Evaluation of Glyphosate Resistant Giant Ragweed

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EVALUATION OF GLYPHOSATE RESISTANT

GIANT RAGWEED (AMBROSIA TRIFIDA) IN OHIO

SOYBEAN (GLYCINE MAX) FIELDS

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree

Master of Science in the Graduate School of

The Ohio State University

James D. Bethel B.S. Agricultural Education

Horticulture and Crop Science Graduate Program

The Ohio State University 2013

Committee: Dr. Mark M. Loux, Advisor Dr. Emilie Regnier Dr. David Barker

Copyrighted by

James D. Bethel

2013

Abstract

Studies were conducted in 2011 and 2012 to evaluate glyphosate resistant giant

ragweed in Ohio soybean (Glycine max) fields. Specific objectives of the studies were as

follows:

1) Determine the spatial distribution and incidence of giant ragweed (Ambrosia trifida)

infestations in Ohio soybean fields at the end of the growing season;

2) Characterize the response of Ohio giant ragweed populations to foliar application of

glyphosate and cloransulam-methyl;

3) Determine the effect of postemergence herbicide management strategies on control

and fecundity of giant ragweed in glyphosate-resistant soybeans and

4) Compare the effectiveness of University-recommended strategies with those used by

growers, for management of giant ragweed.

Driving surveys were conducted in 2011 and 2012 to determine spatial

distribution of giant ragweed infestations across the state of Ohio. Infestations were

evaluated using a visual rating system (0 = field free of giant ragweed; 3 = dense

infestation or clusters of plants spread across the field). Fields receiving ratings of 2 or 3

were designated to be infested. Infested fields accounted for approximately 4% of fields

surveyed, and occurred in 50 to 67% of counties surveyed. Results of the surveys

indicated that giant ragweed infestations were localized occurrences, spread throughout

the state. Infested counties appeared to be distributed in north-central and west-central

Ohio. ii

Greenhouse dose response screens were conducted in early 2012 with seed

collected from 75 infested fields during the 2011 driving survey. Giant ragweed was

treated with glyphosate at 0, 0.84, and 3.3 kg acid equivalent (ae)/ha and cloransulam at

0, 16.8 and 67 g active ingredient (ai)/ha. Treatments were applied when giant ragweed

reached an average height of 10 to 20 cm. Treatment effectiveness was evaluated using

plant mortality, percentage biomass reduction, and biomass percentage of untreated

plants. Mortality was calculated by dividing the number of plants within a population

exhibiting total necrosis at harvest by the total number of plants treated within the

population. Populations were then grouped into the categories of 0, 25, 50, 75, or 100%

mortality. Biomass was presented two ways: 1) the mean biomass was calculated for the

populations in each mortality category; and 2) biomass reduction percentage per

population was determined using the formula,

% Biomass Reduction = (A-B)/A,

where A equals nontreated weight, and B equals treated weight.

The giant ragweed population response to glyphosate indicated that the distribution of populations among mortality classes tend to differ depending on the application rate of glyphosate. With glyphosate applied at 0.84 kg/ha, populations were distributed mostly in the 0 to 50% mortality category indicating most of the populations surveyed had total resistance or low-level resistance to glyphosate. At the 3.3 kg/ha glyphosate rate, most populations fell into the 75 to 100% mortality category indicating that most of the populations surveyed were controlled by glyphosate applied at a high

iii

rate. Differences in the distribution of populations across plant mortality categories for

different glyphosate rates indicated that giant ragweed with low levels of glyphosate

resistance in Ohio are likely to be controlled by glyphosate applied at high rates.

Distribution of populations across biomass reduction classes was similar. A greater

proportion of the populations surveyed were distributed in the 80 to 100% biomass

reduction class when glyphosate was applied at 3.3 kg ae/ha compared to when it was

applied at 0.84 kg ae/ha. When glyphosate was applied at 0.84 kg ae/ha rate a greater

proportion of the populations were distributed in the 61 to 100% biomass reduction categories compared to the 3.3 kg/ha rate.

Giant ragweed response to cloransulam was variable within and between populations. At both the 16.8 and 67 g ai/ha rates, population mortality appeared to follow an almost normal distribution, centered at 25 to 75% mortality. Lack of distribution differences across plant mortality categories in response to cloransulam application indicated that cloransulam-resistant giant ragweed has similar levels of resistance to both high and low rates of application. A similar response was obsereved for giant ragweed growth: within biomass reduction categories, both cloransulam rates had approximately equal numbers of populations in each category.

Comparisons of grower and University control of giant ragweed took place during field studies conducted in 2011 and 2012. In 2011, treatments with two postemergence applications attained 97% control of giant ragweed compared to 79 to

84% control for single postemergence treatments. Growers achieved 60% control of giant ragweed in 2011. In 2012, two postemergence applications attained 80 to 98% control compared to 0 to 7% control with single postemergence applications. Growers

achieved 70% control of giant ragweed in 2012. Grower control of giant ragweed was lower than University treatments due to delays in herbicide application allowing giant ragweed plants to increase in size. Multiple postemergence applications also lowered giant ragweed survival at harvest, 2 to 6% compared to 8.5 and 17% survival attained by single postemergence treatments. Giant ragweed fecundity was lower in plots receiving two postemergence herbicide applications compared to single postemergence treatments.

In order to effectively control giant ragweed throughout the growing season, multiple postemergence herbicide applications must occur to control multiple flushes of giant ragweed seedlings during the season.

Giant ragweed is spread across the soybean growing areas of Ohio, however, problematic infestations only occur in approximately 4% of Ohio soybean fields. These infestations are primarily located in north-central and west-central Ohio. While a vast majority of these infestations are cloransulam resistant, in most populations, glyphosate still has the ability to provide some level of control of giant ragweed when used at high rates. In fields with low level glyphosate resistant giant ragweed, it is recommended that multiple herbicide applications featuring high rates of glyphosate be used to manage the spread of resistance. In addition to high rates of glyphosate, it is encouraged to use other herbicides in addition to glyphosate to provide increased control and to slow the spread of glyphosate resistance.

ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Mark Loux, for taking me on as a graduate student. His support and guidance the past two and a half years have been invaluable as I worked to complete this thesis and discover my passion for agronomic field research. I would also like to thank Dr. Emilie Regnier and Dr. David Barker for their input during my defense examination and especially in suggesting revisions for/reviewing, the final draft of this thesis. I would like to thank Dr. Doug Doohan for his service on my advisory committee and his contributions in the early stages of designing my studies. I also need to thank Dr. Wendy Klooster for her support throughout the writing process.

I would like to thank both Tony Dobbels and Bruce Ackley for their assistance and mentorship as I worked to implement my projects and analyze the data that resulted.

I also need to thank Nate Miller, Jason Parrish, Bryan Reeb, Sami Konkle and Tyler

Johnson for riding along in the truck and helping with numerous spray days across Ohio.

Finally, I would like to thank my parents, brothers Dave and Tom, as well as, family and friends for all of their support the past two and a half years as I worked to achieve this goal.

VITA

June 22, 1988...... Born - Columbus, OH

March 2010...... B.S. Agriculture Education,

The Ohio State University

September 2010 to present...... Graduate Research Associate,

Department of Horticulture and Crop

Science, The Ohio State University

PUBLICATIONS

Bethel, J.D., M. M. Loux. 2011. Comparing University and Grower Practices for

Management of Giant Ragweed in Soybeans. North Central Weed Sci. Soc.

Abstr. 20. North Central Weed Sci. Soc., Milwaukee, Wisconsin.

(December 2011).

Bethel, J.D., M.M. Loux, J.T. Parrish. 2012. Survey of Giant Ragweed Infestation Levels

in Ohio Soybean Fields. North Central Weed Sci. Soc. Abstr. 12. North Central

Weed Sci. Soc., St. Louis, Missouri. (December 2012).

Bethel, J.D., M.M. Loux, S. Prochaska. 2012. Comparing Farmer and University

Practices for Controlling Giant Ragweed. North Central Weed Sci. Soc. Abstr.

114. North Central Weed Sci. Soc., St. Louis, Missouri. (December 2012).

vii

FIELD OF STUDY

Major Field: Horticulture and Crop Science

Specialization: Weed Science

viii

TABLE OF CONTENTS

Abstract ...... ii

Acknowledgements ...... vi

Vita ...... vii

List of Figures ...... xi

List of Tables...... xiii

Chapter 1: Literature Review and Introduction ...... 1

Chapter 2: Spatial evaluation of giant ragweed (Ambrosia trifida) infestations in soybean

(Glycine max) fields throughout Ohio.

Objectives ...... 11

Materials and Methods ...... 12

Results and Discussion ...... 12

Chapter 3: Assessment of glyphosate and ALS resistance in Ohio giant ragweed

(Ambrosia trifida) populations.

Objectives ...... 26

Materials and Methods ...... 26

Results and Discussion ...... 28

Chapter 4: Comparisons between grower and University prescribed control of glyphosate

resistant giant ragweed (Ambrosia trifida)

Objectives ...... 43

Materials and Methods ...... 43

Results and Discussion ...... 46

Thesis Bibliography ...... 65

Appendix

Appendix A: List of counties surveyed in 2011 with the number of fields within

each rating category listed...... 68

Appendix B: List of counties surveyed in 2012 with the number of fields within

each rating category listed...... 70

LIST OF FIGURES

Chapter 2:

2.1 Distribution of counties in 2011 with fields receiving a rating of 0 ...... 17

2.2 Distribution of counties in 2011 with fields receiving a rating of 1 ...... 18

2.3 Distribution of counties in 2011 with fields receiving a rating of 2 ...... 19

2.4 Distribution of counties in 2011 with fields receiving a rating of 3 ...... 20

2.5 Distribution of counties in 2012 with fields receiving a rating of 0 ...... 21

2.6 Distribution of counties in 2012 with fields receiving a rating of 1 ...... 22

2.7 Distribution of counties in 2012 with fields receiving a rating of 2 ...... 23

2.8 Distribution of counties in 2012 with fields receiving a rating of 3 ...... 24

Chapter 3

3.1 Mortality of Ohio giant ragweed populations in response to foliar application of

glyphosate. Results represent the mean of two experiments. (p<0.05)...... 36

3.2 Effect of glyphosate on the biomass of surviving plants within each plant

mortality category. Results represent the mean of all populations within a

category...... 37

3.3 Effect of foliar glyphosate application on the biomass of Ohio giant ragweed

populations. Results represent the mean of two experiments...... 38

3.4 Mortality of Ohio giant ragweed populations in response to foliar application of

cloransulam. Results represent the mean of two experiments. (p<0.05) .... 39

3.5 Effect of cloransulam on the biomass of surviving plants within each plant

mortality category. Results represent the mean of all populations within a

category ...... 40

3.6 Effect of foliar cloransulam application on the biomass of Ohio giant ragweed

populations. Results represent the mean of two experiments ...... 41

xii

LIST OF TABLES

Chapter 2:

2.1 Number of fields surveyed categorized by ratings and displayed as percentages of

total fields surveyed in 2011 and 2012 ...... 15

2.2 Number of counties each field rating category was present in for years 2011 and

2012 ...... 16

Chapter 3:

3.1 Herbicides and rates used in greenhouse study ...... 33

3.2 Effect of glyphosate rate on mortality and biomass of giant ragweed populations

under greenhouse conditions ...... 34

3.3 Effect of cloransulam rate on mortality and biomass of giant ragweed populations

under greenhouse conditions ...... 35

Chapter 4:

4.1 Grower treatments for field sites in 2011 ...... 50

4.2 Grower treatments for field sites in 2012 ...... 51

4.3 University postermergence herbicides and rates used in field study ...... 52

4.4 Summary of responses to survey sent to recipients of the C.O.R.N.

newsletter ...... 53

xiii

4.5 Summary of analysis of variance for treatment and year effects for control of

giant ragweed at harvest. Values taken in 2011 and 2012 ...... 54

4.6 Summary of analysis of variance for treatment and location effects for control of

giant ragweed at harvest in 2011 ...... 55

4.7 Effect of postemergence herbicide treatments on control of giant ragweed in 2011

...... 56

4.8 Summary of analysis of variance for treatment and location effects for control of

giant ragweed at harvest 2012 ...... 57

4.9 Effect of postemergence treatment on control of giant ragweed. Location was a

significant main effect due to the lack of treatment differences among treatments

at location A12 ...... 58

4.10 Summary of analysis of variance for treatment and year effects on giant ragweed

survival after initial herbicide application in 2011 and 2012 ...... 59

4.11 Effect of initial herbicide application on giant ragweed survival 21 days after

treatment ...... 60

4.12 Giant ragweed survival percentages at harvest with mean separation applied for

all sites with years combined ...... 61

4.13 Summary of analysis of variance for treatment and location effects for giant

ragweed seed count/ha in 2012 ...... 62

4.14 Summary of analysis of variance for the effect of treatment on giant ragweed

fecundity in 2012 ...... 63

xiv

4.15 Effect of postemergence herbicide application on giant ragweed fecundity at

location C12 in 2012. Mean separation was calculated using log transformation.

Untransformed mean estimates are shown ...... 64

Chapter 1

Literature Review and Introduction

Giant ragweed (Ambrosia trifida) is a member of the Asteraceae family, which is the largest family within the plant kingdom with over 24,000 species. Giant ragweed is generally considered native to North America (USDA, 2012), but other sources list it as a native of Europe (Bryson & DeFelice, 2010). Possible habitat for giant ragweed includes fields, ditches, roadsides and floodplains (Bryson & DeFelice, 2010). While favoring areas on the periphery of agricultural fields, giant ragweed has gradually become a noxious weed to farmers throughout the United States. In disturbed sites, such as fields with fall tillage, giant ragweed is the dominant plant within the community due to its early spring emergence (Bassett, 1982). However, large-seeded broadleaf weeds like giant ragweed are less dominant in no-till cropping systems because they are not incorporated into the seedbed through tillage (Buhler, Hartzler, & Forcella, 1997).

A summer annual, giant ragweed can grow to a height of 5 m. The stem of the giant ragweed plant is covered in a “rough pubescence”, and features opposite leaf orientation along the stem with palmately divided leaves (Bryson & DeFelice, 2010).

Giant ragweed is an outcrossing species and will cross-pollinate with neighboring plants, thus providing greater genetic diversity within a population or biotype. Seeds are large with a hard, spiny cover, which range from 5 to 10 mm long and 2 to 3 mm wide (Bryson

1 & DeFelice, 2010). Seed production per plant decreases as the population density of giant ragweed increases, with an average of 160 seeds/plant at densities of 500 plants/m2

to around 1400 seeds/plant when giant ragweed population density was as low as 4

plants/m2 (Abul-Fatih, Bazzaz, & Hunt, 1979).

As stated previously, giant ragweed dominates any plant community in which it has the opportunity to become established. The rapid growth rate of giant ragweed diverts resources away from corn (Zea mays) or soybean biomass production, reducing yields. Yield reductions in soybeans increased as giant ragweed population density increased (Baysinge & Sims, 1991). At a density of two giant ragweed plants per 9-m of row, soybean yields were reduced up to 46%, and a density of 16 ragweed plants decreased yield up to 92% in 1988 (Baysinger & Sims, 1991). Soybeans maintained free of giant ragweed for six weeks after soybean emergence did not show a significant yield difference when compared to a plot with no weed control. There were no differences in yield between plots maintained weed free for the entire growing season and plots with a weed free period of 10 weeks (Baysinger & Sims, 1991). When emerging with corn, giant ragweed densities of 1 plant/m2 caused a 50% yield reduction, but this decreased to

a less than 10% yield reduction when ragweed emergence was delayed four weeks

(Harrison, Regnier, Schmoll, & Webb, 2001).

When insufficient weed control practices are implemented, giant ragweed can be

very difficult to control. Giant ragweed appears to have a biphasic emergence pattern

which includes a phase of early emerging plants, followed by a second phase one to two

months later (Schutte et al., 2008). In Ohio, the first phase tended to occur during April,

while the second phase occurred from June into July. Some farmers choose to wait until

2 after the second emergence phase before applying postemergence herbicide. Giant ragweed from the first emergence phase can grow past prescribed label heights if herbicide application is delayed to control plants from the second emergence phase.

Numerous other weeds commonly found in crop production also exhibit prolonged emergence patterns, including common waterhemp (Amaranthus rudis) and palmer amaranth (Amaranthus palmeri). Hartzler et al. (1999) reported the mean time of emergence for common waterhemp to range between 49 and 71 days after April 27, over a three-year period. Palmer amaranth emerged from May until October with multiple phases of emergence, the frequency of which depended upon rainfall events (Norsworthy

& Jha, 2010). Norsworthy and Jha (2010) concluded that this extended emergence overlapped with planting dates for soybeans and cotton in the southeastern United States.

Waterhemp, palmer amaranth and giant ragweed are noxious weeds in Ohio. Populations of these weeds in Ohio have been identified with resistance to at least one herbicide site of action. The prolonged emergence pattern of these weed species presents challenges for their control in cropping systems that rely upon glyphosate-resistant crops.

When attempting to control weeds with prolonged emergence patterns, Ogg and

Dawson (1984) report that it is important to know when weeds will begin to emerge so that cropping practices optimize the ability of a farmer to control problem weeds. The example provided is to avoid planting crops in fields where weed species have a similar emergence period as the crop unless selective herbicides are available for use in that crop

(Ogg & Dawson, 1984). In conventional tillage situations, weeds can be destroyed soon after germination with soil cultivation before seedlings emerge (Ogg & Dawson, 1984).

To control flushes of weeds that emerge later in the season in no-tillage soybeans,

3 residual herbicides should be included with pre-plant burndown herbicide applications

(Loux et al., 2011). When adequate and timely rainfall occurs, preemergence herbicides can control up to 80% of later-emerging giant ragweed (Baysinger & Sims, 1992).

However; as residual herbicide activity decreases over time, postemergence applications should be made to control late emerging weeds (Loux et al., 2011).

Glyphosate is a non-selective herbicide that is capable of controlling a broad spectrum of weed species, and is considered by some to be the “herbicide of the century”

(Duke). Introduced in 1974 by Monsanto, glyphosate was initially largely used in preemergence herbicide applications in reduced tillage situations due to its lack of selectivity. With the introduction of Roundup Ready soybeans in 1996, glyphosate was no longer restricted to preemergence use, and it has become a key herbicide in weed control for over 90% of the soybean acres in the United States (Duke & Powles, 2008).

Glyphosate-resistant corn comprises over 65% of all corn acres in the United States, with other glyphosate resistant crops including, canola (Brassica napus L.), cotton (Gossypuim hirsutum), sugarbeets (Beta vulgaris), and alfalfa (Medicago sativa) (Duke, 2009).

Initially, only one genetically modified trait had been expressed by genetically modified crops. However, a trend towards placing multiple traits in a genetically modified crop variety has become the industry standard with companies coupling herbicide resistance traits with insect tolerance traits, or multiple herbicide resistance traits.

A major obstacle for farmers using glyphosate extensively in their weed control programs is the gradual emergence of biotypes of weed species that exhibit various levels of tolerance or resistance to glyphosate. The introduction of Roundup Ready® soybeans in 1996, caused a decline in both the number of herbicide active ingredients, and

4 herbicide sites of action used by farmers in their weed control programs (Duke, 2009).

The result of the shift to glyphosate-resistant cropping systems has been “strong and

persistent” selection pressure for weeds that are tolerant or resistant to glyphosate (Duke,

2009).

While the adoption of glyphosate-resistant crops has been rapid in the United

States and Canada, weeds that have evolved resistance to glyphosate were not found in

Canada until 2008 (Heap, 2012). This delay could be attributed to the fact that crop rotations in Canada have historically been based on cereal grain (Campbell, 1990) and canola production. Canola is genetically modified to be resistant to glufosinate and

various imidazolinones. This provides an advantage to Canadian farmers, allowing the

use of a diverse array of herbicide mode of actions, and limiting the evolution of weeds

towards glyphosate resistance (Duke, 2009). With the anticipated release of soybean

varieties that are resistant to plant growth regulators and hydroxyphenyl-pyruvate-

dioxygenase (HPPD) inhibitors, it is hoped that farmers in the U.S. will diversify their

weed control programs so that the utility of glyphosate and the other herbicides used in

these varieties can be maintained into the future.

Instances of weed species becoming resistant to multiple herbicide sites of action

are increasing in frequency. In Ohio, herbicide resistance has been found in 10 weed

species, with four of those species demonstrating resistance to two different sites of

action (SOA) (Heap, 2012). Resistance to multiple SOA occurs in giant ragweed,

common ragweed (Ambrosia artemisiifolia), horseweed (Conyza canadensis), and

common lambsquarters (Chenopodium album) (Heap, 2012). There have been reports of

glyphosate resistant palmer amaranth migrating into the Portsmouth, Ohio region via

5 grass and forb seed used in conservation seedings (Loux, 2012). Giant ragweed in Ohio

developed resistance to acetolactase-synthase (ALS) inhibiting herbicides in 1998 (Heap,

2012), limiting postemergence herbicide options to glyphosate and cell membrane

disruptors such as fomesafen and lactofen. Glyphosate resistance in giant ragweed within

Ohio developed in 2004 (Heap, 2012)

When giant ragweed plants survive herbicide applications, plants pass on the resistance to that herbicide to their offspring. A method for measuring resistance is the use of LD50 values to determine the median lethal dose of a herbicide required to kill half of the plants in a tested population. GR50 values are used to determine the herbicide dose required to “reduce shoot weight by 50% relative to the untreated plants” (Heap,

2005). In a dose-response study conducted at Iowa State University with cloransulam, the LD50 for susceptible biotypes was 4.3 g active ingredient (ai)/ha, while LD50 values for resistant biotypes was 339 g ai/ha (Zelaya & Owen, 2004). When using a

combination of primisulfuron and prosulfuron, the LD50 value for susceptible biotypes was 6.7 g ai/ha, and with resistant biotypes the LD50 values was 686 g ai/ha (Zelaya &

Owen, 2004). In comparison, the LD50 value for glyphosate susceptible giant ragweed from Tennessee was 407 g/ha, while resistant biotypes attained an LD50 of 2,176 g/ha

(Norsworthy, 2010). When comparing levels of resistance based on LD50, ALS resistant biotypes have shown resistance to herbicide rates hundreds of times larger than prescribed on the herbicide label. For glyphosate resistant biotypes, the observed resistance herbicide rate was slightly over 4 times the rate applied to the susceptible biotype. The lack of parity between the LD50 ratios of resistant and susceptible biotypes

6 in the glyphosate and ALS studies suggest that giant ragweed biotypes can demonstrate lower levels resistance to glyphosate than to ALS-inhibiting herbicides.

While giant ragweed has been documented as glyphosate resistant, resistance levels can vary among biotypes (Stachler, 2008). In a study at The Ohio State University that utilized four glyphosate resistant giant ragweed populations from Ohio and Indiana,

GR50 values ranged from 8.3 to 23.9 kg ae/ha of glyphosate (Stachler, 2008). When glyphosate was applied at equal rates, some biotypes exhibited higher levels of regrowth after application. Stachler (2008) and numerous others have noted that multiple glyphosate applications can consistently control giant ragweed, while single glyphosate applications have variable results. Results from the Ohio State University study indicated that control of resistant populations increased from 64% to 95% when comparing one versus two postemergence applications.

As more weed species become resistant to glyphosate, farmers face increased pressure to control these weeds. Some farmers reapply glyphosate to weeds unaffected by previous glyphosate applications. University weed scientists recommend that farmers utilize herbicide partners with different SOA to improve weed control and reduce selection for resistance (Nandula, 2005). It is also recommended that farmers use a more integrated approach to weed management by scouting fields early to detect weeds and utilizing various crop production techniques to disturb the natural cycle of weed growth.

This can include rotating between glyphosate-resistant and non-glyphosate resistant crops to delay the onset of resistant weeds, while also utilizing mechanical methods of weed control (Nandula, 2005).

7 One indicator that a weed population may be developing herbicide resistance is sporadic survival of weeds throughout a field following an effective herbicide application. An effective application involves applying herbicides when plants are small, not in drought stress conditions, and when application equipment is properly calibrated

(Loux et al., 2011). Depending on the amount of rainfall present during the spring and summer, preemergence applications should control the initial population of giant ragweed that emerges. A second application could be required to control later emerging giant ragweed. Some farmers will wait until soybeans are near the closure of the crop canopy before spraying, in order to make one postemergence application. As a result, weed size can vary from cotyledon stage to plants taller than the restrictions listed on the herbicide label. The label for Roundup Powermax® prescribes maximum plant heights for giant

ragweed at different product rates per acre. Giant ragweed should not be taller than six

inches when applying glyphosate at the rate of 16 ounces per acre or 0.63 kg acid

equivalent/ha (Monsanto, 2012). When spraying 0.84 kg ae/ha glyphosate at a rate of 22

ounces product per acre, giant ragweed plants should not be over 12 inches in height.

Farmers have the power to make decisions regarding weed control practices in

their fields. When farmers see weeds in their fields, they tend to focus on controlling the

weeds present, instead of attempting to prevent weeds from becoming established at the

outset (Wilson, Tucker, Hooker, LeJeune, & Doohan, 2008). Wilson found that the

majority of farmers, 87-90%, believe “natural elements” such as wind, or wildlife are key

in the introduction of new weed species. Fewer farmers, 67-77%, believe that tillage

implements or harvest equipment are to blame (Wilson et al., 2008). Also, the study

8 determined farmers believed that the negative impacts of weeds within fields are decreases in crop yield and quality, and increased operating costs.

Modern soybean production utilizes no-till and conventional tillage practices.

Each system provides advantages and disadvantages regarding weed control.

Conventional tillage soybean production involves intensive tillage of the seed-bed that eliminates all weeds present before planting. Postemergence herbicide applications are required to control late emerging weed species. No-till soybean production typically requires a preplant herbicide application to remove weeds present before planting.

Almost all preplant herbicide applications include glyphosate, and some include additional herbicides with foliar activity on emerged weeds. This creates an intense selection pressure for weeds that are glyphosate resistant. Some preplant herbicide partners also include residual activity for control of later-emerging weeds. Residual herbicides can be used in both a conventional tillage and no-till soybean cropping system to control weed species with multiple emergence phases.

Approximately 50% of soybean acres use no-till production practices (Duke,

2009). No-till soybean production increases dependence on glyphosate for weed control before and after planting. This reliance on glyphosate has produced giant ragweed biotypes that are increasingly difficult to control and will continue to replenish the seed bank with glyphosate resistant offspring. Strategies must be implemented to reduce the number of seeds being returned to the soil seed bank in order to lower giant ragweed population densities in the future. Weed control strategies using two postemergence herbicide applications have shown to provide better control of glyphosate resistant giant

9 ragweed populations. However, it is unknown if farmers use two postemergence herbicide application to control giant ragweed. Objectives of this research were to:

1) Determine the spatial distribution and incidence of giant ragweed infestations

in Ohio soybean fields at the end of the growing season

2) Characterize the response of Ohio giant ragweed populations to foliar

application of glyphosate and cloransulam-methyl

3) Determine the effect of postemergence herbicide management strategies on

control and fecundity of giant ragweed in glyphosate-resistant soybeans

4) Compare the effectiveness of University strategies with those used by growers

for management of giant ragweed.

Chapter 2

Spatial evaluation of giant ragweed (Ambrosia trifida)

infestations in Soybean (Glycine max) fields throughout Ohio

Statement of problem

Giant ragweed infestations occurring late in the growing season are a result of

ineffective control management. Studies conducted at Purdue University in 2004

estimated that giant ragweed was present in 26% of fields surveyed, while economic

infestations occurred in 20% of soybean fields at harvest (Johnson, Barnes, Gibson, &

Weller, 2004). No studies have been conducted in Ohio to determine the spatial

distribution of giant ragweed infestations occurring late in the growing season. These

giant ragweed populations represent either late-emerging giant ragweed or giant ragweed biotypes that are difficult to control for other reasons. Results of this study allow for mapping of the distribution of giant ragweed infestations in Ohio. It is hypothesized that giant ragweed infestations are spread throughout Ohio.

Objective

Determine the spatial distribution and incidence of giant ragweed infestations in Ohio

soybean fields at the end of the growing season.

Materials and Methods

Surveys were conducted in 2011 and 2012 to determine the levels of giant ragweed infestations in Ohio soybean fields in late September; approximately two weeks before soybean harvest. The survey procedure in 2011 was to follow transects diagonally across each county, and every 16 km (10 miles), assess the infestation level in the next five soybean fields. The area surveyed in 2011 covered 52 counties representing 90 percent of the Ohio soybean acres planted (NASS, 2012). In 2012, the infestation level was assessed in all soybean fields encountered (GPS coordinates were not recorded).

Ratings were compiled, arranged by county and then analyzed using ArcGIS software to determine in which counties giant ragweed infestations were present. The surveyed fields were assigned a field number, and GPS coordinates were recorded using a Garmin

Oregon 400T®. Ratings and GPS coordinates were compiled, and analyzed with ArcGIS software to provide the spatial distribution of giant ragweed infestations in 2011.

The level of giant ragweed infestation was assessed using the following scale: 0 - field free of giant ragweed; 1 – a few single giant ragweed plants in the field; 2 - a few clusters of plants; 3 – dense infestation or clusters of plants spread across the field.

Fields receiving a rating of 2 or 3 were considered to be infested with giant ragweed, and a representative seed sample was collected from these fields for use in subsequent assessment of herbicide resistance.

Results and Discussion In 2011, the survey was conducted in 52 Ohio counties and covered 1010 fields, or an average of 19.4 fields per county (Table 2.1, Table 2.2). Giant ragweed was absent

from 763 fields, approximately 75% of fields surveyed. Fields devoid of giant ragweed

occurred in all counties surveyed (Figure 2.1). Fields receiving a rating of one were

present in 46 of the 52 counties and accounted for 18% of fields surveyed (Figure 2.2).

Fields receiving a rating of two were found in 27 counties and 4.2% of all fields surveyed

(Figure 2.3). Fields receiving a rating of three were found in 16 counties and made up

only 2.2% of all fields rated (Figure 2.4). Infested fields receiving ratings of 2 or 3 were found in 30 counties, which were located primarily in the western half of the state.

In 2012, 3,993 fields in 51 counties were surveyed accounting for 89% of Ohio soybean acres planted (NASS, 2012), and 90% of fields were devoid of giant ragweed

(Figure 2.5). Fields that received a rating of one were found in 50 counties and accounted for 8.2% of all fields surveyed (Figure 2.6). Soybean fields with a rating of two were found in 34 counties and represented 1.7% of fields rated (Figure 2.7). Fields

with a rating of three were found in 14 counties and accounted for only 0.4% of all fields

rated (Figure 2.8).

Above average precipitation (279 mm from April 1, through May 31, compared to

the 30-year mean, 203 mm) in the early part of the growing season in 2011 delayed

planting (Enloe & Crouch, 2012; OARDC, 2012). A delay in planting could have

reduced the use of residual herbicides in soybeans, increasing reliance on postemergence

applications for weed control. Increased precipitation may have also delayed herbicide

applications throughout the growing season, which could have resulted in reduced early

season weed control. Herbicide applications that are delayed would allow for the control

of giant ragweed during its later period of emergence. Weed control in 2012 was affected

by drought for much of the growing season (300 mm during May through September

2011, compred to the 30-year mean, 513 mm) (Enloe & Crouch, 2012; OARDC 2012).

Drought could have slowed late season giant ragweed emergence, reducing the need for

postemergence herbicide applications to control late emerging weeds. Herbicide

treatments applied to drought stressed weeds have reduced effectiveness due to a

decrease in photosynthetic activity.

With surveys from each year combined, late-season infestations of giant ragweed

across the state of Ohio were variable and widespread with significant infestations of

giant ragweed occurring in approximately 4% of Ohio soybean fields. At the end of the

growing season giant ragweed was present in approximately 10 to 25% of soybean fields

rated. In 2011, infestations occurred in 28 counties, the majority of which were located

in west-central Ohio. In 2012, infestations occurred in 35 counties, with distribution

located in north-central and west-central Ohio. The change in survey protocol in 2012,

which stipulated every field along the transect be rated, provided a more accurate

representation of giant ragweed infestation levels in Ohio.

Fields where giant ragweed is established at population densities classified as

infested, are spread throughout Ohio (Appendix 2.1). Differences in survey protocol

between years and the fact that some soybean fields in 2011 were rotated to corn in 2012

do not allow for any assumptions regarding the spread of giant ragweed from 2011 and

2012. Infested fields should be classified as having glyphosate or ALS resistant giant

ragweed biotypes and should be managed to reduce dependence on glyphosate or ALS-

inhibiting herbicides for season long weed control.

Table 2.1 Number of fields surveyed categorized by ratings and displayed as percentages of total fields surveyed in 2011 and 2012. 2011 Survey 2012 Survey Rating Number of Percentage of Number of Percentage of Fields Total Fields Total 0 763 75 3578 89.6

1 181 18 328 8.2

2 43 4.2 67 1.7

3 23 2.2 17 0.4

Table 2.2 Number of counties for which each field rating category was present,for years 2011 and 2012. 2011 2012 Ratings Number of Percentage of Number of Percentage of counties counties counties counties surveyed surveyed 0 52 100 51 100 1 46 88 50 98 2 27 50 34 67 3 16 30 14 27

Figure 2.1. Distribution of all counties receiving a 0 rating in 2011.

Figure 2.2. Distribution of counties with soybean fields receiving a rating of 1 in 2011.

Figure 2.3. Distribution of counties with soybean fields receiving a 2 rating in 2011.

Figure 2.4. Distribution of counties with soybean fields receiving a 3 rating in 2011.

Figure 2.5. Distribution of all counties receiving a rating of 0 in 2012

Figure 2.6. Distribution of counties with soybean fields receiving a rating of 1 in 2012

Figure 2.7. Distribution of counties with soybean fields receiving a rating of 2 in 2012.

Figure 2.8. Distribution of counties with soybean fields receiving a rating of 3 in 2012.

Chapter 3

Assessment of glyphosate and ALS resistance

in Ohio giant ragweed (Ambrosia trifida) populations

Statement of problem

The occurrence in Ohio of giant ragweed populations with resistance to glyphosate and

ALS-inhibiting herbicides has been previously confirmed (Johnson et al., 2012). Glyphosate-

resistant giant ragweed was confirmed in Ohio in 2006 (Heap, 2012). Previous research

confirmed the presence of glyphosate-resistant giant ragweed at three locations in Ohio and one

location in Indiana (Stachler, 2008). The mechanism of resistance in giant ragweed is unknown, however, due to the occurrence of several symptoms displayed by giant ragweed after glyphosate application it is believed that there are multiple mechanisms of resistance present (Stachler,

2008). Rapid necrosis is a symptom observed after glyphosate application results in the rapid

death of leaves that have glyphosate applied to them, which stops the translocation of glyphosate

into other parts of the plant (Personal observation). Other resistance symptoms appear to result

in a temporary inhibition of plant growth in response to treatment with glyphosate, without the

rapid necrosis symptoms. Prior to the study described here, there has not been a comprehensive

screening of giant ragweed populations in Ohio to determine the frequency of resistance to

glyphosate or cloransulam. It was anticipated that the sampled populations would have varying

responses to treatment with two different rates of glyphosate and cloransulam-methyl.

25 Objective

Characterize the response of Ohio giant ragweed populations to foliar application of glyphosate and cloransulam-methyl.

Materials and methods

Seed was collected during the fall of 2011 from 75 soybean fields infested with giant ragweed. Low seed germination rates for several populations resulted in their exclusion from the subsequent study. The fields were identified during the 2011 survey conducted to determine spatial distribution of giant ragweed infestations in Ohio. The seed samples consisted of the combined seed from four to five plants per field. The populations were predominately from fields that were rated 2 or 3 during the survey, indicating moderate to dense infestations in soybeans at the end of the growing season. Fields with a rating of 2, which indicated a cluster of giant ragweed within a field, accounted for 31 of the populations. Fields with a rating of 3, which indicated extensive giant ragweed infestation, accounted for 17 populations. Two of the populations were from fields that received a 0 rating, which indicated almost no giant ragweed present at the end of the growing season. Educators with the Ohio State University Extension provided another 15 populations for herbicide resistance screening, and it was assumed that these populations originated from fields with an extensive giant ragweed infestation. Seeds were separated from other plant material, rinsed in a 1:500 solution of X3 fungicide and placed in plastic mesh bags. The bags were then buried in wet sand for cold priming at 4 to 5 °C for three weeks. Seeds were then removed from mesh bags and placed on germination paper, which was then rolled and placed in a growth chamber at 20 °C to induce germination. Lighting in the growth chamber was set to provide a 12-hr day length. Following germination, seeds from each

26 population were planted individually in a soil media mix into 25 separate, “5.00 AZ Tall” pots, which were then placed in the greenhouse. Pots were arranged on greenhouse benches in a randomized complete block design and organized into four replications based on plant height.

Soil moisture within pots was maintained through the use of an automated sprinkler system set to distribute water three times daily.

Prior to herbicide application, degraded plant material was placed into pots to simulate bacterial/fungal conditions present in soil during actual field herbicide applications. Herbicides were applied when giant ragweed seedlings reached a height of 10 to 20 cm. Herbicide treatments consisted of foliar application of glyphosate or cloransulam at rates corresponding to one and four times the recommended rate for giant ragweed, and these were compared with nontreated controls. Roundup Powermax® with ammonium sulfate was used for the glyphosate treatments, while FirstRate® with crop oil concentrate was used for the cloransulam treatments

(Table 3.1). Herbicides were applied in a volume of 141 L/ha using a mechanized spray chamber with Teejet 8001EVS nozzles and a pressure of 3.86 bars. The experiment was conducted twice. Experiment 1 began on January 27 and ended on February 23, 2012.

Experiment 2 was initiated one week after Exp. 1 was completed, and ended on April 5, 2012.

Supplemental lighting in the greenhouse was provided through metal halide lamps set to provide a 16-hr day length. Longer day length or higher temperatures during Exp. 2 may have contributed to increased levels of herbicide activity.

Data Collection and Analysis

Giant ragweed control was visually evaluated 7 and 14 days after treatment (DAT) using a scale of 0 to 100, where 0 represented no symptoms observed and 100 represented plant death.

27 At 14 DAT, the percent mortality for each population was calculated by dividing the number of

plants within a population exhibiting total necrosis by the total number of plants treated within

the population. Populations were then grouped into the categories of 0, 25, 50, 75, or 100%

mortality (Trainer, 2005). The distribution of plant mortality for high and low rates of

glyphosate and cloransulam, were compared with χ2-analysis using Excel. The number of

populations (averaged for the two experiments) occurring within each mortality category (0, 25,

50, 75, and 100% mortality) (total = 68 or 69 in respective experiments) were compared with χ 2- analysis. Significance was determined at the 5% significance level. Biomass was measured 15

DAT by harvesting and weighing all plant material present above the soil surface. Biomass was presented two ways: 1) the biomass of surviving treated plants as a percentage of untreated plants; and 2) biomass reduction percentage of surviving plants per population. Biomass reduction was determined using the formula, % Biomass Reduction = (A-B)/A, where A was

nontreated weight, and B was treated weight. Plants receiving glyphosate treatments were

observed for multiple symptoms of glyphosate resistance, including growth inhibition, chlorosis, and the rapid necrosis of treated leaves. Populations exhibiting rapid necrosis were recorded in

Exp. 1 only, and presented here as the number of populations exhibiting rapid necrosis.

Results and Discussion

The giant ragweed populations exhibited a range of responses to glyphosate in Exp. 1.

The 0.84 kg ae/ha rate of glyphosate resulted in two populations with 100 percent giant ragweed

mortality, four populations with 75% mortality, 15 populations with 50% mortality, 22

populations with 25% mortality, and 25 populations for which all plants survived (Table 3.2).

Application of glyphosate at 3.3 kg/ha resulted in a trend for increased mortality. At that rate,

28 there were 23 populations with 100% giant ragweed mortality, 16 populations with 75% mortality, seven populations with 50% mortality, 12 populations with 25% mortality, and 10 populations with all plants surviving. Mean plant biomass of surviving plants decreased as mortality category increased, demonstrating that populations with low levels of resistance exhibit an increased response to glyphosate application. The 3.3 kg/ha rate resulted in lower biomass compared with the 0.84 kg/ha treatment, for populations within the 50 and 75% mortality categories. Rapid necrosis symptoms were observed in 25 populations; in 17 populations receiving 0.84 kg/ha glyphosate, and in 18 populations receiving 3.3 kg/ha glyphosate. Ten populations exhibited rapid necrosis at both glyphosate rates. Multiple populations exhibited both rapid necrosis and temporary growth inhibition resistance symptoms.

In Exp. 2, fewer plants survived at both glyphosate rates compared Exp. 1. Higher plant mortality could possibly be attributed to increased glyphosate activity due to longer day length and increased temperatures when this experiment was conducted. Supplemental lighting was maintained at 16 hr day length during Exp. 2, in addition to longer day lengths associated with the progression of the seasons. The 0.84 kg/ha rate resulted in 14 populations with 100% plant mortality, 11 populations with 75% mortality, 19 populations with 50% mortality, 11 populations with 25% mortality, and 14 populations with all plants surviving (Table 3.2). The 3.3 kg/ha rate resulted in, 28 populations with 100% plant mortality, 20 populations with 75% mortality, 11 populations with 50% mortality, 5 populations with 25% mortality, and 5 populations with all plants surviving. Mean biomass of surviving plants decreased as plant mortality categories increased. Biomass was lower for populations receiving 3.3 kg/ha glyphosate compared with the

0.84 kg/ha rate except at 75% plant mortality.

29 On average, populations exhibited decreased mortality at the 0.84 kg/ha glyphosate rate compared with the 3.3 kg/ha rate (Figure 3.1). The majority of populations treated with glyphosate at 3.3 kg/ha exhibited 75 to 100% plant mortality. Differences in the distribution of populations within plant mortality categories were validated (p < 0.001) using χ2 -analysis at the

5% significance level (Figure 3.1). Differences in population distribution demonstrate that giant ragweed with low levels of glyphosate resistance will still respond to the application of glyphosate at high rates. Biomass of surviving plants decreased as plant mortality increased

(Figure 3.2). At 75% mortality, the mean biomass of surviving plants amounted to less than 10% of nontreated plant biomass. The reduction in biomass increased with increasing glyphosate rate

(Figure 3.3). A diverse range of resistance symptoms were present in both experiments. These included temporary growth inhibition, and rapid necrosis. Multiple populations were observed demonstrating both resistance symptoms. Rapid necrosis was present in at least one plant for 24 of the 68 populations screened during this study.

Cloransulam did not effectively control all plants and had varying control within populations in Exp. 1. Cloransulam at 16.8 g ai/ha resulted in three populations with 100% plant mortality, four populations with 75% mortality, 20 populations with 50% mortality, 22 populations with 25% mortality, and 17 populations with all plants surviving (Table 3.3). The 67 g/ha rate resulted in four populations with 100% plant mortality, 16 populations with 75% mortality, 16 populations with 50% mortality, 20 populations with 25% mortality, and 15 populations with all plants surviving. Mean biomass for plants surviving the 67 g/ha rate was lower at all plant mortality categories compared with the 16.8 g/ha rate.

Increased day length and greenhouse temperatures may have caused increased cloransulam activity during Exp. 2. Cloransulam applied at 16.8 g/ha resulted in four

30 populations with 100% plant mortality, 19 populations with 75% mortality, 19 populations with

50% mortality, 16 populations with 25% mortality, and nine populations with all plants surviving. The 67 g/ha rate resulted in nine populations with 100% mortality, 21 populations with 75% mortality, 12 populations with 50% mortality, 13 populations with 25% mortality, and

10 populations with all plants surviving. Biomass accumulation of surviving plants decreased as plant morality category increased. Biomass of surviving plants was higher for the 67 g/ha cloransulam rate at all morality categories with the exception of 75% plant mortality compared to the 16.8 g/ha rate.

The response of giant ragweed to cloransulam application was extremely variable within and between populations. The majority of populations at both cloransulam rates demonstrated plant mortality ranging from 25 to 75% (Figure 3.4). No differences (p = 0.15) in the population distribution across plant mortality categories for either cloransulam rate were found using χ2 - analysis at the 5% significance level. A lack of differences in the distribution of populations among plant mortality categories suggests that the populations studied have similar levels of resistance to both treatment rates. Biomass accumulation of surviving plants as a percentage of untreated plants increased as mortality decreased (Figure 3.5). However, differences in population distribution among plant moratlity categories were not observed between cloransulam rates for biomass accumulation as percentage of untreated plants. A majority of populations treated with cloransulam resulted in approximately 81 to 100% reduction in biomass compared to untreated plants (Figure 3.6).

This study indicates that resistance to glyphosate and/or ALS-inhibiting herbicides is present in the majority of Ohio soybean fields where giant ragweed infestations occur at the end of the growing season. The plant mortality distribution of populations treated with cloransulam

31 was not different between the 16.8 g/ha and 67 g/ha rates indicating similar levels of resistance at both rates. Cloransulam resistance was absent from only 5 to 10% of the giant ragweed populations screened, while partial resistance was present in 90-95% of the populations sampled.

The confirmation of resistance to ALS-inhibiting herbicides in a majority of soybean fields infested with giant ragweed supports previous literature stating that ALS-resistant giant ragweed is present in Ohio (Johnson et al., 2004). Glyphosate applied at the 3.3 kg/ha rate provided higher levels of plant mortality than the 0.84 kg/ha rate which indicated that some populations with low levels of glyphosate-resistance will be controlled by glyphosate applied at a high rate.

Varying levels of mortality and biomass reduction seem to indicate that variations in levels of resistance can exist within a population. The presence of rapid necrosis and temporary plant growth inhibition symptoms after glyphosate application indicates that multiple mechanisms of resistance may be present in Ohio. Populations completely resistant to glyphosate accounted for

28% of populations screened at the 0.84 kg/ha rate compared to 10% of populations screened at

the 3.3 kg/ha rate. Non-resistant populations accounted for 11% of populations tested at the 0.84

kg/ha rate compared to 37% of the populations tested at the 3.3 kg/ha rate.

The surveys conducted in 2011 and 2012 estimated that giant ragweed infestations

occurring at the end of the growing season were present in 4% of Ohio soybean fields. The

results of this greenhouse study suggest that 90 to 95% of giant ragweed infested soybean fields

will exhibit some level of cloransulam resistance. Based on the greenhouse study complete

glyphosate-resistance should be expected in approximately 28% of infested soybean fields,

while lower levels of resistance could be expected in 60% of infested fields.

32 Table 3.1 Herbicides and rates used in greenhouse study Herbicide 1x 4x

Glyphosate 0.84 kg/ha + 3.3 kg/ha +

2.5% v/v AMS 2.5% v/v AMS

Cloransulam 16.8 g/ha + 67 g/ha +

1.2% v/v COC 1.2% v/v COC

Table 3.2. Effect of glyphosate rate on mortality and biomass of giant ragweed populations under greenhouse conditions Number of Mean biomass Mean biomass populations (g) (% of nontreated)

Mortality Rate Exp. 1 Exp. 2 Exp. 1 Exp. 2 (kg ae/ha) 0% 0.84 25 14 6.7 5.6 19.7 0% 3.3 10 5 4.3 2.5 10.9 25% 0.84 22 11 5.2 3.9 14.6 25% 3.3 12 5 4 1.7 9.1 50% 0.84 15 19 4.1 2.6 10.7 50% 3.3 7 11 3 1.9 7.9 75% 0.84 4 11 2.5 1.5 6.3 75% 3.3 16 20 3.2 1.7 7.9 100% 0.84 2 14 0 0 0 100% 3.3 23 28 0 0 0 0% Nontreated 68 69 33.1 29.9 *Biomass measurements were determined using plants that survived herbicide application.

Table 3.3. Effect of cloransulam rate on mortality and biomass of giant ragweed populations under greenhouse conditions Number of Mean biomass Mean biomass populations (g) (% of nontreated) Mortality Rate Exp. 1 Exp. 2 Exp. 1 Exp. 2 (g ai/ha) 0% 16.8 17 9 13.8 16.8 39.8 0% 67 15 10 9.8 18.6 37 25% 16.8 22 16 7.7 7.6 20 25% 67 20 13 7.5 10.3 23.1 50% 16.8 20 19 10.5 7.2 23 50% 67 16 12 3.6 8.6 15.9 75% 16.8 4 19 2.4 9.3 15.3 75% 67 16 21 2.1 6.4 10.9 100% 16.8 3 4 0 0 0 100% 67 4 9 0 0 0 0% Nontreated 46.9 29.9 *Biomass measurements were determined using plants that survived herbicide application.

Figure 3.1 Mortality of Ohio giant ragweed populations in response to foliar application of glyphosate. Results represent the mean of two experiments. (p<0.05)

Figure 3.2. Effect of glyphosate on the biomass of surviving plants within each mortality category. Results represent the mean of all populations within a category.

Figure 3.3. Effect of foliar glyphosate application on the biomass of Ohio giant ragweed populations. Results represent the mean of two experiments.

Figure 3.4. Mortality of Ohio giant ragweed populations in response to foliar application of cloransulam. Results represent the mean of two experiments. (p<0.05).

Figure 3.5. Effect of cloransulam on the biomass of surviving plants within each mortality category. Results represent the mean of all populations within a category.

Figure 3.6. Effect of foliar cloransulam application on the biomass of Ohio giant ragweed populations. Results represent the mean of two experiments.

Chapter 4

Comparisons between grower applied and University prescribed

control of glyphosate resistant giant ragweed (Ambrosia trifida)

Statement of Problem

Giant ragweed is a problematic weed in corn and soybean production in the eastern Corn

Belt (Johnson et al., 2012). A survey completed in 2003 at Purdue University showed that 30% of growers using a corn and soybean crop rotation found giant ragweed to be of concern in their fields (Gibson, Johnson, & Hillger, 2006). Control of giant ragweed with ALS-inhibiting herbicides has decreased in Ohio causing reliance upon glyphosate in postemergence herbicide applications in soybeans. Increased reliance on glyphosate has promoted the selection of glyphosate resistant giant ragweed in Ohio and the rest of the Midwest. Research at The Ohio

State University demonstrated that giant ragweed can display varying levels of sensitivity to glyphosate application (Stachler, 2008). Populations with low level glyphosate resistance had decreased plant survival when higher rates of glyphosate were applied (Stachler, 2008). It was recommended that high rates of glyphosate be partnered with other to slow the rate of spread of the resistant population. The purpose of this research was to compare control practices recommended by the University with those used by growers for the control of giant ragweed populations that exhibited a reduced response to glyphosate. The hypothesis was that the combination of glyphosate with another effective postemergence herbicide would provide more effective control of giant ragweed than glyphosate alone.

Objectives

1. Determine the effect of postemergence herbicide management strategies on control and

fecundity of giant ragweed in glyphosate-resistant soybeans.

2. Compare the effectiveness of University strategies with those used by growers for

management of giant ragweed.

Materials and Methods

Field studies were conducted in 2011 and 2012 in fields where growers or consultants

had reported that giant ragweed populations had become less responsive to glyphosate. In 2011,

three field sites were located in Champaign County Ohio, as follows:

- Field A11, 4.8 km west of Urbana, Ohio

- Field B11, 1.6 km north of Rosewood, Ohio

- Field C11, 1.6 km northwest of West-Liberty, Ohio.

In 2012, three field sites were located throughout Ohio in Union, Clark, and Crawford counties as follows:

- Field A12, 3.2 km east of Woodstock, Ohio

- Field B12, 1.6 km north of Bucyrus, Ohio

- Field C12, 4.8 km northwest of South Charleston, Ohio.

Four treatments were arranged with three replications in a randomized complete block

design with plots measuring 3 x 15 m. Postemergence applications were applied with a

pressurized backpack sprayer calibrated to deliver 140 L/ha. The cooperating growers planted

glyphosate-resistant soybeans with row spacing ranging from 19 to 38 cm in either no-till or

conventional till cropping systems. Farmers applied preemergence herbicides to the entire field prior to research plot establishment, and also applied postemergence herbicides to the field with the exception of the research area (Table 4.1, Table 4.2).

University postemergence treatments were selected to determine the differences in giant ragweed control between treatments that consisted of glyphosate only, compared with a combination of glyphosate and fomesafen or lactofen, and to determine the effect of single versus multiple postemergence applications. Initial University postemergence treatments were applied when giant ragweed reached a height of 15 cm, and second treatments were applied three weeks later. Single postemergence treatments included glyphosate alone and the combination of glyphosate and fomesafen (Table 4.3). Treatments with two postemergence applications were glyphosate applied twice and the combination of glyphosate and fomesafen followed by the combination of glyphosate and lactofen. All treatments contained 5% v/v ammonium sulfate.

The fomesafen treatments contained 1% v/v methylated seed oil, and lactofen treatments contained 0.5% v/v crop oil concentrate. In 2011, growers applied postemergence treatments consisting of only glyphosate at rates of 0.84 to 1.06 kg ae/ha. Growers applied postemergence herbicide treatments 6 to 12 days after the initial University treatments were applied. In 2012, growers applied glyphosate at rates of 0.75 kg to 1.5 kg ae/ha, and applied 14 to 25 days after initial University treatments.

To determine which practices farmers use when controlling giant ragweed in soybeans, a link to an internet survey was provided in an article emailed to over 1,400 recipients of the

Crop Observation and Recommendation Network (C.O.R.N.) newsletter in 2012. Recipients of the C.O.R.N newsletter include farmers, agricultural consultants, and agrichemical dealers.

Surveys were intended to provide an indication of specific control practices growers are

implementing to control giant ragweed for comparison with University weed control

recommendations. Surveys were administered using surveymonkey.com to ensure

respondent anonymity. Of the 1,400 individuals who received the newsletter, 66 participated

in the survey, corresponding to a 4.7% response rate. Survey results are presented as received

with no statistical analysis performed (Table 4.4). Survey results indicate that 70% of

respondents have giant ragweed present in their soybean fields, and 60% of respondents have

experienced increasing difficulty in controlling giant ragweed over the past decade. Survey

results indicate that 9% of respondents apply only glyphosate on all of their spring burndown

acres; 52% of respondents apply glyphosate with an additional herbicide on 100% of spring

burndown acres. The number of postemergence applications varied, with 51% of respondents

making one postemergence application and 42% making two postemergence herbicide

applications. Giant ragweed size at the time of herbicide application was variable among

respondents, 51% applied herbicides when giant ragweed reached a height of 15 to 30 cm,

and 22% when giant ragweed was larger than 30 cm. Initial glyphosate application rate

averaged 1.17 kg/ha, with 51% of respondents applying between 1.02 to 1.27 kg/ha. The rate

of 0.84 kg/ha, which is recommended by the Roundup Powermax® label for giant ragweed

less than or equal to 30cm in height, was used by 33% of survey responders. The maximum

label rate for a single postemergence application of Roundup Powermax®, 1.64 kg, was used

by 14% of respondents (giant ragweed plant height not specified on label).

Data Collection and Analysis

Evaluation of herbicide effectiveness was determined through visual observation of control, using a scale of 0 to 100 where 0 represented no control and 100 represented complete

control. This occurred at 14 and 21 days after the initial and second herbicide applications, and

one week before harvest. Giant ragweed survival was calculated by flagging 20 plants within each plot prior to initial treatment, and determining the number of plants surviving at the time of

the second postemergence application and prior to soybean harvest. For comparison of giant

ragweed survival between University and grower treatments, 20 plants were flagged in portions

of the field where the farmer applied glyphosate.

In 2012, seed was collected from all plants surviving within the plot area, except for field

C12 where a sub-sample of plants within an area one-meter square was collected and

extrapolated to the entire plot due to high giant ragweed densities.

Data were analyzed with the UNIVARIATE procedure in SAS, using the arcsin

transformation to satisfy normality assumptions. Untransformed means were used for

representation in all tables and charts. The MIXED procedure in SAS was used for mean

separation at p = 0.05. Treatment, location, year, and their interactions were treated as fixed

effects with replications as a random factor.

Results and Discussion

Giant ragweed control at harvest differed between 2011 and 2012 (Table 4.5). In 2011,

differences among treatments occurred, but there was no effect of location or any interaction

between location and treatment (Table 4.6). Averaged over locations, the multiple-application

treatments consisting of only glyphosate or glyphosate + fomesafen followed by glyphosate +

lactofen resulted in higher levels of giant ragweed control at harvest when compared with the

single-application treatments (Table 4.7). Control exceeded 95% for the former, but was less

than 85% for the latter. Control in the rest of the field in response to management by the grower

averaged 60 to 70% in 2011 and 2012, respectively. Where postemergence herbicides were

applied twice, combining other herbicides with glyphosate did not improve control compared

with glyphosate applied alone.

In 2012, there was an interaction between location and treatment for giant ragweed

control at harvest, due to the lack of an effect of treatment at location A12 (Tables 4.8 and 4.9).

The multiple-application treatments at locations B12 and C12 resulted in more effective control

than the single-application treatments. Control with multiple applications ranged from 68 to

98%, while control for single applications was less than 20%. In multiple-application treatments,

the addition of fomesafen or lactofen did not significantly improve control compared with glyphosate applied alone. Reduced control of giant ragweed with glyphosate indicates herbicide resistance within the population may be increasing (Stachler, 2008). Multiple herbicide applications can effectively control giant ragweed (Johnson et al. 2012). Control of giant ragweed with low levels of glyphosate resistance is best accomplished with high levels of glyphosate partnered with other effective herbicides (Johson et al. 2012, Stachler, 2008).

Planting date played a role in the effectiveness of single-application treatments in both

2011 and 2012. Extreme amounts of precipitation during the spring of 2011 caused planting and herbicide applications to be postponed until after the initial phase of giant ragweed emergence

(Enloe and Crouch, 2012). Giant ragweed emergence in Ohio occurs in two phases, the initial emergence occurs in April and decreases through May and June, while the second major phase of emergence occurs in July (Schutte et al., 2008). Delays in single-application treatment caused plants from the later phase of giant ragweed emergence to be controlled, providing increased effectiveness at the end of the growing season. Early planting in 2012 allowed for a second phase of emergence after initial postemergence treatments resulting in the need for a second

herbicide application. The inability to control late emerging giant ragweed caused reduced levels of end of season control for single-application treatments.

Giant ragweed survival 21 days after the initial herbicide application varied with treatment, but there was no effect of year or an interaction between treatment and year (Table

4.10). Averaged over years, giant ragweed survival after the initial application was lower for treatments that combined glyphosate with fomesafen at 12 to 16%, compared with glyphosate treatments, which averaged 37% (Table 4.11). The lower survival that occurred with glyphosate and fomesafen demonstrates that where a grower intends to attempt control of giant ragweed with a single postemergence glyphosate treatment, the inclusion of another effective herbicide can improve late-season control. However, giant ragweed survival at the end of the season showed a trend of lower survival for treatments with two herbicide applications compared to single-application treatments (p = 0.05) (Table 4.12). This supports research conducted in 2008 demonstrating that multiple postemergence herbicide applications result in lower giant ragweed survival compared to single-application treatments (Stachler, 2008).

Data regarding giant ragweed fecundity in 2012 showed a significant treatment by location interaction (Table 4.13). Location was a significant effect; with sites A12 and B12 showing no treatment differences (Table 4.14). Treatment had an effect at site C12, where giant ragweed fecundity was lower for treatments with multiple herbicide applications compared to single-application treatments (Table 4.15). Glyphosate applied twice and glyphosate with fomesafen followed by glyphosate with lactofen resulted in no seeds while glyphosate alone and glyphosate with fomesafen had 27 million, and 35 million seeds/ha respectively. Single herbicide applications were unable to control later emerging giant ragweed that produced most of the seed.

Results of this field study confirm previous research demonstrating strategies for control

of giant ragweed with low level glyphosate resistance (Stachler, 2008). Control is most effective

if herbicides are applied when giant ragweed are small and multiple herbicide applications with effective partner herbicides are conducted (Stachler, 2008). Due to the biphasic emergence

pattern of giant ragweed, multiple postemergence herbicide applications are more likely to

provide effective season-long control of giant ragweed than single applications. It is estimated

that 56% of C.O.R.N. newsletter recipients only use one postemergence herbicide application to

control giant ragweed (Table 4.4). Herbicide applications targeted at giant ragweed with low

level glyphosate-resistance should be applied when weeds are 15-25 cm tall (Johnson et al.

2012). The growers in this study experienced lower levels of giant ragweed control due to delayed herbicide applications. Grower treatments were applied 6 to 12 days after University herbicide applications in 2011 and 14 to 25 days later in 2012, allowing giant ragweed to increase in size. The online survey sent to subscribers of the C.O.R.N. newsletter indicates that

73% of growers wait to apply postemergence herbicide treatments until giant ragweed has reached heights over 15 cm, which is within the application height range recommended by

University extension publications for control of giant ragweed with reduced response with glyphosate (Johnson et al. 2012). Survival of giant ragweed at harvest was highest in plots receiving a single-application of glyphosate. Lower survival was also observed in plots receiving multiple-applications (p = 0.05). Plant size restrictions on herbicide labels must be followed and use of the maximum allowable glyphosate rates are recommended on populations suspected of being glyphosate resistant. In fields where giant ragweed population densities are high, giant ragweed seed production can be reduced with multiple herbicide applications.

Table 4.1: Grower treatments for field sites in 2011.

Field Preemergence Planting Date Postemergence A11 4/14/11 5/13/11 6/27/11 Glyphosate - 780 g ae/ha., Glyphosate - 950 g/ha 2,4-D - 640 g/ha, Flumioxazin - 64 g/ha. + chlorimuron - 21 g/ha B11 6/3/11 6/3/2011 7/7/11 Metribuzin - 135 g/ha Glyphosate - 840 g/ha + chlorimuron - 22 g/ha Glyphosate - 560 g/ha

C11 6/5/11 6/6/2011 7/6/11 Glyphosate - 1180 g/ha Glyphosate - 1070 g/ha Flumioxazin - 64 g/ha. Chlorimuron - 47 g/ha + chlorimuron - 21 g/ha 7/20/11 – Spot spray - Fomesafen - 970 g/ha

Table 4.2: Grower treatments for field sites in 2012.

Field Preemergence Planting Postemergence Date A12 5/1/12 5/7/2012 6/22/12 Glyphosate - 840 g ae/ha Glyphosate - 1270 g/ha 2, 4-D - 410 g/ha Fomesafen - 350 g/ha Flumioxazin - 64 g/ha. + chlorimuron - 21 g/ha B12 4/3/12 5/15/2012 6/30/12 Metribuzin - 180 g/ha Glyphosate - 840 g/ha + chlorimuron - 30 g/ha 2,4-D - 540 g/ha

C12 5/31/12 6/3/2012 7/6/2012 Glyphosate - 1680 g/ha Glyphosate - 1680 g/ha Metribuzin - 180 g/ha Clethodim - 76 g/ha + chlorimuron - 30 g/ha Flumioxazin - 30 g/ha Saflufenacil - 20 g/ha

Table 4.3. University postemergence herbicide treatments

Treatments Chemical formulation

Glyphosate 1740 g ae/ha glyphosate + 5% v/v AMS

Glyphosate 1740 g/ha glyphosate + 5% v/v AMS, fb glyphosate followed by 840 g/ha glyphosate + 5% v/v AMS

Glyphosate + 1740 g/ha glyphosate + 350 g/ha fomesafen + 5% v/v AMS, fomesafen + 1% v/v MSO

Glyphosate +fomesafen 1740 g/ha glyphosate + 350g/ha fomesafen + 5% v/v AMS, fb Glyphosate +lactofen + 1% v/v MSO Followed by 840 g/ha glyphosate + 105 g/ha lactofen + 5% v/v AMS, + 0.5% v/v COC. Note: fb (followed by)

Table 4.4. Summary of responses to survey sent to recipients of the C.O.R.N. newsletter. Question n % Is giant ragweed a problem in your fields? Yes 47 71

No 19 28 Have you had more difficulty controlling giant Yes 41 62 ragweed the past 10 years? No 25 38

List % of acres that receive glyphosate only Over 50% 13 20 during burndown. Less than 50% 53 80

What is the most common size of giant 5 to 15 cm 16 24 ragweed at the time of first postemergence application? 15 to 30 cm 34 51 30 to 45 cm 11 16 Over 45 cm 4 6

What is the most common rate of glyphosate Less than or equal to 13 20 for the first postemergence application? 0.84 kg ae/ha 0.9 to 1.27 kg ae/ha 31 47

1.46 to 1.64 kg ae/ha 9 13 Did not respond 13 20

What is the most common rate of glyphosate 0.84 kg ae/ha 9 13 for the second postemergence application? 1.02 to 1.27 kg 14 21 ae/ha

1.64 kg ae/ha 4 6

Did not apply second 39 60 POST

List the percentage of Roundup Ready 0 to 25% 40 60 soybean acres where other herbicides are added to the postemergence glyphosate 26 to 50% 12 18 application. Over 50% 12 18

How many postemergence applications of 1 POST 37 56 glyphosate are required to control giant ragweed? 2 POST 29 43

Table 4.5. Summary of analysis of variance for treatment and year effects for control of giant ragweed at harvest values taken in 2011 and 2012.

Effect DF F Value Pr > F

Year 1 30.28 <0.0001

Treatment 3 19.55 <0.0001 Treatment x 3 7.12 0.0003 Year

Table 4.6. Summary of analysis of variance for treatment and location effects for control of giant ragweed at harvest in 2011.

Effect DF F Value Pr > F

Treatment 3 8.47 0.0003

Location 3 2.70 0.0638 Location x Treatment 9 1.32 0.2694

Table 4.7. Effect of postemergence herbicide treatments on control of giant ragweed in 2011. Treatment Giant ragweed control (mean ± SE*)

Glyphosate + fomesafen 97 ± 3 a fb Glyphosate + lactofen

Glyphosate 96 ± 3 a fb Glyphosate Glyphosate 84 ± 3 b

Glyphosate + fomesafen 79 ± 3 b

*Means separated by letters indicates significant differences among giant ragweed control at harvest (p<0.05).

Table 4.8. Summary of analysis of variance for treatment and location effects for control of giant ragweed at harvest in 2012.

Effect DF F Value Pr > F

Treatment 3 67 <0.0001

Location 2 46 <0.0001

Location x 6 12 <0.0001 Treatment

Table 4.9. Effect of postemergence treatment on control of giant ragweed. Location was a significant main effect due to the lack of differences among treatments at location A12 Location Treatment Giant ragweed control Pr > F (mean ± SE*) A12 Glyphosate + fomesafen 98 ± 5 0.1207 fb Glyphosate + lactofen

Glyphosate fb Glyphosate 98 ± 5 Glyphosate + fomesafen 87 ± 5 Glyphosate 81 ± 5

B12 Glyphosate + fomesafen 89 ± 12 a 0.0086 fb Glyphosate + lactofen

Glyphosate fb Glyphosate 68 ± 12 a Glyphosate + fomesafen 20 ± 12 b Glyphosate 7 ± 12 b

C12 Glyphosate + fomesafen 98 ± 0.50 a < 0.0001 fb Glyphosate + lactofen

Glyphosate fb Glyphosate 98 ± 0.50 a Glyphosate 5 ± 0.50 b Glyphosate + fomesafen 5 ± 0.50 b *Means separated by letters indicates significant differences among giant ragweed control at harvest (p<0.05).

Table 4.10. Summary of analysis of variance for treatment and year effects on giant ragweed survival after initial herbicide application in 2011 and 2012.

Effect DF F Value Pr > F Treatment 3 5.15 0.0028

Year 1 1.99 0.1631

Year x Treatment 3 0.87 0.4602

Table 4.11. Effect of initial herbicide application on giant ragweed survival 21 days after treatment. Treatment Giant ragweed survival Pr >F (mean ± SE*) Glyphosate 37.14 ± 5.99a 0.0046 Glyphosate fb Glyphosate 37.0 ± 6.14a Glyphosate + fomesafen 16.33 ± 5.99b fb Glyphosate + lactofen Glyphosate + fomesafen 12.80 ± 5.99b *Means separated by letters indicates significant differences among giant ragweed control at harvest (p<0.05).

Table 4.12. Survival of giant ragweed percentages at harvest with mean separation applied for all sites with years combined. Treatment Giant ragweed survival Pr >F (mean ± SE) Glyphosate 17.1 ± 4a 0.05 Glyphosate + fomesafen 8.4 ± 4ab Glyphosate fb Glyphosate 5.9 ± 4ab Glyphosate + fomesafen 1.9 ± 4b fb Glyphosate + lactofen *Means separated by letters indicates significant differences among giant ragweed control at harvest (p≤0.05).

Table 4.13. Summary of analysis of variance for treatment and location effects for giant ragweed seed count/ha in 2012.

Effect DF F Value Pr > F

Treatment 3 28.59 <0.0001

Location 2 42.19 <0.0001 Location x 6 8.95 <0.0001 Treatment

Table 4.14. Summary of analysis of variance for the effect of treatment on giant ragweed fecundity in 2012.

Location DF F Value Pr > F

A12 3 2.18 0.1917

B12 3 3.83 0.0760 C12 3 4937.34 <0.0001

Table 4.15. Effect of postemergence herbicide application on giant ragweed fecundity at Location C12 in 2012. Mean separation was calculated using log transformation. Untransformed mean estimates are shown.

Treatment Giant ragweed seed production per Pr > F hectare (mean ± SE*) Glyphosate + fomesafen 35,000,000 ± 421,000 a 0.0019

Glyphosate 27,000,000 ± 421,000 a Glyphosate + fomesafen 0 ± 421,000 b fb Glyphosate + lactofen

Glyphosate fb Glyphosate 0 ± 421,000 b

*Means separated by letters indicates significant differences in giant ragweed fecundity (p<0.05).

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Appendices

Appendix A: List of counties surveyed in 2011 with the number of fields within each rating category listed.

0 – Field free of giant ragweed 1 – A few single giant ragweed plants in the field 2 – A few clusters of plants in the field 3 – Dense infestation of plants spread across the field

County 0 1 2 3 Adams 17 2 0 0 Allen 11 4 0 0 Ashland 11 4 0 0 Auglaize 25 0 0 0 Brown 19 2 0 1 Champaign 10 3 1 1 Clark 12 6 4 0 Clermont 31 7 3 2 Crawford 10 5 0 0 Darke 20 7 2 1 Defiance 18 2 0 0 Deleware 6 3 1 1 Erie 2 3 0 0 Fayette 20 5 0 0 Franklin 8 2 1 0 Fulton 9 5 1 0 Greene 15 7 2 1 Hamilton 19 6 1 0 Hancock 31 4 0 0 Hardin 4 4 1 1 Henry 13 3 0 0 Highland 20 5 2 1 Hocking 1 0 0 0 Huron 12 2 1 0 Jackson 17 1 0 0 Knox 19 3 1 1 Logan 31 4 2 1 Lorain 11 4 0 0 Madison 5 5 0 0 Marion 27 13 3 3

County 0 1 2 3 Medina 5 0 0 0 Mercer 18 2 0 0 Miami 8 3 1 3 Montgomery 20 4 4 3 Morrow 11 5 2 0 Ottawa 11 0 1 0 Paulding 10 0 0 0 Pickaway 20 2 0 0 Pike 12 3 1 1 Preble 15 3 2 0 Putnam 6 3 1 0 Richland 18 5 0 0 Ross 18 1 0 0 Sandusky 15 0 0 0 Scioto 7 5 2 0 Seneca 13 5 1 0 Shelby 11 4 2 0 Union 20 7 2 1 Van Wert 17 2 1 0 Williams 17 2 0 0 Wood 20 2 1 0 Wyandot 22 7 0 0

Appendix B: List of counties surveyed in 2012 with the number of fields within each rating category listed.

0 – Field free of giant ragweed 1 – A few single giant ragweed plants in the field 2 – A few clusters of plants in the field 3 – Dense infestation of plants spread across the field

County 0 1 2 3 Allen 62 9 3 1 Ashland 87 5 1 0 Auglaize 64 15 1 0 Brown 127 2 0 0 Butler 59 2 0 0 Champaign 55 6 1 0 Clark 42 7 1 0 Clermont 71 2 0 0 Crawford 25 16 1 0 Darke 81 8 0 0 Defiance 53 8 3 0 Deleware 63 4 1 2 Erie 43 7 5 1 Fairfield 92 7 1 0 Fayette 56 5 2 0 Franklin 80 6 2 0 Fulton 69 10 2 0 Greene 57 3 1 0 Hancock 74 4 0 0 Hardin 87 15 4 0 Henry 101 4 0 0 Highland 98 5 1 1 Huron 51 2 1 0 Knox 58 12 0 0 Licking 50 7 1 0 Logan 64 4 0 0 Lorain 71 8 1 1 Lucas 65 4 0 0 Madison 100 2 4 1 Marion 65 11 4 1 Mercer 123 9 0 0 Miami 58 8 1 0 Montgomery 84 2 0 1 Morrow 55 7 3 1

County 0 1 2 3 Ottawa 47 6 2 1 Paulding 62 4 0 0 Pickaway 99 3 1 0 Preble 108 4 1 1 Putnam 95 2 0 0 Richland 71 14 3 1 Ross 81 6 2 0 Sandusky 57 6 5 1 Seneca 68 9 1 0 Shelby 44 9 2 2 Union 105 13 1 0 Van Wert 72 6 0 0 Warren 57 0 0 0 Wayne 52 5 0 0 Williams 48 4 2 0 Wood 60 2 0 0 Wyandot 62 10 3 0