01) 86.1 (3.9) 25 34 47 Inverse exponential Dry weight 0.9 (4.0) 105.8 (6.6) 0.08 (0.01) 31 42 59 Density 0.01 (3.3) 102.4 (6.6) 0.1 (0.02) 16 21 28 z Abbreviation: WAA, weeks after application y Parameters: a, upper asymptote (Exponential to maximum) or lower asymptote (Inverse exponential); b, magnitude; c, slope; ED, the effective dose for 90, 95, and 98% control or reduction in dry weight or reduction in density compared to the control
79 Table 3.4- Regression parameters of exponential to a maximum and inverse exponential equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, dry weight, and density for metribuzin plus saflufenacil plus glyphosate field dose experiments conducted in 2014 and 2015 in Ontario, Canada Z
Regression Parameters y (se) Metribuzin dose (g a.i. ha-1) Exponential to maximum a b c ED90 ED95 ED98 Weed Control 1 WAA 2.85 x 10-11 99.3 (2.5) 0.2 (0.1) 5 6 9 (0)x 2 WAA 2.72 x 10-11 (0) 95.6 (2.4) 0.5 (1.9) 6 11 - 4 WAA 99.8 (0.6) 3.1 (0.6) 0.003 (0.001) - - 217 8 WAA 99.3 (2.6) 11.8 (3.0) 0.004 (0.003) 61 261 572 Inverse exponential Dry weight 0.5 (8.7) 35.3 (8.8) 0.003 (0.002) 523 820 1251 Density 4.4 (10.9) 53.3 (15.1) 0.008 (0.007) 283 557 - z Abbreviation: WAA, weeks after application y Parameters: a, upper asymptote (Exponential to maximum) or lower asymptote (Inverse exponential); b, magnitude; c, slope; ED, the effective dose for 90, 95, and 98% control or reduction in dry weight or reduction in density compared to the control x SAS 9.4 does not have the computing power to determine the standard error for very small parameter estimates
80 Chapter 4: Control of glyphosate-resistant Canada fleabane with saflufenacil plus tankmix partners in soybean
4.1 Abstract
Glyphosate plus saflufenacil applied preplant (PP) has provided excellent control of glyphosate-resistant (GR) Canada fleabane in soybean in previous studies, but more recent research and commercial results in growers’ fields has demonstrated variable control of GR
Canada fleabane with this tankmix. The objective of this study was to determine if the level and consistency of GR Canada fleabane control with glyphosate plus saflufenacil could be improved with the addition of a third tankmix partner. Six field trials were conducted over a two-year period (2014, 2015) in fields with confirmed populations of GR Canada fleabane in ON. GR
Canada fleabane interference reduced soybean yield by 73% in this study compared to the weed free control. At 4 and 8 weeks after application (WAA), glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) provided 99 and 88% control of GR Canada fleabane respectively, and at 8 WAA, reduced GR Canada fleabane density by 96% and biomass 89% compared to the untreated control. Glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) improved the control of GR Canada fleabane to 100 and 97% at 4 and 8 WAA, respectively. At 8 WAA, glyphosate plus saflufenacil plus amitrole (2000 g a.i. ha-1) reduced GR Canada fleabane density and biomass 99 and 97%, respectively. At 8 WAA, glyphosate plus saflufenacil plus dicamba at
300 or 600 g a.i. ha-1 reduced GR Canada fleabane biomass 97 and 98%, respectively. There was no improvement in the control of GR Canada fleabane when 2,4-D, metribuzin, flumetsulam, cloransulam-methyl, chlorimuron, glufosinate or paraquat was added to the tankmix of glyphosate plus saflufenacil. Tank-mixing dicamba with glyphosate plus saflufenacil applied pre-plant in soybean improved control of GR Canada fleabane, however, this caused 14 and 46% crop injury at 2 and 4 WAA respectively. Soybean yield for saflufenacil alone and saflufenacil
81 tankmix treatments was similar to the weed free control (3.25 tonnes ha-1) with the exception of tank-mixing dicamba (600 g a.i. ha-1), that was less (2.39 tonnes ha-1) and similar to the glyphosate alone treatment.
4.2 Introduction
Canada fleabane is distributed throughout the Canadian provinces, except Newfoundland, and can be observed most frequently in the eastern parts of Canada (Weaver 2001; Cici and Van
Acker 2009). Canada fleabane is most commonly found on well-drained, coarse-textured, and minimally disturbed soils (Bhowmik and Bekech 1993; Weaver 2001). Highest Canada fleabane germination (61%) was reported at 24/20 C day/night temperatures and 13 hour photoperiod; however, it can also germinate (15%) under dark conditions (Nandula et al. 2006). Although
Canada fleabane can germinate and emerge throughout the year (Buhler and Owen 1997), it most frequently emerges between late August and October in Canada (Weaver 2001). Fall-emerging plants form a rosette in the fall and the stems elongate the following spring; a smaller portion of the population emerge in the spring/summer and have a summer annual growth habit (Weaver
2001). A 1.5-m tall Canada fleabane plant can produce up to 230,000 wind-dispersed seeds
(Weaver 2001), with 99% of them landing within 100 m of the mother plant, which can result in a rapid increase in population (Dauer et al. 2007).
Glyphosate is a systemic, broad-spectrum herbicide that was commercialized by
Monsanto in 1974 (Franz et al. 1997). Transgenic GR soybean and canola were commercialized in 1996 and since then, their adoption has been rapid (Dill 2005; Duke and Powles 2008). This rapid adoption can be attributed to improved crop safety, simplicity and broad-spectrum weed control with glyphosate applied postemergence (POST) (Dill 2005). Consequently, the use of glyphosate has increased tremendously since it can be applied pre-plant (PP) or POST in GR crops (Nandula et al. 2005). The use of diverse weed management strategies has decreased due
82 to the repeated use of glyphosate in GR crops (Powles 2008), which has resulted in intense selection pressure for glyphosate resistant biotypes (Powles et al. 1998; Nandula et al. 2005).
The first documented GR weed species was rigid ryegrass (Lolium rigidum Gaud.) in 1996
(Powles et al. 1998). As of 2015, there were 32 GR weed species globally including Canada fleabane (Heap 2016). In Canada, GR Canada fleabane was first documented in Essex county
Ontario in 2010 (Byker et al. 2013c). By 2012, it was reported in eight Ontario counties and its distribution in the province was expected to increase (Byker et al. 2013c).
GR Canada fleabane is a highly competitive weed species. Soybean yield can be reduced up to 93% (Byker et al. 2013b), while corn yield can be reduced up to 69% due to GR Canada fleabane interference if no control strategies are implemented (Ford et al. 2014a). Canada fleabane interference (25 plants m-2) can reduce cotton lint yield by 46% (Steckel and Gwathmey
2009). Overall, GR Canada fleabane can have a large impact on crop yields if not controlled.
In conventional tillage crop production systems, control of Canada fleabane using fall or spring tillage can be effective (Brown and Whitwell 1988; Kapusta 1979); however, only small plants are controlled with tillage (Shrestha et al. 2008). In no-tillage systems herbicides are used to control Canada fleabane (Bruce and Kells 1990). Pre-emergence (PRE) herbicides in soybean should be used to control GR Canada fleabane because POST herbicide options have limited control (Loux et al. 2006). Using a PRE herbicide with residual activity on Canada fleabane in soybean is desirable (Loux et al. 2006), since Canada fleabane can emerge after a non-residual burndown application such as glyphosate (Buhler and Owen 1997).
Saflufenacil is a protoporphyrinogen-oxidase (PPO) inhibiting herbicide (Grossman et al.
2010). When tankmixed with glyphosate, saflufenacil provides broad-spectrum control of grass and broadleaf weeds (Mellendorf et al. 2013). Saflufenacil can be used as a PP herbicide or as a desiccant in soybean in eastern Canada (Anonymous 2014a). GR Canada fleabane and other
83 herbicide resistant weeds such as ALS resistant prickly lettuce (Lactuca serriola L.) can be controlled by saflufenacil (Liebl et al. 2008; Soltani et al. 2010; Trolove et al. 2011); however, control of specific broadleaf weeds depends on application rate (Geier et al. 2009; Soltani et al.
2012). Furthermore, Mellendorf et al. (2013) found that the control of GR Canada fleabane increased as the rate of saflufenacil increased from 25 to 50 g a.i. ha-1 (80 to 95% control respectively), in a non-crop field study. At 7 and 30 days after planting, saflufenacil applied PP at 25 and 50 g a.i. ha-1 controlled GR Canada fleabane plants (cm) greater than 90% (Owen et al.
2011).
GR Canada fleabane control with glyphosate plus saflufenacil can be variable. For instance, at 28 days after POST application of glyphosate alone and glyphosate plus saflufenacil (25 g ai ha-1), GR Canada fleabane was controlled 37 and 57% respectively (Ikley 2012). Mellendorf et al. (2013) suggested a third herbicide with a different mode of action should be added to glyphosate plus saflufenacil for more consistent control of GR Canada fleabane. Therefore, the objective of this study was to determine the level of GR Canada fleabane control and soybean crop injury with glyphosate and saflufenacil plus a third tankmix partner applied PP in soybean.
It is hypothesised that full season, residual control of GR Canada fleabane can be achieved in soybean when an effective tankmix partner is added to glyphosate plus saflufenacil.
4.3 Materials and Methods
Six field trials were conducted over a two-year period (2014, 2015) at three field locations in southwestern Ontario with previously confirmed GR Canada fleabane populations to determine the efficacy of glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) plus a third tankmix partner applied PP in soybean. The experiments were arranged as a randomized complete block design with four replications in each field. The plots were 2.25 m wide with 3 soybean rows spaced 0.75 m apart and 8 m in length. All herbicide treatments were applied PP
84 -1 using a CO2 pressurized backpack sprayer that was calibrated to deliver 200 L ha of spray mixture at 240 kPa. The boom was 1.5-m wide with four ULD 120-02 nozzles (Hypro, New
Brighton, MN) spaced 50-cm apart. Each replication included an untreated (weedy) and weed- free control. The weed-free control was maintained weed free with a glyphosate (1800 g a.e. ha-
1), saflufenacil (25 g a.i. ha-1) and metribuzin (400 g a.i. ha-1) tankmix applied PP followed by hand hoeing as required. A cover spray of quizalofop-p-ethyl (36 g a.i. ha-1) in 2014 and glyphosate (900 g a.i. ha-1) in 2015 was applied to remove potentially confounding effects of other weed species. Location, soil characteristics, seeding date, herbicide application date and
Canada fleabane height and density at time of application are listed in Table 4.1. Herbicide treatments and rates applied are listed in Table 4.2.
Soybean injury was assessed visually 2 and 4 weeks after soybean emergence (WAE) on a scale of 0 (no injury) to 100% (plant death). Canada fleabane control was assessed visually 4 and 8 weeks after application (WAA) on a scale of 0 to 100% where 0 was no control and 100 was plant death. Canada fleabane density and dry weight were determined 8 WAA by counting the plants in two, 0.25 m2 quadrants per plot. The same plants were cut at the soil surface, placed in paper bags, and dried at 60°C to a constant moisture and weighed (Byker et al. 2013a).
Soybean seed yield was determined at maturity from a 2-m length of the center row of each plot by the use of a stationary threshing machine. The grain moisture content and weight were recorded for each plot. Soybean grain yield is reported in tonnes ha-1 at a grain moisture of 13%.
Data were analyzed using PROC GLIMMIX in SAS (Ver. 9.4, SAS Institute Inc., Cary,
NC). Variances were partitioned into random effects (environment within year and location, replication within environment, and the environment by treatment interaction), and fixed effects
(herbicide treatments). A likelihood ratio was used to test the significance of environment, replication within environment, and environment by treatment interactions from zero (Dr. S.
85 Bowley, Personal Communication). There was no significant environment by treatment interaction so all environments were combined for analysis. The significance of fixed effects was tested using the F-test. Residual plots were used to ensure that the variances were randomly distributed, independent, and homogeneous across treatments. Means were separated using the
Tukey-Kramer multiple range test at P<0.05 using the pdmix800 SAS macro (Saxton 1998).
The estimation method used was the Laplace method which obtains solutions to the likelihood equations through integral approximation of the log-likelihood and provides unbiased covariance parameter estimates when the number of observations per subject is small, compared to the pseudo-likelihood approach (Gbur et al. 2012). Weed control data at 4 and 8 WAA were analysed by specifying a beta distribution and a cumulative complementary log-log link function due to controls (Gbur et al. 2012; Dr. S. Bowley, Personal Communication). Also, untreated and weed free controls were adjusted by adding or subtracting 1.0 x 10-10 to lie within the beta distribution. Weed density and weed biomass data were analyzed by specifying a gamma distribution and the default log link function because it is flexible and can accommodate many distributional shapes and skewed responses (Gbur et al. 2012). Soybean yield was analysed by specifying a normal distribution and the default identity link function.
4.4 Results and Discussion
There was no crop injury with the exception of glyphosate plus saflufenacil plus dicamba
(data not shown). At 2 and 4 WAA, glyphosate plus saflufenacil plus dicamba (300 g a.i. ha-1) caused 11 and 37% soybean injury, respectively. With the higher rate of dicamba, glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) caused 14 and 46% soybean injury, respectively.
The injury included stunted plants, leaf cupping and dead plants caused by the dicamba and the short interval between spraying and soybean seeding. This injury was expected because dicamba-resistant soybean was not used in this study.
86 At 4 WAA, glyphosate and glyphosate plus saflufenacil provided 25.5 and 98.8% control of GR Canada fleabane, respectively (Table 4.2), which is similar to the results reported by
Byker et al. (2013b); however, this contrasts to Ikley (2012) who found 57% control with glyphosate plus saflufenacil (25 g a.i. ha-1) in a greenhouse study. The addition of a tankmix partner to glyphosate plus saflufenacil did not increase GR Canada fleabane control with the exception of dicamba (600 g a.i. ha-1) that provided 99.8% control. The results from this study are similar to the 99, 94, 94, 97% control of Canada fleabane with chloransulam-methyl, chlorimuron, flumetsulam, and 2,4-D at 4 WAA, respectively (Tardif and Smith 2003). In contrast to the results from this study where glyphosate plus saflufenacil plus metribuzin provided 99.5% control, Tardif and Smith (2003) reported metribuzin (1120 g ai ha-1) provided only 73% control of Canada fleabane while Byker et al. (2013a) reported 99% control of GR
Canada fleabane with glyphosate (900 g ai ha-1) plus metribuzin (1120 g ai ha-1). These results are similar with either glyphosate plus 2,4-D (840 g a.i. ha-1) or glyphosate plus dicamba (280 g a.i. ha-1) tankmixes that both provided over 90% control at 4 WAA, but in contrast to glyphosate plus metribuzin (420 g a.i. ha-1) that provided 58% control of GR Canada fleabane (Eubank et al.
2008).
At 8 WAA, glyphosate and glyphosate plus saflufenacil provided 98.8 and 87.9% control of GR Canada fleabane, respectively (Table 4.2.). There was a numeric increase in GR Canada fleabane control by adding a tankmix partner to glyphosate plus saflufenacil, but differences were not always statistically significant. Glyphosate plus saflufenacil plus amitrole, and glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) provided 96.7 and 97.5% control respectively, which was equivalent to the weed free control. Glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) was the only treatment that provided statistically greater control than glyphosate plus saflufenacil. These results are similar to Byker et al. (2013b) where glyphosate
87 plus dicamba (600 g a.i. ha-1) applied PP, followed by glyphosate applied POST, provided 97% control of GR Canada fleabane. In contrast, Byker et al. (2013b) found that glyphosate plus 2,4-
D (560 g a.i. ha-1) applied PP, followed by glyphosate applied POST, provided 77% control 4
WAA. Loux et al. (2006) reported that glyphosate plus 2,4-D plus chlorimuron or chloransulam, glyphosate plus 2,4-D, or glyphosate plus metribuzin provided the best control of GR Canada fleabane. The results from this study are similar to Loux (2014) where glyphosate plus saflufenacil plus 2,4-D, dicamba, metribuzin or chloransulam-methyl are very effective for the control GR Canada fleabane.
At 8 WAA, glyphosate reduced GR Canada fleabane density by 28%, which was equivalent to the untreated control (Table 4.2). Glyphosate plus saflufenacil reduced GR Canada fleabane density by 96%. Davis et al. (2010) reported that saflufenacil applied at 50 and 100 g a.i. ha-1 reduced GR Canada fleabane densities by 78 and 86% compared to the untreated control respectively, in a no-till fallow field study. The addition of a tankmix partner to glyphosate plus saflufenacil did not reduce GR Canada fleabane density with the exception of amitrole, which reduced GR Canada fleabane density by 99%. All the other three-way tankmixes resulted in GR
Canada fleabane density that was equivalent to glyphosate plus saflufenacil. The results from this study are similar to observations by Eubank et al. (2008), who found glyphosate plus 2,4-D (840 g a.i. ha-1), and glyphosate plus dicamba (280 g a.i. ha-1), reduced GR Canada fleabane density by 99 and 98%, respectively. In contrast, Eubank et al. (2008) found glyphosate plus metribuzin
(420 g a.i. ha-1) reduced GR Canada fleabane density by 66%.
At 8 WAA, glyphosate alone and glyphosate plus saflufenacil reduced GR Canada fleabane biomass 28 and 89%, respectively, compared to the untreated control (Table 4.2).
Similarly, Byker et al. (2013a) reported 99% reduction in Canada fleabane biomass at 4 WAA with glyphosate plus saflufenacil in soybean under field conditions. In contrast Ikley (2012)
88 reported glyphosate plus saflufenacil provided a 45% reduction in GR Canada fleabane biomass in a greenhouse study 4 WAA. The addition of another tankmix partner to glyphosate plus saflufenacil did not reduce GR Canada fleabane biomass compared to the untreated control with the exception of adding amitrole, dicamba at either 300 g a.i. ha-1 or 600 g a.i. ha-1, which reduced biomass by >97%. These results are similar to Byker et al. (2013a; 2013b), who reported glyphosate plus amitrole (4 WAA), and glyphosate plus dicamba (300 or 600 g a.i. ha-1) (8
WAA), reduced GR Canada fleabane biomass by greater than 97%. The addition of flumetsulam, glufosinate and paraquat to glyphosate plus saflufenacil reduced GR Canada fleabane biomass by
87, 89 and 86%, respectively, which was less than when amitrole or dicamba (300 or 600 g a.i. ha-1) were added to glyphosate plus saflufenacil. The addition of 2,4-D ester, metribuzin, or cloransulam-methyl reduced GR Canada fleabane biomass by 93%, 92%, and 93%, respectively, which was equivalent to the addition of amitrole or dicamba (300 g a.i. ha-1). These results are similar to Byker et al. (2013a) where glyphosate plus 2,4-D (500 g a.i. ha-1), and glyphosate plus chloransulam-methyl reduced GR Canada fleabane biomass by 95 and 93%, respectively.
GR Canada fleabane interference reduced soybean yield by 73% compared to the weed free control (Table 4.2). This is similar to the 82% yield reduction between the untreated control and the most efficacious treatment found by Eubank et al. (2008) that was glyphosate plus 2,4-D
(840 g a.i. ha-1). In this study, glyphosate alone resulted in an average soybean yield (1.53 tonnes ha-1), similar to the untreated control (1.02 tonnes ha-1). The addition of saflufenacil to glyphosate resulted in soybean yield equivalent to the weed free control (2.71 and 3.25 tonnes ha-1 respectively). The addition of a third tankmix partner to glyphosate plus saflufenacil resulted in soybean yield that was equivalent to the weed free control (3.25 tonnes ha-1), with the exception of dicamba (600 g a.i. ha-1), that resulted in a 37% reduction in soybean yield compared to the weed-free control. This yield reduction was expected because of the observed
89 injury from the addition of dicamba to glyphosate and saflufenacil in non-dicamba-resistant soybean.
4.5 Conclusions
Based on these results, the most efficacious treatments for the control of GR Canada fleabane were glyphosate plus saflufenacil plus amitrole or dicamba (600 g a.i. ha-1). The application of amitrole applied PP is no longer allowed in Ontario; therefore, this herbicide is not an option for soybean growers for the control of GR Canada fleabane. Also, dicamba is not an option for Ontario soybean growers if the soybean cultivar is not dicamba-resistant, due to the risk of crop injury and yield reduction from dicamba. The other tankmix partners investigated provided similar control of GR Canada fleabane. The tankmix partners 2,4-D ester, metribuzin, and chloransulam-methyl provided excellent control of GR Canada fleabane and reduced density and biomass similar to amitrole. However, multiple-resistant Canada fleabane (glyphosate and chloransulam-methyl) has been reported in Ontario (Byker et al. 2013c), suggesting that 2,4-D ester and metribuzin are better tankmix partners with glyphosate plus saflufenacil in non- dicamba resistant soybean. The application of glyphosate with saflufenacil plus either, 2,4-D ester or metribuzin, can provide full-season residual control of GR Canada fleabane.
90 Table 4.1- Location, agronomic information, height and density of glyphosate-resistant Canada fleabane during field experiments conducted in Ontario, Canada in 2014 and 2015
Soil Characteristics (0-15cm) Seeding Spray Canada fleabaneZ Location Year Closest Texture OM (%) pH Date Date Size Density Year Town (cm) (# m-2) S1 2014 Mull Loam 3.1 6.6 June-10 June-9 up to 13 1344 S2 2014 Blenheim Sandy Loam 2.9 6.5 June-20 June-9 up to 11 652 S3 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-30 up to 14 234 S4 2015 Mull Loam 2.6 6.0 June-12 June-11 up to 9 1141 S5 2015 Blenheim Sandy Loam 4.2 6.2 June-6 June-4 up to 11 537 S6 2015 Harrow Sandy Loam 2.5 6.1 May-29 May-28 up to 14 153 Z Canada fleabane size and density at time of treatment application from untreated control plots
91 Table 4.2- Percent control of GR Canada fleabane at 4 and 8 weeks after treatment application (WAA) and density and biomass at 8 WAA of glyphosate plus saflufenacil plus tankmix partners and soybean yield during field experiments conducted across six locations in Ontario, Canada in 2014 and 2015
Percent Control Percent Control Weed Density Weed Biomass Soybean Rate 4 WAA 8 WAA 8 WAA 8 WAA Yield TreatmentZ (g a.i/a.e. ha-1) plants m-2 g m-2 tonnes ha-1 Untreated Control 0 0 708 a 291.9 a 1.02 c Weed Free Control 100 a 100 a 0 0 3.25 a G 900 25.5 d 9.5 d 510 a 208.8 a 1.53 bc G + S 900+25 98.8 c 87.9 c 31 b 33.5 b 2.71 a G + S + amitrole 900+25+2000 99.5 abc 96.7 abc 8 c 7.9 de 3.02 a G + S + 2,4-D ester 900+25+500 99.3 bc 94.6 bc 19 bc 19.1 bcd 2.98 a G + S + metribuzin 900+25+400 99.5 abc 96.4 bc 12 bc 22.2 bcd 2.86 a G + S + flumetsulam 900+25+70 99.4 abc 95.3 bc 24 bc 36.6 bc 2.90 a G + S + cloransulam-methyl 900+25+35 99.1 bc 94.8 bc 17 bc 21.0 bcd 2.87 a G + S + chlorimuron 900+25+9 98.6 c 89.9 bc 30 b 29.1 bc 2.71 a G + S + glufosinate 900+25+500 98.6 c 93.4 bc 27 bc 32.9 b 2.83 a G + S + paraquat 900+25+1100 99.3 bc 93.6 bc 22 bc 39.7 b 3.13 a G + S + dicamba 900+25+300 99.6 abc 94.8 bc 15 bc 8.9 cde 2.70 a G + S + dicamba 900+25+600 99.8 ab 97.5 ab 15 bc 4.8 e 2.39 ab Z Abbreviations: G= glyphosate and S= saflufenacil a-e Means followed by the same letter are not statistically different with the Tukey-Kramer multiple range test at P<0.05
92 Chapter 4: Control of glyphosate-resistant Canada fleabane with saflufenacil plus tankmix partners in soybean
4.1 Abstract
Glyphosate plus saflufenacil applied preplant (PP) has provided excellent control of glyphosate-resistant (GR) Canada fleabane in soybean in previous studies, but more recent research and commercial results in growers’ fields has demonstrated variable control of GR
Canada fleabane with this tankmix. The objective of this study was to determine if the level and consistency of GR Canada fleabane control with glyphosate plus saflufenacil could be improved with the addition of a third tankmix partner. Six field trials were conducted over a two-year period (2014, 2015) in fields with confirmed populations of GR Canada fleabane in ON. GR
Canada fleabane interference reduced soybean yield by 73% in this study compared to the weed free control. At 4 and 8 weeks after application (WAA), glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) provided 99 and 88% control of GR Canada fleabane respectively, and at 8 WAA, reduced GR Canada fleabane density by 96% and biomass 89% compared to the untreated control. Glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) improved the control of GR Canada fleabane to 100 and 97% at 4 and 8 WAA, respectively. At 8 WAA, glyphosate plus saflufenacil plus amitrole (2000 g a.i. ha-1) reduced GR Canada fleabane density and biomass 99 and 97%, respectively. At 8 WAA, glyphosate plus saflufenacil plus dicamba at
300 or 600 g a.i. ha-1 reduced GR Canada fleabane biomass 97 and 98%, respectively. There was no improvement in the control of GR Canada fleabane when 2,4-D, metribuzin, flumetsulam, cloransulam-methyl, chlorimuron, glufosinate or paraquat was added to the tankmix of glyphosate plus saflufenacil. Tank-mixing dicamba with glyphosate plus saflufenacil applied pre-plant in soybean improved control of GR Canada fleabane, however, this caused 14 and 46% crop injury at 2 and 4 WAA respectively. Soybean yield for saflufenacil alone and saflufenacil
81 tankmix treatments was similar to the weed free control (3.25 tonnes ha-1) with the exception of tank-mixing dicamba (600 g a.i. ha-1), that was less (2.39 tonnes ha-1) and similar to the glyphosate alone treatment.
4.2 Introduction
Canada fleabane is distributed throughout the Canadian provinces, except Newfoundland, and can be observed most frequently in the eastern parts of Canada (Weaver 2001; Cici and Van
Acker 2009). Canada fleabane is most commonly found on well-drained, coarse-textured, and minimally disturbed soils (Bhowmik and Bekech 1993; Weaver 2001). Highest Canada fleabane germination (61%) was reported at 24/20 C day/night temperatures and 13 hour photoperiod; however, it can also germinate (15%) under dark conditions (Nandula et al. 2006). Although
Canada fleabane can germinate and emerge throughout the year (Buhler and Owen 1997), it most frequently emerges between late August and October in Canada (Weaver 2001). Fall-emerging plants form a rosette in the fall and the stems elongate the following spring; a smaller portion of the population emerge in the spring/summer and have a summer annual growth habit (Weaver
2001). A 1.5-m tall Canada fleabane plant can produce up to 230,000 wind-dispersed seeds
(Weaver 2001), with 99% of them landing within 100 m of the mother plant, which can result in a rapid increase in population (Dauer et al. 2007).
Glyphosate is a systemic, broad-spectrum herbicide that was commercialized by
Monsanto in 1974 (Franz et al. 1997). Transgenic GR soybean and canola were commercialized in 1996 and since then, their adoption has been rapid (Dill 2005; Duke and Powles 2008). This rapid adoption can be attributed to improved crop safety, simplicity and broad-spectrum weed control with glyphosate applied postemergence (POST) (Dill 2005). Consequently, the use of glyphosate has increased tremendously since it can be applied pre-plant (PP) or POST in GR crops (Nandula et al. 2005). The use of diverse weed management strategies has decreased due
82 to the repeated use of glyphosate in GR crops (Powles 2008), which has resulted in intense selection pressure for glyphosate resistant biotypes (Powles et al. 1998; Nandula et al. 2005).
The first documented GR weed species was rigid ryegrass (Lolium rigidum Gaud.) in 1996
(Powles et al. 1998). As of 2015, there were 32 GR weed species globally including Canada fleabane (Heap 2016). In Canada, GR Canada fleabane was first documented in Essex county
Ontario in 2010 (Byker et al. 2013c). By 2012, it was reported in eight Ontario counties and its distribution in the province was expected to increase (Byker et al. 2013c).
GR Canada fleabane is a highly competitive weed species. Soybean yield can be reduced up to 93% (Byker et al. 2013b), while corn yield can be reduced up to 69% due to GR Canada fleabane interference if no control strategies are implemented (Ford et al. 2014a). Canada fleabane interference (25 plants m-2) can reduce cotton lint yield by 46% (Steckel and Gwathmey
2009). Overall, GR Canada fleabane can have a large impact on crop yields if not controlled.
In conventional tillage crop production systems, control of Canada fleabane using fall or spring tillage can be effective (Brown and Whitwell 1988; Kapusta 1979); however, only small plants are controlled with tillage (Shrestha et al. 2008). In no-tillage systems herbicides are used to control Canada fleabane (Bruce and Kells 1990). Pre-emergence (PRE) herbicides in soybean should be used to control GR Canada fleabane because POST herbicide options have limited control (Loux et al. 2006). Using a PRE herbicide with residual activity on Canada fleabane in soybean is desirable (Loux et al. 2006), since Canada fleabane can emerge after a non-residual burndown application such as glyphosate (Buhler and Owen 1997).
Saflufenacil is a protoporphyrinogen-oxidase (PPO) inhibiting herbicide (Grossman et al.
2010). When tankmixed with glyphosate, saflufenacil provides broad-spectrum control of grass and broadleaf weeds (Mellendorf et al. 2013). Saflufenacil can be used as a PP herbicide or as a desiccant in soybean in eastern Canada (Anonymous 2014a). GR Canada fleabane and other
83 herbicide resistant weeds such as ALS resistant prickly lettuce (Lactuca serriola L.) can be controlled by saflufenacil (Liebl et al. 2008; Soltani et al. 2010; Trolove et al. 2011); however, control of specific broadleaf weeds depends on application rate (Geier et al. 2009; Soltani et al.
2012). Furthermore, Mellendorf et al. (2013) found that the control of GR Canada fleabane increased as the rate of saflufenacil increased from 25 to 50 g a.i. ha-1 (80 to 95% control respectively), in a non-crop field study. At 7 and 30 days after planting, saflufenacil applied PP at 25 and 50 g a.i. ha-1 controlled GR Canada fleabane plants (cm) greater than 90% (Owen et al.
2011).
GR Canada fleabane control with glyphosate plus saflufenacil can be variable. For instance, at 28 days after POST application of glyphosate alone and glyphosate plus saflufenacil (25 g ai ha-1), GR Canada fleabane was controlled 37 and 57% respectively (Ikley 2012). Mellendorf et al. (2013) suggested a third herbicide with a different mode of action should be added to glyphosate plus saflufenacil for more consistent control of GR Canada fleabane. Therefore, the objective of this study was to determine the level of GR Canada fleabane control and soybean crop injury with glyphosate and saflufenacil plus a third tankmix partner applied PP in soybean.
It is hypothesised that full season, residual control of GR Canada fleabane can be achieved in soybean when an effective tankmix partner is added to glyphosate plus saflufenacil.
4.3 Materials and Methods
Six field trials were conducted over a two-year period (2014, 2015) at three field locations in southwestern Ontario with previously confirmed GR Canada fleabane populations to determine the efficacy of glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) plus a third tankmix partner applied PP in soybean. The experiments were arranged as a randomized complete block design with four replications in each field. The plots were 2.25 m wide with 3 soybean rows spaced 0.75 m apart and 8 m in length. All herbicide treatments were applied PP
84 -1 using a CO2 pressurized backpack sprayer that was calibrated to deliver 200 L ha of spray mixture at 240 kPa. The boom was 1.5-m wide with four ULD 120-02 nozzles (Hypro, New
Brighton, MN) spaced 50-cm apart. Each replication included an untreated (weedy) and weed- free control. The weed-free control was maintained weed free with a glyphosate (1800 g a.e. ha-
1), saflufenacil (25 g a.i. ha-1) and metribuzin (400 g a.i. ha-1) tankmix applied PP followed by hand hoeing as required. A cover spray of quizalofop-p-ethyl (36 g a.i. ha-1) in 2014 and glyphosate (900 g a.i. ha-1) in 2015 was applied to remove potentially confounding effects of other weed species. Location, soil characteristics, seeding date, herbicide application date and
Canada fleabane height and density at time of application are listed in Table 4.1. Herbicide treatments and rates applied are listed in Table 4.2.
Soybean injury was assessed visually 2 and 4 weeks after soybean emergence (WAE) on a scale of 0 (no injury) to 100% (plant death). Canada fleabane control was assessed visually 4 and 8 weeks after application (WAA) on a scale of 0 to 100% where 0 was no control and 100 was plant death. Canada fleabane density and dry weight were determined 8 WAA by counting the plants in two, 0.25 m2 quadrants per plot. The same plants were cut at the soil surface, placed in paper bags, and dried at 60°C to a constant moisture and weighed (Byker et al. 2013a).
Soybean seed yield was determined at maturity from a 2-m length of the center row of each plot by the use of a stationary threshing machine. The grain moisture content and weight were recorded for each plot. Soybean grain yield is reported in tonnes ha-1 at a grain moisture of 13%.
Data were analyzed using PROC GLIMMIX in SAS (Ver. 9.4, SAS Institute Inc., Cary,
NC). Variances were partitioned into random effects (environment within year and location, replication within environment, and the environment by treatment interaction), and fixed effects
(herbicide treatments). A likelihood ratio was used to test the significance of environment, replication within environment, and environment by treatment interactions from zero (Dr. S.
85 Bowley, Personal Communication). There was no significant environment by treatment interaction so all environments were combined for analysis. The significance of fixed effects was tested using the F-test. Residual plots were used to ensure that the variances were randomly distributed, independent, and homogeneous across treatments. Means were separated using the
Tukey-Kramer multiple range test at P<0.05 using the pdmix800 SAS macro (Saxton 1998).
The estimation method used was the Laplace method which obtains solutions to the likelihood equations through integral approximation of the log-likelihood and provides unbiased covariance parameter estimates when the number of observations per subject is small, compared to the pseudo-likelihood approach (Gbur et al. 2012). Weed control data at 4 and 8 WAA were analysed by specifying a beta distribution and a cumulative complementary log-log link function due to controls (Gbur et al. 2012; Dr. S. Bowley, Personal Communication). Also, untreated and weed free controls were adjusted by adding or subtracting 1.0 x 10-10 to lie within the beta distribution. Weed density and weed biomass data were analyzed by specifying a gamma distribution and the default log link function because it is flexible and can accommodate many distributional shapes and skewed responses (Gbur et al. 2012). Soybean yield was analysed by specifying a normal distribution and the default identity link function.
4.4 Results and Discussion
There was no crop injury with the exception of glyphosate plus saflufenacil plus dicamba
(data not shown). At 2 and 4 WAA, glyphosate plus saflufenacil plus dicamba (300 g a.i. ha-1) caused 11 and 37% soybean injury, respectively. With the higher rate of dicamba, glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) caused 14 and 46% soybean injury, respectively.
The injury included stunted plants, leaf cupping and dead plants caused by the dicamba and the short interval between spraying and soybean seeding. This injury was expected because dicamba-resistant soybean was not used in this study.
86 At 4 WAA, glyphosate and glyphosate plus saflufenacil provided 25.5 and 98.8% control of GR Canada fleabane, respectively (Table 4.2), which is similar to the results reported by
Byker et al. (2013b); however, this contrasts to Ikley (2012) who found 57% control with glyphosate plus saflufenacil (25 g a.i. ha-1) in a greenhouse study. The addition of a tankmix partner to glyphosate plus saflufenacil did not increase GR Canada fleabane control with the exception of dicamba (600 g a.i. ha-1) that provided 99.8% control. The results from this study are similar to the 99, 94, 94, 97% control of Canada fleabane with chloransulam-methyl, chlorimuron, flumetsulam, and 2,4-D at 4 WAA, respectively (Tardif and Smith 2003). In contrast to the results from this study where glyphosate plus saflufenacil plus metribuzin provided 99.5% control, Tardif and Smith (2003) reported metribuzin (1120 g ai ha-1) provided only 73% control of Canada fleabane while Byker et al. (2013a) reported 99% control of GR
Canada fleabane with glyphosate (900 g ai ha-1) plus metribuzin (1120 g ai ha-1). These results are similar with either glyphosate plus 2,4-D (840 g a.i. ha-1) or glyphosate plus dicamba (280 g a.i. ha-1) tankmixes that both provided over 90% control at 4 WAA, but in contrast to glyphosate plus metribuzin (420 g a.i. ha-1) that provided 58% control of GR Canada fleabane (Eubank et al.
2008).
At 8 WAA, glyphosate and glyphosate plus saflufenacil provided 98.8 and 87.9% control of GR Canada fleabane, respectively (Table 4.2.). There was a numeric increase in GR Canada fleabane control by adding a tankmix partner to glyphosate plus saflufenacil, but differences were not always statistically significant. Glyphosate plus saflufenacil plus amitrole, and glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) provided 96.7 and 97.5% control respectively, which was equivalent to the weed free control. Glyphosate plus saflufenacil plus dicamba (600 g a.i. ha-1) was the only treatment that provided statistically greater control than glyphosate plus saflufenacil. These results are similar to Byker et al. (2013b) where glyphosate
87 plus dicamba (600 g a.i. ha-1) applied PP, followed by glyphosate applied POST, provided 97% control of GR Canada fleabane. In contrast, Byker et al. (2013b) found that glyphosate plus 2,4-
D (560 g a.i. ha-1) applied PP, followed by glyphosate applied POST, provided 77% control 4
WAA. Loux et al. (2006) reported that glyphosate plus 2,4-D plus chlorimuron or chloransulam, glyphosate plus 2,4-D, or glyphosate plus metribuzin provided the best control of GR Canada fleabane. The results from this study are similar to Loux (2014) where glyphosate plus saflufenacil plus 2,4-D, dicamba, metribuzin or chloransulam-methyl are very effective for the control GR Canada fleabane.
At 8 WAA, glyphosate reduced GR Canada fleabane density by 28%, which was equivalent to the untreated control (Table 4.2). Glyphosate plus saflufenacil reduced GR Canada fleabane density by 96%. Davis et al. (2010) reported that saflufenacil applied at 50 and 100 g a.i. ha-1 reduced GR Canada fleabane densities by 78 and 86% compared to the untreated control respectively, in a no-till fallow field study. The addition of a tankmix partner to glyphosate plus saflufenacil did not reduce GR Canada fleabane density with the exception of amitrole, which reduced GR Canada fleabane density by 99%. All the other three-way tankmixes resulted in GR
Canada fleabane density that was equivalent to glyphosate plus saflufenacil. The results from this study are similar to observations by Eubank et al. (2008), who found glyphosate plus 2,4-D (840 g a.i. ha-1), and glyphosate plus dicamba (280 g a.i. ha-1), reduced GR Canada fleabane density by 99 and 98%, respectively. In contrast, Eubank et al. (2008) found glyphosate plus metribuzin
(420 g a.i. ha-1) reduced GR Canada fleabane density by 66%.
At 8 WAA, glyphosate alone and glyphosate plus saflufenacil reduced GR Canada fleabane biomass 28 and 89%, respectively, compared to the untreated control (Table 4.2).
Similarly, Byker et al. (2013a) reported 99% reduction in Canada fleabane biomass at 4 WAA with glyphosate plus saflufenacil in soybean under field conditions. In contrast Ikley (2012)
88 reported glyphosate plus saflufenacil provided a 45% reduction in GR Canada fleabane biomass in a greenhouse study 4 WAA. The addition of another tankmix partner to glyphosate plus saflufenacil did not reduce GR Canada fleabane biomass compared to the untreated control with the exception of adding amitrole, dicamba at either 300 g a.i. ha-1 or 600 g a.i. ha-1, which reduced biomass by >97%. These results are similar to Byker et al. (2013a; 2013b), who reported glyphosate plus amitrole (4 WAA), and glyphosate plus dicamba (300 or 600 g a.i. ha-1) (8
WAA), reduced GR Canada fleabane biomass by greater than 97%. The addition of flumetsulam, glufosinate and paraquat to glyphosate plus saflufenacil reduced GR Canada fleabane biomass by
87, 89 and 86%, respectively, which was less than when amitrole or dicamba (300 or 600 g a.i. ha-1) were added to glyphosate plus saflufenacil. The addition of 2,4-D ester, metribuzin, or cloransulam-methyl reduced GR Canada fleabane biomass by 93%, 92%, and 93%, respectively, which was equivalent to the addition of amitrole or dicamba (300 g a.i. ha-1). These results are similar to Byker et al. (2013a) where glyphosate plus 2,4-D (500 g a.i. ha-1), and glyphosate plus chloransulam-methyl reduced GR Canada fleabane biomass by 95 and 93%, respectively.
GR Canada fleabane interference reduced soybean yield by 73% compared to the weed free control (Table 4.2). This is similar to the 82% yield reduction between the untreated control and the most efficacious treatment found by Eubank et al. (2008) that was glyphosate plus 2,4-D
(840 g a.i. ha-1). In this study, glyphosate alone resulted in an average soybean yield (1.53 tonnes ha-1), similar to the untreated control (1.02 tonnes ha-1). The addition of saflufenacil to glyphosate resulted in soybean yield equivalent to the weed free control (2.71 and 3.25 tonnes ha-1 respectively). The addition of a third tankmix partner to glyphosate plus saflufenacil resulted in soybean yield that was equivalent to the weed free control (3.25 tonnes ha-1), with the exception of dicamba (600 g a.i. ha-1), that resulted in a 37% reduction in soybean yield compared to the weed-free control. This yield reduction was expected because of the observed
89 injury from the addition of dicamba to glyphosate and saflufenacil in non-dicamba-resistant soybean.
4.5 Conclusions
Based on these results, the most efficacious treatments for the control of GR Canada fleabane were glyphosate plus saflufenacil plus amitrole or dicamba (600 g a.i. ha-1). The application of amitrole applied PP is no longer allowed in Ontario; therefore, this herbicide is not an option for soybean growers for the control of GR Canada fleabane. Also, dicamba is not an option for Ontario soybean growers if the soybean cultivar is not dicamba-resistant, due to the risk of crop injury and yield reduction from dicamba. The other tankmix partners investigated provided similar control of GR Canada fleabane. The tankmix partners 2,4-D ester, metribuzin, and chloransulam-methyl provided excellent control of GR Canada fleabane and reduced density and biomass similar to amitrole. However, multiple-resistant Canada fleabane (glyphosate and chloransulam-methyl) has been reported in Ontario (Byker et al. 2013c), suggesting that 2,4-D ester and metribuzin are better tankmix partners with glyphosate plus saflufenacil in non- dicamba resistant soybean. The application of glyphosate with saflufenacil plus either, 2,4-D ester or metribuzin, can provide full-season residual control of GR Canada fleabane.
90 Table 4.1- Location, agronomic information, height and density of glyphosate-resistant Canada fleabane during field experiments conducted in Ontario, Canada in 2014 and 2015
Soil Characteristics (0-15cm) Seeding Spray Canada fleabaneZ Location Year Closest Texture OM (%) pH Date Date Size Density Year Town (cm) (# m-2) S1 2014 Mull Loam 3.1 6.6 June-10 June-9 up to 13 1344 S2 2014 Blenheim Sandy Loam 2.9 6.5 June-20 June-9 up to 11 652 S3 2014 Harrow Sandy Loam 2.1 6.6 June-2 May-30 up to 14 234 S4 2015 Mull Loam 2.6 6.0 June-12 June-11 up to 9 1141 S5 2015 Blenheim Sandy Loam 4.2 6.2 June-6 June-4 up to 11 537 S6 2015 Harrow Sandy Loam 2.5 6.1 May-29 May-28 up to 14 153 Z Canada fleabane size and density at time of treatment application from untreated control plots
91 Table 4.2- Percent control of GR Canada fleabane at 4 and 8 weeks after treatment application (WAA) and density and biomass at 8 WAA of glyphosate plus saflufenacil plus tankmix partners and soybean yield during field experiments conducted across six locations in Ontario, Canada in 2014 and 2015
Percent Control Percent Control Weed Density Weed Biomass Soybean Rate 4 WAA 8 WAA 8 WAA 8 WAA Yield TreatmentZ (g a.i/a.e. ha-1) plants m-2 g m-2 tonnes ha-1 Untreated Control 0 0 708 a 291.9 a 1.02 c Weed Free Control 100 a 100 a 0 0 3.25 a G 900 25.5 d 9.5 d 510 a 208.8 a 1.53 bc G + S 900+25 98.8 c 87.9 c 31 b 33.5 b 2.71 a G + S + amitrole 900+25+2000 99.5 abc 96.7 abc 8 c 7.9 de 3.02 a G + S + 2,4-D ester 900+25+500 99.3 bc 94.6 bc 19 bc 19.1 bcd 2.98 a G + S + metribuzin 900+25+400 99.5 abc 96.4 bc 12 bc 22.2 bcd 2.86 a G + S + flumetsulam 900+25+70 99.4 abc 95.3 bc 24 bc 36.6 bc 2.90 a G + S + cloransulam-methyl 900+25+35 99.1 bc 94.8 bc 17 bc 21.0 bcd 2.87 a G + S + chlorimuron 900+25+9 98.6 c 89.9 bc 30 b 29.1 bc 2.71 a G + S + glufosinate 900+25+500 98.6 c 93.4 bc 27 bc 32.9 b 2.83 a G + S + paraquat 900+25+1100 99.3 bc 93.6 bc 22 bc 39.7 b 3.13 a G + S + dicamba 900+25+300 99.6 abc 94.8 bc 15 bc 8.9 cde 2.70 a G + S + dicamba 900+25+600 99.8 ab 97.5 ab 15 bc 4.8 e 2.39 ab Z Abbreviations: G= glyphosate and S= saflufenacil a-e Means followed by the same letter are not statistically different with the Tukey-Kramer multiple range test at P<0.05
92 Chapter 5: Efficacy of saflufenacil for control of glyphosate-resistant Canada fleabane [Conyza canadensis] as affected by height, density and time of day
5.1 Abstract
Control of glyphosate-resistant (GR) Canada fleabane in soybean with glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) has been variable. The objective of this research was to determine the effect of GR Canada fleabane height and density, and time of day (TOD) of application on saflufenacil plus glyphosate efficacy in soybean. All experiments were completed six times during a two-year period (2014, 2015) in fields previously confirmed with GR Canada fleabane. Applications from 09:00- 21:00 h provided optimal control of GR Canada fleabane 8
WAA. Soybean yield paralleled GR Canada fleabane control with the highest yield of 3.0 t ha-1 at 15:00 h, and the lowest yield of 2.4 t ha-1 at 06:00 h. The height and density of GR Canada fleabane at application had minimal effect on saflufenacil efficacy. Saflufenacil provided >99% control of GR Canada fleabane when applied to small plants and low densities; however, control decreased to 95% where the weed was >25 cm tall, and to 96% in densities >800 plants m-2 at 6
WAA due to some plant regrowth. TOD of application had a greater influence on GR Canada fleabane control with saflufenacil than height or density. To optimize control of GR Canada fleabane, saflufenacil should be applied during daytime hours to small plants at low densities.
Optimizing GR Canada fleabane control minimizes weed seed return and weed interference.
5.2 Introduction
The first GR broadleaf weed in the world was Canada fleabane; its existence was confirmed first in Delaware, USA in 2000 (VanGessel 2001). In Canada, GR Canada fleabane was first reported in Essex County, Ontario in 2010 and by 2012 it had been documented in eight counties within Ontario (Byker et al. 2013c). Some biotypes of Canada fleabane in Ontario are
93 resistant to glyphosate and cloransulam-methyl and are classified as multiple-resistant biotypes
(Byker et al. 2013c). The spread of resistant biotypes is aided by the morphology of Canada fleabane. Self-compatibility and the ability to produce a large number of wind-dispersed seeds per plant allows resistant biotypes to easily spread to neighbouring areas (Weaver 2001; Zelaya et al. 2004). Since Canada fleabane can germinate in undisturbed soils, in crop and non-crop land there is a large area for resistant biotypes to establish and spread (Nandula et al. 2005).
Canada fleabane is a winter or summer annual weed in the Asteraceae family (Frankton and Mulligan 1987). The seeds of Canada fleabane are very small, 1 to 2 mm long (Frankton and
Mulligan 1987), with an attached pappus that aids in wind dispersal (Royer and Dickenson
1999). Canada fleabane has small, ovate shaped, hairless cotyledons that are 1 to 2-mm wide and
2- to 3.5-mm long (Royer and Dickenson 1999). If plants germinate in the fall, a basal rosette forms with spatula-shaped, hairy leaves with coarsely-toothed margins; the following spring the stem elongates while the rosette deteriorates (Frankton and Mulligan 1987). A basal rosette is not formed by spring-germinated Canada fleabane (Bhowmik and Bekech 1993). The mature leaves of Canada fleabane have an alternate arrangement on the stem, are oblong to lance shaped, bristly haired, and range from 2- to 10-cm in length (Royer and Dickenson 1999). The margins of mature leaves are slightly toothed or smooth and the upper leaves are shorter in length (Royer and Dickenson 1999). Canada fleabane has a single erect stem that is densely covered in hairs
(Loux et al. 2006), and can grow up to 180 cm in height (Frankton and Mulligan 1987). As the plant matures, numerous flowering branches emerge containing many small flower heads on branched terminal clusters (Frankton and Mulligan 1987; Royer and Dickenson 1999). Each of these flowers are self-compatible (Weaver 2001) and primarily self-pollinated (Smisek 1995).
The number of flower heads and corresponding seed production by a Canada fleabane plant is correlated with stem height (Regehr and Bazzaz 1979; Smisek 1995). A 40-cm tall Canada
94 fleabane plant can produce approximately 2,000 seeds, while a plant 1.5-m in height can produce
230,000 seeds (Weaver 2001).
Postemergent (POST) herbicides in soybean (Glycine max L. Merr.) are not effective for control of GR Canada fleabane (Davis et al. 2009). PP herbicides have been shown to provide effective control of GR Canada fleabane. At 8 weeks after application (WAA), glyphosate (900 g a.i. ha-1) plus 2,4-D at 560 and 1120 g a.i. ha-1 controlled GR Canada fleabane 73-95 and 85-
95%, respectively. (Byker et al. 2013b). At 8 WAA, glyphosate (900 g a.i. ha-1) plus metribuzin
(1120 g a.i. ha-1) applied PP, controlled GR Canada fleabane 97-99% (Byker et al. 2013b); however, soybean injury can occur from high rates of metribuzin, especially on coarse-textured, high pH soils.
Saflufenacil is a group 14, protoporphyrinogen IX oxidase (PPO) inhibiting herbicide in the pyrimidinedione chemical family (Grossman et al. 2010) that can provide effective control of
GR Canada fleabane. Byker et al (2013b) reported that saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) provided 88-100% control 4 WAA in soybean. Variable results were reported by Ford et al. (2014b), where saflufenacil (50 g a.i. ha-1) provided 98-100% control 8
WAA at four of five sites, but only 49% control was obtained at the fifth site. Other’s have reported variable control of GR Canada fleabane with saflufenacil in grower’s fields and published literature. For example, Ikley (2012) reported saflufenacil (25 g a.i. ha-1) applied with methylated seed oil (MSO) at 1% v/v and ammonium sulphate (AMS) at 2% v/v provided 35, 32 and 30% control of GR Canada fleabane at 7, 14, and 28 DAA, respectively in a greenhouse study. The addition of glyphosate (900 g a.i. ha-1) to saflufenacil (25 g a.i. ha-1) plus MSO (1% v/v) and AMS (2% v/v) improved the control to 61, 67, and 57% at 7, 14, and 28 DAA, respectively (Ikley 2012). Reports from published literature and grower’s fields indicate variable control of GR Canada fleabane with saflufenacil.
95 The time of day (TOD) of herbicide application can affect herbicide efficacy, although this is dependent on the herbicide and weed species (Stewart et al. 2009). Several factors that change throughout the day such as: air temperature, relative humidity (RH) and light intensity cause plant physiological changes that may account for variable weed control due to TOD
(Stewart et al. 2009). Cuticular wax and plasma membrane fluidity increases with air temperature, which results in greater herbicide uptake (Johnson and Young 2002). RH can affect weed control as reported by Coezter et al. (2001), where redroot pigweed (Amaranthus retroflexus L.) control with glufosinate increased as RH was increased from 35-90% from studies conducted in growth chambers. In contrast, Stewart et al. (2009) did not observe a correlation between RH and herbicide efficacy. During early morning and late evening applications, heavy dew on plant leaves has been suspected of causing herbicide runoff and/or dilution (Doran and
Andersen 1976); however, Stewart et al. (2009) concluded that dew did not contribute to the
TOD effect as much as other factors. Some weed species have been reported to change their leaf angle in response to light availability, such as redroot pigweed, common lambsquarters
(Chenopodium album L.) and velvetleaf (Abutilon theophrasti Medic.) (Andersen and Koukkari
1979; Kraatz and Andersen 1980). Leaf angle changes could decrease herbicide efficacy due to less leaf area to intercept the herbicide, as Stewart et al. (2009) suggested leaf angle variations may have contributed to greater weed control between 09:00 and 18:00 h.
Due to the long period of Canada fleabane germination and emergence (Weaver 2001) there can be a range of plant heights and densities at the time of PP herbicide application. In one year of a two-year study by Mellendorf et al. (2013), control of GR Canada fleabane with saflufenacil (25 g a.i. ha-1) applied with 1% v/v crop oil concentrate, was greater than 98% for plants 5 to 45 cm in height. In the first year of the Mellendorf et al. (2013) study, GR Canada fleabane control was decreased by 1% for each 8 cm increase in plant height with glyphosate
96 (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1); however, control was still greater than 94%.
Reduced control of GR Canada fleabane with saflufenacil (25 g a.i. ha-1) alone has been attributed to extensive regrowth from large rosettes (Ikley 2012). Ikley (2012) suggested that saflufenacil likely did not translocate past the apical meristem and toward the roots because plants in the rosette form have other growing points protected by the apical meristem. Having optimal criteria for foliar herbicide applications is important to avoid variability in weed control, such as: optimal spray coverage, proper adjuvant system, optimal environmental conditions (i.e. adequate sunlight and warm air temperatures), and relatively small weed size; these factors promote optimal foliar activity of a PPO-inhibiting herbicide such as saflufenacil (Mellendorf et al. 2013).
GR Canada fleabane can result in soybean yield losses of up to 93% where no weed management tactics are employed (Byker et al. 2013b). Poor control of GR Canada fleabane results in increased seed production (Weaver 2001) and wide spread wind-dispersal of 550 km from the parent plant (Shields et al. 2006). Since POST herbicides in soybean do not provide consistent control of GR Canada fleabane, PP or PRE herbicides must be used especially in reduced/no-till soybean production (Loux et al. 2006; Davis et al. 2009). Excellent control of GR
Canada fleabane can sometimes be achieved with saflufenacil (Byker et al. 2013a); however, an inquiry into the factors that cause variable control is needed. The objective of these studies was to determine the effect of TOD of application and the effect of GR Canada fleabane height and density, on control with saflufenacil in soybean. It is hypothesized that GR Canada fleabane can be controlled in soybean with saflufenacil by optimizing the time of application.
5.3 Materials and Methods
Three separate field studies were conducted to evaluate the effect of GR Canada fleabane height and density, and TOD on control with glyphosate plus saflufenacil. These studies were
97 conducted in field sites previously confirmed with GR Canada fleabane. Each study had six location-years over a two-year period (2014, 2015), totaling 18 field trials. A randomized complete block design with four replications was used for the TOD trial. The trial plots were
2.25-m wide by 8-m in length with 3 rows of soybean spaced 0.75-m apart. The treatments were saflufenacil applied at seven different times of the same day for each location year, from 06:00- to 24:00 h at three hour intervals. A weedy and weed-free control were included in each replicate.
The height and density trials had four untreated and four treated replicates, each 38-m long and 3-m wide, alternating in sequence. The treatments for the height trial were seven different categories of GR Canada fleabane plant heights from up to one cm, to greater than 25 cm. The height trial had 10 Canada fleabane plants (subsamples) per treatment, randomly distributed throughout one half of the trial replicates, totaling 70 plants. This was repeated for the other half of the replicate for a total of 140 plants per trial. Each plant in the height trial was marked with a wire flag that was angled in the same direction as the spray would be applied, to avoid interference with spray deposition. The GR Canada fleabane density trial had seven density treatments ranging from 1-20 Canada fleabane plants m-2, up to greater than 800 plants m-2. Each treatment had ten, 0.25 m-2 quadrants, randomly distributed throughout half of the replicate totalling 70 quadrants. This was repeated for the other half of the replicate for a total of
140 quadrants per trial. Quadrants and flags were randomly distributed throughout the trial area; however, GR Canada fleabane heights and densities needed to represent the associated treatment so placement was random, but representative of the treatment. Spray application occurred after all quadrants or flags were placed.
A backpack sprayer calibrated to deliver 200 L ha-1 of spray mixture at 240 kPa was used to apply all herbicide treatments. All applications for the TOD study were PP to soybean, while
98 the height and density trials were conducted in a non-crop area with the herbicide applied POST to the GR Canada fleabane. The sprayer boom was 1.5-m wide with four ULD120-02 nozzles
(Hypro, New Brighton, MN) spaced 50- cm apart. Untreated (weedy) and weed-free controls were included in each replicate of the TOD trials. The weed-free control was treated with a PP tankmix of glyphosate (1800 g a.i. ha-1), saflufenacil (25 g a.i. ha-1), and metribuzin (400 g a.i. ha-1), followed by hand hoeing if required. The herbicide application for all trials across all treatments was a tankmix of glyphosate (900 g a.i. ha-1), saflufenacil (25 g a.i. ha-1), and Merge© surfactant (1 L ha-1). Quizalofop-p-ethyl (36 g a.i. ha-1) and glyphosate (900 g a.i. ha-1) were applied as cover sprays for the TOD trial in 2014 and 2015 respectively to remove potentially confounding effects of other weed species. Pictures were taken of randomly marked plants in
TOD trials to visually inspect leaf angle variation across application times; leaf angle was not measured. Information on herbicide application dates, herbicide application times, seeding dates and soil characteristics are listed in Table 5.1. Details on the environmental conditions at each application for all studies are listed in Table 5.2.
Control of GR Canada fleabane was visually assessed at 1, 2, and 4 WAA for all studies,
6 WAA in the height and density studies, and 8 WAA in the TOD study. A scale of 0 to 100% was used where 0 was no control and 100 was plant death. In the TOD study, at 8 WAA, GR
Canada fleabane dry weight and density were measured by counting the number of plants in two,
0.25 m2 quadrants per plot, and then cutting them at the soil surface. Cut plants were placed into paper bags and dried to a constant weight at 60°C and then weighed. Dry weight was similarly measured in the density study; however, all marked quadrants were harvested at 6 WAA; the height trial was similar but only involved the individually marked plants for dry weight. Soybean yield was measured for the TOD study by pulling a 2-m length from the center row and threshing it in a stationary threshing machine at soybean maturity. The weight and moisture content of the
99 harvested grain was recorded for each plot. The soybean grain yield is presented in t ha-1 at 13% grain moisture.
The PROC NLIN procedure in SAS (Ver. 9.4, SAS Institute Inc., Cary NC) was used to analyze the responses in all studies. For analysis all environments were combined for each study
(Chapter 4). The weed-free and weedy controls were not included in the regression analysis. For the TOD study, all parameters were fit to a quadratic parabolic curve model (Equation [1]) and
PROC REG was used to test for lack of fit. The lack of fit for the linear and quadratic terms was not significant, confirming the appropriateness of the quadratic parabolic model. Since Equation
[1] includes an estimated parameter by the regression output, a one-sided hypothesis test could be conducted to determine confidence bounds (UCLA: Statistical Consulting Group). Equation
[4] was used to calculate the critical value for the estimate of the parameter maximum/minimum to determine treatment differences (UCLA: Statistical Consulting Group). If the parameter estimate for a treatment is within the bounds between estimate maximum/minimum and the critical value, it is not significantly different (P>0.05).
The GR Canada fleabane height study used a linear model (Equation [2]) for all parameters because the height treatments were equally spaced with the exception of the up to 1 cm treatment, and lack of fit was not significant (>0.05). In the GR Canada fleabane density study, all parameters were fit to an exponential (Equation [3]) or linear model. If lack of fit was not significant for the linear model, then it was used. When lack of fit was significant for the linear model it was tested for the exponential model, and if not significant the exponential model was applied. The predicted values in Tables 3-5 were generated using the appropriate regression model based on lack of fit.
In the TOD study, all parameters were regressed against time of application, represented by TIME in the equation. For the height study, all parameters were regressed against GR Canada
100 fleabane height at application, represented by HEIGHT in the equation. In the density study, all parameters were regressed against GR Canada fleabane density at application, represented by
DENSITY in the equation.
Quadratic parabolic curve model Y= c (TIME – a)2 + b Equation [1]
Where a is the TOD at the vertex, b is the predicted value at the vertex, and c is the
constant.
Linear model Y= a (HEIGHT) + b Equation [2]
Where a is the slope, and b is the intercept.
Exponential model Y= a e(b DENSITY) Equation [3]
Where a is the magnitude and b is the slope.
Critical value C = d – (tobs x se) Equation [4]
Where d is the estimate of the parameter maximum/minimum, tobs is the value from a
one-sided T table that corresponds to P (0.05) and degrees of freedom associated with the
residual sum of squares, and se is the standard error of the parameter.
5.4 Results and Discussion
5.4.1 Study 1. Effect of Time of Day on Glyphosate plus Saflufenacil Efficacy
Control of GR Canada fleabane was consistently greater than 90% at 1 and 2 WAA at all times of day (TOD) with glyphosate plus saflufenacil. At 1 WAA, glyphosate plus saflufenacil provided greater than 97% control of GR Canada fleabane across all TODs (Table 5.3). The highest control of 99% was from 06:00-18:00 h. At 2 WAA, glyphosate plus saflufenacil provided 92-96% control of GR Canada fleabane with the greatest control from 09:00- 21:00 h.
Glyphosate plus saflufenacil provided the lowest control of 93 and 92% when applied at 06:00 and 24:00 h, respectively. The results from this study are similar to Byker et al. (2013a) who
101 reported that saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) provided 98% and greater than 90% control at 1 and 2 WAA, respectively.
At 4 WAA, glyphosate plus saflufenacil provided 89 to 97% control of GR Canada fleabane with the greatest control from 09:00- 18:00 h (Table 5.3). Control was less at 06:00 h and from applications at 21:00 h and later, decreasing to 93% control at 06:00 h and 89% control at 24:00 h. At 4 WAA, Byker et al. (2013a) found that saflufenacil (25 g a.i. ha-1) plus glyphosate (900 g a.i. ha-1) provided greater than 95% control which is consistent to these findings when applied between 9:00 and 18:00 h. A higher dose of saflufenacil (50 g a.i. ha-1) with glyphosate (900 g a.i. ha-1) was used by Ford et al. (2014b), and consistent with the results of this study they reported greater than 93% control of GR Canada fleabane at 4 WAA at four sites, however in contrast, one site had 44% control. Where Ford et al. (2014b) had excellent control the TOD of application was 10:00 h at two sites and 17:00 h at the other two; the site with 44% control was sprayed at 10:00 h.
At 8 WAA, the largest range in control of GR Canada fleabane was observed due to TOD application of glyphosate plus saflufenacil. Saflufenacil applied at 15:00 h provided 89% control of GR Canada fleabane (Table 5.3), with applications at 6:00 and 24:00 h providing 80 and 78%, respectively. In contrast, Ford et al. (2014b) reported greater than 98% control of GR Canada fleabane at four sites with glyphosate plus saflufenacil, however the rate was 50 g a.i. ha-1; in contrast, at one site only 49% control was obtained. Ford et al. (2014b) stated the site with less control may be due to the GR Canada fleabane present was primarily fall-germinated and plants were relatively larger at application than at the other four sites. Similarly to the maximum control in this study, Chapter 4 reported 88% control of GR Canada fleabane with glyphosate plus saflufenacil applied from 06:30- 10:00 h across six sites.
102 The density of GR Canada fleabane was lowest when glyphosate plus saflufenacil was applied from 09:00- 21:00 h, which is an inverse of the control data (Table 5.3). The density of
GR Canada fleabane was greater for applications at 06:00 and 24:00 h, with 153 plants m-2 and
185 plants m-2 respectively. There was a decrease in GR Canada fleabane density of 69% when glyphosate plus saflufenacil was applied at 24:00 h compared to 15:00 h. Where Ford et al.
(2014b) had excellent control with glyphosate plus saflufenacil (50 g a.i. ha-1), GR Canada fleabane density was less than one plant m-2 across four sites but was 38 plants m-2 where control was low (49% control at 8 WAA). In contrast, Mellendorf et al. (2013) reported excellent control of GR Canada fleabane with a 91 and 97% reduction in density when saflufenacil was applied at
25 and 50 g a.i. ha-1, respectively.
At 8 WAA, the GR Canada fleabane dry weight followed the same trend as the density data. Dry weight data was lowest at the 15:00 h application timing (Table 5.3). Earlier and later application times had greater dry weight with 62 g m-2 at 06:00 h and 78 g m-2 at 24:00 h. There was a 50% decrease in dry weight when glyphosate plus saflufenacil was applied at 15:00 compared to 24:00 h (39 g m-2 at 15:00 h and 78 g m-2 at 24:00 h). Where Ford et al (2014b) had excellent control of GR Canada fleabane with glyphosate plus saflufenacil (50 g a.i. ha-1) at four sites, dry weight was one g m-2 or less, and 183 g m-2 at one site with low control (49% control at
8 WAA) which is a much wider range in dry weight than observed in this study.
The 09:00- 21:00 h TOD range resulted in the greatest control of GR Canada fleabane at
8 WAA and also resulted in the highest soybean yield. The soybean yield maximum of 3.0 t ha-1 was obtained when glyphosate plus saflufenacil was applied at 15:00 h (Table 5.3). Applications earlier or later than 15:00 h resulted in incrementally less soybean yield of 2.4 and 2.6 t ha-1 when glyphosate plus saflufenacil was applied at 6:00 and 24:00 h, respectively. The reason for differences in soybean yield among herbicide application timings can be attributed to differences
103 in weed interference as indicated by the control, density and dry weight data. The difference in soybean yield due to TOD was 0.6 t ha-1 or 20% (3.0 t ha-1 at 15:00 h and of 2.4 t ha-1at 6:00 h).
Similarly, Stewart et al. (2009) reported reduced corn yield for some herbicides when applied early in the day (glufosinate, diflufenzopyr/dicamba, and bromoxynil) or late in the day
(glufosinate, diflufenzopyr/dicamba, bromoxynil, atrazine, glyphosate, and nicosulfuron).
Where weed control was greatest, air temperature was greatest, cloud cover was lowest, relative humidity was lowest and dew was not present (Table 5.2). None of these factors were individually controlled so we were unable to determine the relative influence of each factor on weed control. Stewart et al. (2009), concluded that air temperature had the largest influence on herbicide efficacy while relative humidity and dew did not impact herbicide efficacy appreciably.
Upon visual inspection of the images taken of marked plants at each application time, leaf angle for Canada fleabane leaves did not appear to change throughout the day (not shown).
In contrast, velvetleaf, common lambsquarters, and redroot pigweed can have differing leaf angles throughout the day, resulting in reduced herbicide interception and retention with herbicides applied either early or late in the day (Stewart et al. 2009).
5.4.2 Study 2. Effects of GR Canada Fleabane Height on Glyphosate plus
Saflufenacil Efficacy
At 1 WAA, glyphosate plus saflufenacil provided 98% control of GR Canada fleabane for plant heights up to one cm (Table 5.4). As the height of GR Canada fleabane increased to greater than 25 cm, control was 91% meaning there was more living tissue on large plants. At 2
WAA, glyphosate plus saflufenacil provided 95 to 100% control of GR Canada fleabane with the lower control for the larger plants at the time of application and death of most small plants. In a field study by Mellendorf et al. (2013), saflufenacil (25 g a.i. ha-1) alone provided greater than
104 94% control of GR Canada fleabane that was 5 to 45 cm in height at the time of application.
They reported a decrease in control of 1% for every 8 cm increase in GR Canada fleabane height, which was similar to the results from this study where glyphosate was tankmixed. In the second year of their study, Mellendorf et al. (2013) found control was not affected by height. Where saflufenacil (25 g a.i. ha-1) was tankmixed with glyphosate, Mellendorf et al. (2013) found no differences in control due to differences in GR Canada fleabane height at application which is in contrast to the results from this study where plant death occurred for most plants less than 5 cm in height, and some regrowth was noted on larger plants.
At 4 WAA, glyphosate plus saflufenacil provided 94 to 99% control of GR Canada fleabane (Table 5.4). The control of GR Canada fleabane with glyphosate plus saflufenacil resulted in plant death for most small plants and some regrowth in larger plants. Similarly control of GR Canada fleabane with saflufenacil at 6 WAA was maximized for plants up to 5 cm in height with 99% control due to death of most plants and minimal green tissue; as height increased, control decreased to 95% for plants greater than 25 cm in height as less plants were completely dead and more regrowth occurred. In a field study for GR Canada fleabane control in corn with 2,4-D choline/ glyphosate DMA at various rates, no difference in control was found due to varying Canada fleabane height at application as similar rates were predicted to control
10, 20 or 30 cm plants (Ford et al. 2014c).
The percent dry weight of saflufenacil-treated GR Canada fleabane plants at 6 WAA was least at 12% for plants up to 1 cm in height (Table 5.4). For GR Canada fleabane >1 cm, the percent dry weight relative to untreated plants increased up to 26% for plants greater than 25 cm tall.
105 5.4.3 Study 3. Effects of GR Canada Fleabane Density on Glyphosate plus
Saflufenacil Efficacy
At 1, 2 and 4 WAA, glyphosate plus saflufenacil provided complete control of GR
Canada fleabane with densities of 1-40 plants m-2 (Table 5.5). With density >800 plants m-2, control was 97, 96 and 96% at 1, 2, and 4 WAA, respectively due to some plants with regrowth.
Byker et al. (2013a) reported 99 to 100% control of GR Canada fleabane with glyphosate plus saflufenacil (25 g a.i. ha-1) at sites with densities of 7, 29-81, and 48-183 plants m-2 and 95 and
88% control at sites with 92-103, and 158-184 plants m-2, respectively; perhaps the control seen by Byker et al. (2013a) across the various densities of GR Canada fleabane indicates that density was not a key factor in the degree of saflufenacil efficacy. At 6 WAA, glyphosate plus saflufenacil provided 100% control of GR Canada fleabane with densities of 1-20 plants m-2. For densities that were greater than 20 plants m-2, control of GR Canada fleabane with saflufenacil decreased to 96%, as there were plants present with regrowth. In contrast, Chapter 4 found 88% control of GR Canada fleabane with saflufenacil (25 g a.i. ha-1) plus glyphosate at 8 WAA where densities at application ranged from 153-1344 plants m-2 across six sites.
For 21-40 plants m-2 of GR Canada fleabane, the dry weight at 6 WAA was 1 g m-2 or 3% of the untreated control (Table 5.5). Where control of GR Canada fleabane with saflufenacil was lowest (401-800, >800 plants m-2), dry weight of GR Canada fleabane at 6 WAA was 20 and 25 g m-2 or 11 and 13% of the untreated control, respectively; the larger amount of dry weight for high GR Canada fleabane densities is due to plant regrowth that occurred after application. This is similar to Chapter 4 where GR Canada fleabane control with saflufenacil (25 g a.i. ha-1) plus glyphosate resulted in a dry weight of 33.5 g m-2 or 11% of the untreated control due to plant regrowth. In contrast, Byker et al. (2013a) reported dry weight of GR Canada fleabane 4 WAA
106 of less than 1 g m-2 or 1% of the untreated control where densities at glyphosate plus saflufenacil
(25 g a.i. ha-1) application ranged from 7-184 plants m-2 across five sites.
5.5 Conclusions
Glyphosate plus saflufenacil can provide excellent control of GR Canada fleabane; however, control appears to be influenced by TOD of application. In this study, the greatest control of GR Canada fleabane with glyphosate plus saflufenacil was achieved when applied between 09:00- 21:00 h. Interestingly, GR Canada fleabane control was reduced from applications of glyphosate plus saflufenacil at 06:00 and 24:00 h. The GR Canada fleabane density measurement at 8 WAA follows the same pattern as the percent control with the least density from applications between 09:00- 21:00 h. The dry weight measurement of GR Canada fleabane was greatest from applications at 06:00, 21:00 and 24:00 h, which follows a similar trend as the other data.
In the TOD study, there appeared to be a relationship between GR Canada fleabane control and soybean yield. Where no weed control tactics are used, GR Canada fleabane interference can reduce soybean yield up to 93% (Byker et al. 2013b). The difference between the highest and lowest yield for different TOD saflufenacil applications was 0.6 t ha-1. The average cash price for soybean in Canadian dollars from September 2013 to December 2015 was
$465.40 t-1 (Personal Communication-Rob Bos- Hensall District Coop). Soybean at $465.40 t-1 with a 0.6 t ha-1 difference in yield from GR Canada fleabane interference due to the time of saflufenacil application equated to a yield loss of $279.25 ha-1 just because it was sprayed at
06:00 h instead of 15:00 h; across 100 ha of soybean, that would be a loss of $27,925. It is very interesting how not only is there an effect from the TOD when glyphosate plus saflufenacil is applied on the control of GR Canada fleabane, but how much it could directly cost farmers in respect to gross returns per hectare.
107 There appears to be relationships among environmental conditions such as air temperature, cloud cover, relative humidity, and dew presence throughout the day and control of
GR Canada fleabane with glyphosate plus saflufenacil. The importance of each of these environmental factors individually could not be determined in this study as none were independently controlled. Future research may investigate the roles of these factors on GR
Canada fleabane control with glyphosate plus saflufenacil.
The height and density of GR Canada fleabane at application did not have as large an impact on control with glyphosate plus saflufenacil as TOD of application. Greater than 90% control of GR Canada fleabane was obtained with glyphosate plus saflufenacil across all heights and densities evaluated in this study. Conditions at application may have been favourable for excellent control of GR Canada fleabane in this study; the TOD of application for the height and density studies was within the range of optimal control times as shown in the TOD study (Table
1). Where Ford et al. (2014b) had poor control (49% at 8 WAA) of GR Canada fleabane with saflufenacil (50 g a.i. ha-1) plus glyphosate, it was suggested to be due to primarily having fall- germinated Canada fleabane which were large at the time of application; in this study there were few sites with fall-germinated Canada fleabane and where present, was less than 20% of the population (Budd- personal observation). The excellent control obtained in the height and density studies may be due to predominantly spring-germinated GR Canada fleabane and that the
TOD of application was within the optimal range as determined by the TOD study. Future research could examine the effect of spring vs fall-germinated GR Canada fleabane on control with glyphosate plus saflufenacil.
This research concludes that control of GR Canada fleabane with glyphosate plus saflufenacil is influenced by TOD while there is minimal impact of GR Canada fleabane height or density at optimal TOD applications. Improved control of GR Canada fleabane with
108 glyphosate plus saflufenacil is obtained with applications made during the daytime hours. Given the competitiveness with soybean and large seed distribution potential of GR Canada fleabane, optimal control is required to prevent yield loss, and manage resistant populations to reduce the spread of GR biotypes.
109 Table 5.1- Location and agronomic information for factors influencing glyphosate-resistant Canada fleabane control with glyphosate plus saflufenacil studies in Ontario, Canada in 2014 and 2015
Spray Time Soil Characteristics (0-15 cm) Location YearZ Spray Date Seeding Date Year Closest Town (h) Texture OM (%) pH T1 June-9 - June-10 H1 July-3 08:00 - 2014 Mull Loam 3.1 6.6 D1 July-9 08:45 - T2 June-9 - June-20 H2 June-9 20:45 - 2014 Blenheim Sandy Loam 2.9 6.5 D2 June-5 21:15 - T3 May-30 - June-2 H3 June-3 11:45 - 2014 Harrow Sandy Loam 2.1 6.6 D3 May-30 18:15 - T4 June-11 - June-12 H4 June-23 20:50 - 2015 Mull Loam 2.6 6.0 D4 June-11 18:15 - T5 June-4 - June-6 H5 June-23 21:20 - 2015 Blenheim Sandy Loam 4.2 6.2 D5 June-4 18:15 - T6 May-28 - May-29 H6 June-5 11:17 - 2015 Harrow Sandy Loam 2.5 6.1 D6 May-28 15:15 - Z T1-T6, Location years for time of day of glyphosate plus saflufenacil application study; H1-H6, Location years for height of Canada fleabane at glyphosate plus saflufenacil application study; D1-D6, Location years for density of Canada fleabane at glyphosate plus saflufenacil application study
110 Table 5.2- Environmental measurements at each application for the effect from time of day of glyphosate plus saflufenacil application on glyphosate-resistant Canada fleabane control study from 2014 and 2015 in Ontario, Canada
Time of Relative Cloud Wind Canada fleabaneZ Air Temperature Dew Application Day Humidity Cover Velocity Height Density °C Presence (h) (%) (%) (km h-1) (cm) (# m-2) T1 06:00 10.7 94 100 7.2 Y 9-June-2014 09:00 14.0 88 50 8.0 Y 12:00 23.0 56 35 4.0 N 15:00 26.0 51 30 3.1 N up to 13 1826 18:00 23.3 56 40 3.7 N 21:00 19.4 77 60 1.6 N 24:00 18.8 77 90 0 Y
T2 06:00 10.6 100 100 5.6 Y 9-June-2014 09:00 17.2 77 50 3.4 N 12:00 23.7 58 40 2.3 N 15:00 23.5 41 30 2.8 N up to 15 301 18:00 24.0 54 10 7.7 N 21:00 22.5 66 30 0 N 24:00 17.8 80 90 0 Y
T3 06:00 12.7 100 0 0 Y 30-May- 09:00 18.5 81 0 3.6 Y 2014 12:00 20.6 58 0 3.3 N up to 14 87 15:00 27.7 28 0 2.3 N 18:00 27.0 31 0 1.6 N 21:00 22.3 60 0 0 N 24:00 15.7 92 0 0 Y
T4 06:00 13.9 100 25 0 Y 11-June- 09:00 20.4 91 20 1.1 Y up to 12 382 2015
111 12:00 23.8 38 70 1.8 N 15:00 23.4 58 95 0 N 18:00 22.3 63 90 3.7 N 21:00 19.2 77 100 3.9 N 24:00 17.4 90 100 2.1 N
T5 06:00 13.5 94 100 5.7 Y 4-June-2015 09:00 16.1 94 90 5.5 N 12:00 20.8 56 40 4.9 N 15:00 23.2 49 75 4.5 N up to 15 579 18:00 19.6 68 50 6.1 N 21:00 18.1 84 30 2.3 N 24:00 15.5 100 0 4.0 Y
T6 06:00 14.9 100 0 0 Y 28-May- 09:00 19.2 93 0 2.7 Y 2015 12:00 23.4 82 0 1.1 N up to 18 77 15:00 27.5 37 0 2.2 N 18:00 26.9 41 0 1.8 N 21:00 19.6 70 0 0 N 24:00 16.9 86 0 2.0 Y Z Canada fleabane height and density on the day of treatment application from untreated control plots
112 Table 5.3- Regression parameters of parabolic curve equation for glyphosate-resistant Canada fleabane control 1, 2, 4, and 8 WAA, density, dry weight, and soybean yield for time of day of glyphosate plus saflufenacil application study conducted in 2014 and 2015 in Ontario, CanadaZ
Regression ParametersY (se) Critical Predicted values for each time of day Variable a b c Value 06:00 h 09:00 h 12:00 h 15:00 h 18:00 h 21:00 h 24:00 h Weed Control (%) 1 WAA 11.6 (1.9) 99.4 (0.4) -0.02 (0.01) 99 99 99 99 99 99 98X 97 2 WAA 14.3 (1.2) 95.9 (0.9) -0.04 (0.02) 94 93 95 96 96 95 94 92 4 WAA 13.5 (0.9) 97.3 (1.0) -0.07 (0.02) 96 93 96 97 97 96 93 89 8 WAA 14.6 (1.3) 88.9 (2.8) -0.12 (0.06) 84 80 85 88 89 87 84 78
Density (plants m- 121 14.4 (1.6) 56.3 (39.3) 1.39 (0.82) 153 96 64 57 75 117 185 2)W Dry Weight (g m- 56 13.8 (1.6) 38.5 (10.4) 0.38 (0.22) 62 47 40 39 45 58 78 2)W Yield (t ha-1) -0.006 2.7 15.7 (1.6) 3.0 (0.2) 2.4 2.7 2.9 3.0 2.9 2.8 2.6 (0.004) Z Abbreviation: WAA, weeks after application Y Parameters: a, time of day at vertex; b, predicted value at vertex; c, constant X Numbers in bold are significantly less than the parameter maximum, or significantly greater than the parameter minimum as defined by the critical value that is the one-sided confidence bound where outside of P<0.05 W Measured at 8 WAA
113 Table 5.4- Regression parameters of linear equation for glyphosate-resistant Canada fleabane control 1, 2, 4, and 6 WAA, dry weight of treated plants, and percent dry weight of treated plants of untreated plants for height at application of glyphosate plus saflufenacil study conducted in 2014 and 2015 in Ontario, CanadaZ
Regression ParametersY Predicted values for each height range (se) Variable Up to 1 a b 2-5 cm 6-10 cm 11-15 cm 16-20 cm 21-25 cm >25 cm cm Weed Control (%) 1 WAA -1.18 99.0 (1.1) 98 97 95 94 93 92 91 (0.24) 2 WAA -0.87 101.0 (0.7) 100 99 98 98 97 96 95 (0.15) 4 WAA -0.90 100.4 (0.8) 99 99 98 97 96 95 94 (0.19) 6 WAA -0.67 100.1 (0.8) 99 99 98 97 97 96 95 (0.17) Dry Weight (g plant-1) Treated Plants 0.17 (0.04) -0.34 (0.17) 0 0 0 0 1 1 1 % of Untreated 2.4 (3.2) 9.2 (14.4) 12 14 16 19 21 24 26 Plants Z Abbreviation: WAA, weeks after application Y Parameters: a, slope; b, intercept
114 Table 5.5- Regression parameters of exponential and linear equations for glyphosate-resistant Canada fleabane control 1, 2, 4, and 6 WAA, dry weight of treated plants, and percent dry weight of treated plants of untreated plants for density at application of glyphosate plus saflufenacil study conducted in 2014 and 2015 in Ontario, CanadaZ
Regression ParametersX (se) Predicted values for each density range 1-20 21-40 41-100 101-200 201-400 401-800 >800 VariableY a b plants m- plants m- plants m- plants m- plants m- plants m- plants m- 2 2 2 2 1 2 2 Weed Control (%) 1 WAA -0.005 101.1 (0.3) 101 100 99 99 98 98 97 (0.0007) 2 WAA* -0.71 (0.09) 101.1 (0.4) 100 100 99 98 98 97 96 4 WAA -0.007 100.9 (0.3) 100 100 99 98 98 97 96 (0.0008) 6 WAA -0.008 101.0 (0.3) 100 99 99 98 97 96 96 (0.0008) Dry Weight (g m-2) Treated Plants* 4.7 (0.6) -8.1 (2.9) -3 1 6 11 16 20 25 % of Untreated 2.1 (0.4) -1.7 (1.8) 0 3 5 7 9 11 13 Plants* Z Abbreviation: WAA, weeks after application Y * Indicates linear equation used, otherwise exponential X Parameters: a, intercept; b, slope (For exponential); a, slope; b, intercept (For linear)
115 Chapter 6: General Discussion 6.1 Contributions
This research provided updated information on the distribution of GR Canada fleabane in
Ontario. A survey was conducted from 2013-2015 and confirmed GR Canada fleabane in 22 more counties since 2012 for a total of 30 counties in Ontario. This survey also showed that multiple-resistant Canada fleabane, resistant to glyphosate and cloransulam-methyl can be found in 17 additional counties since 2012 for a total of 22 counties in Ontario. From this survey farmers know that GR Canada fleabane is throughout Ontario and this knowledge should be used when planning weed management strategies.
The biologically effective rate of saflufenacil was investigated for the control of GR
Canada fleabane. At 8 WAA, the rate of saflufenacil, required for 90 and 95% control of GR
Canada fleabane was 25 and 36 g a.i. ha-1, respectively; saflufenacil alone did not provide 98% control 8 WAA with the rate range examined. When applied in combination with glyphosate
(900 g a.i. ha-1), the rate of saflufenacil required for 90, 95 and 98% control of GR Canada fleabane was 25, 34, and 47 g a.i. ha-1, respectively at 8 WAA. From a herbicide resistance management standpoint, the biologically effective rate of metribuzin as a tankmix partner with saflufenacil (25 g a.i. ha-1) and glyphosate (900 g a.i. ha-1) was investigated for the control of GR
Canada fleabane. At 8 WAA, when applied in combination with glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1), the rate of metribuzin required for 90, 95 and 98% control of GR
Canada fleabane was 61, 261, and 572 g a.i. ha-1, respectively.
Several soybean herbicides were investigated for efficacy on GR Canada fleabane as tankmix partners with glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1). The best tankmix partners identified were amitrole (2000 g a.i. ha-1) and dicamba (600 g a.i. ha-1).
However, subsequent to the initiation of this research, the registration of amitrole has changed so
116 that it may no longer be used PP in soybean, and dicamba will cause a high degree of crop injury to non-dicamba-resistant soybean. The tankmix partners 2,4-D, metribuzin, flumetsulam and cloransulam-methyl were found to provide excellent control of GR Canada fleabane as well, however with multiple-resistant Canada fleabane having resistance to group 2 herbicides, flumetsulam and cloransulam-methyl are not recommended.
The optimal time of day (TOD) to apply saflufenacil for the control of GR Canada fleabane was identified in this research. At 8 WAA, for optimal control of GR Canada fleabane it was found that glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) should be applied between 09:00-21:00 h; applications made outside of this range provided poorer control resulting in weed interference that reduced soybean yield.
The effect of GR Canada fleabane height and density on the efficacy of glyphosate (900 g a.i. ha-1) plus saflufenacil (25 g a.i. ha-1) was investigated. GR Canada fleabane height and density had very little effect on the efficacy of glyphosate plus saflufenacil. Excellent GR
Canada fleabane control was observed at all heights and densities evaluated, showing that height and density were minor factors influencing glyphosate plus saflufenacil efficacy.
6.2 Limitations
This was a very intensive program including seven different field experiments, across 3-4 locations, over two years for a total of 45 field experiments and a three-year province wide survey. Many factors were examined to answer the research objectives, but regardless there are always areas for improvement for future work.
The three-year survey to update the maps on the distribution of GR Canada fleabane across the province was no small feat. Scouting for Canada fleabane escapes involved waiting until the plants were near seed maturity and had to be collected prior to soybean harvest. If the survey was delayed, the GR Canada fleabane would have been cut near the soil surface with the
117 cutter bar of the combine, making seed collection nearly impossible. Consequently, there was a small window of opportunity spanning a month to collect GR Canada fleabane seed from across the province, while during the same time period yield data had to be collected for all field experiments as well. It was very difficult for one person to scout for Canada fleabane escapes from all soybean-producing areas of the province thoroughly enough within that narrow window of time. As a result, this survey underestimates the number of sites within Ontario counties with
GR Canada fleabane. The objective of this survey was not to determine the actual number of sites with GR Canada fleabane, but to determine the geographical spread of resistant biotypes in the province. However, due to time constraints there is a chance that a county not currently identified with GR Canada fleabane, may have some present.
The most effective herbicide active ingredients for the control of GR Canada fleabane in
Ontario were examined as tankmix partners with glyphosate plus saflufenacil. This was a limitation of the study, since only registered herbicides were evaluated. It would have been interesting to include experimental herbicides, so that this study could have identified current and future options for GR Canada fleabane control in Roundup Ready soybean.
The primary objective of the TOD study was to examine how TOD of glyphosate plus saflufenacil application affected control of GR Canada fleabane. There were seven treatments at three hour intervals from 06:00-24:00 h. Since TOD was the primary objective, perhaps the
03:00 h time should have been included so that TOD was completely examined. Reduced control from the 06:00 and 24:00 h application times would allow one to assume that the same would be found for 03:00 h, but it was not examined and assumptions should not be made.
The height and density studies were a new venture for the Ridgetown campus team.
Much thought was put into how to setup these trials to best capture height and density as factors in GR Canada fleabane control with glyphosate plus saflufenacil. The weed control evaluations
118 were difficult for the height study. The scale used was percent necrosis of each plant however it becomes difficult to interpret whether regrowth of the same size on a large (25 cm) plant compared to a five cm plant was equivalent control or not. Evaluations were similar for the density study where the regrowth of a few plants in a small density treatment compared to a large density treatment became difficult to directly compare. Future weed height and density studies should determine an evaluation procedure that best compares control across different heights and densities.
6.3 Future Research
The GR Canada fleabane present in this research was primarily spring-germinated and control of fall-germinated plants could not be examined. Future studies should focus on control of fall- versus spring-germinated GR Canada fleabane. Fall-germinated GR Canada fleabane plants will have a larger root system in the spring compared to spring-germinated plants. The larger root system may allow GR Canada fleabane plants to re-grow from burndown herbicide applications such as saflufenacil and therefore may be also be a factor in variable control.
The density study examined how the number of GR Canada fleabane plants in a given area impacts saflufenacil efficacy. In high plant density situations, herbicide coverage becomes difficult due to the overlapping of plant leaves. An investigation of the degree of herbicide coverage on GR Canada fleabane with different herbicides would be interesting to provide growers recommendations on water volume, nozzle type, spray pressure and ground speed to optimize applications.
The primary focus of future research should be on integrated GR Canada fleabane management. This is an area of weed management that can often be overlooked, yet provide several benefits for weed control. Crop rotation, tillage, crop row spacing and seeding rate, use of cover crops and crop residue management all influence weed management. All trials in this
119 research were managed on fields that had soybean grown the year before with the exception of the Harrow site in 2014 where there was corn across one-half of the site. Where there was corn residue the amount of Canada fleabane growing the following spring was less than where there was soybean residue, perhaps due to shading. This is just one observation of how different cultural control practices can have an impact on weed management. Future research that examines several practices could provide farmers with various recommendations of different methods to incorporate for managing GR Canada fleabane.
120 Chapter 7: Literature Cited
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129 Chapter 8: Appendixies 8.1 Chapter 5.4.1 Additional Figures
100.0%
90.0%
80.0%
70.0%
60.0%
50.0%
40.0%
30.0% Weed$Control$1$WAA$(%)$ 99.4%%Maximum,% 20.0% Equation%[1]% 10.0% Critical%Value=%99%%
0.0% 6.00% 9.00% 12.00% 15.00% 18.00% 21.00% 24.00% Time$of$Day$(h)$
Figure 5.4.1.1. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 1 week after application for six combined sites in ON across 2014 and 2015.
130 100%
90%
80%
70%
60%
50%
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30% Weed$Control$2$WAA$(%)$ 95.9%%Maximum,% 20% Equation%[1]%% Critical%Value=%94%% 10%
0% 6.00% 9.00% 12.00% 15.00% 18.00% 21.00% 24.00% Time$of$Day$(h)$
Figure 5.4.1.2. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 2 weeks after application for six combined sites in ON across 2014 and 2015.
131 100%
90%
80%
70%
60%
50%
40%
30% Weed$Control$4$WAA$(%)$ 97.3%%Maximum,% 20% Equation%[1]%% 10% Critical%Value=%96%%
0% 6.00% 9.00% 12.00% 15.00% 18.00% 21.00% 24.00% Time$of$Day$(h)$
Figure 5.4.1.3. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 2 weeks after application for six combined sites in ON across 2014 and 2015.
132 100%
90%
80%
70%
60%
50%
40%
Weed$Control$8$WAA$(%)$ 30% 88.9%%Maximum,% 20% Equation%[1]% Critical%Value=%84%% 10%
0% 6.00% 9.00% 12.00% 15.00% 18.00% 21.00% 24.00% Time$of$Day$(h)$
Figure 5.4.1.4. Control of glyphosate-resistant Canada fleabane with saflufenacil with different time of day applications, 8 weeks after application for six combined sites in ON across 2014 and 2015.
133 200%
180% )$
?2 160%
140%
120%
100%
80%
60% 56.3%Minimum,% 40% Camada$;leabane$Density$(plants$m Equation%[1]% Critical%Value=%121% 20%
0% 6.00% 9.00% 12.00% 15.00% 18.00% 21.00% 24.00% Time$of$Day$(h)$
Figure 5.4.1.5. Reduction in glyphosate-resistant Canada fleabane density with saflufenacil with different time of day applications, 8 weeks after application for six combined sites in ON across 2014 and 2015.
134 90%
80% )$
?2 70%
60%
50%
40%
30%
20% 38.5%Minimum,% Canada$;leabane$Dry$Weight$(g$m Equation%[1]% Critical%Value=%56% 10%
0% 6.00% 9.00% 12.00% 15.00% 18.00% 21.00% 24.00% Time$of$Day$(h)$
Figure 5.4.1.6. Reduction in glyphosate-resistant Canada fleabane dry weight with saflufenacil with different time of day applications, 8 weeks after application for six combined sites in ON across 2014 and 2015.
135 3.5%
3%
)$ 2.5% ?1
2%
1.5%
3.0%Maximum,%% Soybean$Yield$(tonne$ha 1% Equation%[1]% Critical%Value=%2.7% 0.5%
0% 6.00% 9.00% 12.00% 15.00% 18.00% 21.00% 24.00% Time$of$Day$(h)$
Figure 5.4.1.7. Reduction soybean yield from glyphosate-resistant Canada fleabane interference due to different time of day applications of saflufenacil, for six combined sites in ON across 2014 and 2015.
136 8.2 Chapter 5.4.2 Additional Figures
100%
90%
80%
70%
60% Equation%[2]% 50%
40%
Weed$Control$1$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Height$Treatment$
Figure 5.4.2.1. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 1 week after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm.
137 100%
90%
80%
70%
60% Equation%[2]% 50%
40%
Weed$Control$2$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Height$Treatment$
Figure 5.4.2.2. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 2 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm.
138 100%
90%
80%
70%
60% Equation%[2]% 50%
40%
Weed$Control$4$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Height$Treatment$
Figure 5.4.2.3. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 4 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm.
139 100%
90%
80%
70%
60% Equation%[2]% 50%
40%
Weed$Control$6$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Height$Treatment$
Figure 5.4.2.4. Control of glyphosate-resistant Canada fleabane at different heights at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm.
140 5%
4% )$ ?1 3%
2%
Dry$Weight$(g$plant$ Equation%[2]%
1%
0% 1% 2% 3% 4% 5% 6% 7% Height$Treatment$
Figure 5.4.2.5. Reduction in glyphosate-resistant Canada fleabane dry weight at different Canada fleabane heights at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm.
141 100%
90%
80%
70%
60%
50%
40% Equation%[2]% 30%
20% Percent$Dry$Weight$of$Untreated$Control$(%)$ 10%
0% 1% 2% 3% 4% 5% 6% 7% Height$Treatment$
Figure 5.4.2.6. Percent dry weight of untreated control for glyphosate-resistant Canada fleabane at different Canada fleabane heights at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: up to 1cm; 2: 2-5 cm; 3: 6-10 cm; 4: 11-15 cm; 5: 16-20 cm; 6: 21-25 cm; 7: >25 cm.
142 8.3 Chapter 5.4.3 Additional Figures
100%
90%
80%
70%
60% Equation%[3]% 50%
40%
Weed$Control$1$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Density$Treatment$
Figure 5.4.3.1. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 1 week after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2.
143 100%
90%
80%
70%
60% Equation%[2]% 50%
40%
Weed$Control$2$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Density$Treatment$
Figure 5.4.3.2. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 2 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2.
144 100%
90%
80%
70%
60% Equation%[3]% 50%
40%
Weed$Control$4$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Density$Treatment$
Figure 5.4.3.3. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 4 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2.
145 100%
90%
80%
70%
60% Equation%[3]% 50%
40%
Weed$Control$6$WAA$(%)$ 30%
20%
10%
0% 1% 2% 3% 4% 5% 6% 7% Density$Treatment$
Figure 5.4.3.4. Control of glyphosate-resistant Canada fleabane at different densities at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2.
146 50%
45%
40%
35%
)$ Equation%[2]% ?2 30%
25%
20% Dry$Weight$(g$m
15%
10%
5%
0% 1% 2% 3% 4% 5% 6% 7% Density$Treatment$
Figure 5.4.3.5. Reduction in glyphosate-resistant Canada fleabane dry weight at different Canada fleabane densities at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2.
147 100%
90%
80%
70%
60%
50%
40%
30% Equation%[2]% 20% Percent$Dry$Weight$of$Untreated$Control$(%)$ 10%
0% 1% 2% 3% 4% 5% 6% 7% Density$Treatment$
Figure 5.4.3.6. Percent dry weight of untreated control for glyphosate-resistant Canada fleabane at different Canada fleabane densities at saflufenacil application, 6 weeks after application for six combined sites in ON across 2014 and 2015; Treatment 1: 1-20 plants m-2; 2: 21-40 plants m-2; 3: 41-100 plants m-2; 4: 101-200 plants m-2; 5: 201-400 plants m-2; 6: 401-800 plants m-2; 7: >800 plants m-2.
148 8.4 SAS Code for Analyzing Regressions in Chapter 3 title 'BER Saflufenacil Stats Analysis'; data first; input env trt dose rep plot phyto14 phyto28 wc7 wc14 wc28 wc56 den drwt yld; *analvar=wc7; *analvar=wc14; analvar=wc28; *analvar=wc56; *analvar=phyto14; *analvar=phyto28; *analvar=den+0.00000001; *analvar=pden; *analvar=drwt+0.00000001; *analvar=pdrwt *analvar=yld; *analvar=pyld;
*Saflufenacil alone; if trt=2 then delete;
*Saflufenacil plus glyphosate; *if trt=1 then delete; *if trt=2 then delete;
*Saflufenacil plus glyphosate plus metribuzin; *if trt=1 then delete; *if trt=2 then delete; *if trt=3 then delete;
*for inverse exponential; *if dose=0 then dose=0.1; cards; (enter data here) ; proc sort data=first; by dose env rep; run; proc plot; plot analvar*dose; run;
*Control data used one of the exponential to maximum equations;
*Density and dry weight used inverse exponential equation; */ **exponential to maximum - ; **a=intercept , b=magnitude , c=slope? (dose constant); **???make dose=0.1 for trt 1???; proc nlin method=marquardt; *by timing; parameters a=0 b=100 c=0.01; bounds a>=0;
149 bounds a<=0; *if dose=0 then model analvar=0; model analvar=a+b*(1-exp(-c*dose)); output out=b predicted=bp run; proc corr; var analvar bp; run; */
**alternate 1 exponential to maximum - ; **a=upper asymptote, b=magnitude constant, c=slope (dose constant); proc nlin method=marquardt; *by timing; parameters a=100 b=100 c=0.001; *if dose=0 then model analvar=0; model analvar=a-b*(exp(-c*dose)); output out=b predicted=cp run; proc corr; var analvar cp; run; */ /* **alternate 2 exponential to maximum - ; **a=upper asymptote, b=dose constant, c=magnitude constant); proc nlin method=marquardt; *by timing; parameters a=100 b=0.001 c=100; model analvar=a-c*(b**dose); output out=b predicted=dp run; proc corr; var analvar dp; run; */ **Inverse exponential - for density and dry weights; **a=lower asymptote, b=reduction in y from int to a, c=slope from int to a; **???make rate=0.1 for trt 1; /* proc nlin method=marquardt; parameters a=0 b=40 c=0.02; bounds a>=0; model analvar=a+b*exp(-c*dose); output out=b predicted=ep run; proc corr; var analvar ep; run;
150 8.5 SAS Code for Analyzing Means Comparisons in Chapter 4 title 'Saflufenacil New Tankmixes Stats Analysis'; data first; input env trt rep plot phyto14 phyto28 wc28 wc56 den drwt yld; if wc28=0 then wc28=0.0000000001; if wc28=1 then wc28=0.9999999999; if wc56=0 then wc56=0.0000000001; if wc56=1 then wc56=0.9999999999;
*for control; if trt=1 then delete;
*for density and dry weight; *if trt=2 then delete; cards; (enter data here) ; title2'wc56;link=ccll;dist=beta'; proc sort data=first; by env trt rep; run;
*for control and phyto data, dist=beta, link=ccll
*for density and dry weight data, dist=gamma, link=log
*for yield data, dist=normal, log-identity proc glimmix data=first method=laplace; class trt rep env; model wc56=trt/ dist=beta link=ccll; random env rep(env) env*trt; *lsmeans trt/pdiff adjust=tukey; covtest 'test of env =0' 0 . . . , 1 -1; covtest 'test of rep=0' . 0 . . , 1 -1; covtest 'test of trt x env =0' . . 0 . , 1 -1; covtest 'test of residuals=0' . . . 0 , 1 -1; output out=second student=studentresid residual=resid predicted=pred; lsmeans trt/ ilink adjust=tukey pdiff; ods output diffs=ppp lsmeans=mmm; *ods html exclude lsmeans diffs; run; %include'C:\Users\buddc\Documents\GRCF14\Stats\Macros\pdmix800.sas';
151 %pdmix800 (ppp,mmm,alpha=0.05,sort=no); run; proc sgscatter data=second; plot studentresid*(pred trt rep env); run; proc sort data=second; by studentresid; proc print; run;
152 8.6 SAS Code for Analyzing Regressions in Chapter 5 title 'Saflufenacil TOD Regression Analysis'; data first; input env trt tod airtemp rh cloud rep plot wc7 wc14 wc28 wc56 den pden drwt pdrwt yld pyld;
*analvar=wc7; *analvar=wc14; *analvar=wc28; *analvar=wc56; *analvar=den; *analvar=pden; *analvar=drwt; *analvar=pdrwt; analvar=yld; *analvar=pyld;
*Saflufenacil alone; if trt=1 then delete; if trt=2 then delete;
*for exponential; *if dose=0 then dose=0.1;
cards; (enter data here) ; data second; set first; trt2=trt*trt; run; proc sort data=second; by env tod rep; run;
*/ **linear; **for height trial proc nlin method=marquardt; parameters a=1 b=0.1; model analvar=(a*trt)+b; output out=b predicted=p; run; proc corr; var analvar p; run; proc plot; plot p*tod/vref=100;
153 run; proc reg data=b; model analvar= trt / lackfit; run;
**exponential; **for density trial proc nlin method=marquardt; parameters a=1 b=0.1; model analvar=a e^(b*trt); output out=b predicted=p; run; proc corr; var analvar p; run; proc plot; plot p*tod/vref=100; run; proc reg data=b; model analvar= trt / lackfit; run;
**parabolic curve; ** for TOD study **c=constant (positive=U shaped, negative=hump), a=x value for vertex, b=y- value for vertex; proc nlin method=marquardt data=second; parameters a=12 b=97 c=-0.2; model analvar=c*((tod-a)*(tod-a))+b; output out=b predicted=p; run; proc corr; var analvar p; run; proc plot; plot p*tod/vref=100; run; proc reg data=b; model analvar= trt / lackfit; model analvar= trt2 / lackfit; run;
154