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This is to certify that the

dissertation entitled

HERBICIDE TOLERANCE AND WEED CONTROL
IN CORN [Zea maze L.] WITH ISOXAFLUTOLE

presented by

Christy Leigh Sprague

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Crop and Soil Sciences

 

 

MW)

/ Majofprofessoi

Date January 27, 1999

 

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ma W14

HERBICIDE TOLERANCE AND WEED CONTROL
IN CORN [Zea mays L.] WITH ISOXAFLUTOLE

By

Christy Leigh Sprague

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Crop and Soil Sciences

1999

ABSTRACT

HERBICIDE TOLERANCE AND WEED CONTROL
IN CORN [Zea mays L.] WITH ISOXAFLUTOLE

By

Christy Leigh Sprague

In conventional tillage corn, isoxaflutole (5-cyclopropyl isoxazol-4-yl-2-mesyl-4-
trifluoromethylphenyl ketone) applied preemergence controlled common lambsquarters

(Chenopodi'um album L.), redroot pigweed (Amaranthus retroflexus L), common ragweed
(Ambrosia artemisiifolia L.), and velvetleaf (Abutilon theophrasti Medicus) in years with
adequate rainfall. In no-tillage corn, weed control was more variable from herbicide
treatments applied early preplant compared with the preemergence application timing.
Isoxaflutole controlled common lambsquarters and velvetleaf, but redroot pigweed and
common ragweed control varied among years and application timings. Giant foxtail (Setaria
faberi Herrm.) control in both tillage systems was rate, timing, and year dependent. Selected
tank-mixtures with isoxaflutole increased giant foxtail control. In some cases, isoxaflutole
injured corn in conventional tillage and no-tillage studies. Corn injury was most commonly
observed in coarse textured soils with low clay and organic matter and was more severe with
higher rates of isoxaflutole. Injury to corn from isoxaflutole was not unique to any tillage
system and was site, year, and rate dependent.

Field studies were conducted to determine whether isoxaflutole and isoxaflutole tank-
mixtures could be used for weed control after corn had emerged. Isoxaflutole applied

postemergence controlled common lambsquarters, common ragweed, redroot pi gweed, and

velvetleaf, regardless of application timing. However, giant foxtail control varied between
years and application timings. Severe corn injury was observed when isoxaflutole tank-
mixed with metolachlor/benoxacor was applied to 2-leaf and 4-leaf corn. Increased
isoxaflutole absorption and retention was the basis for the increased corn injury observed

when isoxaflutole tank-mixed with metolachlor/benoxacor was applied to emerged corn.

GR50 (50% growth reduction) values indicated that the corn hybrids Pioneer 3751 and
Pioneer 3737 were less tolerant to isoxaflutole than the hybrids Pioneer 3394 and Pioneer
3963. Differences in hybrid tolerance were primarily due to differential herbicide metabolism
rates. Increased uptake of isoxaflutole was also a contributing factor to the sensitivity of the
hybrid Pioneer 373 7.

R-29148 was the most effective of five antidotes evaluated to protect corn against
isoxaflutole injury. MON-13900 was also an effective antidote. Technical R-29l48 applied
at rates greater than 90 g ha" provided excellent protection against isoxaflutole injury and
also prevented injury to corn from diketonitrile, the active metabolite of isoxaflutole.
Enhanced metabolism resulting in inactivation of isoxaflutole was the protective mechanism
of R—29148. The mixed fimction oxidase inhibitor, piperonyl butoxide, increased
isoxaflutole activity in corn, suggesting that oxidative reactions may be involved in the

metabolism of isoxaflutole in corn.

ACKNOWLEDGMENTS

Sincere appreciation is extended to Dr. James J. Kells and Dr. Donald Penner for
granting me the opportunity to pursue a Ph.D. degree at Michigan State University, for
guiding my thoughts, and challenging me with new ideas and research perspectives. The
opportunities and experiences I have had during this degree have helped me learn and grow
as a person and a professional.

I would also like to extend a special thank you to Dr. Matthew Zabik and Dr. James
Baird for serving on my committee and for providing guidance throughout my research. A
special thanks to Andy Chomas and Frank Roggenbuck for their friendships, technical
support, and advise on the many questions I was faced with throughout my graduate degree.

Special thank you’s are extended to the many people who participated in my research
and fi'om whom I have learned so much: Julie Root, Brent George, Stephanie Eickholt, Gabe
Corey, and Pat O’Boyle. I am also appreciative for the discussions, insights, and friendships
provided by present and past graduate students and technicians: Brent Tharp, Joe Simmons,
Jason Fausey, Kelly Nelson, Matt Rinella, Corey Ransom, Sherry White, Kyle Poling, Nate
Kemp, Chad Lee, Caleb Dalley, Paul Knoerr, Beau McSparin, Terry Wright, and Gary
Powell.

Finally, without the support and encouragement from my friends and family I never
would have been able to achieve this degree. So special thanks goes to my parents Larry and

Janet, my sister Holly, and my brother Matt.

iv

TABLE OF CONTENTS

LIST OF TABLES .................................................... vii

LIST OF FIGURES .................................................... x

INTRODUCTION ...................................................... 1
CHAPTER 1

WEED CONTROL AND CORN (Zea mays) TOLERANCE FROM SOIL-APPLIED

ISOXAFLUTOLE ...................................................... 4

ABSTRACT ...................................................... 4

INTRODUCTION ................................................. 6

MATERIALS AND METHODS ...................................... 7

RESULTS AND DISCUSSION ..................................... 10

Crop Response ............................................. 10

Weed Control .............................................. 12

Corn Grain Yield ........................................... 15

LITERATURE CITED ............................................ 19
CHAPTER 2

WEED CONTROL AND CORN (Zea mays) TOLERANCE AS AFFECTED BY THE

TIMING OF ISOXAFLUTOLE APPLICATION ........................... 29

ABSTRACT ..................................................... 29

INTRODUCTION ................................................ 3 1

MATERIALS AND METHODS ..................................... 33

Field Experiments .......................................... 33

Greenhouse Experiments ..................................... 34

RESULTS AND DISCUSSION ..................................... 40

Field Experiments .......................................... 40

Greenhouse Experiments ..................................... 42

LITERATURE CITED ............................................ 48
CHAPTER 3

PHYSIOLOGICAL BASIS FOR DIFFERENTIAL CORN (Zea mays) TOLERANCE

OF FOUR CORN HYBRIDS TO ISOXAFLUTOLE ........................ 60

ABSTRACT ..................................................... 60

INTRODUCTION ................................................ 61

MATERIALS AND METHODS ..................................... 62

Corn Tolerance ............................................. 62

TABLE OF CONTENTS (cont.)

Uptake, Translocation, and Metabolism ......................... 63

Statistical Analysis .......................................... 65

RESULTS AND DISCUSSION ..................................... 66

Corn Tolerance ............................................. 66

Herbicide Uptake ........................................... 66

Translocation .............................................. 66

Metabolism ............................................... 67

LITERATURE CITED ............................................ 70
CHAPTER 4

ENHANCING THE MARGIN OF SELECTIVITY OF ISOXAFLUTOLE IN CORN

(Zea mays) WITH ANTIDOTES ......................................... 76

ABSTRACT ..................................................... 76

INTRODUCTION ................................................ 78

MATERIALS AND METHODS ..................................... 80

Protecting Corn Against Isoxaflutole Injury ...................... 80

R-29148 Rate Response Study ................................. 82

Basis for R-29148 Efficacy ................................... 82

Statistical Analysis .............................. < ............ 86

RESULTS AND DISCUSSION ..................................... 86

Protecting Corn Against Isoxaflutole Injury ...................... 86

R-29l48 Rate Response Study ................................. 87

Basis for R-29l48 Eflicacy ................................... 88

LITERATURE CITED ............................................ 91

SUMMARY .......................................................... 98

vi

LIST OF TABLES

CHAPTER 1
WEED CONTROL AND CORN (Zea mays) TOLERANCE FROM SOIL-APPLIED
ISOXAFLUTOLE

Table 1. Conventional tillage and no-tillage site descriptions. .............. 21

Table 2. Rainfall distribution over 7-d intervals up to 28 d after application. . . 22

Table 3. Corn injury, weed control, and com grain yield with isoxaflutole and
isoxaflutole combinations applied preemergence. ....................... 23

Table 4. Corn injury, weed control, and corn grain yield with isoxaflutole and
isoxaflutole combinations applied preemergence. ....................... 24

Table 5. Corn injury, weed control, and corn grain yield with isoxaflutole and
isoxaflutole combinations applied preemergence. ....................... 25

Table 6. Corn injury, weed control, and corn grain yield with isoxaflutole and
isoxaflutole combinations applied preemergence. ....................... 26

Table 7. Corn injury, weed control, and corn grain yield with isoxaflutole and
isoxaflutole combinations applied preemergence. ....................... 27

Table 8. Weed control and corn grain yield with isoxaflutole and isoxaflutole
combinations applied preemergence. ................................. 28

CHAPTER 2

WEED CONTROL AND CORN (Zea mays) TOLERANCE AS AFFECTED BY THE

TIMING OF ISOXAFLUTOLE APPLICATION
Table I . Herbicide application information for field studies in 1996 and 1997. 50
Table 2. Rainfall distribution over 7-d intervals up to 28 d after planting. . . . . 51
Table 3. Influence of isoxaflutole and isoxaflutole tank-mixtures applied at four

different timings on corn injury, corn height, and corn grain yield for field studies
in 1996 and 1997. ................................................ 52

vii

LIST OF TABLES (cont.)

Table 4. Giant foxtail control 60 DAP with isoxaflutole and isoxaflutole tank-
mixtures applied at four different timings in field studies in 1996 and 1997. . . 53

Table 5. Corn injury and height reductions from isoxaflutole and isoxaflutole plus
metolachlor/benoxacor from foliar and foliar plus soil applications at three different
corn stages in a greenhouse study. ................................... 54

Table 6. Corn injury and height reduction from isoxaflutole and
isoxaflutole + acetochlor/MON-l3900 applied at four different timings in a
greenhouse study. ................................................ 55

Table 7. Absorption, translocation, and metabolism of isoxaflutole alone and in
combination with metolacholor/benoxacor at three corn stages. ............ 56

Table 8. Spray retention of foliar applications of isoxaflutole alone and in
combination with metolachlor/benoxacor to corn at 3 different growth stages. . 57

Table 9. Magnitude of increase of corn injury, '4 C-isoxaflutole absorption, and

retention due to the presence of metolachlor/benoxacor in the spray solution in
greenhouse studies. ............................................... 58

CHAPTER 3
PHYSIOLOGICAL BASIS FOR DIFFERENTIAL CORN (Zea mays) TOLERANCE
OF FOUR CORN HYBRIDS TO ISOXAFLUTOLE

Table 1. Differences in corn hybrid tolerance to isoxaflutole. .............. 72

Table 2. Uptake, translocation, and metabolism of isoxaflutole in four corn

hybrids. ........................................................ 73
Table 3. Factors contributing to hybrid sensitivity to isoxaflutole. .......... 74
CHAPTER 4

ENHANCING THE MARGINS OF SELECTIVITY OF ISOXAFLUTOLE IN CORN
(Zea mays) WITH ANTIDOTES

Table 1. Protection of herbicide/antidote combinations against isoxaflutole injury to
four different corn hybrids (14 DAT). ................................ 93

Table 2. Protection of corn from isoxaflutole injury to four different corn hybrids
with varying rates of R-29148. ...................................... 94

viii

LIST OF TABLES (cont.)

Table 3. Protection of the corn hybrids Pioneer 3751 and Pioneer 3963 from
isoxaflutole and diketonirile injury with the antidote, R-29148. ............ 95

Table 4. Absorption of metabolism of '4 C-isoxaflutole in corn alone and in the
presence of the antidote, R-29148 (18 DAT). ........................... 96

Table 5. Influence of the mixed function oxidase inhibitor, piperonyl butoxide, on the
activity of foliar applied isoxaflutole in four corn hybrids. ................ 97

ix

LIST OF FIGURES

INTRODUCTION
Figure 1. The degradation of isoxaflutole in plants. ....................... 3

CHAPTER 2

WEED CONTROL AND CORN (Zea mays) TOLERANCE AS AFFECTED BY THE
TIMING OF ISOXAFLUTOLE APPLICATION

Figure 1. F oliar absorption of '4 C-isoxaflutole over time in the presence and
absence of metolachlor/benoxacor. ................................... 59

CHAPTER 3

PHYSIOLOGICAL BASIS FOR DIFFERENTIAL CORN (Zea mays) TOLERANCE
OF FOUR CORN HYBRIDS TO ISOXAFLUTOLE

Figure 1. Metabolism of isoxaflutole over time in Pioneer 3394, Pioneer 3963,
Pioneer 3751, and Pioneer 3737. .................................... 75

INTRODUCTION

Isoxaflutole, an isoxazole herbicide, is a selective soil-applied herbicide used for
control of both broadleaf and grass weed species in corn. In susceptible species, isoxaflutole
causes a bleaching symptomology that is similar to other carotenoid biosynthesis inhibitors
that inhibit phytoene desturase. This enzyme catalyzes the first two desaturation steps in the
conversion of the colorless carotenoid precursor, phytoene, into colored carotenoids.
Isoxaflutole indirectly disrupts this pathway in carotenoid biosynthesis by inhibiting 4-
hydroxyphenylpyruvate dioxygenase (HPPD). Inhibition of HPPD decreases levels of
platoquinone, which turns out to be an essential cofactor of phytoene desaturase.

In soil and in plants, isoxaflutole is rapidly converted into a diketonitrile derivative
by opening the isoxazole ring. Diketonitrile is the active inhibitor of HPPD. In treated plants,
diketonitrile undergoes degradation to a benzoic acid derivative (Figure 1). The extent and
rate of this degradation has been correlated to the degree of species susceptibility.

The recent introduction of isoxaflutole provides additional alternatives for early
preplant and preemergence weed control in conventional tillage and no-tillage corn. Previous
research has shown that isoxaflutole provides excellent control of several broadleaf and grass
weed species. However, as with other selective herbicides, isoxaflutole may not adequately
control all weed species. The use of herbicide tank-mixtures with isoxaflutole could help
improve weed control and broaden the spectrum of weeds controlled. Further research needs

to be conducted to determine how isoxaflutole best fits in Michigan agriculture.

Equipment failures, excessive rainfall, and warmer growing conditions have been the
cause of untimely delays in applications of preemergence herbicides. These delays may result
in applications after corn has emerged. A number of preemergence herbicides can be applied
after corn emergence without causing corn injury or reducing weed control. However, little
research has been done to show the effects of isoxaflutole applications to emerged corn.

It has been reported that under certain conditions preemergence applications of
isoxaflutole can cause significant corn injury. It is important to understand the factors that
are associated with isoxaflutole injury to corn, and then be able to identify management
practices that would allow for the effective use of isoxaflutole without causing injury to corn.
One strategy that is already extensively used with a number of preemergence herbicides is
the use of antidotes to protect crops from herbicide injury. The major mechanism by which
currently developed antidotes protect crops from herbicidal injury is by the enhancement of
herbicide detoxification. Further research needs to be conducted to determine if herbicide
antidotes are effective in protecting corn from isoxaflutole injury.

Field, greenhouse, and laboratory experiments were initiated to: identify weed control
strategies utilizing isoxaflutole alone and with selected tank-mixtures in conventional tillage
and no-tillage corn, evaluate the effect of isoxaflutole application timing on corn tolerance
and weed control, determine the physiological basis for increased activity of foliar applied
isoxaflutole tank-mixtures with metolachlor/benoxacor, determine the physiological basis
for differences in tolerance among corn hybrids, evaluate antidotes as possible herbicide
protectants against isoxaflutole injury to corn, and determine the basis for the protective

action of effective antidotes.

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CHAPTER 1
WEED CONTROL AND CORN (Zea mays) TOLERANCE FROM
SOIL-APPLIED ISOXAFLUTOLE
Abstract: Field experiments were conducted in 1996, 1997, and 1998 to evaluate weed
control and corn tolerance from soil-applied isoxaflutole. Isoxaflutole treatments alone and
in tank-mixtures with other com herbicides were applied preemergence at two locations with
conventional tillage, and at early preplant and preemergence application timings in no-tillage
corn. Isoxaflutole was applied alone and in tank-mixtures with one-half the typical field rates
of other preemergence corn herbicides. In conventional tillage experiments in 1996, 1997,
and at one location in 1998, all treatments containing isoxaflutole provided greater than 90%
control of common lambsquarters, redroot pi gweed, common ragweed, and velvetleaf. In two
no—tillage experiments, common lambsquarters and velvetleaf control was greater than 90%,
regardless of application timing. However, control of redroot pi gweed and common ragweed
varied between years and application timings. Weed control was more variable from
herbicide treatments applied early preplant compared with the preemergence application
timing. Giant foxtail control in both tillage systems was rate, timing, and year dependent.
Isoxaflutole rates higher than 79 g ha’l controlled giant foxtail greater than 85%, at three of
five locations. At one location, tank-mixtures with isoxaflutole increased giant foxtail
control. Corn injury occurred in one of two conventional tillage locations and at the no—
tillage location in both 1996 and 1997. Injury was most commonly observed in coarse

textured soils with low clay and organic matter and was more severe with higher rates of

isoxaflutole. Com injury from isoxaflutole occurred at application rates above the proposed
rates for use on corn. In some cases, severe injury to corn reduced corn yield. Injury to corn
from isoxaflutole was not unique to any tillage system and was site, year, and rate dependent.
Nomenclature: Isoxaflutole, S-cyclopropyl isoxazol-4-yl-2-mesyl-4-trifluoromethylphenyl
ketone; common lambsquarters, Chenopodium album L. # CHEAL‘; common ragweed,
Ambrosia artemisiifolia L. # AMBEL; giant foxtail, Setariafaberi Herrm. # SETFA; redroot
pigweed, Amaranthus retroflexus L. # AMARE; velvetleaf, Abutilon theophrasti Medicus
# ABUTH; corn, Zea mays L.

Abbreviations: HPPD, 4-hydroxyphenylpyruvate dioxygenase; DAP, days after planting.

 

' Letters following this symbol are a WSSA— approved computer code from
Composite List of Weeds, Revised 1989. Available from WSSA.

5

INTRODUCTION

Isoxaflutole is a soil-applied herbicide used for selective broadleaf and grass weed
control in corn. Isoxaflutole is an isoxazole herbicide that inhibits 4-hydroxyphenylpyruvate
dioxygenase (HPPD, EC 1.13.11.27) (Pallett et al. 1998). It causes a bleaching
symptomology in susceptible species that is similar to herbicides that disrupt carotenoid
biosynthesis by inhibiting phytoene desturase (Lee et al. 1997; Luscombe and Pallett 1996;
Pallett et al. 1997).

Isoxaflutole provides a new alternative for preemergence (Bhowmik and Prostak
1996; Curvey and Kapusta 1996; Geier and Stahlman 1997; Luscombe et al. 1994; Mosier
et al. 1995; Obermeier et al. 1995; Veilleux et al. 1995; Vrabel et al. 1995; Wrucke et a1.
1997; Young et al. 1998) and early preplant (Mosier et al. 1995; Simkins et al. 1995) weed
control in corn. Researchers have reported that isoxaflutole has residual activity for at least
6 weeks after application in conventional tillage and no-tillage corn (Bhowmik and Prostak
1996; Luscombe et al. 1994; Mosier et a1. 1995) and provides excellent control of several
weed species at rates ranging from 53 g ha'l to 158 g ha’l (Bhowmik and Prostak 1996;
Curvey and Kapusta 1996; Geier and Stahlman 1997; Luscombe et al. 1994; Mosier et al.
1995; Obermeier et al. 1995; Simkins et al. 1995; Veilleux et al. 1995; Vrabel et al. 1995;
Wrucke et al. 1997; Young et al. 1998). However, at higher rates there have been some
reports of corn injury (Curvey and Kapusta 1996; Geier and Stahlman 1997). For instance,
Bhowmik and Prostak (1996) and Obermeier et al. (1995) observed corn injury when
isoxaflutole was applied at 158 g ha“, but complete recovery occurred by 42 days after
application.

Preemergence herbicide tank-mixtures have been utilized to improve weed control

and broaden the spectrum of weeds controlled (Johnson et al. 1997; Schuh and Harvey
1989). Tank-mixtures may also help reduce the selection for herbicide resistant weeds
(Shaner et al. 1997). Increased weed control has been reported when other preemergence
herbicides have been tank-mixed with isoxaflutole (Bhowmik and Prostak 1996; Obermeier
et al. 1995; Veilleax et al. 1995; Young et al. 1998). For example, Young et al. (1998)
observed a significant increase in common cocklebur (Xanthium strumarium L.) control
when one-half the normal use rate of atrazine (6-chloro-N-ethyl-N’—(1-methylethyl)-1,3,5-
triazine-2,4-diamine) was tank-mixed with isoxaflutole.

The objectives of this research were to (a) evaluate corn tolerance and yield response
from applications of isoxaflutole alone and in several tank-mixtures, and (b) identify weed

control strategies utilizing isoxaflutole in conventional tillage and no-tillage corn.

MATERIALS AND METHODS

Conventional tillage and no-tillage field experiments were conducted in 1996, 1997,
and 1998 to evaluate weed control and corn tolerance from soil-applied isoxaflutole.
Conventional tillage experiments were conducted at two locations each year; the Michigan
State University Crop and Soil Science Research Farm at East Lansing and the Michigan
State University Horticultural Experiment Station at Clarksville. No-tillage experiments were
conducted at the East Lansing location. The East Lansing soil was a Capac (fine-loamy,
mixed mesic Aerie Ochraqualfs), and the soil at Clarksville was a Lapeer (coarse-loamy,
mixed, mesic Mollie Haplaquepts). Specific soil texture, soil pH and (%) organic matter are
listed by year and location in Table l.

Tillage for the conventional tillage experiments consisted of moldboard plowing in

the fall prior to spring disking and field cultivation. Prior to spring cultivation, 320 kg ha‘l
of 46-0-0 fertilizer was applied broadcast. At planting, 140 kg ha" of 6-24-24 fertilizer was
applied as a banded treatment 5 cm below and 5 cm beside the corn seed. Pioneer 35732 corn
was planted at rate of 62 000 seeds/ha at East Lansing, and 66 690 seeds/ha at Clarksville for
the conventional tillage experiments.

Garst 87003 corn was planted into soybean [Glycine max (L.) Merr.] stubble at a rate
of 65 500 seeds/ha in the no-tillage experiments. Prior to planting, 333 kg ha" of 34-0-0
fertilizer was broadcast. At planting, 278 kg ha" of 6-24-24 fertilizer was applied as a banded
treatment 5 cm below and 5 cm beside the corn seed. Planting dates for all experiments are
listed in Table 1.

The experimental design for all experiments was a randomized complete block. Each
plot was 10.6 m long and consisted of 4 rows spaced 76 cm apart. The conventional tillage
experiments and the 1998 no-tillage experiment were replicated 3 times, and the 1996 and
1997 no-tillage experiments were replicated 4 times. The treatments in the 1996 and 1997
no-tillage experiments were arranged as a factorial. The factors consisted of herbicide
application timing (early preplant and preemergence) and herbicide treatment.

In all experiments, isoxaflutole was applied alone and in combination with other
preemergence corn herbicides. In 1996, herbicide treatments included isoxaflutole alone at
79, 105, and 132 g ha‘1 in conventional tillage and 105, 132, and 158 g ha‘l in no-tillage. In
1997, the isoxaflutole rates were reduced to 53, 79, and 105 g ha‘1 in conventional tillage and

79, 105, and 132 g ha’1 in no-tillage because of the excessive corn injury observed in 1996.

 

2 Pioneer Hi-Bred International, Inc., Des Moines, IA.
3 Garst Seed Company, 2369 330‘h Street, Slater IA.

8

In 1998, the isoxaflutole rates in no-tillage were reduced to correspond to the conventional
tillage rates. In each year, isoxaflutole was applied alone and in tank mixtures with 1.1 kg
ha’1 of metolachlor/benoxacor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-
methylethyl)acetamide) / (4-dichloroacetyl)-3,4-dihydro-3-methyl-2H—1,4-benzoxazine); 0.9
kg ha" of acetochlor/dichlormid (2-chloro-N-(ethoxymethyl)-N—(2-ethyl-6-
methylphenyl)acetamide) / (2,2-dichloro-N-N-di-2-propenylacetamide); 0.65 kg ha‘1 of
dimethenamid (2-chloro-N(2,4-dimethyl-3-thienyl)-N—(2-methoxy—1-methylethyl)-
acetamide); 0.84 kg ha" of pendimethalin (N-(l-ethylpropyl)-3,4-dimethyl-2,6-
dinitrobenzamine); 0.44 kg ha‘1 of BAYFOE 5043/metribuzin (4-fluoro-N-isopropyl-2-(5-
trifluoromethyl-l ,3,4—thiadiazol-2—yloxy) / (4-amino-6(1 , l -dimethylethyl)-3-(methylthio)-
1,2,4-triazin-5(4H)-one); and 1.1 kg ha" of atrazine. The isoxaflutole component of the tank
mixture was applied at 105 g ha‘1 in 1996 and 79 g ha" in 1997 and 1998. Additional
treatments included an atrazine plus metolachlor/benoxacor (1.1 and 2.2 kg ha")
preemergence herbicide standard; an untreated plot and a weed-free plot. Early preplant
herbicide treatments in the no-tillage experiments contained 1.0 % v/v of crop oil
concentrate“ and were applied 26 and 24 (1 prior to planting in 1996 and 1997, respectively.
All no-tillage experiments were treated with a preemergence application of glyphosate (N;
phosphonomethyl)glycine) plus 2,4-D ester (2,4-dichlorophenoxy)acetic acid) plus
ammonium sulfate at 420 g ae ha“, 560 g ha‘1 and 20 g L" , respectively, to eliminate existing
vegetation. All herbicides were applied with a tractor mounted, compressed-air sprayer

calibrated to deliver 2.6 L ha" at 207 kPa using 8003 flat-fan nozzless. Herbicide application

 

4Herbimax, a crop oil concentrate product of Loveland, Inc., Greeley, CO 80632.
5 Spraying Systems Co., PO. Box 7900, Wheaten, IL 60189.

9

dates and soil temperatures at application are presented in Table 1.

Weed control by species and com injury were evaluated visually with 0 representing
no visible injury and 100 representing complete plant death. Corn injury and weed control
were evaluated 30 and 60 d afier planting (DAP), respectively, in all experiments. Corn grain
yield was determined by harvesting the center two rows of each plot with a plot combine.
Seed weight was adjusted to 15% moisture.

Data were subjected to analysis of variance and mean separation using Fisher’s
Protected LSD test at a = 0.05. Data were combined over locations when treatment by
location interactions were not significant at a = 0.05. Non-transformed means for com injury
and weed control are presented since arcsine and square root transformations did not alter
the interpretation of the data. Results are presented by year due to changes in isoxaflutole

rates and significant year by treatment interactions.

RESULTS AND DISCUSSION
Crop Response. Corn tolerance data are presented separately by year, location, and tillage
systems due to changes in isoxaflutole rates and differences in crop response. Crop injury
consisted of bleaching of newly developed tissue followed by stunting, common symptoms
associated with isoxaflutole (Lusombe and Pallett 1996). Corn density was not affected at

any location or tillage system.

Conventional Tillage. There was a major difference in crop response to isoxaflutole at the
two conventional tillage locations. There were also significantly different amounts of rainfall

alter herbicide application between these two locations (Table 2). No herbicide injury to com

10

was evident at the East Lansing location in 1996 with any of the isoxaflutole treatments
(Table 3). However, at the Clarksville location all herbicide treatments containing
isoxaflutole injured corn. Isoxaflutole injured com 30%, 33% and 55% at 79, 105, and 132
g ha“, respectively, 30 DAP. All tank-mixtures with isoxaflutole injured corn. Increases in
corn injury were observed when isoxaflutole was combined with acetochlor/dichlormid,
dimethenamid, and BAYFOE 5043/metribuzin. Other studies have also shown significant
corn injury from high rates of isoxaflutole (Bhowmik and Prostak 1996; Obermeier et al.
1995). In addition to differences in soil type (Table 1) between the two locations, corn injury
at Clarksville may have resulted from increased amounts of rainfall after herbicide
application (Table 2). Research with other preemergence herbicides have also shown
enhanced crop injury from excessive amounts of rainfall closely after herbicide application
(Rowe and Penner 1990; Sprague et al. 1997; Wright et al. 1995).

Isoxaflutole was applied at 53, 79, and 105 g ha“ at both conventional tillage
locations in 1997. Significant corn injury occurred only at Clarksville when isoxaflutole was
applied at the highest rate of 105 g ha‘l and when it was tank-mixed with
acetochlor/dichlormid (Table 4).

In 1998, isoxaflutole rates and tank-mixtures were consistent with herbicide
treatments applied in 1997. There was no significant corn injury from any herbicide

treatment at either the East Lansing or Clarksville locations (Table 5).

No-Tillage. Differences in crop response were observed between early preplant and
preemergence applications of isoxaflutole in 1996. There was no corn injury from

isOxaflutole applied 26 d before planting (Table 6). However, all herbicide treatments

11

containing isoxaflutole applied preemergence injured corn. Corn injury increased with
increasing rates of isoxaflutole. Isoxaflutole injured com 18%, 28% and 60% at 105, 132,
and 158 g ha“, respectively, 30 DAP. Tank-mixtures with either acetochlor/dichlormid or
BAYFOE 5043/metribuzin increased corn injury when compared with isoxaflutole alone at
105 g ha".

As with the conventional tillage studies, isoxaflutole rates were reduced in the 1997
no-tillage trial. There was no injury to corn from isoxafiutole applied early preplant (Table
7). However, isoxaflutole applied preemergence at 132 g ha‘I injured corn 5%, 30 DAP. This
corn injury may be related to poor seed fiirrow closure when the corn was planted.

Herbicide treatments were applied only at the preemergence application timing in
1998. Isoxaflutole rates were reduced from rates applied in 1997, to 53, 79, and 105 g ha".

Corn was not injured from any of the herbicide treatments (Table 8).

Weed Control. Weed control data are presented separately by year and tillage systems due
to changes in isoxaflutole rates. In conventional tillage experiments, weed control data has
been combined over locations when there were no location by treatment interactions. Weed

control data are presented by application timing in no-tillage experiments.

Conventional Tillage. Giant foxtail control was greater than 87% with all herbicide
treatments in 1996 (Table 3). Isoxafiutole tank-mixtures with metolachlor/benoxacor,
acetochlor/dichlormid, dimethenamid, pendimethalin, and BAYFOE 5043/metribuzin
increased late season control of giant foxtail to greater than 96%. Other researchers have also

observed enhanced giant foxtail control from tank-mixtures with isoxaflutole (Young et al.

12

1998). Giant foxtail control with isoxaflutole tank-mixtures was comparable to
metolachlor/benoxacor plus atrazine. Control of common lambsquarters, redroot pigweed,
and common ragweed was greater than 96% with all herbicide treatments. All treatments
containing isoxaflutole controlled velvetleaf greater than 98% compared with 62% control
with metolachlor/benoxacor plus atrazine.

Isoxaflutole controlled giant foxtail 77%, 84%, and 85% at 53, 79, and 105 g ha",
respectively, in 1997 (Table 4). There was no benefit in tank-mixing other preemergence
herbicides with isoxaflutole at 79 g ha'l for giant foxtail control. Giant foxtail control was
similar to metolachlor/benoxacor plus atrazine with all treatments containing isoxaflutole
greater than 79 g ha", except when tank-mixed with pendimethalin. Common lambsquarters,
redroot pigweed, and common ragweed control was greater than 90% with all herbicide
treatments. Control of velvetleaf was greater than 92%, with all treatments containing
isoxaflutole compared to 47% control with metolachlor/benoxacor plus atrazine.

Weed control data in 1998 were presented by location due to differences in control
between East Lansing and Clarksville. At East Lansing, control of giant foxtail, common
lambsquarters, redroot pigweed, and velvetleaf was less than 50% with isoxaflutole at 53,
79, and 105 g ha’l (Table 5). Herbicide tank-mixtures containing atrazine increased common
lambsquarters control to 80% and 90% with metolachlor/benoxacor and isoxaflutole,
respectively. Redroot pigweed control was increased to 62% when isoxaflutole was tank-
mixed acetochlor/dichlormid. At Clarksville, control of common lamsquarters and redroot
pigweed was greater than 89% with all herbicide treatments. Precipitation was less than 17
mm at both locations within 28 d after herbicide treatment (Table 2). Inadequate weed

centrol at the East Lansing location was probably due to limited rainfall after herbicide

13

application, decreasing herbicide uptake. Curvey and Kapusta (1996) reported inconsistent
weed control with isoxaflutole when there was no significant rainfall within 14 d afier
application. Differences in weed control between the two locations could be due to
differences in weed germination related to the amount of moisture in the soil at planting. At
East Lansing, 14 (1 prior to planting there was 81 mm of rainfall and at Clarksville there had

only been 8 mm of rain.

No-Tillage. Season-long weed control evaluations were made 60 DAP, which was equivalent
to 86 days afier the early preplant application in 1996. Early preplant and preemergence
applications of isoxaflutole at 105 g ha'l controlled giant foxtail 70% and 88%, respectively
(Table 6). There was generally no benefit in tank-mixing other herbicides with isoxaflutole
for control of giant foxtail. However, when atrazine was tank-mixed with isoxaflutole there
was increased control of giant foxtail when applied at the early preplant timing. Giant foxtail
control was greater than 80% with the two higher rates of isoxaflutole and was independent
of application timing. Common lambsquarters control was greater than 80% with all
herbicide treatments regardless of application timing. Preemergence treatments containing
isoxaflutole controlled common lambsquarters greater than 93%, which was significantly
greater than the metolachlor/benoxacor : atrazine combination. All herbicide treatments
containing isoxaflutole controlled redroot pi gweed greater than the standard of
metolachlor/benoxacor plus atrazine at the early preplant application timing. However, only
isoxaflutole at 158 g ha‘I and the isoxaflutole : atrazine tank-mixture provided adequate
control (>80%) of redroot pigweed at this timing. Redroot pigweed control was 85% or

greater with all herbicide treatments applied preemergence. Control of common ragweed was

14

inadequate (<80%) with all herbicide treatments, except the isoxaflutole : atrazine tank-
mixture at the early preplant application timing. Preemergnce applications of all herbicide
treatments containing isoxaflutole controlled common ragweed equal to the
metolachlor/benoxacor : atrazine tank-mixture.

Early preplant herbicide treatments were applied 25 days before planting in 1997
(Table 1). Isoxaflutole at all rates controlled giant foxtail, common lambsquarters, and
velvetleaf (>90%), regardless of application timing (Table 7). Tank-mixing other herbicides
with isoxaflutole did not benefit control of any of these weed species. Control of common
lambsquarters and velvetleaf with isoxaflutole was superior to the metolachlor/benoxacor :
atrazine tank-mixture. All herbicide treatments controlled common ragweed (>80%),
regardless of application timing. Common ragweed control was excellent (>90%) with all
isoxaflutole treatments applied preemergence. Preemergence applications of isoxaflutole
needed to be applied at 130 g ha“l or in tank-mixtures with metoalchlor, acetochlor,
dimethenamid, BAYFOE 5043/metribuzin, or atrazine to adequately control (>80%) redroot
pigweed. However, season-long redroot pigweed control from isoxaflutole applied early
preplant required tank-mixtures with atrazine (Table 7).

In the 1998 no-tillage trial, herbicide treatments were applied only at a preemergence
timing. The predominate weed species throughout this trial was velvetleaf. Due to limited
rainfall after herbicide application (Table 2), velvetleaf control was highly variable (Table
8). Control was greatest when isoxaflutole was applied at 105 g ha‘1 and when 79 g ha“ of

isoxaflutole was tank-mixed with atrazine.

COrn Grain Yield. Corn grain yield was presented separately by tillage system, location,

15

and herbicide application timing.

Conventional Tillage. In 1996, corn grain yield was higher at the East Lansing location.
Heavy infestations of giant foxtail and velvetleaf severely decreased yield of the untreated
check at East Lansing (Table 3). At this location, corn grain yield was reduced compared
with the weed-free check when isoxaflutole was applied at 105 and 132 g ha" and when
isoxaflutole was tank-mixed with acetochlor/dichlormid, even though visible corn injury was
not apparent. However, at Clarksville, reductions in corn yield resulted from severe corn
injury. Yields were reduced when isoxafiutole was applied at 132 g ha‘1 and when
isoxaflutole was tank-mixed with either acetochlor/dichlormid, pendimethalin, or BAYFOE
5043/metribuzin.

Corn grain was harvested only at the East Lansing location in 1997. Yields from all
herbicide treatments were similar to the weed-free check (Table 4). The only significant
reduction in corn yield was from the untreated check, due to weed competition.

Weed competition reduced corn yields in 1998 at the East Lansing location (Table
5). Limited rainfall at this location probably decreased herbicide uptake resulting in poor
weed control. However, excellent weed control at Clarksville resulted in yields from all

treatments being similar to the weed-free check.

No-Tillage. Generally, corn grain yields were higher from herbicide treatments applied
preemergence than with treatments applied early preplant. In 1996, herbicide treatments
applied 26 (1 prior to planting resulted in decreased season long control of giant foxtail,

redroot pigweed, and common ragweed (Table 6). Reductions in control of these weed

16

species probably resulted in greater weed competition causing decreases in com grain yield.
However, reductions in yield from preemergence treatments occurred when corn was
severely injured. Treatments that resulted in yield reductions were isoxaflutole at 158 g ha",
and the isoxaflutole tank-mixtures with acetochlor/dichlormid or atrazine.

Early preplant treatments in 1997 that did not adequately control redroot pigweed
resulted in reductions in corn yield (Table 7). Early preplant treatments that did not reduce
corn yield were isoxaflutole at 105 g ha‘l and 132 g ha“, isoxaflutole plus BAYFOE
5043/metribuzin, isoxaflutole plus atrazine, and atrazine plus metolachlor/benoxacor.
Preemergence applications of isoxaflutole alone at 79 g ha’1 and metolachlor/benoxacor plus
atrazine resulted in reduced yield compared with the weed-free check. This was likely due
to inadequate control of redroot pigweed with the lowest rate of isoxaflutole and the lack of
velvetleaf control with metolachlor/benoxacor plus atrazine.

Herbicide treatments in 1998 that did not adequately control velvetleaf resulted in
reduced corn yield (Table 8). Treatments that did not reduce corn yield were isoxaflutole at
79 g ha“1 and 105 g ha" and isoxafiutole tank-mixed with either BAYFOE 5043/metribuzin
or atrazine.

Collectively, these experiments demonstrated that preemergence applications of
isoxaflutole provided excellent control of a number of weed species, except under limited
rainfall. In some cases, tank-mixing other herbicides with isoxaflutole increased giant foxtail
and redroot pigweed control. In no-tillage corn, herbicide treatments applied preemergence
provided more consistent weed control than treatments applied early preplant. This is similar
to what other researchers have shown with early preplant applications of herbicides (Buhler

1991; Ritter 1985). Johnson et al. (1997) observed more variable weed control when

17

herbicide treatments were applied more than 15 d prior to planting. Under certain conditions,
corn was injured and yield was reduced when isoxaflutole rates were above the proposed
rates for use on corn. Injury was most commonly observed in coarse textured soils with low
clay and organic matter and was more severe with higher rates of isoxaflutole. Injury to corn

from isoxaflutole was not unique to any tillage system and was site, year, and rate dependent.

18

LITERATURE CITED

Bhowmik, P. C. and R. G. Prostak. 1996. Activity of EXP 31130A in annual weed control
in field corn. Weed Sci. Soc. Am. Abstr. 36:13.

Buhler, D. D. 1991. Early preplant atrazine and metolachlor in conservation tillage corn (Zea
mays L.). Weed Technol. 5:66-71.

Curvey, S. E. and G. Kapusta. 1996. Corn Weed Control with EXP31130A. North Cent.
Weed Sci. Soc. 51:57-58.

Geier, P. W. and P. W. Stahlman. 1997. Efficacy of Isoxaflutole alone and in combinations
in corn. North Cent. Weed Sci. Soc. 52:81.

Johnson, W. G., M. S. DeFelice, and C. S. Holman. 1997. Application Timing Affects Weed
Control with Metolachlor Plus Atrazine in No-Till Corn (Zea mays). Weed Technol. 1 1:207-
21 1 .

Lee, D. L., M. P. Prisbylla, T. H. Cromartie, D. P. Dagarin, S. W. Howard, W. M. Provan,
M. K. Ellis, T. Fraser, and L. C. Mutter. 1997. The discovery and structural requirements of
inhibitors of p-hydroxyphenylpyruvate dioxygenase. Weed Sci. 45:601-609.

Luscombe, B. M. and K. E. Pallett. 1996. Isoxaflutole for weed control in maize. Pestic.
Outlook. 29-32.

Luscombe, B. M., T. E. Vrabel., M. D. Paulsgroves, S. Cramp, P. Cain, A. Gamblin, and J.
C. Millet. 1994. RPA 201772: A new broad spectrum preemergence herbicide for com. Proc.
North Cent. Weed Sci. Soc. 59:57-58.

Mosier, D. G., W. Duckworth, K. K. Watteyne, L. L. King, and M. A. Wrucke. 1995.
Efficacy of EXP31 130A in conventional and no-till corn. North Cent. Weed Sci. Soc. 50:74.

Obermeier, M. R., C. H. Slack, J. R. Martin, and W. W. Witt. 1995. Evaluations of
EXP3113OA - A new preemergence corn herbicide. Proc. North Cent. Weed Sci. Soc. 50:25.

Pallett, K. E., J. P. Little, M. Sheekey, and P. Veerasekaran. 1998. The mode of action of

isoxaflutole 1. Physiological effects, metabolism, and selectivity. Pestic. Biochem. Physiol.
62:1 13-124.

Pallett, K. E., J. P. Little, P. Veerasekaran, and F. Viviani. 1997. Extended summary new
perspective in mechanisms of herbicide action. Pestic. Sci. 50:83-84.

Ritter, R. L. 1985. Control systems for triazine-resistant smooth pigweed (Amaranthus

hybridus L.) in corn (Zea mays L.) and soybeans [Glycine max (L.) Merr.]. Weed Sci.
33:400—404.

19

Rowe, L. and D. Penner. 1990. Factors affecting choloracetanilide injury to corn. Weed
Technol. 4:904—906.

Schuh, J. F. and R. G. Harvey. 1989. Woolly cupgrass (Erichloa villosa) control in (Zea
mays) with pendimethalin/triazine combinations and cultivation. Weed Sci. 37:405-411.

Shaner, D. L., D. A. F eist, and E. J. Retzinger. 1997. SAMOA: one company’s approach to
herbicide-resistant weed managment. Pesticide Sci. 5 1 2367-3 70.

Simkins, G .S., V. H. Lengkeek, W. Duckworth, and T. E. Vrabel. 1995. Effect of
application timing on performance of EXP 31130A for field corn weed control. Proc. North
Cent. Weed Sci. Soc. 50:25.

Sprague, C. L., E. W. Stoller, and S. E. Hart. 1997. Preemergence broadleaf control and crop
tolerance in imidazolinone-resistant and -susceptible corn (Zea Mays). Weed Technol.
1 1 :1 18-122.

Veilleux, D. P., J. D. Lavoy, W. Duckworth, and M. L. Christian. 1995. Efficacy of
EXP31130A tank mixtures in conventional and no-till corn. North Cent. Weed Sci. Soc.
50:75.

Vrabel, T. E., J. O. Jensen, M. A. Wrucke, C. Hicks. 1995. EXP31130A: A new preemergent
herbicide for corn. Proc. North Cent. Weed Sci. Soc. 50:24-25.

Wright, T. R., A. G. Ogg Jr., and E. P. Fuerst. 1995. Dissipation and water activation of
UCC-C4243. Weed Sci. 43:149-155.

Wrucke, M. A., L. L. King, and D. P. Veilleux. 1996. Effect of cultivation on performance
of isoxaflutole in corn. North Cent. Weed Sci. Soc. 51:11.

Young, B. G., S. E. Hart, and F. W. Simmons. 1998. Performance of preemergence
applications of isoxaflutole in corn. Weed Sci. Soc. Am. Abstr. 38: 1.24.

20

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88 88 o: 8 8 8 8 8 8 8 8 8 o o E _ + 88 0:88 + 28222:
88 a8 8 8 8 8 8 8 2: 2: 8 2: o o E _ + 8 288. + 22:88:
28.55:: \
:8: 88 2: 8 8 8 8 8 2: 2: 2: 8 _ o 8: + 8 88 88% + 22:88:
88 88 8 8 2: 8 8 8 8 8 8 8 _ o :2: + 8 2882625 + 22:88:
$8 88 2: 8 2: 8 8 8 8 2: 2: 8 _ o :8 + 8 285826 + 22:88:
28 88 8 8 8 a 8 8 2: 8 2: 2: o o :8 + 8 82028.: + 22:88:
88 88 2: 8 8 8 2: 8 2: 8 8 8 N o E _ + 8 1282202 + 22:88:
88 88 8 8 8 8 8 8 2: 8 2: 8 m o 8: 22:88:
8 8 88 2: 8 8 2: 8 8 2: 2: 8 8 o o 2: 22:88:
: _ 8 88 8 8 8 8 8 s 8 8 8 8 N o 8 22:88:
I .2: M2. II 2. 2. I .2. w I
8: 8: m2: 8: :2: 8: mm: 8: mm: .3: m2: 8: 8: x8: 8: 2228:
22> 220 :59: 5:52 5232 28:6 <88, 22::
28:80 863

 

82 82:32

626908082 Com—mum 88:82:88 2025888 28 232.2388 53: 22> 58w 88 ER .888 303 N 2328

27

Table 8. Weed control and corn grain yield with isoxaflutole and isoxaflutole combinations
applied preemergence.

 

No-Tillage 1998

 

 

 

 

Herbicide Rate Injurya Velvetleaf Grain Yield
Controlb
—gha"——%— % kgha"———
Isoxaflutole 53 0 48 7664
Isoxaflutole 79 0 80 9349
Isoxaflutole 105 0 93 10156
Isoxaflutole + metolachlorc 79 + 1121 0 65 8856
Isoxaflutole + acetochlord 79 + 897 0 73 8704
Isoxaflutole + dimethenamid 79 + 650 0 58 8630
Isoxaflutole + pendimethalin 79 + 841 O 68 8391
Isoxaflutole + BAYFOE 5043 79 + 437 0 86 9762
/ metribuzin
Isoxaflutole + atrazine 79 + 1121 0 93 10185
Metolachlor + atrazine 2242 + 1121 0 45 8255
Untreated check 0 0 2848
Weed-free check 0 100 10544
LSD 0.05 —NS— — 17— —1664——

 

' Corn injury was evaluated 30 DAT.
bVelvetleaf control was evaluated 60 DAT.

° Contained the herbicide safener benoxacor.
“ Contained the herbicide safener dichlormid.

28

CHAPTER 2
WEED CONTROL AND CORN (Zea mays) TOLERANCE AS AFFECTED
BY THE TIMING OF ISOXAFLUTOLE APPLICATION
Abstract. Field studies were conducted in 1996 and 1997 to determine whether isoxaflutole
and isoxaflutole tank-mixtures could be used for weed control after corn had emerged.
Isoxaflutole at 105 g ha‘l and tank-mixed metolachlor/benoxacor or atrazine were applied
preemergence, and to spike, 2-leaf, and 4-leaf corn. Herbicide treatments over the four
application timings provided greater than 90% common lambsquarters, common ragweed,
redroot pigweed, and velvetleaf control. However, giant foxtail control varied between years
and application timings. Severe corn injury, 70% and 40%, was observed when isoxaflutole
tank-mixed with metolachlor/benoxacor was applied to 2~leaf and 4-leaf corn, respectively.
Greenhouse studies confirmed increased corn injury from delayed applications of
isoxaflutole tank-mixed with metolachlor/benoxacor. Similarly, increased corn injury was
observed from postemergence applications of isoxaflutole tank-mixed with acetochlor/MON-
13900. Herbicide absorption, translocation, metabolism, and retention studies were
conducted to determine the physiological basis for the observed corn injury from delayed
applications of the isoxaflutole tank-mixture with metolachlor/benoxacor.
Metolachlor/benoxacor increased radiolabeled isoxaflutole absorption when applied to spike,
2-leaf and 4-leaf corn. Isoxaflutole translocation and metabolism did not explain enhanced
corn injury. However, isoxaflutole retention increased 5-fold when metolachlor/benoxacor

was present in the spray solution and applied to 2-1eaf and 4-1eaf corn. Increased isoxaflutole

29

absorption and retention appeared to be the basis for increased corn injury when tank-mixed

with metolachlor/benoxacor and applied to emerged corn.

Nomenclature: Acetochlor, 2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-
methylphenyl)acetamide); atrazine, 6—chloro-N-ethyl-N’ -( 1-methylethyl)-l ,3 ,5-triazine-2,4-
diamine; benoxacor, (4-dichloroacetyl)-3,4-dihydro-3-methyl-2H-1,4—benzoxazine;
isoxaflutole, 5-cyclopropyl isoxazol-4-y1-2-mesyl-4-trifluoromethylphenyl ketone;
metolachlor, 2-chloro-N-(2-ethyl-6—methylphenyl)-N-(2-methoxy- 1 -methylethyl)acetamide;
MON-13900, (:)-3-dichloroacetyl-5-(2-furanyl)-2,2-dimethyl oxazolidine; common
lambsquarters, Chenopodium album L. # CHEAL"; common ragweed, Ambrosia
artemisiifolia L. # AMBEL; giant foxtail, Setariafaberi Herrm. # SETFA; redroot pi gweed,
A maranthus retroflexus L. # AMARE; velvetleaf, Abutilon theophrasti Medicus # ABUTH;
corn, Zea mays L.

Abbreviations: DAP, days afier planting; DAT, days after treatement; HAT, hours after

treatment; HPPD, 4-hydroxyphenylpyruvate dioxygenase; LSS, liquid scintillation

spectrometry; TLC, thin layer chromatography.

 

' 6 Letters following this symbol are a WSSA- approved computer code from
Composite List of Weeds, Revised 1989. Available from WSSA.

3O

INTRODUCTION

Isoxaflutole is a soil applied herbicide used for selective broadleaf and grass weed
control in corn. Isoxaflutole, an isoxazole herbicide, is a competitive inhibitor of the 4-
hydroxyphenylpyruvate dioxygenase enzyme (HPPD, EC 1.13.11.27) (Pallett et al. 1998).
Inhibition of HPPD reduces levels of plastoquinone, a cofactor of phytoene desaturase, a key
enzyme in carotenoid biosynthesis (Lee et al. 1997; Pallett et al. 1997). Isoxaflutole causes
a bleaching symptomology in susceptible species that is similar to herbicides that disrupt
carotenoid biosynthesis by inhibiting the phytoene desaturase enzyme (Lee et al. 1997;
Luscombe and Pallett 1996; Pallett et al. 1997).

Isoxaflutole provides new alternatives for preemergence weed control in corn
(Bhowmik and Prostak 1996; Curvey and Kapusta 1996; Geier and Stahlman 1997;
Luscombe et al. 1994; Mosier et al. 1995; Obermeier et al. 1995; Veilleux et al. 1995; Vrabel
et al. 1995; Wrucke et al. 1997; Young et al. 1998). Researchers have reported that
isoxaflutole has residual activity for at least 6 weeks after application in conventional tillage
and no-tillage corn (Bhowmik and Prostak 1996; Luscombe et al. 1994; Mosier et al. 1995)
and provides excellent control of several weed species at rates ranging from 53 g ha'l to 158
g ha’l (Bhowmik and Prostak 1996; Curvey and Kapusta 1996; Geier and Stahlman 1997;
Luscombe et al. 1994; Mosier et al. 1995; Obermeier et al. 1995; Simkins et a1. 1995;
Veilleux et al. 1995; Vrabel et al. 1995; Wrucke et al. 1997; Young et al. 1998). There have
also been some reports of greater weed control when other preemergence herbicides have
been tank-mixed with isoxaflutole (Bhowmik and Prostak 1996; Obermeier et al. 1995;
Veilleax et al. 1995; Young et al. 1998). For instance, Young et al. (1998) observed

significant increases in common cocklebur (Xanthium strumarium L.) and giant foxtail

31

control when one-half the normal use rates of atrazine and metolachlor/benoxacor were tank-
mixed with isoxaflutole, respectively.

Equipment failure, excessive rainfall, and warm growing conditions may cause
untimely delays in applications of preemergence herbicides resulting in applications after
corn has emerged. These herbicide applications are often referred to as delayed preemergence
or early postemergence applications. Two concerns with these types of applications are the
potential for corn injury and herbicidal effectiveness on emerged weeds. A number of
preemergence herbicides including: atrazine, alachlor (2-chloro-N-(2,6'-diethylphenyl)-N-
(methoxymethyl)acetamide), metolachlor/benoxacor, acetochlor/dichlormid (2-chloro-N-
(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)acetamide)/(2,2-dichloro-N-N—di-2-
propenylacetamide), acetochlor/MON-13900, cyanazine (2-[[4-chloro-6-(ethylamino)-1,3,5-
triazin-2-yl]amino]-2-methylpropanenitrile), pendimethalin (N—( 1-ethylpropyl)-3 ,4-dimethyl-
2,6-dinitrobenzamine), python (N-(2,6-difluorophenyl)-5-methyl[ 1 ,2,4]triazolo[1 ,5-
a]pyrimidine-2-sulfonamide), and a number of combinations of these herbicides can be
applied after corn has emerged (Kells 1998). However, there has been little research on
isoxaflutole or isoxaflutole tank-mixtures applied to emerged corn.

The objectives of this research were to (a) evaluate the effect of application timing
on corn tolerance for isoxaflutole alone and in tank-mixtures with metolachlor/benoxacor or
atrazine, (b) examine weed control from delayed isoxaflutole applications, (c) confirm the
enhanced activity of isoxaflutole and metolachlor/benoxacor on corn injury fi'om delayed
applications in the greenhouse, (d) compare the increased activity of the isoxaflutole :
metolachlor/benoxacor tank-mixture with a tank-mixture of isoxaflutole and another

chloroacetamide herbicide, acetochlor/MON-13900 on corn injury, and (e) determine the

32

physiological basis for the enhanced corn injury observed from delayed application timings

of isoxaflutole tank-mixtures with metolachlor/benoxacor.

MATERIALS AND METHODS
Field Experiments

Conventional tillage experiments were conducted in 1996 and 1997 to evaluate weed
control and corn tolerance from isoxaflutole and isoxaflutole tank-mixtures applied at four
different timings. Experiments were conducted at the Michigan State University Crop and
Soil Science Research Farm at East Lansing on a Capac sandy clay loam soil (fine-loamy,
mixed mesic Aeric Ochraqualfs) containing 2.4% organic matter with a pH of 6.1 in 1996
and 2.2% organic matter with a pH of 6.4 in 1997.

Tillage consisted of moldboard plowing in the fall prior to spring disking and field
cultivation. Prior to spring cultivation, 320 kg ha" of 46-0-0 fertilizer was applied broadcast.
At planting, 140 kg ha‘1 of 6—24-24 fertilizer was applied as a banded treatment 5 cm below
and 5 cm beside the corn seed. Pioneer 35737 corn was planted on May 17 and May 13 in
1996 and 1997, respectively, at a rate of 62 000 seeds ha". Each plot was 10.6 m long and
consisted of 4 rows spaced 76 cm apart.

Experiments were conducted as a randomized complete block design in a factorial
arrangement with 3 replications. The factors consisted of herbicide application timing and
herbicide treatment. Herbicide application timings were preemergence and when com was
at the spike, 2-1eaf and 4-leaf stages. Com leaf stages are described as the number of visible

leaves. Herbicide treatments included isoxaflutole alone at 105 g ha‘1 and in combination

 

7Corn, Pioneer Hi-Bred International, Inc., Des Moines, IA.

33

with 1.1 kg ha“l of metolachlor/benoxacor; or 1.1 kg ha" of atrazine. Additional treatments
not included in the factorial arrangement were an untreated check and a weed-free check. All
herbicides were applied with a tractor mounted, compressed-air sprayer calibrated to deliver
2.6 L ha'l at 207 kPa using 8003 flat-fan nozzless. Herbicide application times, corn stages,
weed heights, and densities are presented in Table l. Rainfall data for both years are
presented in Table 2.

Corn tolerance was evaluated 30 DAP by visually evaluating plants for bleaching
and necrotic symptoms and also by measuring corn height (base to highest portion of the
plant). Weed control by species was visually evaluated 60 DAP. Visual evaluations were
based on a scale of 0 (no effect) to 100% (complete weed or crop death). Corn grain yield
was determined by harvesting the center two rows of each plot with a plot combine. Seed
weight was adjusted to 15% moisture.

Data were subjected to analysis of variance and means separated using Fisher’s
Protected LSD test at a = 0.05. Data were combined over years when treatment and/or
application timing by year interactions were not significant at a = 0.05. Non-transformed
means for corn injury and weed control are presented since arcsine and square root
transformations did not alter the interpretation of the data. Corn height and yield results were

converted to a percent of the weed-free treatment after mean separation.

Greenhouse Experiments

General Plant Culture. Pioneer 3573 corn seeds were planted two per pot, 2.54 cm deep in

pots (875 ml) containing a Spinks loamy sand (sand, mixed, mesic Psammentic Hapludalfs).

 

8Spraying Systems Co., PO. Box 7900, Wheaton, IL 60189.

34

Corn plantings were staggered to ensure the appropriate corn stage at time of herbicide
application. Plants were grown in a greenhouse maintained at 25:2 C. Natural sunlight was
supplemented with light from sodium vapor lamps, which provide a total midday light
intensity of 1,000 umol m'2 s‘I photosynthetic photon flux at plant height during a 16-h

photoperiod. Plants were watered and fertilized as needed to promote optimum growth.

Corn Tolerance. Two separate greenhouse experiments were established to evaluate corn
tolerance to early postemergence applications of isoxaflutole alone and in combination with
acetanilide herbicides. In the first experiment, herbicide treatments consisted of 105 g ha‘l
of isoxaflutole; 1.1 kg ha" of metolachlor/benoxacor; and a tank mixture of the two
herbicides. Herbicides were applied when com was at three different growth stages; spike,
2-leaf, and 4-leaf (5, 8, and 15 cm tall, respectively). In addition to herbicide treatment and
application timing, foliar absorption and foliar plus root absorption were evaluated. Root
uptake of herbicides was prevented by covering the soil surface with vermiculite before
herbicide application. The vermiculite was removed immediately after application.

In a second study, herbicide treatments consisted of 105 g ha’l of isoxaflutole; 1.8 kg
ha" of acetochlor/MON-13900; and a tank mixture of the two herbicides. Herbicide
applications were made preemergence and when com was at the spike, 1-leaf, and 2-leaf
stages (4, 6, and 17 cm tall, respectively). Immediately after the preemergence application,
herbicides were incorporated by overhead irrigation (1.0 to 2.0 cm).

In both studies, herbicides were applied through an 8003 E flat fan nozzle delivering
234 L ha“ at a pressure of 172 kPa. Corn tolerance was evaluated 10 d after treatment

(DAT) by visually evaluating plants for bleaching and necrotic symptoms and also by

35

measuring corn height (base to the highest portion of the plant as they stood). Visual corn
injury ratings were based on a scale from 0 to 100, with 0 indicating no effect and 100
indicating plant death. Corn height was recorded in cm and presented as a percent of the non-
treated plants, with 0 indicating total reduction in plant height and 100 indicating height

equal to the non-treated plants.

Absorption. A study was conducted to compare absorption of isoxaflutole alone and in
combination with metolachlor/benoxacor in corn at the spike, 2-leaf, and 4-leaf growth
stages. Corn was planted and grown in soil media as previously described but with one plant
per pot. The youngest fiilly developed leaf (with a collar) was selected for l“C-isoxaflutole
treatment. This was the 1St true leaf on the 2-leaf corn and 2“d true leaf on the 4-leaf corn. For
corn at the spike stage the l“C-isoxaflutole was applied to the abaxial side of what would
develop into the 1St true leaf. Leaf area of the leaves targeted for ”C-isoxaflutole was
measured with a transparent belt conveyor accessory for a portable leaf area meter". Previous
research conducted by Hart et a1. (1992) was used to determine that 10 u g of spray solution
was retained per cm2 of leaf. Each plant was treated with at a minimum of 333 Bq of phenyl-
U-labeled l4C-isoxaflutole (1,889 kBq mg‘l specific activity, 99.3% purity). Separate spotting
solutions contained the appropriate amounts of l“C-isoxaflutole, unlabeled isoxaflutole,
formulation blank, and water alone and in combination metolachlor/benoxacor to simulate
105 g ha“1 of isoxaflutole and 1.1 kg ha" of metolachlor/benoxacor at a spray volume of 234
L ha" for the appropriate leaf size.

Unabsorbed "C-isoxaflutole was removed by gently swirling the treated leaf in a 20-

 

9Portable leaf area meter, Li-Cor Inc., PO. Box 4425, Lincoln, NE 68504.

36

ml scintillation vial containing 3 ml of 50% methanol solution for 60 sec. Treated leaves
were then rinsed with 0.5 ml of 50% methanol as they were removed from the scintillation
vial. The efficiency of this procedure was 99% removal of the applied l4C-isoxaflutole.
Unabsorbed l“C-isoxaflutole was quantified using liquid scintillation spectrometry (LSS).
The amount of MC-isoxaflutole absorbed was calculated as the difference between the
amount of 1“C applied and the amount I"C recovered in the 50% methanol wash. Absorption

of l"C-isoxaflutole was sampled at 0, 1, 4, 12, and 24 h.

T ranslocation and Metabolism. A study similar to the absorption experiment was designed
to compare translocation and metabolism of isoxaflutole alone and in combination with
metolachlor/benoxacor in corn at the spike, 2-leaf, and 4-1eaf stages. Isoxaflutole absorption
was also evaluated in this experiment. Corn plants were grown in the same manner as in the
absorption study. When corn plants were at the appropriate stages, the leaf targeted for MC
application was covered with cellophane and the remainder of the plant treated with
unlabeled isoxaflutole at 105 g ha'I or isoxaflutole tank-mixed with metolachlor/benoxacor
at 105 g ha" and 1.1 kg ha“, respectively. Herbicides were applied with the same conditions
and equipment used for the corn tolerance experiments.

Radiolabeled solutions were applied the same as in the absorption study. However,
to achieve sufficient absorption of radioactivity for metabolism research, a minimum of 3.3
kBq was needed per treatment. Treated leaves were excised from the plant at 12 and 72 h
after treatment (HAT). Absorption was determined from these plants using the same
procedures previously described. Plant parts were sectioned into treated leaf, above treated

leaf, below treated leaf, and roots. These parts were immediately frozen and stored at -30

37

C until further analysis. All plant parts excluding the treated leaf were combusted in a
biological sample oxidizerlo using carbon-l4 cocktail to trap evolved C02. Samples were
radioassayed by LSS. Harvested treated leaves were used to determine metabolism of
isoxaflutole. Treated leaves were ground in a tissue homogenizer“ with 20 ml of acetone.
The homogenate was vacuum filtered'2 and the residue rinsed with an additional 20 m1 of
acetone. The rinsate volume was recorded, and two l-ml aliquots were radioassayed with
LSS to determine total extractable 1“C. The residue along with the filter paper, was air dried
and combusted to determine unextractable radioactivity. The filtrate was evaporated to a
volume of 1 ml with a rotary evaporator at 40 C. The solution was transferred into a test tube,
pH adjusted to <30, and partitioned with 1 ml of ethyl acetate. If an aqueous fraction was
present it was removed and the acetone : ethyl acetate fraction was concentrated to 100 to
150 [21 under a stream of air in a water bath (40 C). Twenty-five microliters of the
concentrated extract containing 15 to 50 Bq of radioactivity was spotted onto 20- by 20-cm
silica gel thin layer chromatography (TLC) plates'3 for metabolite separation. Plates were
developed to a 13-cm solvent front in ethyl acetate : methanol : acetic acid (92:5:3 v/v/v).

Radioactive positions, proportions, and their corresponding Rf values were determined by

10Biological sample oxidizer, R.J. Harvey Instruments Corp., 123 Patterson St.,
Hillsdale, NJ 07642.

“Tissue homogenizer, Sorvall Omni-mixer. Sorvall, Inc., Newton, CT.
”Vacuum filter, Whatman #1. Whatman International Ltd., Maidstone, England.

13Plates, Whatman® Linear-K Preadsorbant Silica Gel 150A, Whatman
International Ltd., Maidstone, England.

38

 

scanning TLC plates with a radiochromatogram scanner”.

Herbicide absorption was calculated as the total 14C recovered in the plant divided
by the total MC applied. l“C translocation out of the treated leaf was calculated as the amount
l“C recovered in the plant parts, excluding the treated leaf, divided by the total l“C recovered
in the plant. Herbicide metabolism in the treated leaf was calculated by dividing the
extractable 14C-total active which included isoxaflutole and the metabolite diketonitrile (2-
cyclopropyl-3 -(2-mesyl-4-trifluoromethylphenyl)-3-oxopropanenitrile) by the total 1“C in the
treated leaf. In the radiolabeled translocation and metabolism experiment average l4C-

recovery over all harvest times was 95%.

Spray Retention. A foliar spray retention study was conducted to determine the effect of
metolachlor/benoxacor on foliar retention of isoxaflutole in corn at the spike, 2-1eaf, and 4-
leaf stages. A version of the technique reported by Boldt and Putnum (1980) was followed.
Herbicide treatments examined were isoxaflutole at 105 g ha‘l alone and in combination with
1.1 kg ha‘1 of metolachlor/benoxacor. Spray treatments, including Chicago sky blue dyeIS
(2.5 g L“), were applied to corn at the three different stages. Immediately after application,
the whole plant was harvested and rinsed with distilled water containing nonionic surfactant
(M8)” at 0.25% (v/v). Absorbance of the rinsate was determined spectrophotometrically

(625 nm). Dye retention (ug plant") was calculated from a standard curve.

 

MRadiochromatogram scanner, Arnbis Systems, Inc., 3939 Ruffin Road, San
Diego, CA 92123.

15Blue dye, Sigma Chemical Co., St. Louis, MO 63187.

16Nonionic surfactant, X-77 Valent U.S.A. Corp., 1333 N. California Blvd., PO.
Box 8025, Walnut Creek, CA. 94596-8025.

39

Statistical Analysis. All experiments were conducted twice as completely randomized
designs in factorial arrangements with four replications. Data were subjected to analysis of
variance and means separated using Fisher’s Protected LSD test at a = 0.05. Statistical
analysis indicated no experimental run interactions, so the data were combined and reported
as the means of the two experiments. Non-transformed means are presented since arcsine

and square root transformations did not alter the interpretation of the data.

RESULTS AND DISCUSSION
Field Experiments
Crop Response. Corn tolerance data were combined over years since there were no
application timing and/or treatment by year interactions. When corn injury occurred it
consisted of bleaching of newly developed tissue followed by stunting common symptoms
associated with isoxaflutole (Luscombe and Pallett 1996).

Corn was tolerant to all preemergence applications of isoxaflutole alone and in
combination with metolachlor/benoxacor or atrazine each at 1.1 kg ha‘l (Table 3). However,
when these treatments were applied when com was at the spike stage there was a synergistic
injury effect on corn from the combination of isoxaflutole and metolachlor/benoxacor. This
combination also significantly reduced corn height, but had no affect on yield at the end of
the season. When this treatment was applied at the 2-leaf stage it severely injured corn 72%
and reduced corn height 52%. This severe injury resulted in a 33% decrease in yield
compared with the weed-free check. Com was tolerant to applications of isoxaflutole alone
or in combination with atrazine at the 2-leaf stage. All herbicide treatments injured corn

when applications where made to corn at the 4-leaf stage. Corn injury was 6%, 39%, and

40

15% fiom isoxaflutole alone, isoxaflutole tank-mixed with metolachlor/benoxacor, and
isoxaflutole tank-mixed with atrazine, respectively. The isoxaflutole : metolachlor/benoxacor
tank-mixture was the only treatment that resulted in reductions in corn height and corn grain
yield. Overall, when isoxaflutole was tank-mixed with metolachlor/benoxacor and applied
after corn had emerged the corn was injured and in some cases corn yield was severely
reduced. The enhanced activity of isoxaflutole when metolachlor/benoxacor was added to
the spray solution was similar to the synergistic response that Scott et al. (1998b) reported
from early postemergence applications of dimethenamid (2-chloro-N-(2,4-dimethyl-3-
thienyl)-N-(2-methoxy—1-methylethyl)acetamide) tank-mixtures with either imazethapyr ((:)-
2-[4,5-dihydro-4-methyl-4-(I-methylethyl)-5-oxo-1H—imidazol-2-yl]~5-ethyl-3-
pyridinecarboxylic acid) or sethoxydirn (2-(1-ethoxyimino)butyl]—5-[2-(ethylthio)propyl]-3—

hydroxy-2-cyclohexen-1-one) on several weed species.

Weed Control. Because there were no application timing and/or treatment by year
interactions, data were combined over years for broadleaf weed control. However, giant
foxtail control data were presented by year due to interactions.

All treatments containing isoxaflutole provided excellent control (>95%) of common
lambsquarters, redroot pigweed, common ragweed, and velvetleaf, regardless of application
timing (data not shown). Giant foxtail control was greater than 85% with all herbicide
treatments in 1996, regardless of application timing (Table 4). The tank-mixtures with
metolachlor/benoxacor at any application timing and with atrazine applied when com was
at the 4-leaf stage increased giant foxtail control. Control of giant foxtail was variable in

1997 (Table 4). All preemergence treatments provided excellent control of giant foxtail.

41

Control of giant foxtail decreased as applications were made later with isoxaflutole alone and
in combination with atrazine. Applications of isoxaflutole tank-mixed with
metolachlor/benoxacor at the spike and 2-leaf corn stages controlled giant foxtail (>80%).
Giant foxtail control increased to 99% when this treatment was applied at 4-leaf stage corn.
Differences in giant foxtail control between 1996 and 1997 may be attributed to the
differences in giant foxtail densities between the 2 years at the different application timings
(Table l). The amount of rainfall after herbicide applications may also be a contributing
factor in the differences in giant foxtail control between the 2 years (Table 2). In 1996, there
was significantly more rainfall for herbicide incorporation after spike, 2-leaf and 4-leaf

applications compared with the 1997 growing season.

Greenhouse Experiments

Corn Tolerance. Com was tolerant to foliar applied isoxaflutole at the spike, 2-leaf, and 4-
leaf stages (Table 5). However, foliar plus root uptake of isoxaflutole injured corn from
applications at the 2-leaf stage. Significant reductions in corn height were also seen fi'om this
treatment applied at the spike and 2-leaf corn stages. Com was not injured from applications
of metolachlor/benoxacor at any of the three growth stages. However, foliar plus root uptake

of metolachlor/benoxacor at the spike and 2-leaf stage reduced corn height. The combination

of isoxaflutole and metolachlor/benoxacor whether applied at the spike, 2-leaf, or 4-leaf
stage injured corn and reduced corn height, regardless if applied to only the foliage or foliar
plus soil. Injury was greatest from herbicide uptake by both foliar tissue and the root. Corn
at the 2-leaf stage was more sensitive to this treatment than 4—leaf corn and corn at the spike

stage. lsoxaflutole plus metolachlor/benoxacor applied at the 2-leaf stage caused 56% and

42

83% injury to corn from foliar uptake compared with foliar plus root uptake, respectively.
This shows that there was some significant activity from foliar applications of isoxaflutole
when tank-mixed with metolachlor/benoxacor. Young and Hart (1998) reported increased
activity from foliar applications of isoxaflutole on giant foxtail with the addition of a spray
adjuvant.

In the second corn tolerance study, corn injury was less than 10% from isoxaflutole
alone, regardless of application timing (Table 6). Acetochlor/MON-l3900 alone did not
injure corn. However, isoxaflutole tank-mixtures with acetochlor/MON-13900 injured com
19%, 33%, and 54% from applications at the spike, l-leaf, and 2-leaf stages, respectively.
Severe reductions in corn height were also observed from this treatment at the different corn
stages. Similar to the study when isoxaflutole was tank-mixed with metolachlor/benoxacor,
the chloroacetamide herbicide, acetochlor with the safener MON-13900 increased
isoxaflutole injury to corn. This is similar to the observations of Scott et al. (19983) that the
chloroacetamide herbicides dimethenamid, acetochlor, and metolachlor synergistically
enhanced grass control when tank-mixed with reduced rates of fluazifop-P ((R)-2-[4-[[5-

(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid), imazethapyr, or sethoxydim.

Absorption. Foliar absorption of l"C from the l4C-isoxaflutole applied ranged between 8 and
15%, l h after treatment (HAT) with isoxaflutole alone or in combination with
metolachlor/benoxacor for corn treated at the spike, 2-leaf, and 4-leaf stages (Figure 1).
Significant increases in foliar absorption occurred 4 HAT in corn at the spike stage, and 12
HAT in corn at the 2-leaf and 4-leaf stages. The addition of metolachlor/benoxacor increased

foliar absorption of MC from the I“C-isoxaflutole applied by com treated at all three stages.

43

Absorption was 55%, 43%, and 34% at 24 HAT by com treated with the combination of
isoxaflutole and metolachlor/benoxacor at the spike, 2-leaf, and 4-leaf stages, respectively,
compared with 37%, 32%, and 29% absorption from isoxaflutole applied alone.

In a separate study conducted to determine translocation and metabolism, isoxaflutole
absorption was also evaluated. Absorption of '4C from the ”C-isoxaflutole applied peaked
at 12 HAT, when isoxaflutole was applied alone at the spike, 2-leaf, and 4—leaf stages (Table
7). However, the addition of metolachlor/benoxacor to isoxaflutole delayed maximum
absorption until 72 HAT when applied to 4-leaf corn. At 72 h, 14C absorption was 29%, 27%,
and 37% when isoxaflutole was tank-mixed with metolachlor/benoxacor and applied at the
spike, 2-leaf, and 4-leaf stages, respectively.

There were differences in absorption patterns between the two studies. In the first
study isoxaflutole absorption was greatest at the spike stage followed by the 2-leaf and 4-leaf
stages (Figure 1). This was different from the second study in that isoxaflutole absorption
was greatest at the 4-leaf stage followed by similar absorption of isoxaflutole at the spike and
2-leaf stages (Table 7). Differences in patterns of isoxaflutole absorption between the two
studies was the result of cooler temperatures in the greenhouse at the time the second study
was conducted. The first absorption study was conducted in October and the second study
was conducted in December. Previous research has shown that warmer temperatures increase
herbicide absorption (Willingham and Graham 1988). In both studies the addition of

metolachlor/benoxacor to isoxaflutole increased isoxaflutole absorption, regardless of corn
stage, However, these increases in isoxaflutole absorption did not sufficiently explain the

enhanced corn injury observed from delayed applications of isoxaflutole tank-mixed with

metolachlor/benoxacor.

44

Translocation. Translocation of 14C from the l4C-isoxaflutole applied was less than 10% at
12 h (Table 7). Translocation of isoxaflutole and isoxaflutole combined with
metolachlor/benoxacor were similar at 12 h and 72 h when com was treated at the spike
stage. Corn treated at the 2-1eaf stage with isoxaflutole alone translocated more 14C than corn
treated with the combination of isoxaflutole and metolachlor/benoxacor. However, when
corn was treated at the 4-leaf stage, the combination of isoxaflutole and
metolachlor/benoxacor translocated more MC than corn treated with isoxaflutole alone.
Greater l4C translocation did not consistently correlate with treatments that resulted in the
greatest corn injury. Therefore, translocation of isoxaflutole does not appear to be a
significant factor in the enhancement of corn injury when isoxaflutole is tank-mixed with

metolachlor/benoxacor.

Metabolism. Two distinct metabolites of 14C-isoxaflutole (Rf: 0.91) were separated from the
parent herbicide. Both of these metabolites were present at the 12 h harvest. The metabolites
detected had R, values of 0.62 and 0.3, respectively. The R jvalues of these metabolites

corresponded to the Rf values of the metabolites, diketonitrile and a benzoic acid derivative
(2-mesyl-4-trifluoromethylphenyl benzoic acid), reported by Pallett et al. (1998).
Radiolabeled standards also confirmed the Rf values of isoxaflutole and diketonitrile. The
metabolite diketonitrile is the potent inhibitor of the HPPD enzyme, therefore it is an
herbicidally active component of isoxaflutole (Pallett et al. 1998). In the following discussion
the term active herbicide refers to the sum of isoxaflutole and diketonitrile.

At the 12 h harvest, 10% or less of the active herbicide had been metabolized,

45

regardless of corn stage or herbicide treatment (Table 7). However, by 72 h differences in
herbicide metabolism became apparent. The amount of active herbicide present at 72 h was
73%, 65%, and 67% when com was treated with isoxaflutole at the spike, 2-leaf, and 4-leaf
stages, respectively. The addition of metolachlor/benoxacor enhanced metabolism or the
inactivation of the active herbicide in corn that was treated at the different stages. For
example, corn treated at the spike stage retained 77% of the active herbicide when
isoxaflutole was applied alone compared with 36% with the addition of
metolachlor/benoxacor. It can not be determined from this study if increased metabolism was
due to the herbicide, metolachlor, or the herbicide safener, benoxacor. However, previous
research has shown that benoxacor has enhanced the rate of metabolism of other herbicides
(Rowe et al. 1991). Since the addition of metolachlor/benoxacor increased metabolism of the
active herbicide in corn treated at the three different stages, decreased metabolism does not
explain the enhanced corn injury from foliar applied combinations of isoxaflutole and
metolachlor/benoxacor. This is contradictory to previous research which has identified
isoxaflutole metabolism as the primary basis for differential selectivity between tolerant and
susceptible species (Pallett et al. 1998), as well as differences in tolerance among corn

hybrids (Sprague and Penner 1998).

Spray Retention. The amount of isoxaflutole retained on the surface of corn plants was 0.05,
0.09, and 0.3 Lug when treated with isoxaflutole alone at the spike, 2-leaf, and 4-leaf stages,
respectively (Table 8). Isoxaflutole retention was greatest at the 4-leaf stage, since a portion
of the spray solution was trapped in the whorl of the corn plant. The addition of

metolachlor/benoxacor to the isoxaflutole spray solution increased isoxaflutole retention 5-

46

.v‘ ‘
4. V1,)
.1 All»

- 1
”rs.

‘1'.“ "'1"
*1.“ .115

m x

I
u-~ '9

a)... 0

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1 fun
diswrb

- ‘
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fold when it was applied to 2-leaf and 4-leaf corn. Increased isoxaflutole retention appears
to be a major factor contributing to enhanced corn injury from the isoxaflutole tank-mixture
with metolachlor/benoxacor applied to 2-leaf and 4-leaf corn.

The results of this research indicate that application timing preemergence through 4-
leaf stage corn, had little effect on broadleaf weed control with isoxaflutole. Control of giant
foxtail with isoxaflutole became more variable with later application timings, except when
tank-mixed with metolachlor/benoxacor. This tank-mixture greatly reduced corn tolerance
when applied after corn emergence. Enhanced corn injury from these treatments was the
result of increased isoxaflutole absorption and retention (Table 9). Previous research has
shown that when spray adjuvants were added to isoxaflutole activity increased due to
increased isoxaflutole absorption and retention (Young and Hart 1998). This suggests that
metolachlor/benoxacor may act similar to a spray adjuvant by increasing isoxaflutole

absorption and retention resulting in corn injury from isoxaflutole after corn had emerged.

47

 

LITERATURE CITED

Bhowmik, P. C., R. G. Prostak. 1996. Activity of EXP 31130A in annual weed control in
field corn. Weed Sci. Soc. Am. Abstr. 36:13.

Boldt, P. F. and A. R. Putnam. 1980. Selectivity mechanisms for foliar application of
diclofop-methyl, I. Retention, absorption, and volatility. Weed Sci. 28:474-477.

Curvey, S. E. and G. Kapusta. 1996. Corn Weed Control with EXP31130A. North Cent.
Weed Sci. Soc. 51257-58.

Geier, P. W. and P. W. Stahlman. 1997. Efficacy of Isoxaflutole alone and in combinations
in corn. North Cent. Weed Sci. Soc. 52:81.

Hart, S. E. 1992. Agronomic, physiological, and genetic studies involving sulfonylurea
herbicides. Ph. D. dissertation. Michigan State University. East Lansing, MI. p. 167.

Kells, J. J. 1998. Delayed herbicide application in corn. Crop Advisory Team Alert. Vol. 13,
No. 5. Michigan State University Extension. pp. 6-7.

Lee, D. L., M. P. Prisbylla, T. H. Cromartie, D. P. Dagarin, S. W. Howard, W. M. Provan,
M. K. Ellis, T. Fraser, and L. C. Mutter. 1997. The discovery and structural requirements of
inhibitors of p—hydroxyphenylpyruvate dioxygenase. Weed Sci. 45:601-609.

Luscombe, B. M. and K. E. Pallett. 1996. lsoxaflutole for weed control in maize. Pestic.
Outlook. 29-32.

Luscombe, B. M., T. E. Vrabel. M. D. Paulsgroves, S. Cramp, P. Cain, A. Garnblin, and J.
C. Millet. 1994. RPA 201772: A new broad spectrum preemergence herbicide for corn. Proc.
North Cent. Weed Sci. Soc. 59:57-58.

Mosier, D. G., W. Duckworth, K. K. Watteyne, L. L. King, and M. A. Wrucke. 1995.
Efficacy of EXP31 130A in conventional and no-till corn. Proc. North Cent. Weed Sci. Soc.
50:74

Obermeier, M.R., C.H. Slack, J.R. Martin, and W.W. Witt. 1995. Evaluations of
EXP31 130A - A new preemergence corn herbicide. Proc. North Cent. Weed Sci. Soc. 50:25.

Pallett, K. E., J. P. Little, P. Veerasekaran, and F. Viviani. 1997. Extended summary new
perspective in mechanisms of herbicide action. Pestic. Sci. 50:83-84.

Pallett, K. E., J. P. Little, M. Sheekey, and P. Veerasekaran. 1998. The mode of action of
isoxaflutole 1. Physiological effects, metabolism, and selectivity. Pestic. Biochem. Physiol.
62:113-124.

Rowe, L., J. J. Kells, and D. Penner. 1991. Efficacy and mode of action of CGA-154281, a

48

protectant for corn (Zea mays) from metolachlor injury. Weed Sci. 39:78-82.

Scott, R. C., D. R. Shaw, W. B. O’Neal, and T. D. Klingaman. 1998a. Spray adjuvant,
formulation, and enviorrnental effects on synergism from post-applied tank mixtures of SAN
582H with Fluazifop-P, Imazethapyr, and Sethoxydim. Weed Technol. 12:463-469.

Scott, R. C., D. R. Shaw, R. L. Ratliff, and L. J. Newsom. 1998b. Synergism of grass weed
control with postemergence combinations of SAN 582 and fluazifop-P, imazethapyr, or
sethoxydim. Weed Technol. 12:268-274.

Simkins, G .S., V. H. Lengkeek, W. Duckworth, and T. E. Vrabel. 1995. Effect of
application timing on performance of EXP31 130A for field corn weed control. Proc. North
Cent. Weed Sci. Soc. 50:25.

Sprague, C. L. and D. Penner. 1998. Basis for differential tolerance of four corn hybrids to
isoxaflutole. Proc. North Cent. Weed Sci Soc. 53.

Veilleux, D. P., J. D. Lavoy, W. Duckworth, and M. L. Christian. 1995. Efficacy of
EXP31130A tank mixtures in conventional and no-till corn. North Cent. Weed Sci. Soc.
50:75.

Vrabel, T. E., J. O. Jensen, M. A. Wrucke, and C. Hicks. 1995. EXP31130A: A new
preemergent herbicide for corn. Proc. North Cent. Weed Sci. Soc. 50224-25.

Willingham, G. L. and L. L. Graham. 1988. Influence of environmental factors and adjuvants
on the foliar penetration of acifluorofen in velvetleaf (Abutilon theophrasti): an analysis

using the fractional factorial design. Weed Sci. 36:824-829.

Wrucke, M. A., L. L. King, and D. P. Veilleux. 1996. Effect of cultivation on performance
of isoxaflutole in corn. North Cent. Weed Sci. Soc. 51:11.

Young, B. G. and S. E. Hart. 1998. Optimizing foliar activity of isoxaflutole on giant foxtail
(Setariafaberi) with various adjuvants. Weed Sci. 46:397-402.

Young, B. G., S. E. Hart, and F. W. Simmons. 1998. Performance of preemergence
applications of isoxaflutole in corn. Weed Sci. Soc. Am. Abstr. 38:1 .24.

49

 

 

 

 

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Amount of Rainfall
Days after planting 1996 1997
mm
0-7 53 29
8-14 1 5
15-21 25 19
22-28 14 0
Total 93 53

 

 

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52

Table 4. Giant foxtail control 60 DAP with isoxaflutole and isoxaflutole tank-mixtures
applied at four different timings in field studies in 1996 and 1997.

 

 

1996 1997
Application Isoxaflutole“ Isoxaflutole
timing alone + metolachlorb + atrazine alone + metolachlor + atrazine

 

 

 

% control

 

 

 

 

 

Pre 87 98 9o 96 98 94 .
Spike 96 100 92 75 81 80

2-Leaf 92 99 95 53 82 71

4-Leaf 95 100 99 65 99 65

LSD,” 4 9

 

' Herbicide rates: isoxaflutole at 105 g ha"; metolachlor/benoxacor at 1.1 kg ha“; atrazine at 1.1 kg
ha".
5 The formulation of metolachlor contained the herbicide safener benoxacor.

53

 

 

 

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Table 7. Absorption, translocation, and metabolism of isoxaflutole alone and in combination
with metolachlor/benoxacor at three corn stages.

 

Harvest time

 

12h 72h

 

Isoxaflutole Isoxaflutole Isoxaflutole Isoxaflutole
Stage + metolachlora + metolachlor

 

 

 

Foliar absorption (% of applied)

Spike 1 1 32 7 29
2-Leaf 1 7 3O 1 7 27
4-Leaf 7 l6 1 2 3 7
LSDOAOS 5

 

 

 

Translocation (% of absorbed)b

Spike 1 2 6 5
2-Leaf 7 3 14 8
4-Leaf 3 7 1 8 25
LSDOI05 3

 

 

 

 

Metabolism (% active)c

Spike 9O 92 73 36
2-Leaf 92 93 65 44
4-Leaf 92 9O 67 54
LSDO.05 6

 

 

" Formulation of metolachlor includes the herbicide safener benoxacor.

b Translocation of '4C out of the treated leaf.

° Metabolism is expressed as the amount of the active compounds in the extractable
component of the shoot.

56

 

 

Table 8. Spray retention of foliar applications of isoxaflutole alone and in combination with
metolachlor/benoxacor to corn at three different growth stages.

 

 

 

 

 

Growth Stage
Herbicide Spike 2-Leaf 4-Leaf
u g of isoxaflutole / plant
lsoxaflutole‘ 0.05 0.09 0.3 1
lsoxaflutole + metolachlor/benoxacor 0.07 0.41 1.53
LSD 0‘0, 0.18

 

 

 

‘ Herbicide rates: isoxaflutole at 105 g ha"

; metolachlor/benoxacor at 1.1 kg ha“.

57

 

Table 9. Magnitude of increase of corn injury, l4C-isoxaflutole absorption, and retention due
to the presence of metolachlor/benoxacor in the spray solution in greenhouse studies.

 

Application timing Injury Absorption (72 h) Retention

 

 

 

fold increase

Spike 2 5 1
2-Leaf 9 2 5
4-Leaf 29 3 5

 

58

 

 

Ti:

 

 

100

 

 

 

 

 

80 LSD 0.05 1:
g, T
“c5.
0.
(U
“5 60 ~
2%
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.9
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O
U)
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(U
0
5!. 20 _
0 l l I l
o 1 4 12 24

Time (h)

Figure I. Foliar absorption of l4C-isoxaflutole over time in the presence and absence of
metolachlor/benoxacor. Solid lines (—) indicate isoxaflutole applied alone, dashed lines (---)
are the combination of isoxaflutole and metolachlor/benoxacor. Corn treated at the different
stages are represented by different symbols: spike (O), 2-leaf (A), and 4-leaf (I) com.

59

 

CHAPTER 3

PHYSIOLOGICAL BASIS FOR DIFFERENTIAL CORN (Zea mays)
TOLERANCE OF FOUR CORN HYBRIDS TO ISOXAFLUTOLE

Abstract. Greenhouse and laboratory experiments were conducted to determine the
physiological basis for differential tolerance of four Pioneer corn hybrids to isoxaflutole.
Differences in corn tolerance were quantified by determining the herbicide rate required to
injure and reduce corn height 50% (GRSO). GR,0 values indicated that the corn hybrids
Pioneer 3751 and Pioneer 3737 were less tolerant to isoxaflutole than the hybrids Pioneer
3394 and Pioneer 3963. Experiments using l4C-isoxaflutole were conducted to determine if
hybrid sensitivity was due to differential uptake, translocation, or metabolism of the
herbicide. Differences in hybrid tolerance were primarily due to differential herbicide
metabolism rates. The time required for 50% inactivation (Tm) of isoxaflutole was 42 h and
52 h for the more tolerant corn hybrids, Pioneer 3394 and Pioneer 3963, respectively. The
time required was 66 h and 93 h for the more sensitive hybrids Pioneer 3751 and Pioneer
3737, respectively. Increased uptake of isoxaflutole was also a contributing factor to the
sensitivity of the hybrid Pioneer 373 7.

Nomenclature: Isoxaflutole, 5-cyclopropyl isoxazol-4-yl-2-mesyl-4-trifluoromethylphenyl
ketone; corn, Zea mays L.

Abbreviations: GRSO, rate causing 50% growth reduction; Tm, time required to metabolize
50% of active herbicide; HPPD, 4-hydroxyphenylpyruvate dioxygenase; DAT, days after

treatment.

60

INTRODUCTION

Isoxaflutole is a member of a new class of isoxazole herbicides used for selective
preemergence control of both annual grass and broadleaf weed species in corn. Isoxaflutole
exerts its herbicidal activity by competitively inhibiting 4-hydroxyphenylpyruvate
dioxygenase (HPPD, EC 1.13.11.27) (Luscombe and Pallett 1996; Pallett et al. 1998; Viviani
et al. 1998). Inhibition of HPPD reduces the levels of plastoquinone, a cofactor of phytoene
desturase, a key enzyme of carotenoid biosynthesis (Lee et al. 1997; Pallett et al. 1997).
Typical symptomology from isoxaflutole is similar to other carotenoid biosynthesis
inhibitors, bleaching of newly developed tissue followed by growth cessation and necrosis.

Differential tolerance of crop cultivars, including com, to several herbicides has been
reported. Corn hybrids have demonstrated differential tolerance to herbicides including:
alachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(methoxymethyl)acetamide) (N arsaiah and
Harvey 1977; Rowe and Penner 1990), butylate (S-ethyl bis(2-methylpropyl)carbamotioate)
(Wright and Rieck 1 973), nicosulfuron (2-[[[[(4,6-dimethoxy-2-
pyrimidinyl)amino]carbonyl]amino]sulfonyl]-N,N-dimethyl-3-pyridinecarboxamide)
rimsulfuron (N-[[(4,6-dimethoxy—2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-
pyridinesulfonamide) (Doohan et al. 1998), imazaquin (2-[4,5-dihydro-4-methyl-4-(1-
methylethyl)-5-oxo-1H-imidazol-Z-yl]-3-quinolinecarboxylic acid) (Renner et al. 1988),
metolachlor (2-chloro-N-(2-ethyl-6—methylphenyl)-N-(2-methoxy-1-methylethyl)acetarnide)
(Cottingham and Hatzios 1992; Rowe and Penner 1990), and rimsulfuron (Green and Ulrich
1994). Corn injury from isoxaflutole applied preemergence has been reported in several
research trials. Obermeier et al. (1995) reported corn injury from isoxaflutole applied at 132

and 158 g ha". Bhowmik and Prostak (1996) and Sprague et al. (1996) also observed injury

61

to corn from preemergence applications of isoxaflutole. Differences in tolerance to
isoxaflutole among corn hybrids has not been reported.

The four corn hybrids used in this study were selected from a screen of 15 hybrids
based on their differential responses to isoxaflutole. The objectives of this research were to
I) examine the differences in tolerance of four Pioneer corn hybrids to isoxaflutole and 2)

determine the physiological basis for the differential tolerance observed.

MATERIALS AND METHODS

Corn Tolerance

Two seeds per pot of four corn hybrids, Pioneer 3394, Pioneer 3963, Pioneer 3751, and
Pioneer 373717 were planted 2.54 cm deep in pots (875 ml) containing a Spinks loamy sand
(sand, mixed, mesic Psammentic Hapludalfs). Immediately after planting, the soil surface
was treated with isoxaflutole at 52.56, 105, 210, and 420 g ai ha“. Herbicides were applied
through an 8003 E flat fan nozzle at 234 L ha" and 172 kPa to the soil surface. Herbicide
treatments were incorporated by overhead irrigation (1.0 to 2.0 cm) after herbicide
application. Plants were grown in the greenhouse at 25:2 C and sunlight was supplemented
with sodium vapor lighting to provide a total midday light intensity of 1,000 ,umol m'zs‘l
photosynthetic photon flux at plant height in a 16-h day. Plants were watered and fertilized
as needed to promote optimum growth. Corn tolerance was evaluated 14 d after treatment
(DAT) by visually evaluating plants for bleaching and necrotic symptoms and also by
measuring corn height (soil surface to the highest portion of the plant). Visual com injury

ratings were based on a scale from 0 to 100, with 0 indicating no effect and 100 indicating

 

l7Corn, Pioneer Hi-Bred International, Inc., Des Moines, IA.

62

plant death. Corn height was recorded in cm and presented as a percent of nontreated plants,
with 0 indicating total reduction in plant height and 100 indicating height equal to the
nontreated plants.
Uptake, Translocation, and Metabolism

A single experiment was designed to compare uptake, translocation, and metabolism of
isoxaflutole between four different com hybrids, Pioneer 3394, Pioneer 3963, Pioneer 3751,
and Pioneer 3737. Corn hybrids were exposed to l‘lC-radiolabeled isoxaflutole and all three
measurements were determined fi'om the same plant.

Corn seeds of each hybrid were germinated in propagating flats (68 L) containing
vermiculite. Flats were watered as needed and placed in the greenhouse under the same
conditions described previously. Four day old seedlings were transferred into 60-ml glass
vials wrapped in aluminum foil, exposing the roots of these seedlings to 50 m1 of 1“C-
isoxaflutole hydroponic solution for 8 h. Each vial of radiolabeled hydroponic solution
contained 2.9 kBq of phenyl-U-labeled l4C-isoxaflutole (1,889 kBq mg" specific activity,
99.3% purity), with 0.5% v/v of acetone and the appropriate amounts of unlabeled
isoxaflutole, formulation blank, and water to equal 0.25 ag ai ml'1 of isoxaflutole.

After 8 h, roots were rinsed with deionized water. Plants for the O h harvest were
sectioned into roots, seeds, and shoots. Plant parts were immediately frozen and stored at -30
C until further analysis. The remainder of the seedlings were transplanted into plastic cones
(4 cm diameter by 21 cm height) containing a Spinks loamy sand soil described previously.
These plants were watered and fertilized as needed until harvested. The remainder of the
harvests were at 24 h, 72 h and 168 h after the radiolabeled pulse. The final volume of

hB’Clroponic solutions were measured, and two l-ml aliquots were radioassayed by liquid

63

..“..-

 

 

scintillation spectrometry (LSS) to determine the amount of unabsorbed isoxaflutole.
All plant parts excluding the shoot were combusted in a biological sample oxidizer”, the
14C02 trapped, and radioassayed by LSS.

To determine the metabolism of isoxaflutole, shoots were ground in a tissue
homogenizer19 with 20 ml of acetone. The homogenate was vacutun filtered20 and the residue
rinsed with an additional 20 ml of acetone. The rinsate volume was recorded, and two l-ml
aliquots were radioassayed with LSS to determine total extractable l“C. The residue along
with the filter paper, was air dried and combusted to determine unextractable radioactivity.
The filtrate was evaporated to a volume of 1 ml with a rotary evaporator at 40 C. The
solution was transferred into a test tube, pH adjusted to <30, and partitioned with 1 m1 of
ethyl acetate. If an aqueous fraction was present it was removed and the acetone : ethyl
acetate fraction was concentrated to 100 to 150 Ml under a stream of air in a water bath (40
C).

Twenty-five microliters of the concentrated extract containing 15 to 50 Bq of
radioactivity was spotted onto 20- by 20-cm silica gel thin layer chromatography (TLC)
plates21 for metabolite separation. Plates were developed to a 13-cm solvent front in ethyl
acetate : methanol : acetic acid (92:5:3 v/v/v). Radioactive positions, proportions, and their

corresponding Rf values were determined by scanning TLC plates with a radiochromatogram

 

l8Biological sample oxidizer, R.J. Harvey Instruments Corp., 123 Patterson St.,
Hillsdale, NJ 07642.

19Tissue homogenizer, Sorvall Omni-mixer. Sorvall, Inc., Newton, CT.
20Vacuum filter, Whatman #1. Whatman International Ltd., Maidstone, England.

21Plates, Whatman® Linear-K Preadsorbant Silica Gel 150A, Whatman
International Ltd., Maidstone, England.

64

w'

scanner”.

Herbicide uptake was calculated as the total 14C recovered in the plant divided by the total
1“C in the hydroponic solution. 14C translocation into the shoot was calculated as the amount
1“C in the extractable and unextractable fractions of the shoot divided by the total l“C in the
plant. Herbicide metabolism in the shoot was calculated by dividing the extractable MC of
the total active component of isoxaflutole and the metabolite diketonitrile (2-cyclopropyl-3-
(2-mesyl-4-trifluoromethylphenyl)-3-oxopropanenitrile) by the total MC in the shoot.
Metabolism data was also presented as the total amount of MC in the shoot. This included the
amount of l4C-isoxaflutole, l4C-diketonitrile, l"C-benzoic acid derivative (2-mesyl-4-
trifluoromethylphenyl benzoic acid), and the insoluable residue of the shoot.

Statistical Analysis
All studies were conducted twice and arranged as completely randomized designs with
four replications. GR50 values for corn tolerance and Tl,2 values for metabolism were
determined using the log-logistic dose response model, y = a + ((oc - a) / (1 + exp(fl ln(x /
GR50)))) + e, where x is the rate of application or time for metabolism, 5 is the lower
asymptote, a is the upper asymptote, and ,6 relates to the rate of change near the inflection
point (Tharp et al. 1998). Differences in GR50 and T1,2 values between hybrids were tested
for significance with the sum of squares reduction test.
Uptake, translocation, and metabolism data were subjected to analysis of variance and
mean separation using Fisher’s Protected LSD test at a = 0.05. Statistical analysis indicated

no interactions between experiments, so the data were combined and reported as the means

 

22Radiochromatogram scanner, Arnbis Systems, Inc., 3939 Ruffin Road, San
Diego, CA 92123.

65

of the two experiments. Non-transformed means are presented since arcsine and square root
transformations did not alter the interpretation of the data. In the radiolabeled experiments

l“C-recovery averaged over all harvest times and experiments was 94%.

RESULTS AND DISCUSSION

Corn Tolerance

Corn injury consisted of stunting and bleaching of leaf tissue, common symptoms
associated with isoxaflutole (Luscombe and Pallet 1996). Com tolerance to isoxaflutole was
determined by comparing GR50 values of the four corn hybrids. Pioneer 3394 and Pioneer
3963 were the most tolerant corn hybrids, isoxaflutole rates that caused 50% com injury to
these hybrids were 351 and 208 g ha", respectively (Table 1). Isoxaflutole rates that caused
50% com injury were 140 and 120 g ha‘l for the more sensitive corn hybrids, Pioneer 3751
and Pioneer 3737. Reductions in corn height were consistent with visual injury
determinations (Table 1).
Herbicide Uptake

Root absorption of l4C-isoxaflutole was equal for the corn hybrids Pioneer 3394,
Pioneer 3963, and Pioneer 3751 (Table 2) after the 8 h pulse into the radiolabeled hydroponic
solution. There was an increase in herbicide uptake in one of the more sensitive corn hybrids,
Pioneer 3737. This increase in herbicide uptake could likely contribute to the increased
sensitivity of Pioneer 3737 to isoxaflutole. However, herbicide uptake cannot account for the
differences in com tolerance of the hybrids Pioneer 3394, Pioneer 3963, and Pioneer 3751.
Translocation

Isoxaflutole movement in the plant was determined by measuring l4C translocation

66

 

into the shoot. Equal amounts of l“C-isoxaflutole were translocated into the shoot of all four
Pioneer corn hybrids afier the first harvest time (Table 2). There was a slight increase in
translocation of l“C-isoxaflutole in Pioneer 3737 when compared with Pioneer 3963 and
Pioneer 3751 at the 24 h harvest. However, any differences observed in the translocation of
l4C-isoxaflutole between hybrids were not apparent at the 72 h and 168 h harvests.
Translocation of l“C-isoxaflutole increased with each hybrid over time, with peak
translocation at the last harvest time, 168 h. The data indicates that translocation of
isoxaflutole did not contribute to the differences in tolerance of the four Pioneer corn
hybrids.

Metabolism

Two distinct metabolites of l“C-isoxaflutole (Rf = 0.91) were separated from the
parent herbicide. Both of these metabolites were present in all four corn hybrids after the first
harvest, 8 h after initiation of '4C pulse. The metabolites detected had Rfvalues of 0.62 and
0.3, respectively. The Rf values of these metabolites corresponded to the R, values of the
metabolites diketonitrile and a benzoic acid derivative, reported by Pallett et a1. (1998).
Radiolabeled standards also confirmed R, values of isoxaflutole and diketonitrile. The
metabolite diketonitrile is the potent inhibitor of the HPPD enzyme, therefore it is the
herbicidally active component of isoxaflutole (Pallett et al. 1998; Viviani et al. 1998). In the
fOllowing discussion the term active herbicide refers to the sum of isoxaflutole plus
diketonitrile.
After the 8 h pulse, the amount of active herbicide present was greater than 80 % for

all four corn hybrids (Table 2). The active herbicide was greatest in the sensitive Pioneer

3 737 hybrid. However, it was not different from the tolerant hybrid, Pioneer 3963. Within

67

 

 

1r

 

24 h, the more tolerant hybrid Pioneer 3394 was the only hybrid that significantly
metabolized the active herbicide. Between 24 h and 72 h all four hybrids significantly
metabolized the active herbicide. Again, the amount of active herbicide present was greatest
in the most sensitive hybrid, Pioneer 37 37. At the last harvest, 168 h afier the radiolabeled
pulse, less than 42 % of the active herbicide was lefi in all four corn hybrids.

At the first harvest, the majority of the active herbicide was diketonitrile, less than
1 O % remained as isoxaflutole (Figure 1). This rapid conversion took place during the 8 h
pulse of the corn plants in the I“C hydroponic solution. The amount of diketonitrile present
at the first harvest ranged between, 69 % and 82 % in the four corn hybrids. The appearance
of this metabolite was consistent for the four corn hybrids. However, the rate at which
diketonitrile was metabolized to the inactive benzoic acid derivative was different among the
hybrids.

The time required to metabolize 50 % of the active herbicide was similar for the two
more tolerant corn hybrids, Pioneer 3394 and Pioneer 3963 (Table 2). Pioneer 3394 and
Pioneer 3963 metabolized the active herbicide 50 % in 42 h and 52 h, respectively.
Metabolism of the more sensitive corn hybrids was slower. Pioneer 3751 and Pioneer 3737
metabolized the active herbicide in 66 h and 93 h, respectively.

The rate of herbicide metabolism was a major factor in determining the differential
tOIeI‘ances of the four corn hybrids. Corn hybrids more tolerant to isoxaflutole were able to
metabolize it more rapidly than the more sensitive hybrids. Previous research by Pallett et
al. ( 1 998) has identified isoxaflutole metabolism as the primary basis for differential

sel'E’CtiVity between the tolerant species corn and a susceptible species velvetleaf (A butilon

theophrasti Medicus)-

68

 

The results of this research indicate that there are differences in corn tolerance to
isoxaflutole. The physiological basis for this differential tolerance is primarily due to the rate
in which the active component of isoxaflutole is metabolized to the inactive benzoic acid

derivative (Table 3). Isoxaflutole uptake can also play a role in differences in corn tolerance.

69

 

 

LITERATURE CITED

Bhowmik, P. C. and R. G. Prostak. 1996. Activity of EXP 31130A in annual weed control
in field corn. Weed Sci. Soc. Am. Abstr. 36:13.

Cottingham, C. K. and K. K. Hatzios. 1992. Basis of differential tolerance of two corn
hybrids. Weed Sci. 40:359-363.

Doohan, D. J ., J. A. Ivany, R. P. White, and W. Thomas. 1998. Tolerance of early maturing
corn (Zea Mays) hybrids to DPX-97406. Weed Technol. 12:41-46.

Green, J. M. and J. F. Ulrich. 199. Response of maize (Zea mays) inbreds and hybrids to
rimsulfuron. Pestic. Sci. 40:187-191 .

Lee, D. L., M. P. Prisbylla, T. H. Cromartie, D. P. Dagarin, S. W. Howard, W. M. Provan,
M. K. Ellis, T. Fraser, and L. C. Mutter. 1997. The discovery and structural requirements of

inhibitors of p-hydroxyphenylpyruvate dioxygenase. Weed Sci. 45:601-609.

Luscombe, B. M. and K. E. Pallett. 1996. Isoxaflutole for weed control in maize. Pestic.
Outlook. 29-32.

Narsaiah, D. B., and R. J. Harvey. 1977. Differential responses of corn inbreds and hybrids
to alachlor. Crop Sci. 17:657-659.

Obermeier, M. R., C. H. Slack, J. R. Martin, and W. W. Witt. 1995. Evaluations of
EXPB 1 130A - A new preemergence corn herbicide. Proc. North Cent. Weed Sci. Soc. 50:25.

Pallett, K. E., J. P. Little, M. Sheekey, andP. Veerasekaran. 1998. The mode of action of
isoxaflutolel. Physiological effects, metabolism, and selectivity. Pestic. Biochem. Physiol.

62:113124.

Pallett, K. E., J. P. Little, P. Veerasekaran, and F. Viviani. 1997. Extended Summary: New
perspective in mechanisms of herbicide action. Pestic. Sci. 50:83-84.

Renner, K. A., W. F. Meggitt, and D. Penner. 1988. Response of corn (Zea mays) cultivars
to‘ lmazaquin. Weed Sci. 36. 625- 628.

fiowe L. and D. Penner. 1990. Factors affecting choloracetanilide injury to corn. Weed
ec=hnol. 4:904-906.

188p ragUe C. L., J. J. Kells, and D. Penner. 1996. Weed control and corn tolerance with
Oxaflutole. Proc. North Cent. Weed Sci. Soc. 51: 50- 51.

:1: ftp, B. E., O. Schabenberger, andJ. J. Kells. 1998. Response of annual weed specres to
osinate and glyphosate. 1998. Weed Technol. (Submitted).

70

 

Viviani F., J. P. Little, and K. E. Pallett. 1998. The mode of action of isoxaflutole II.
Characterization of the inhibition of carrot 4-hydroxyphenylpyruvate dioxygenase by the
diketonitrile derivative of isoxaflutole. Pestic. Biochem. Physiol. 62: 125-134.

Vrabel, T. E., J. O. Jensen, M. A. Wrucke, and C. Hicks. 1995. EXP31130A: A new
preemergent herbicide for corn. Proc. North Cent. Weed Sci. Soc. 50:24-25.

Wright, T. H. and C. E. Rieck. 1973. Differential butylate injury to corn hybrids. Weed Sci.
21:194-196.

71

 

Table 1. Differences in corn hybrid tolerance to isoxaflutole.a

 

 

 

 

 

 

GRSOb
Hybrid Visual Injury Height

g ha"
Pioneer 3394 351 a 348 a
Pioneer 3963 208 b 211 b
Pioneer 3751 140 c 156 c
Pioneer 3737 120 d 116 d

' GRso values, within a column, followed by the same letter are not significantly different at the 0.05

probability level.
b GR50 values were determined by visual injury and by percent height reduction using the log-logistic

regression analysis model y = 6 + ((a - 6) / (1 + exp(flln(x / GR,0)))) + e.

72

 

Table 2. Uptake, translocation, and metabolism of isoxaflutole in four corn hybrids.

 

Hybrids

 

Harvest timesal Pioneer 3394 Pioneer 3963 Pioneer 3751 Pioneer 3737

 

 

 

Root absorption (%)b
O h 3 3 3 5

 

 

 

 

Translocation (% in shoot)c

O h 47 47 42 44

24 h 73 72 71 79

72 h 81 80 77 81

168 h 88 86 84 87
LSDo.os 7

 

 

 

Metabolism (% active)d

 

 

o h 81 87 81 92

24 h 71 81 82 89

72 h 51 51 59 71

168 h 39 37 41 42
LSDM, 10

 

Rate of Metabolism (Tm) (h)"
Time (h)f 42 a 52 a 66 b 93 c

 

 

‘ Harvest times are after an 8 h pulse in the l“C hydroponic solution.

b Root absorption expressed as a percentage of l‘C in hydroponic solution.

c Translocation to shoot is expressed as a percent of the total amount of 1“C absorbed.

d Metabolism is expressed as the amount of active in the extractable component of the shoot.
° Time required for 50% of the active herbicide to be metabolized.

f T172 values followed by the same letter are not significantly different at the 0.05 probability level.

73

I

Table 3. Factors contributing to hybrid sensitivity to isoxaflutole.

 

 

 

 

Sensitivity to F actors“
Corn Hybrids Isoxaflutole Absorption Translocation Metabolism
Pioneer 3394 low — — _
Pioneer 3963 low — — _
Pioneer 3751 high —— _ *4:
Pioneer 3737 high ** — **
a ** = contributes significantly, — = does not contribute significantly.

74

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75

CHAPTER 4

ENHANCING THE MARGIN OF SELECTIVITY OF ISOXAFLUTOLE
IN CORN (Zea mays) WITH ANTIDOTES

Abstract. The antidotes dichlonnid, MON-4660, benoxacor, R-29148, and MON-13900

were tested for protecting corn against isoxaflutole injury under greenhouse conditions. The
antidotes were evaluated by tank-mixing isoxaflutole with the labeled rates of a
commercially available herbicide/antidote combination. Preemergence applications of high
rates of isoxaflutole alone at 105 g ha‘1 and 210 g ha" injured the corn hybrids Pioneer 3751,
Pioneer 3737, Pioneer 3394, and Pioneer 3963 more than 30%. R-29l48 was the most
effective of the five antidotes evaluated and provided excellent protection of all four corn
hybrids against isoxaflutole. MON-13900 was also effective. Dichlonnid, MON-4660, and
benoxacor were less effective as isoxaflutole antidotes and offered only partial protection to
the four corn hybrids. Another study evaluated acetolactate synthase (ALS)-inhibiting
herbicides as potential protectants against isoxaflutole injury to com. Isoxaflutole injury was
not altered by the ALS-inhibitors. Technical R-29148 applied at rates greater than 90 g ha‘l
provided excellent protection against isoxaflutole injury and also prevented injury to corn
from diketonitrile, the active metabolite of isoxaflutole. In laboratory studies, R-29148 did
”Qt alter root absorption of ”C-isoxaflutole; however, R-29148 significantly enhanced the
rate of isoxaflutole metabolism in corn. Enhanced metabolism resulting in inactivation of
isoxaflutole appeared to be the protective mechanism of R-29148. The mixed function

oxrdase inhibitor, piperonyl butoxide (PBO), did not affect corn growth. However, P30

76

 

1‘-

increased isoxaflutole activity on the corn hybrids Pioneer 3751, Pioneer 373 7, Pioneer 3394,
and Pioneer 3963. These results demonstrate that com tolerance to isoxaflutole can be
enhanced with the use of antidotes such as R-29148 and MON-13900, that R—29148 protects
corn from isoxaflutole by the enhancement of isoxaflutole metabolism, and that oxidative
reactions may be involved in the metabolism of isoxaflutole in corn.

Nomenclature: Benoxacor, (4 -dichloroacetyl)-3 ,1 -dihydro-3-methyl-2H-1 ,4-benzoxazine;
dichlonnid, 2,2-dichloro-N, N-di-2-propenylacetamide; diketonitrile, 2-cyclopropyl-3-(2-

mesyl-4—trifluoromethylphenyl)-3-oxopropanenitrile; isoxaflutole, 5-cyclopropyl isoxazol—4-

 

yl-2-mesyl-4-trifluoromethylphenyl ketone; MON-4660, 4-(dichloroacetyl-1-oxa-4-azaspiro-

‘1

(4,5)-decane; MON-13900, 3-dichloroacetyl-5-(2-furanyl)—2,2-dimethyl-oxazolidine;
piperonyl butoxide, o<[2-2(-butoxyethoxy)-ethoxy]-4,5-methylenedioxy-2-propyltoluene; R-
29148, 3-(dichloroacetyl)-2,2,5—trimethyl-1,3-oxazolidine; corn, Zea mays L.

Abbreviations: ALS, acetolactate synthase; DAP, days afler planting; DAT, days after

treatment; HPPD, 4-hydroxyphenylpyruvate dioxygenase; PBO, piperonyl butoxide.

77

INTRODUCTION

Isoxaflutole is an isoxazole herbicide registered for selective preemergence use in
corn. Isoxaflutole, used either alone or in combination with other preemergence herbicides
such as chloroacetarnides or atrazine (6-chloro-N-ethyl-N’ -(1-methylethyl)-1 ,3 ,5-t1iazine-2,4-
diamine) has a wide spectrum of activity on a number of annual grass and broadleaf weed
species when applied at rates ranging from 53 g ha’l to 158 g ha'l (Bhowmik and Prostak
1996; Curvey and Kapusta 1996; Geier and Stahlman 1997; Luscombe et al. 1994; Mosier
et al- 1995; Obermeier et al. 1995; Simkins et al. 1995; Veilleux et al. 1995; Vrabel et al.
1995; Wrucke et al. 1997; Young et al. 1998).

Under certain conditions preemergence applications of isoxaflutole have injured corn
(Curvey and Kapusta 1996; Geier and Stahlman 1997; Sprague et al. 1996). For example,
Bhowmik and Prostak (1996) and Obermeier et al. (1995) observed corn injury when
isoxaflutole was applied at 158 g ha". There have also been reports of differences in corn
tolerance to isoxaflutole with different corn hybrids. Sprague and Penner (1998) reported that
the corn hybrids Pioneer 3751 and Pioneer 3737 were less tolerant to isoxaflutole than the
hybrids Pioneer 3394 and Pioneer 3963.

In 1962, Hoffman (1962) introduced the concept of increasing crop selectivity to
herbicides with antidotes (also known as safeners). Since this discovery, a number of
compounds have been identified as antidotes to protect crops such as corn, grain sorghum

[sorghum bicolor (L.) Moench], rice (Oryza sativa L.), and winter cereals from applications
or thiOcarbamate, chloroacetanilide, sulfonylurea, imidazolinone, cyclohexanedione, and
isovaOIidinone herbicides (Hatzios et al. 1997; Kreuz 1993). The major mechanism by

whlch currently developed antidotes protect crops from herbicidal injury is by the

78

 

ll

enhancement of herbicide detoxification. Antidotes enhance glutathione conjugation of the
chloroacetanilide herbicides; and enhance activity of degradative enzymes such as

cytochrome P450 mono-oxygenases and UDP-glucosyl transferases in grass crops protecting

them from aryloxyphenoxypropionate, sulfonylurea and imidazolinone herbicide injury
(Hatzios et al. 1997). For example, Rowe et al. (1991) reported that the antidote benoxacor
protected corn from metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N—(2-methoxy-l-
methylethyl)acetamide) injury by enhancing the rate of metolachlor metabolism. Novosel
(1997) has reported increased metolachlor metabolism in giant foxtail (Setaria faberi
Herrm.) when applied in the presence of an ALS-inhibiting herbicide. The use of an ALS-
inhibitor may also potentially protect corn against herbicide injury.

The mechanism of herbicidal action of isoxaflutole is the inhibition of the 4-
hydroxyphenylpyruvate dioxygenase enzyme (HPPD, EC 1.13.11.27) (Luscombe and Pallett
1 996; Pallett et al. 1998; Viviani et al. 1998). Following foliar or root uptake, isoxaflutole
is rapidly converted to a diketonitrile derivative by opening the isoxazole ring (Pallett et al.
1998). Diketonitrile has been reported by Lee et al. (1997) and Pallett et al. (1998) to be the
actual inhibitor of the HPPD enzyme. Diketonitrile is further degraded to an inactive benzoic
acid derivative (2-mesyl-4-trifluoromethylphenyl benzoic acid). It is not known whether
oxidative reactions by mixed function oxidase enzymes are involved in the metabolism of
isoxaflutole. The activity of mixed function oxidase enzymes can be inhibited by antioxidant

compounds or inhibitors such as piperonyl butoxide (PBO), sesamex (a component of
seSaJTle oil, EDU (N-[2-2-oxo-1-imidazolidinyl]ethyl-N-phenylurea), and n-propyl gallate
(Rubin et al. 1980). Simarmata and Penner (1993) reported piperonyl butoxide enhanced

pri 11) isulfuron (2-[[[[[4,6-bis(difluoromethoxy)-2-

79

pyrimidinyl]amino]carbonyl]amino]sulfonyl] benzoic acid) injury to corn by inhibiting
prirnisulfirron metabolism, which is induced by a cytochrome P-450 mono-oxygenase system
(Fonne-Pfister et al. 1990).
The objectives of this research were to (a) identify effective antidotes against
isoxaflutole injury to corn, (b) determine the dosage rate of antidote required for efficacy, and

(C) determine the basis for the protective action of effective antidotes.

MATERIALS AND METHODS
Protecting Corn Against Isoxaflutole Injury.
General plant culture. Corn seeds of selected hybrids were planted two per pot, 2.54 cm deep
in pots (875 ml) containing a Spinks loamy sand soil (sand, mixed, mesic Psammentic
I‘Iapludalfs). Herbicide applications were made preemergence through an 8003 E flat fan
nozzle at a spray volume of 234 L ha" at 172 kPa and incorporated by overhead irrigation
( 1.0 to 2.0 cm) immediately after herbicide application. Plants were grown in a greenhouse
maintained at 16-h days at 2532 C. Natural sunlight was supplemented with sodium vapor
lighting to provide a total midday light intensity of 1,000 umol m’zs" photosynthetic photon
flux at plant height. Plants were watered and fertilized as needed to promote optimum
growth.
Corn tolerance was evaluated 14 d after treatment (DAT) by visually evaluating

plants for bleaching and necrotic symptoms and also by measuring corn height (soil surface
to the highest portion of the plant). Visual corn injury ratings were based on a scale from 0

to 100, with 0 indicating no effect and 100 indicating plant death.

80

 

Antidote evaluation. Four corn hybrids, previously identified as tolerant or sensitive to
isoxaflutole (Sprague and Penner 1998), were planted and treated under the previously
described conditions. The sensitive hybrids were Pioneer 375123 and Pioneer 3737. The
tolerant hybrids were Pioneer 3394 and Pioneer 3963. Isoxaflutole was applied at 105 g ha‘l
t0 the sensitive hybrids and at 210 g ha‘l to the tolerant hybrids. Isoxaflutole applied at these
rates was used to evaluate the antidotes diclormid, MON—4660, benoxacor, R-29148, and
MON-13900. The antidotes were evaluated by tank-mixing isoxaflutole with the labeled rate
of the commercially available herbicide/antidote combinations. These herbicide/antidote
cOInbinations were metolachlor/benoxacor (30:1) at 2.2 kg ha", acetochlor (2-chloro-N-
(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)acetamide) /dichlormid (6:1) at 1.8 kg ha‘,
acetochlor/MON-466O (10:1) at 1.8 kg ha“, EPTC (S-ethyl dipropyl carbarnothioate) /R-
29148 (24:1) at 4.5 kg ha“, MON-12000 (methyl 5-[[(4,6-dimethoxy-2-
pyrimidinyl)amino]carbonylaminosulfonyl]3-chloro-1-methy1-1-H—pyrazole-4-carboxylate)
/MON-139OO (1:3) at 84 g ha", and acetochlor/MON-139OO (15:1) at 1.8 kg ha". These
herbicide/antidote combinations were also applied alone.

The imidazolinone-resistant corn hybrid, Pioneer 37511R, was planted and treated
under the previously described conditions to determine if ALS-inhibiting herbicides protect
corn from isoxaflutole injury. Preemergence applications of isoxaflutole at 210 g ha" were
applied alone and in combination with 105 g ha‘l of rimsulfuron (N—[[(4,6-dimethoxy-2-
pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide); 8 g hal of

thifensulfuron (3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-

 

23Corn, Pioneer Hi-Bred International, Inc., Des Moines, IA.

81

 

l".-

Y 1)afflinejcarbonyl]amino]sulfonyl]-2-thiophenecarboxylic acid); 63 g ha" of flumetsulam
(IV-(2,6—difluorophenyl)-5-methyl[l ,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide); 127 g ha'l
of MON-13900; and 40 g ha’1 of CGA-152005 (1-(4-methoxy-6-methyl-triazin-2-yl)-3-[2-
(3,3,3-trifluoropropyl)—phenyl-sulfonyl]-urea). These ALS-inhibiting herbicides were also
applied alone.
R-29148 Rate Response Study.
The corn hybrids Pioneer 37 5 1 , Pioneer 3737, Pioneer 3394, and Pioneer 3963 were
planted and treated as previously described to determine the effective R-29148 rates to
protect corn from isoxaflutole injury. Immediately after planting, the soil surface was treated
With isoxaflutole at 105 g ha‘1 to the hybrids Pioneer 3751 and Pioneer 3737, and at 210 g
ha" to the hybrids Pioneer 3394 and Pioneer 3963. Isoxaflutole at the aforementioned rates
Was applied alone and in combination with the antidote, R-29148 at 23, 45, 90, 180, and 360
g ha".
Basis for R-29148 Efficacy.
R-29148 efficacy against diketonitrile. Two corn hybrids were planted and treated as
previously described to determine if the antidote, R-29148, protected corn fi'om diketonitrile
injury. Pioneer 3751 and Pioneer 3963 were treated with preemergence applications of
isoxaflutole; diketonitrile; with or without R-29148 at 180 g ha". Isoxaflutole and
diketonitrile rates were 105 g ha‘l for the hybrid Pioneer 3751 and 210 g ha‘l for the hybrid

Pioneer 3963.

R-29148 effect on isoxaflutole absorption and metabolism. To determine the effects of R-

29148 on isoxaflutole uptake and metabolism, seed of the corn hybrid Pioneer 3737 was

82

 

planted 2.54 cm deep in a 3 dram shell vial“, one seed per vial containing 20 g of a Spinks
loamy sand soil previously described . Immediately after planting 5-m1 of radiolabeled
solutions were added to each vial. Each solution contained 2.5 kBq of phenyl-U-labeled l“C--
isoxaflutole (1 ,889 kBq mg‘l specific activity, 993% purity), with 0.5% v/v of acetone and
the appropriate amounts of unlabeled isoxaflutole, formulation blank, and water to be
equivalent to 105 g ha" of isoxaflutole. In addition to the I“C-isoxaflutole, one solution
Contained the antidote R-29148 at a rate equivalent to 180 g ha“. Treatment vials were placed
in the greenhouse under the previously described conditions. Plants were watered and
fertilized as needed until the appropriate harvest times.
Corn plants were harvested 7, 10, 14 and 18 d after planting (DAP). At each harvest,
plants were sectioned into roots and shoots. Plant parts and soil samples containing 14C-
i soxaflutole were immediately frozen and stored at -30 C until further analysis. Roots and
Soil samples were combusted in a biological sample oxidizer”, the 14CO2 trapped, and
radioassayed by liquid scintillation spectrometry (LSS).
To determine metabolism of isoxaflutole in the presence and absence of R-29148 shoots
were ground in a tissue homogenizer26 with 20 ml of acetone. The homogenate was vacuum
filtered27 and the residue rinsed with an additional 20 ml of acetone. The rinsate volume was

recorded, and two l-ml aliquots were radioassayed with LSS to determine total extractable

¥

24Shell vials, VWR Scientific, P. O. Box 66929, O’Hare AMF, Chicago, IL
60666.

25Biological sample oxidizer, R.J. Harvey Instruments Corp., 123 Patterson St.,
Hillsdale, NJ 07642.

26Tissue homogenizer, Sorvall Omni-mixer. Sorvall, Inc., Newton, CT.
27Vacuum filter, Whatman #1. Whatman International Ltd., Maidstone, England.

83

 

'4C. The residue along with the filter paper, was air dried and combusted to determine
unextractable radioactivity. The filtrate was evaporated to a volume of 1 ml with a rotary
evaporator at 40 C. The solution was transferred into a test tube, pH adjusted to <30, and
partitioned with 1 ml of ethyl acetate. If an aqueous fraction was present it was removed and
the acetonezethyl acetate fraction was concentrated to 50 to 100 121 under a stream of air in
a water bath (40 C).

Twenty-five microliters of the concentrated extract containing 25 to 50 Bq of
radioactivity was spotted onto 20- by 20-cm silica gel thin layer chromatography (TLC)
plates28 for metabolite separation. Plates were developed to a 13-cm solvent front in ethyl
acetatezmethanolzacetic acid (92:5:3 v/v/v). Radioactive positions, proportions, and their
corresponding Rf values were determined by scanning TLC plates with a radiochromatogram
scanner”.

Herbicide uptake was calculated as the total 1“C recovered in the plant divided by the total
14C solution added to the soil vials. Herbicide metabolism in the shoot was calculated by
dividing the extractable l“C of the total active component of isoxaflutole which is the
metabolite diketonitrile by the total 1“C in the shoot. Total l4C-recovery was calculated by
dividing the sum of the amount of 1“C found in the shoot, root, and soil by the total amount

of MC added to the soil. The average l4C-recovery was 96%.

Influence of the mixed function oxidase inhibitor, PBO, on isoxaflutole activity. The corn

 

28Plates, Whatman® Linear-K Preadsorbant Silica Gel 150A, Whatman
International Ltd., Maidstone, England-

29Radiochromatograrn scanner, Arnbis Systems, Inc., 3939 Ruffin Road, San
Diego, CA 92123.

84

 

hybrids Pioneer 3751, Pioneer 373 7, Pioneer 3394, and Pioneer 3963 were used to determine
the effects that the mixed function oxidase inhibitor piperonyl butoxide had on corn treated
with isoxaflutole- Two seeds per pot of the four corn hybrids were planted 2.54 cm deep in
pots (875 ml) containing a commercial potting mix”. Plants were grown in the greenhouse
under the previously described conditions and watered and fertilized as needed to insure
maximum growth. Herbicide treatments were applied when corn plants were at the V1 stage
(15 to 16.5 cm tall). Isoxaflutole and piperonyl butoxide were applied alone and in
combination with each other. Piperonyl butoxide rates were 0.7 and 1.1 kg ha". Isoxaflutole
rates were 210 g ha" for the hybrids Pioneer 3751 and Pioneer 3737 and 420 g ha'I for the
hybrids Pioneer 3394 and Pioneer 3963. These rates were chosen from a previous foliar
applied isoxaflutole rate response experiment. All treatments included 25% (v/v) ethanol and
0.25% (v/v) nonionic surfactant". Ethanol was used to insure that the piperonyl butoxide
remained in the spray solution and the non-ionic surfactant was used to achieve maximum
retention of the herbicide treatments. Herbicide treatments were applied through the
previously described spray system.

Corn tolerance was evaluated 10 DAT by visually evaluating plants for bleaching and
necrotic symptoms and also by measuring corn height (soil surface to the highest portion of
the plant). Visual corn injury ratings were based on a scale from 0 to 100, with 0 indicating

no effect and 100 indicating plant death.

30Baccto, professional planting mix, Michigan peat Co., PO. Box 980129,
Houston, TX. 77098.

3‘Nonionic surfactant, Activator-90, a mixture of alkyl polyoxyethylene ether and
fi-ee fatty acids. Loveland Industries, Inc., PO. Box 1289, Greeley, CO 80632.

85

Statistical Analysis.

All studies were conducted twice and arranged as completely randomized designs with
four replications. All data were subjected to analysis of variance and mean separation using
Fisher’s Protected LSD test at a = 0.05. Statistical analysis indicated no interactions between
experiments, so the data were combined and reported as the means of the two experiments.
Non-transformed means are presented since arcsine and square root transformations did not

alter the interpretation of the data.

RESULTS AND DISCUSSION

Protecting Corn Against Isoxaflutole Injury.

Antidote evaluation. Corn injury consisted of stunting and bleaching of leaf tissue, common
symptoms associated with isoxaflutole (Luscombe and Pallett 1996). The corn hybrids
Pioneer 3751, Pioneer 3737, Pioneer 3394, and Pioneer 3963 were injured 44%, 43%, 30%,
and 54%, respectively, from isoxaflutole applied alone (Table 1). Corn injury in all four corn
hybrids was significantly reduced when any of the herbicide/antidote combinations were
added to isoxaflutole. However, there were varying degrees of protection to corn from the
different herbicide/ antidote combinations. The herbicide/ antidote combinations that protected
com the greatest from isoxaflutole injury were the herbicides that contained the antidotes
MON-13900 and R-29148. The antidote, MON-13900 was combined with the herbicides
MON-12000 and acetochlor, from these combinations there were two hybrids that were not
significantly injured. All four com hybrids were not significantly injured when the EPTC/R—
29148 combination was tank-mixed with isoxaflutole. All herbicide/antidote combinations

applied alone did not significantly injure corn. The herbicide/antidote combination with R-

86

 

 

29148 was the most consistent and effective antidote tested for protecting corn against
isoxaflutole injury. Other researchers have observed that different antidotes vary in their
effectiveness in protecting corn against herbicide injury (Kotoula-Syka and Hatzios 1996;
Leavitt and Penner 1978). For example, Leavitt and Penner (1978) found that the antidote
dichlormid was the most effective of six potential antidotes evaluated in protecting corn from
acetanilide injury.

Pioneer 37511R corn was injured 68% from isoxaflutole applied at 210 g ha" (data
not shown). There were no differences in corn injury when any of the ALS-inhibiting
herbicides: rimsulfuron, thifensulfuron, flumetsulam, MON-12000, or CGA-152005 were
tank-mixed with isoxaflutole. The presence of an ALS-inhibitor did not protect corn from
isoxaflutole injury. This is contrary to observations by Novosel (1997) in which ALS-
inhibitors antagonized giant foxtail control with metolachlor. The basis for this observation

was increased metolachlor metabolism in the presence of ALS-inhibiting herbicides.

R-29148 Rate Response Study.

Since R-29148 was the most effective antidote in protecting corn from isoxaflutole
injury, a rate response study was initiated to determine the effective R-29148 rates.
Isoxaflutole injury to corn ranged between 20% and 30% in all four corn hybrids Pioneer
3751, Pioneer 3737, Pioneer 3394, and Pioneer 3963 (Table 2). All rates of R-29148
significantly reduced isoxaflutole injury to corn. However, when isoxaflutole was applied
with greater than 90 g ha“1 of R-29l48 corn was not significantly injured with any of the four
corn hybrids. Other research has shown that effective protection against herbicide injury is

antidote rate dependent (Kotoula-Syka and Hatzios 1996).

87

 

-‘ eh.

 

 

Basis for R-29148 Efficacy.

R-29148 eflicacy against diketonitrile. Isoxaflutole rapidly converts to its first metabolite,
diketonitrile, in both plants and in the soil. Since the dynamics of this conversion is not well
understood in the soil, a study was conducted to determine if the antidote, R-29148,
protected corn injury from diketonitrile. The corn hybrid Pioneer 3571 was injured 14% and
24% from 105 g ha’l of isoxaflutole and diketonitrile, respectively (Table 3). Injury to the
hybrid Pioneer 3751 was 45% and 58% from applications of isoxaflutole and diketonitrile,
respectively, at 210 g ha“. The addition of 180 g ha" of R—29l48 to isoxaflutole and
diketonitrile reduced corn injury to negligible levels. Thus, R—29148 protected both corn

hybrids from both isoxaflutole and diketonitrile injury.

R-29148 eflect on isoxaflutole absorption and metabolism. Differences in isoxaflutole
absorption and metabolism in the presence and absence of R-29148 were similar for the four
harvest times. Thus, absorption and metabolism data will only be presented from the 18 d
harvest (Table 4).

Root absorption of MC-isoxaflutole was not different in the presence or absence of
R-29148 (Table 4). Therefore, the antidote R-29148 did not prevent isoxaflutole injury to
corn by reducing the amount of isoxaflutole absorbed.

Three distinct areas of radioactivity were found on the TLC plates both in the
presence and absence of R—29148. The R, values for these areas were 0.91, 0.62, and 0.3.
Previous research (Pallett et al. 1998), along with standards included in the study, suggest
that the Rfvalues of 0.91 and 0.62 correspond to the parent compound isoxaflutole and the

first metabolite diketonitrile, respectively. Since diketonitrile is the active component of

88

 

isoxaflutole the sum of isoxaflutole and diketonitrile will be referred to as the active
herbicide in the following discussion. The area detected at the Rf value of 0.3 corresponded
to a derivative of benzoic acid that has been reported as an inactive metabolite of isoxaflutole
(Pallett et al. 1998). Therefore, R-29148 did not prevent isoxaflutole injury to corn by
changing the pathway of isoxaflutole metabolism. In both the presence and absence of R-
29148, isoxaflutole was first converted to diketonitrile and then to the nonphytotoxic
derivative of benzoic acid.

By the 7 d harvest all but 2% of the parent herbicide isoxaflutole had been converted
to the active diketonitrile or the benzoic acid derivative (data not shown). The amount of
active herbicide present at the 18 d harvest was 30% and 50% in the presence and absence
of R-29148, respectively (Table 4). R-29148 increased metabolism of the active herbicide
resulting in protection of corn from isoxaflutole injury. Several researchers have also
identified enhancement of herbicide metabolism as the mode of action of several antidotes
(Hatzios 1997; Rowe et al. 1991; Simarmata and Penner 1993). Previous research has
identified metabolism as the primary difference in isoxaflutole selectivity between and

within species (Pallett et al. 1998; Sprague and Penner 1998).

Influence of the mixed function oxidase inhibitor, PBO, on isoxaflutole activity. Isoxaflutole
injury to the corn hybrids Pioneer 3751, Pioneer 3737, Pioneer 3394, and Pioneer 3963
ranged between 10% and 21% in this study (Table 5). Applications of piperonyl butoxide at
0.7 or 1.1 kg ha‘1 did not affect corn growth. However, the addition piperonyl butoxide at
either rate of isoxaflutole increased the activity of isoxaflutole on all four com hybrids. For

example, the corn hybrid Pioneer 3751 was injured 21% from isoxaflutole applied alone. The

89

 

‘_._

addition of piperonyl butoxide at 0.7 and 1.1 kg ha’1 increased corn injury to 81% and 86%,
respectively.

These results demonstrated that oxidative reactions may be involved in the
metabolism of isoxaflutole by com and may contribute to its selectivity. Increased herbicide
activity by piperonyl butoxide as well as other inhibitors of cytochrome P-450 mono-
oxygenases has been used by several investigators as indirect evidence for the involvement
of oxidative reactions in herbicide metabolism (Kotoula-Syka and Hatzios 1996; Rubin et
al. 1990; Simarmata and Penner 1993).

Overall, the results of this research demonstrate that com tolerance to isoxaflutole can
be enhanced with the use of an antidote. R-29148 was the most effective antidote tested in
protecting corn from isoxaflutole injury, and MON-13900 also had good antidotal activity.
The protective mechanism of R-29148 was the enhancement of isoxaflutole metabolism. The
enhancement of corn tolerance to isoxaflutole with antidotes, as well as, the increased
activity observed with piperonyl butoxide suggests that cytochrome P-450 mono-oxygenases

maybe involved in the metabolism of isoxaflutole by com.

90

 

LITERATURE CITED

Bhownrik, P. C. and R. G. Prostak. 1996. Activity of EXP 31130A in annual weed control
in field corn. Weed Sci. Soc. Am. Abstr. 36:13.

Curvey, S. E. and G. Kapusta. 1996. Corn Weed Control with EXP31130A. North Cent.
Weed Sci. Soc. 51:57-58.

Fonne-Pfister, R., J. Gaudin, K. Kruez, K. Ramisteiner, and E. Ebert. 1990. Hydroxylation
of primisulfirron by an inducible cytochrome P450-dependent monooxygenase systems from
maize. Pestic. Biochem. Physiol. 37:165-173.

Geier, P. W. and P. W. Stahlman. 1997. Efficacy of Isoxaflutole alone and in combinations
in corn. North Cent. Weed Sci. Soc. 52:81.

Hatzios, K. K. 1997. Regulation of xenobiotic degrading enzymes with herbicide safeners.
In K. K. Hatzios, K. K. ed. Regulation of Enzymatic Systems Detoxifying Xenobiotics in
Plants. Netherlands: Kluwer Academic Publishers. 372275-288 pp.

Hoffman, O. L. 1962. Chemical seed treatments as herbicide antidotes. Weeds. 10:32.

Kreuz, K. 1993. Herbicide safeners: Recent advances and biochemical aspects of their mode
of action. Proc. Brighton Crop. Prot. Conf. Weeds. 3: 1249-1258-

Leavitt, J. R. C. and D. Penner. 1978. Potential antidotes against acetanilide herbicide injury
to corn (Zea mays). Weed Res. 18:281-286.

Lee, D. L., M. P. Prisbylla, T. H. Cromartie, D. P. Dagarin, S. W. Howard, W. M. Provan,
M. K. Ellis, T. Fraser, and L. C. Mutter. 1997. The discovery and structural requirements of
inhibitors of p-hydroxyphenylpyruvate dioxygenase. Weed Sci. 45:601-609.

Luscombe, B. M. and K. E. Pallett. 1996. Isoxaflutole for weed control in maize. Pestic.
Outlook. 29-32.

Luscombe, B. M., T. E. Vrabel. M. D. Paulsgroves, S. Cramp, P. Cain, A. Garnblin, and J.
C. Millet. 1994. RPA 201772: A new broad spectrum preemergence herbicide for corn. Proc.
North Cent. Weed Sci. Soc. 59:57-58.

Mosier, D. G., W. Duckworth, K. K. Watteyne, L. L. King, and M. A. Wrucke. 1995.
Efficacy of EXP31 130A in conventional and no-till corn. Proc. North Cent. Weed Sci. Soc.
50:74

Novosel, K. M. 1997. Efficacy of annual grass control by metolachlor as influenced by
. flumetsulam, halosulfirron, and chlorimuron-ethyl. Ph. D. dissertation. Michigan State
University. East Lansing, MI. p. 43-84.

91

 

Obermeier, M.R., C.H. Slack, J.R. Martin, and W.W. Witt. 1995. Evaluations of
EXP31130A - A new preemergence corn herbicide. Proc. North Cent. Weed Sci. Soc. 50:25.

Pallett, K. E., J. P. Little, M. Sheekey, and P. Veerasekaran. 1998. The mode of action of
isoxaflutole 1. Physiological effects, metabolism, and selectivity. Pestic. Biochem. Physiol.
62:1 13-124.

Rowe, L., J. J. Kells, and D. Penner. 1991. Efficacy and mode of action of CGA-154281, a
protectant for com (Zea mays) from metolachlor injury. Weed Sci. 39:78-82.

Rubin, B., J. R. C. Leavitt, D. Penner, and A. W. Saettler. 1980. Interaction of antioxidants
with ozone and herbicide stress. Bull. Environ. Contam. Toxicol. 25:623-629.

Simarmata, M. and D. Penner. 1993. Protection from primisulfuron injury to corn (Zea mays)
and sorghum (Sorghum bicolor) with herbicide safeners. Weed Technol. 7: 174-179.

Simkins, G .S., V. H. Lengkeek, W. Duckworth, and T. E. Vrabel. 1995. Effect of
application timing on performance of EXP31 130A for field corn weed control. Proc. North
Cent. Weed Sci. Soc. 50:25.

Sprague, C. L., J. J. Kells, and D. Penner. 1996. Weed control and corn tolerance with
isoxaflutole. Proc. North Cent. Weed Sci Soc. 51 :50.

Sprague, C. L. and D. Penner. 1998. Basis for differential tolerance of four corn hybrids to
isoxaflutole. Proc. North Cent. Weed Sci Soc. 53.

Veilleux, D. P., J. D. Lavoy, W. Duckworth, and M. L. Christian. 1995. Efficacy of
EXP31130A tank mixtures in conventional and no-till corn. North Cent. Weed Sci. Soc.
50:75.

Viviani F., J. P. Little, and K. E. Pallett. 1998. The Mode of Action of Isoxaflutole II.
Characterization of the inhibition of carrot 4-hydroxyphenylpyruvate dioxygenase by the
diketonitrile derivative of isoxaflutole. Pestic. Biochem. Physiol. 62:125-134.

Vrabel, T. E., J. O. Jensen, M. A. Wrucke, and C. Hicks. 1995. EXP31130A: A new
preemergent herbicide for corn. Proc. North Cent. Weed Sci. Soc. 50224-25.

Wrucke, M. A., L. L. King, and D. P. Veilleux. 1996. Effect of cultivation on performance
of isoxaflutole in corn. North Cent. Weed Sci. Soc. 51:11.

Young, B. G., S. E. Hart, and F. W. Simmons. 1998. Performance of preemergence
applications of isoxaflutole in corn. Weed Sci. Soc. Am. Abstr. 38: 1.24.

92

 

 

Table 1. Protection of herbicide/antidote combinations against isoxaflutole injury to four
different corn hybrids (14 DAT).

 

 

 

 

 

 

Hybrids
Herbicide Rate Pioneer 3751 Pioneer 3737 Pioneer 3394 Pioneer 3963
Isoxaflutolea + kg ha" % inj uryb
None — 44 43 3O 54
Metolachlor/benoxacorc 2.2 23 20 1 7 24
Acetochlor/dichlormid 1 .8 20 23 27 21 F
Acetochlor/MON-4660 1 .8 24 24 14 20 :l
EPTC/R-29l48 4.5 2 4 2 1
MON-12000/MON-13900 0.08 6 6 12 3
Acetochlor/MON-13900 1 .8 4 1 l 7 7 :‘r'
LSDOD, ——5— —4— —5— —7— '

 

‘ Isoxaflutole rates for Pioneer 3751 and Pioneer 3737 were 105 g ha'1 and 210 g ha" for Pioneer
3394 and 3963.

b Corn injury was evaluated 14 DAT.

° Herbicide/antidote combinations applied alone injured corn less than 5%.

93

 

Table 2. Protection of corn from isoxaflutole injury to four different corn hybrids with
varying rates of R-29148.

 

 

 

 

 

 

Hybrids
Treatment Rate Pioneer 3751 Pioneer 3737 Pioneer 3394 Pioneer 3963
Isoxaflutolea + - g ha'l - % injuryb
None — 24 3O 29 3O
R-29148c 23 2 S 5 6
R-29148 45 0 4 5 2
R-29148 9O 0 3 4 1
R-29148 180 0 1 1 0
R-29148 360 O 1 1 O
LSDMS —3— —5— —5— ——5——

 

‘ Isoxaflutole rates for Pioneer 3751 and Pioneer 3737 were 105 g ha" and 210 g ha" for Pioneer
3394 and 3963.

b Corn injury was evaluated 14 DAT.

c Corn was not injured from R-29148 applied alone.

94

 

 

 

at...

 

 

Table 3. Protection of the com hybrids Pioneer 3751 and Pioneer 3963 from isoxaflutole and
diketonitrile injury with the antidote, R-29148.

 

 

 

Pioneer 3751 Pioneer 3963
Herbicide Alone + R-29148a Alone + R-29148
% injuryb ——-— -—————- % injury
Isoxaflutolec 14 1 45 4
Diketonitrile 24 1 58 2
LSDODS 3 7

 

 

 

' R-29148 was applied at 180 g ha".
b Corn injury was evaluated 14 DAT.
° Isoxaflutole and diketonitrile rates for Pioneer 3751 were 105 g ha‘1 and for Pioneer 3963 were 210

g ha".

95

 

Table 4. Absorption and metabolism of l4C-isoxaflutole in corn alone and in the presence of

the antidote, R-29l48 (18 DAT).

 

 

 

 

 

 

 

 

Herbicide interaction Rate Absorption Metabolism
— g ha'I — % of applied — % activea
Isoxaflutole 105 27 50
Isoxaflutole + R-29148 105 + 182 24 3O
LSD0_05 NS 8

 

‘ Active contains both isoxaflutole and diketonitrile.

96

 

 

 

Table 5. Influence of the mixed function oxidase inhibitor, piperonyl butoxide, on the
activity of foliar applied isoxaflutole in four corn hybrids.

 

 

 

 

 

Hybrids
Herbicidea Rate Pioneer 3751 Pioneer 3737 Pioneer 3394 Pioneer 3963
— kg ha" - % injuryb
Isoxaflutole° — 21 20 13 10
Isoxaflutole + PBOd 0.7 81 76 68 79
Isoxaflutole + PBO 1.1 86 81 77 84
LSDoos 5 4 4 4

 

 

 

‘ All herbicide treatments were applied with 25% (v/v) ethanol and 0.25% (v/v) non-ionic surfactant.
"’ Corn injury was evaluated 14 DAT.

° Isoxaflutole rates for Pioneer 3751 and Pioneer 3737 were 210 g ha‘l and 420 g ha’l for Pioneer
3394 and 3963. -

d PBO = piperonyl butoxide; No corn injury was observed from PBO applied alone to any of the four
corn hybrids.

97

 

 

SUMMARY

In field studies, isoxaflutole alone provided excellent control of a number of weed
species, except in conditions with limited rainfall. In some cases, tank-mixing other
herbicides with isoxaflutole increased giant foxtail and redroot pigweed control. In no-tillage
com, herbicide treatments applied preemergence provided more consistent weed control than
treatments applied early preplant. Under certain conditions, applications of isoxaflutole
injured corn, which in some cases resulted in reductions of com yield. Typical com injury
symptoms that were associated with isoxaflutole were bleaching of newly developed tissues
followed by stunting of the entire corn plant. Corn injury from isoxaflutole was most
commonly observed when applications were made on coarse textured soils containing low
clay and organic matter. Injury was more severe with higher rates of isoxaflutole. Injury to
corn from isoxaflutole was not unique to any tillage system and was site, year, and rate
dependent.

In field studies that evaluated the effect of isoxaflutole application timing on corn
tolerance and weed control, the results indicated that the application timing of isoxaflutole
preemergence through 4-leaf stage corn, had little effect on broadleaf weed control.
However, control of giant foxtail with isoxaflutole became more variable with later
application timings, except when tank-mixed with metolachlor/benoxacor. In addition to
enhancing giant foxtail control, this tank-mixture greatly reduced corn tolerance when
applied after corn had emerged, especially to 2-leaf and 4-1eaf corn. Greenhouse studies

Confirmed the increased corn injury that was observed from delayed applications of

98

 

 

 

isoxaflutole tank-mixtures with metolachlor/benoxacor. Similarly, there was an increase in
corn injury from postemergence applications of isoxaflutole tank-mixed with another
chloroacetamide herbicide, acetochlor/MON-13900. Further greenhouse and laboratory
studies were conducted to determine if herbicide absorption, translocation, metabolism, or
retention was the physiological basis for the corn injury observed from delayed applications
of the isoxaflutole tank-mixture with metolachlor/benoxacor. Metolachlor/benoxacor
increased radiolabeled isoxaflutole absorption when applied to spike, 2-leaf and 4—1eafcom.
Even though isoxaflutole absorption was increased, this increase was not enough to explain
the severity of the injury observed from the isoxaflutole : metolachlor/benoxacor tank-
mixture. Isoxaflutole translocation and metabolism were not factors in the increased corn
injury observed. However, isoxaflutole retention increased 5-fold when
metolachlor/benoxacor was present in the spray solution and applied to 2-leaf and 4-leaf
corn. Increased isoxaflutole absorption and retention were the basis for increased corn injury
when tank-mixed with metolachlor/benoxacor and applied to emerged corn. These results
suggest that metolachlor/benoxacor may act similar to a spray adjuvant by increasing
isoxaflutole absorption and retention resulting in corn injury.

In greenhouse studies, results revealed that there were differences in corn hybrid
tolerance to isoxaflutole. GR50 values indicated that the corn hybrids Pioneer 3751 and
Pioneer 3737 were less tolerant to isoxaflutole than the hybrids Pioneer 3394 and Pioneer

3963. Differences in hybrid tolerance was primarily due to the rate in which the active
component of isoxaflutole is metabolized to the inactive benzoic acid derivative. Increased

uptake of isoxaflutole was also a contributing factor to the sensitivity of the hybrid Pioneer

3737.

99

 

In greenhouse studies, the use of an antidote increased corn tolerance to isoxaflutole.
R-29148 was the most effective antidote tested in protecting corn from isoxaflutole injury.
The antidote MON-13900 also provided protection to corn from isoxaflutole injury. The
protective mechanism of R-29148 was the enhancement of isoxaflutole metabolism. The
enhancement of corn tolerance to isoxaflutole with antidotes, as well as, the increased
activity observed with piperonyl butoxide suggests that cytochrome P-450 mono-oxygenases

might be involved in the metabolism of isoxaflutole by corn.

100