1 f'

This is to certify that the
thesis entitled i
MODE OF ACTION OF THE HERBICIDE

ANTIDOTE R-25788 (E)Ef2,2-DICHLOROACETAMIDE) f

presented by

J Robert Carlton Leavitt

 

has been accepted towards fulfillment

of the requirements for
Ph.D .
degree in l

 

 

Department of Crop and Soil Science

<7 K 7

Major professor

Date May 15, 1978

 

0-7639

 

     

MODE OF ACTION OF THE HERBICIDE ANTIDOTE

R-25788 (EififDIALLYL-Z,Z-DICHLOROACETAMIDE)

3?

J Robert Carlton Leavitt

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crap and Soil Science

1978

ABSTRACT

MODE or ACTION OF THE HERBICIDE ANTIDOTE
R-25788 (§,_1[-DIALLYL-2,2-DICHLOROACETAMIDE)

By

J Robert Carlton Leavitt

R—25788 (Eyflrdiallyl-Z,Z-dichloroacetamide) protected corn
(gggumgyg L.) frdm injury caused by two herbicide classes to which it
has structural similarity, the acetanilides and the thiocarbamates.
Rr25788 was the most effective antidote for both acetanilide and
thiocarbamate injury of six compounds tested (R925788, R—29148, CDAA,
1,8-naphthalic anhydride, carboxin, and gibberellin 6A3).

The protective effect of R-25788 was specific for corn, it did not
protect four weed species treated. The structural similarity between
R925788 and the herbicides could be the basis of the protective effect;
Rr25788 could act as a competitive inhibitor to the herbicides at some
unknown site of action specific to corn.

It has been suggested that R—25788 could prevent an herbicide
induced inhibition of gibberellin synthesis or lipid synthesis.
Exogenous application of gibberellin GA3 did not prevent either aceta-
nilide or thiocarbamate injury symptoms indicating that the herbicides
do not act by simply inhibiting gibberellin synthesis. Although EPTC,
a thiocarbamate herbicide, induced epicuticular wax aggregation on corn
leaves which was prevented by R—25788, it did not reduce the total
amount of epicuticular wax on corn leaves. This indicates that EPTC
might not inhibit lipid synthesis in this plant species. Metolachlor,

an acetanilide herbicide, had no observable effect on epicuticular wax

J Robert Carlton Leavitt
on corn.

All three acetanilide herbicides and one thiocarbamate sulfo-
xide tested reacted with glutathione to form herbicide-glutathione
conjugates in an in ziggg, non-enzymatic system. Since R—25788 has
been reported to selectively increase the glutathione content of corn,
R-25788 might protect corn from acetanilide herbicide injury by increa-
sing the rate of herbicide metabolism to non-phytotoxic glutathione
conjugates. However, because R-25788 was required to protect geneti-
cally atrazine-susceptible corn from alachlor injury but not from
thiocarbamate injury it is suggested that R925788 may protect corn from
EPTC injury by increasing the rate of EPTC sulfoxidation followed by
EPTC-sulfoxide conjugation. R-25788 did not protect genetically atrazi-
ne-susceptible corn from atrazine injury, indicating that R-25788 does
not stimulate glutathione7§rtransferase activity or atrazine-GSH

conjugation in corn.

ACKNOWLEDGMENTS

I would like to express my appreciation to Dr. Donald Penner
for his friendship, assistance, and guidance during the completion of
this dissertation. I would also like to thank Dr. Matthew Zabik for
his advice and the gracious use of his equipment, Dr. James Flore and
Dr. Violet Wert for providing various analytical standards, and Dr.
Gary Hooper and Dr. Karen Baker for their help and use of the Electron
Optics Center. I am grateful to Dr. William F. Meggitt, Dr. Matthew
Zabik, Dr. James Flore, and Dr. Bernard Knezek for their service as
guidance committee members.

I extend a very special thanks to my wife Maria for the prepa-

ration of this manuscript.

ii

TABLE OF CONTENTS

L I ST OF TABLES O I O O O I O O O O O O O O O O O O O O 0
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O 0

CHAPTER 1: POTENTIAL ANTIDOTES AGAINST ACETANILIDE HERBICIDE
INJURY TO CORN (ZEA MAYS) . . . . . . . . . . . .

Abstract . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . .
Material and Methods . . . . . . . . . . . . .
Plant culture, herbicide and antidote evaluation .
Chemicals used . . . . . . . . . . . . . . . .
Results and Discussion . . . . . . . . . . . . . . .

Literature cited . . . . . . . . . . . . . . . . . .

CHAPTER 2: PROTECTION OF CORN (ZEA MATS) FROM ACETANILIDE
HERBICIDE INJURY WITH THE ANTIDOTE R925788 . . .

Abstract . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . .
Materials and Method . . . . . . . . . . . . . . . . .
Plant culture and chemical treatment evaluation .
Chemical application . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . .

Literature cited . . . . . . . . . . . . . . . .

iii

Page

vii

10

l9
19
20
21
21
22
23
25

27

CHAPTER 3: PREVENTION OF EPTC-INDUCED EPICUTICULAR WAX

AGGREGATION ON CORN (ZEA HAYS) WITH R—25788 .

Abstract
Introduction . . . . . . . . . . . . . . . . . .
Material and Methods . . . . . . . . . . . . . . .

Plant culture and chemical application . . . .

Wax extraction, cuticular transpiration, SEM, and

GLC O O O O O O O O O O O O I O O O O O O O O 0
Results and Discussion . . . . . . . .
Literature Cited . . . . . . . . . . . . .

CHAPTER 4: THE E! VITRO CONJUGATION OF GLUTATHIONE AND

OTHER THIOLS WITH ACETANILIDE HERBICIDES AND
EPTC SULFOXIDE AND THE ACTION OF THE HERBICIDE

ANTIDOTE R-25788 . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . .

Material and Methods . . . . . . . . . . . .
Reagents and Equipment . . . . . . . . . . . .
Reaction of thiols with herbicides . . . .

Plant culture for herbicide-antidote response
8 tUdy O O O O O O I O O O O O O O O O O O 0

Results and Discussion

Literature Cited . . . . . . . . . . . . . .
CHAPTER 5: SUMMARY . . . . . . . . . . . . . . . .
LIST OF REFERENCES . . . . . . . . . . . . .

APPENDICES O O O O O O O O O O O O O O O O O O O O 0

iv

Page

43
43
44
44

44

46
47

50

58
58
59
60
60

61

62
63
67
76
77

81

LIST OF TABLES
Page
CHAPTER 1

1. Effects of six potential antidotes on corn injury
from 13.44 kg/ha of alachlor, metolachlor, H-22234
md EPTC O O O O O O O O O O O O O O O O O O O O C 12

2. Effects of six potential antidotes on corn injury
from 13.44 kg/ha of 3-26910, acetochlor, and EPTC. 14

3. Effects of CDAA on corn injury from acetochlor. . 16

4. Effects of EPTC or metolachlor, with and without
GA3, on height of plant grown in nutrient solution. 17

5. Effect of CA3 on corn injury caused by metolachlor. 18

CHAPTER 2

1. Protective effect of 1.12 kg/ha of R225788 against
corn injury from three acetanilide and one thio-
carbamate herbicide applied at five rates. . . . . 33

2. Protective effect of 1.12 kg/ha of R925788 against
corn injury from two acetanilide herbicides
applied at five rates . . . . . . . . . . . . . . 36

3. Protective effect of five rates of R925788 against
corn injury from five acetanilide and one thiocar-
bamate herbicides applied at 13.44 kg/ha. . . . . 38

4. Protective effect of 1.12 kg/ha of R925788 applied
as a tank mix with, or sequentially to, H-22234
applied preplant incorporated and pre-emergence at
13.44 kg/ha. . . . . . . . . . . . . . . . . . . . 41

S. A comparison of the protective effect of 1.12 kg/
ha of R225788 against injury from H—26910 between
corn and four weed species . . . . . . . . . . . 42

Page
CHAPTER 3

1. Influence of EPTC and Re25788 on epicuticular wax
deposition and cuticular transpiration of corn
leaves 0 O O O O O O O O I O O O O O I O O O Q I S 2

2. Influence of metolachlor and R-25788 on epicuti-
cular wax deposition and cuticular transpiration
Of com leaves I O I O O O O O O O O O O O O O O 5 3

3. Effect of EPTC and R—25788 on the major corn epi-
cuticular wax component, l-dotriacontanol . . . . 54

4. Predisposition of corn to injury from postemer-
gence-applied paraquat by EPTC and R925788 applied

at planting . . . . . . . . . . . . . . . 55
CHAPTER 4
1. Reaction of GSH with three acetanilide herbicides. 69
2. The pH-dependence of the GSH-alachlor conjugation
reaction . . . . . . . . . . . . . . . . . . . . 72
3. Reaction of alachlor with other thiols . . . . . 73
4. Reaction of GSH with various herbicides . . . . . 74

5. Response of atrazine susceptible, GSHe§7transfe-
rase deficient, corn inbred line GT112 to butyla—
te, EPTC, alachlor, and atrazine in three
experiments . . . . . . . . . . . . . . . . . . 75

vi

LIST OF FIGURES

Page
CHAPTER 2
1. Corn injury symptoms caused by preplant-incorporated
applications of EPTC and their prevention by R—25788 29
2. Corn injury symptoms caused by preplant-incorporated
applications of metolachlor and their prevention by
R-25788 o o o o o o o o o o o o o o o o o o o o o o 31
CHAPTER 3
1. Scanning electron micrographs of corn leaf surface
showing the epicuticular wax aggregation induced
by EPTC and its prevention by R-25788 . . . . . . . 56
CHAPTER 4
1. Structure of GS-acetanilide herbicide conjugates.. . 70

vii

INTRODUCTION

The purpose of this study was to investigate the mode of action
of the herbicide antidote R925788 (Nggrdiallyl-Z,2-dichloroacetamide).
A herbicide antidote is a compound that selectively protects crop
plants from herbicide injury without protecting weeds. R-25788 is the
only herbicide antidote in commercial production today and is used to
protect corn from thiocarbamate herbicide injury. A comparison of the
structure of R-25788 (Appendix B) to a wide variety of known herbicides
reveals that its structure is similar to that of two herbicide classes,
the thiocarbamates and the acetanilides (Appendix A). Because of this
structural similarity it has been suggested (41) that R-25788 prevents
thiocarbamate injury by acting as a competitive inhibitor to the herbi-
cides at some unknown site of action in corn. This hypothesis was
tested by examining the efficacy of R925788 (and related compound-Appen-
dix B) as an antidote to the acetanilide herbicides with which it also
has structural similarity. It has also been reported (12) that the
thiocarbamates inhibit gibberellin synthesis in corn and that R-25788
might reverse this inhibition. This hypothesis was evaluated by apply-
ing exogenous gibberellin to herbicide treated corn in an attempt to
prevent herbicide injury. Thiocarbamate herbicides also inhibit lipid
synthesis in various plant species (14, 17, 28, 44, 46, 47).

The hypothesis that R925788 might prevent this inhibition (45) was

tested by examining the effect of both thiocarbamate and acetanilide

1

herbicides and R925788 on epicuticular wax deposition on corn leaves.
It has also been reported (3, 26, 27) that R-25788 increases the glu-
tathione content of corn which increases the rate of thiocarbamate

metabolism to non-toxic glutathione conjugates. Glutathione conjuga-

tion reactions with various herbicides were therefore investigated.

CHAPTER 1

POTENTIAL ANTIDOTES AGAINST ACETANILIDE HERBICIDE
INJURY TO CORN (ZEA HAYS)

9.11m

R—25788 (2,2-Dichloro-NJN7diallylacetemide) was the most effec-
tive of six potential antidotes evaluated to counter corn (Eggwmazg_L.)
injury from the acetanilide herbicides alachlor, metolachlor, acetochlor,
H—22234 Q§,chloroacety11§f(2,6-diethylphenyl)g1ycine ethyl ester),
H—26910 (Nrchloroacetylfifif(2-methy1-6-ethylphenyl)glycine isopropyl
ester). The other potential antidotes in order of decreasing effecti-
veness were: R—29148 (2,2-dimethy1-5-methy1-dichloroacetyloxazolidine),
NA (1,8—naphthalic anhydride), CDAA (2-chlorof§,§rdiallylacetamide),
Carboxin (2,3-dihydro-5—carboxanilido-6-methy1-1,4-oxathiin) , and gibbe-

rellin GA GA only partly relieved the stunting of corn caused by

3)“ 3

EPTC and metolachlor and did not prevent other herbicide injury symptoms,
suggesting that the mode of action of EPTC and metolachlor is not to
simply block GA3 synthesis. R—25788 protected corn equally well from

acetanilide or EPTC injury.

Introduction

Since the discovery by Hoffman (8) that 4'-chloro-2-hydroxy-
imino-acetanilide selectively protected wheat (Triticum aestivum L.)
from injury caused by subsequant foliar applications of barban, numerous
other compounds have been examined for antidotal activity. Hoffman
(9) later found that seed treatment with NA protected corn from EPTC
injury. Rains and Fletchall (14) confirmed this and also reported
that R-25788 selectively protected corn from EPTC and that NA protected
sorghum (Sorghum‘bicolor (L.) Moench) from alachlor injury. However,
Estin (5) could not substantiate this. Donald and Fawcett (4) reported
that gibberellin (GAB) and R929l48 were effective in preventing EPTC
injury to corn, but Harvey, Chang, and Fletcher (6) reported that GA3
and indoleacetic acid were ineffective protectants against EPTC injury
to corn. Chang, Marsh, and Jennings (2) found that alachlor inhibited

growth of oat (Avena sativa L.) seedlings and that pre-treatment with

 

GA3 overcame this inhibition. Miller and Nalewaja (12) reported that
seed treatments of carboxin and R925788 decreased wheat injury from tri-
allate.

Chang, Stephenson, and Bandeen (1) found that R-25788 was a more
effective EPTC antidote than either NA or CDAA. Lay and Casida (ll)
concluded that four out of 32 compounds tested, including R925788, had
superior antidotal activity, and CDAA had moderate antidotal activity
against EPTC.

Dixon, Stoller, and McGlamery (3) reported that corn has widely

varying tolerance to alachlor and five closely related compounds.

Since Hickey and Krueger (7) reported that NA could reverse an alachlor

injury effect, corn tolerance to some of the more toxic acetanilide
herbicide might be improved with this or other herbicide antidotes.

In this study several chemicals with previously reported anti-
dote activity were evaluated for their efficacy in protecting corn from

five acetanilide herbicides.

Materials and Methods
Plant culture, herbicide and antidote evaluation.
Plants for all studies except those using nutrient solution were
grown in a greenhouse soil (1:1:1 soil, sand, peat) in 946-ml waxed

food cups. The herbicides, R925788, CDAA, and GA were sprayed on the

3
soil as formulated emulsifiable concentrates in an oil-inrwater emul-
sion. The formulated emulsifiable concentrate of R-29l48 was sprayed
on the soil in a 502 water-502 ethanol mixture. The herbicides and the
antidotes were sprayed sequentially with a link belt sprayer at 2.1
kg/cmz pressure in 93S L/ha spray volume and incorporated into the top
2.5 cm of soil. Carboxin and NA were applied as a 0.5% (w/w) seed
treatment without binder. Five DeKalb 315A corn seeds were planted

2.0 cm deep into the soil of each cup. After planting, the cups were
placed in a glass house with supplementary artificial lighting. Tempe-
rature ranged from 200 C at night to 33° C during the day. The plants
in the main-herbicide—antidote and the separate CDAA-acetochlor studies
were fertilized twice (6 and 12 days after planting) with a commercial
fertilizer in solution testing 20:20:20 for NPK. When herbicide and
gibberellin effects were examined the plants were not fertilized to

avoid any possible hormone-fertilizer interactions. Post-emergence

treatment of 6A3 were sprayed at 2.1 kg/cm2 pressure in 310 L/ha spray

volume when the plants were 10 cm tall. EPTC and metolachlor were
applied 24 h later in a 10 m1 soil drench. The number of emerged
seedlings showing visible herbicide injury were counted 10 days after
planting. Twenty-one days after planing, plant heights were measured
and the plants harvested, dried in a forced-air oven at 500 C for 48 h,
and the dry weight determined. The data are expressed as the percent
injured seedlings per cup, the average height in on per corn plant per
cup, and the average dry weight in mg per corn plant per cup. The
percent injury data were converted to their are sines for statistical
analysis.

For the nutrient culture study, plants were grown in Hoagland’s
No. 1 solution which was changed daily. DeKalb 315A corn seeds were
germinated at 240 C on filter paper in the dark for 72 h between sealed
trays. Three corn seedlings were placed in slits cut in 7.5 cm diam.by
1.9 cm deep foam rubber disks. The disks sat in 295-ml plastic tumblers
wrapped in aluminum foil to keep out light. Technical grade EPTC and
metolachlor were solubilized by adding 0.1% ethanol to each solution
except for the ethanol-free control. The gibberellin (GA3) used was
the water-soluble 102 potassium salt. Plants heights were measured 9
days after transfer to the plastic tumblers.

Chemical§_used.

The five antidotes used were R—25788, R929148, CDAA (all at 1.12
kg/ha) and carboxin and NA (0.5% w/w seed treatments). These were
evaluated against 13.44 kg/ha of alachlor, metolachlor and H—22234 in
the first part of the study and H926910 and acetochlor in the second
part. EPTC was included as a reference. Though all of these compounds

are not named as acetanilide herbicides, they all have a 2-chloroaceta-

nilide core. The CDAAracetochlor interaction was further evaluated
with CDAA at 0, 1.12, 2.24 kg/ha against acetochlor at 0, 4.48, 13.44
kg/ha in a two-way factorial experiment.

In the nutrient culture study, EPTC was added to give concentra-

6, 5 x 10-.5 and 5 x 10-.4 M and metolachlor at 5 x 10"6

5

tions of 5 x 10-
and 5 x 10-5 M. GA3 was tested for antidotal effects in the 5 x 10-
M concentration of each herbicide and was given at daily increasing
concentrations from 10"7 M on day 1 to 8 x 10-6 M on day 9. In a fur-
ther herbicide-gibberellin study, the antidotal properties of formulated
6A3 was evaluated at 1.12 kg/ha against 4.48 or 13.44 kg/ha of EPTC and
metolachlor on corn grown in soil culture. Both herbicide and GA3 were
applied pre-plant-incorporated in the first part of this study and
postemergence in the second part. _This study was designed as a two-way
factorial study split between methods of application. All experiments

were repeated and had five replications except for the extended rate

study of the CDAAracetochlor interaction which had four replications.

Results and Discussion

The compounds evaluated in the herbicide-antidote study varied
significantly in their antidotal properties. Rr25788 was the most ef-
fective; it countered visible injury caused by five acetanilide herbi-
cides as well as EPTC (Tables 1 and 2). The height and dry weight of
corn treated with R925788 or R-29148 in combination with all six herbi-
cides was not significantly different from that of the corn treated
with either antidote alone. NA was also an effective antidote, although
it was weak against H-22234 (Tables 1 and 2). CDAA was less effective

as an antidote, and corn plants treated with it in combination with

metolachlor, H-22234, and H-26910 were taller than with the herbicides
alone but significantly shorter than plants treated with CDAA alone or
the controls (Tables 1 and 2). The antidotal properties of CDAA were
not increased by increasing the rate from 1.12 to 2.24 kg/ha (Table 3).
Carboxin had little or no antidotal activity on any of the herbicides
tested, including EPTC.

The antidotes caused some corn injury. As shown in Table 2, the
R-25788, R929148, CDAA, and carboxin used alone significantly decreased
the dry weight of corn below that of the control, but only R929148 had
the same effect shown in Table 1. CDAA, which has partial antidotal
properties, also has herbicidal properties and can damage wheat and
ryegrass seedlings (Jaworski (10) as well as corn (Chang et a1. (1).

0f the herbicides used, alachlor was the least phytotoxic to
corn, although it did decrease plant height and dry weight at the rate
used (Table l). Metolachlor was more phytotoxic than alachlor but not
as phytotoxic as EPTC. Acetochlor and H926010 had approximately equal
phytotoxicity (Table 2). H-22234 was significantly more phytotoxic
than EPTC (Table 1).

In the nutrient culture study, the 0.12 ethanol added to increa-
se the solubility of the herbicides inhibited corn growth (Table 4).
The corn plants treated with EPTC and metolachlor were usually shorter
than the controls containing ethanol and the ethanol-free control
(Table 4). Both herbicides caused greater growth inhibition at increa-
sing rates.

GA3 increased corn growth sufficiently to overcome the stunting
caused by EPTC and metolachlor as compared to the ethanol control

(Table 4) but it did not prevent herbicide injury symptoms such as leaf

stunting, leaf rolling and twisting, and stem swelling. Furthermore,

the GA did not fully overcome the herbicide-induced growth inhibition

3
as the plants receiving the combination were shorter and lighter than
those receiving only the 6A3 (Tables 4 and 5). Since exogenously added
0A3 did not overcome the injury symptoms caused by EPTC and metolachlor,
it is doubtful that the mode of action of these two herbicides is merely

the blocking of GA3 synthesis. Although GA may partially prevent plant

3
stunting due to EPTC and metolachlor injury, its failure to prevent the
other injury symptoms severely limits its effectiveness as an antidote.

The simplest proposed mode of antidotal action is that the anti-
dote inhibits the action of the herbicide because of structural simila-
rity (Stephenson et al.,(15). If so, R929l48 should be a more effective
acetanilide antidote than R925788 because its oxazolidine ring more
closely resembles the phenyl ring of the herbicides than does the allyl
side chains of Re25788.

The dichloroacetamides, Rr25788 and R929148, were more effective
in preventing acetanilide herbicide injury to corn than was the single
monochloroacetamide, CDAA. This is in agreement with Pallos et a1.

(13) who reported that the dichloroacetamides were more effective
thiocarbamate antidotes than the monochloroacetamides.

In conclusion, R925788 was the most effective antidote for aceta-
nilide herbicide injury tested, and R929l48 and NA also had good antido-

tal activity. CDAA only partially overcame acetanilide herbicide

injury, and carboxin and 6A3 had little or no antidotal activity.

10.

11.

12.

10

LITERATURE CITED

Chang, T.C., Stephenson, G.R., and Bandeen, J.D. 1973. . Compara-
tive effects of three EPTC antidotes. weed Sci. 21:292-295.

Chang, F.Y., Marsh, H.V.Jr., and Jennings, P.H. 1975. Effect of
alachlor on Avena seedlings: inhibition of growth and intera-
ction with gibberellic acid and indoleacetic acid. Pestic.
Bio. and Physiol. 5:323—329.

Dixon, G.A., Stoller, E.W., and McGlamery, M.D. 1976. Studies on
alachlor and related herbicides. Proc. N. Cent. Weed Control
Conf. 31:33.

Donald, W.W. and Pawcett, R.S. 1975. Prevention of EPTC injury
to hybrid corn with antidote 29148 and exogenoud GAB’ Proc.
N. Cent. weed Control Conf. 30:28.

Eastin, E.F. 1972. Evaluation of a sorghum seed treatment to
prevent injury from acetanilide herbicides. Agron. J.
64:556-567.

Harvey, M.R., Chang, F.Y., and Fletcher, R.A. 1975. Relationship
between §7ethyldipropylthiocarbamate injury and peroxidase
activity in corn seedlings. Can. J. Bot. 53:225-230.

Hickey, J.S. and Krueger, W.A. 1974. Alachlor and 1,8-naphthalic
anhydride effects on corn cole-ptiles. Weed Sci. 22:250-252.

Hoffman, 0.L. 1962. Chemical seeds treatments as herbicide:anti-
dotes. weeds 10:322.

Hoffman, 0.L. 1969. Seed treatment with 1,8-naphthalic anhydride
protects corn from EPTC. weed Sci. Soc. Amer. Abstr. No. 12.

Jaworski, E.G. 1956. Biochemical action of CDAA, a new herbicide.
Science 123:847-848.

Lay, M.M. and Casida, J.E. 1976. Dichloroacetamide antidotes
enhance thiocarbamate sulfoxide detoxification by elevating
corn root glutathione content and glutathioneeSftransferase
activity. Pestic. Bio. Physiol. 6:442-456.

Miller, S.D. and J.D. Nalewaja. 1976. Herbicide antidotes with
triallate. Proc. N. Cent. weed Cont. Conf. 31:145.

13.

14.

11

Pallos, F.M., Gray, R.A., Arneklev, D.R. and Brokke, M.E. 1975.
Antidotes protect corn from thiocarbamate herbicide injury.
J. Agric. Food. Chem. 23:821-822.

Rains, L.J. and Fletchall, 0.H. 1971. The use of chemicals to
protect crops from herbicide injury. Proc. N. Cent. Weed
Control Conf. 26:42.

Stephenson, G.R., Bunce, N.J., Makowski, R.I., and Currie, J.C.
1978. Structure activity relationships for SrethyleN,N7
dipropylthiocarbamate antidotes in corn. J. Agric. Food.
Chem. 26:137-140.

12

 

 

 

 

 

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n.09mm mam emummlm .
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13

Ifiwfiewam uo: mums mumuuoa umawfiqm sues macauumuouda you can uoouuo name some now mmsmaou manna: memo:

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Ammamauaoov

H 0H can.

14

 

 

 

 

 

 

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15

language uo: mums muouuma umawfiam Aua3 mmoauomuoumu you can uoouwm mama some now measaoo :HAuwa memo:

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16

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:uoo

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m man—mm.

17

 

 

Table 4 - Effects of EPTC or metolachlor, with and without GA3, on
height of plants grown in nutrient solution. ‘
Plant ht, cm
Herbicides - GA3 '+ GA3
(1.12 kg/ha)

None 31 f
None + ethanol 0.12 21 d 38 g
EPTC + ethanol 0.12

5x10‘6M 13b

leo-SM 14b 20d

5 x 10"4 M 9 a
Metolachlor + ethanol 0.1%

5x10-6M 21d

SxIO-SM 17c 28e

 

a. Means within columns with similar letters were not significantly
different at the 52 level by Duncan's Multiple Range Test.

18

 

 

Table 5 - Effects of GA3 on corn injury caused by metolachlor and
a
EPTC
Method of Herbicide GA Plant ht Plant dry wt
Application kg/ha kg/Ha em mg/plant
Preplant None 0 49 e 417 g
Incorporated 1.12 61 f 489 h
EPTC
4.48 0 20 b 224 cd
4.48 1.12 32 c 300 e
13.44 0 6 a 95 a
13.44 1.12 16 b 188 be
Metolachlor
4.48 0 26 c 252 d
4.48 1.12 37 d 346 f
13.44 0 18 b 172 b
13.44 1.12 26 c 253 d
Post-emergenceb None 0 55 e 412 be
1.12 63 f 484 d
EPTC
4.48 0 33 b 348 b
4.48 1.12 45 cd 391 be
13.44 0 22 a 254 a
13.44 1.12 23 a 265 a
Metolachlor
4.48 0 40 be 363 b
4.48 1.12 48 d 440 cd
13.44 0 25 a 238 a
13.44 1.12 37 b 378 bc

 

a. Means within columns with similar letters were not significantly
different at the 52 level by Duncan's Multiple Range Test.

b. Herbicides given as soil drench and GA3 as foliar spray when plants
were 10cm tall. .

CHAPTER 2

PROTECTION OF CORN (ZEA MAYS) FROM ACETANILIDE
HERBICIDAL INJURY WITH THE ANTIDOTE R~25788

Abstract

The antidote R-25788 (NJNfdiallyl-Z,2-dichloroacetamide) protece
ted corn (Zea Mays L. 'DeKalb 315A') seedlings from.injury caused by the
acetanilide herbicides, alachlor (2-chloro-2',6'-diethy1eNr(methoxymethyl)
acetanilide), metolachlor (2-chloroeNf(2-ethyl-6-methylphenyl)fN7(2-
methoxy-l-methylethyl)acetamide), H-22234 (NrchloroacetyleN7(2,6-diethy1-
pheny1)g1ycine ethyl ester), and H—26910 (Nrchloroacetylegr(2-methy1-6-
ethylphenyl)glycine isopropyl ester) in a grenhouse study. R925788,
however, did not protect four weed species tested. R-25788 only partial-
ly protected corn from injury caused by acetochlor (2-chloroeNr(ethoxy-
methy1)-6'-ethy1egraceto—toluidide). R-25788 was an effective antidote
whether applied preemergence, preplant-incorporated, or as a tank mix.
Injury symptoms caused by EPTC (Sfethyl dipropylthiocarbamate) and the
acetanilide herbicides were similar; both caused leaf twisting and rol-

ling, and at high rates leaves failed to emerge through the coleoptile.

19

20

Introduction

Rains and Fletchall (10) reported that R-25788 selectively pro-
tected corn from EPTC injury and Meggitt et a1. (9) and Chang et a1. (2)
confirmed this. Chang et al. (3) found that R925788 significantly redu-
ced the phytotoxicity of ten of twenty-two herbicides tested on corn;
six of these were thiocarbamates. They reported that R925788 was more
effective against EPTC, vernolate (grpropyl-dipropylthiocarbamate), and
butylate (Srethyl diisobutylthiocarbamate) than alachlor. Chang et a1.
(3) suggested that different sites of action and (or) different structu-
re-activity relationship might explain the differential antidotal
activity of R925788 to the different herbicide groups.

Stephenson and Chang (12) synthesized 25 dichloroacetamide
analogues of Re25788 and tested them for antidotal activity against five
thiocarbamate herbicides, including EPTC. The R925788 analogue that was
the best antidote to each of the thiocarbamate herbicides was the one
with side chains identical to the respective herbicides. They speculated
that the dichloroacetamides were competitive inhibitors at the site of
thiocarbamate herbicide action.

The purpose of this study was to investigate more extensively the
antidotal properties of Re25788 by comparing its antidotal effect on
varying rates of five acetanilide herbicides and by testing varying rates
of R925788 against the same herbicides, by examining different methods of
antidote application, and to determine if the antidotal properties of

R925788 against acetanilide herbicides were selective for corn.

21

Materials and Methods
Plant culture and chemical treatment evaluation.

The soil used in all experiments was a greenhouse soil (1:1:1
soil, sand, peat). Plants for the study combining R-25788 with various
rates of herbicides were grown in 473-ml waxed cups. Plants for the stu-
dy of various rates of R925788 and study of the method of application
were grown in 946-ml waxed cups. Plants for the selectivity study were
grown in 15.2 by 30.4 by 5.1 cm styrofoam trays. Formulated emulsifia-
ble concentrates of the herbicides and the R925788 were sprayed on the
soil surface sequentially with a link belt sprayer at 2.16 kg/cmz pres-
sure with 935 L/ha spray volume, and incorporated into the soil sepa-
rately in all but the study of method of application. In the method of
application research some treatments were made preemergence and some
combinations of H-22234 with R-25788 were sprayed as a tank mix. Incor-
poration was done by mixing the top 2.5 cm of soil in each group or tray.
Soil in all cups for control plants was stirred similarly, although no
chemicals were incorporated. Five corn seed were planted 2.0 cm deep
into the soil of each group or tray. Fifteen seeds of pigweed (£22222?
thus retroflexus L.), barnyardgrass (Echinochloa crus:galli (L.) Beauv.),
yellow foxtail (Setaria lutescens (weigel) Hubb.), and green foxtail
(Setaria viridis (L.) Beauv.) were planted 0.28 cm deep in the trays for
the selectivity study. After planting, the cups or trays were placed in
a greenhouse supplemented with artificial lighting to give a 14-h day.
The temperature ranged from 25 C at night to 33 C during the day. All
plants were fertilized twice (6 and 12 days after planting) with a com-

mercial 20:20:20 fertilizer. The number of emerged seedlings showing

visible herbicide injury were counted 10 days after planting for all but

22

the selectivity study. In the study to compare the injury from EPTC and
metolachlor, the plants were harvested and photographed 10 days after
treatment. In the selectivity study the number of emerged seedlings
injury by herbicides was determined 21 days after planting. In all the
other studies plant ht was measured and the plants harvested 21 days
after planting. The plants were dried in a forced air oven at 50 C for
48 h and the dry wt determined. The data are expressed as the percent
injured corn seedlings, the average ht in cm per corn plant and the
average dry wt in mg per corn plant. The percent injury data were con-
verted to their arc sines for statistical analysis.

Chemical application.

The efficacy of a high rate (1.12 kg/ha) of R925788 was tested
against five rates (0.0, 2.24, 4.48, 6.72, 13.44 kg/ha) of the five ace-
tanilide herbicides and one thiocarbamate herbicide. The study was divi-
ded into two parts and both parts repeated with a three-way factorial de-
sign. In the first part of this study, the herbicides used were EPTC,
alachlor, metolachlor, and H-22234. In the second part, the herbicides
H—26910 and acetochlor were applied.

To determine the rate of R-25788 necessary to protect against
a high rate (13.44 kg/ha) of the herbicide, five rates of R925788 (0.0,
0.14, 0.28, 0.56, 1.12 kg/ha) were used. This study was a two-way facto-
rial design. In the method of application study, the herbicide H-22234,
and the antidote R225788 were applied alone and in combination, both
preemergence and preplant-incorporated in a completely randomized design.

The study on selectivity examined the antidotal effect of R-25788
against acetanilide herbicides between corn and four weed species. This

study was a two-way factorial design and was repeated using acetochlor

23

with the same results (data not presented). Because the injury
symptoms of the acetanilide herbicides and EPTC were very similar, the
relative injury was compared photographically. Metolachlor was chosen
because it was similar to EPTC in the rates required to cause identical
symptoms.

The data presented are the means of two experiments with five
replications per experiment except for the selectivity study which had

four replications.

Results

Rr25788 effectively protected corn from injury caused by five
acetanilide herbicides tested (Tables 1, 2). The corn receiving herbi-
cide plus R—25788 showed significantly less visible injury and had
greater ht and dry wt than corn that did not receive the antidote
(Tables 1, 2). R-25788 was as effective in protecting corn from acetani-
lide injury as it was in protecting corn from EPTC (Table l).

The various herbicides examined differed significantly in their
phytotoxicity (Tables 1, 2). Alachlor was least injurious and did not
decrease either corn ht or dry wt significantly at rates less than
13.44 kg/ha, although it did cause significant visible injury when rated
at 10 days (Table l). Metolachlor, acetochlor, and EPTC all signifi-
cantly decreased corn ht at 4.48 kg/ha (Tables 1 and 2). Metolachlor
and acetochlor also significantly decreased corn dry wt and increased
visible injury occured with metolachlor and acetochlor at 4.48 kg/ha,
and EPTC at 6.72 kg/ha (Table l). H-22234 and H-26910 also caused signi-
ficant injury to corn. Both of these herbicides significantly decreased

corn ht at only 2.24 kg/ha (Tables 1, 2). R925788 alone had no signi-

24

ficant effect on corn (Table 3).

Different rates of R925788 were required to protect against
injury from the 13.44 kg/ha rate of the different herbicides. A rate
of 0.14 kg/ha R-25788 was needed to provide protection from 13.44 kg/ha
of alachlor, metolachlor, and EPTC (Table 3). R-25788 applications of
0.56 kg/ha completely prevented the phytotoxic effects of H922234 and
H-26910 (Table 3). In one study R925788 at 1.12 kg/ha prevented visible
injury caused by acetochlor but did not prevent the decrease in both ht
and dry wt caused by acetochlor at 13.44ng/ha (Table 2). In another
study visible injury caused by 13.44 kg/ha acetochlor was also not pre-
vented by R-25788 (Table 3).

H922234 alone caused less corn injury if applied preemergence
rather than preplant incorporated, 64 vs 872, respectively (Table 4).
R925788 applied sequentially or as a tank mix, protected corn from
H-22234 injury (Table 4). R925788 prevented H-22234 injury whether both
were applied preemergence or preplant incorporated (Table 4).

The protective effects of R-25788 against injury from acetanilide
herbicides were selective for corn (Table 5). None of the four weed
species tested, pigweed, barnyardgrass, yellow foxtail, or green foxtail
showed any evidence of being protected from H-26910 injury by R-25788.

Identical injury symptoms were evident on corn treated with EPTC.
and all the acetanilide herbicides tested, though the rates required to
produce symptoms of the same intensity varied widely. Because of their
similar rate responses, EPTC and metolachlor were compared photographi-
cally for injury symptoms with and without R925788 at 1.12 kg/ha (Figu-
res l, 2). The injury symptoms common to both classes of herbicide

include leaf rolling and twisting at low rates, extremely distorted

25

leaves and enlarged stems at higher rates, and complete failure of the
first true leaves to emerge through the coleOptile at the highest rates

examined.

Discussion

The antidote R—25788 effectively protected corn from acetanilide
herbicide injury. A prior report indicating that Rr25788 was less effec-
tive as an acetanilide herbicide antidote than an EPTC antidote was based
on comparison between EPTC and alachlor (3). Since alachlor is not as
phytotoxic to corn as EPTC, it does not provide a good basis for compa-
rison.

Since the plants in all of these studies were grown in the
greenhouse for only 21 days, it is not certain if the protective effects
of R925788 to acetanilide herbicides can be extended to an entire grow-
ing season in the field. The results of these greenhouse studies indi-
cate that H-22234, H—26910, and to a lesser degree metolachlor may cause
sufficient crop injury that the protection offered by R225788 cauld be
beneficial for corn under field conditions.

The similarity between EPTC and the acetanilide herbicide injury
symptoms, as well as the efficacy of R925788 as an antidote for symptoms
of both, could be easily explained if both classes of herbicides have
similar mode of action. The simplest proposed mode of action of R-25788
is that the antidote inhibits the action of the herbicide because it is
structurally similar to them (11). While it is true that R925788 resem-
bles EPTC, it resembles the acetanilide herbicides much less. However,
all of the herbicides, including EPTC, contain a carbamate linkage as

does R-25788. They all have electronegative groups attached to the

26

carbonyl carbon; i.e., §7ethy1 for EPTC, 2-chloromethy1 for R-25788,

and chloromethyl for the five acetanilide herbicides. Therefore, their
chemical reactivities toward certain carbamoyl acceptors such as gluta-
thione may be the same. It has also been proposed that EPTC is metabo-
lized after activation by conjugation to glutathione (1, 5, 7, 8).

There are reports that acetanilide herbicides may also form glutathione
conjugates. Frear and Swanson (4) reported that propacthr (2-chloro—
Nyisopropylacetanilide) and barban (4-chloro-2-butyny1;grchlorocarbani-
late) reacted with glutathione in vitro. They also reported that propar
chlor, barban, and alachlor were inhibitors of glutathione conjugation
with triazine herbicides. Lamoreaux et a1. (6) have isolated a gluta-
thione conjugate of propachlor from corn. If R-25788 increases the
glutathione content and the glutathioneegftransferase activity in corn
and if the thiocarbamate and acetanilide herbicides are both metabolized
by conjugation with glutathione, then Rr25788 could increase the rates
of metabolism of both classes of herbicides and explain the antidote

mode of action.

10.

27

LITERATURE CITED

Casida, J.P., R.A. Gray, and H. Tilles. 1974. Thiocarbamate
sulfoxides: potent, selective, and biodegradable herbicides.
Science 184:573-574.

Chang, F.Y., J.D. Bandeen, and G.R. Stephenson. 1972. A selecti-
ve antidote for prevention of EPTC injury in corn. Can. J.
Plant Sci. 52:704-714.

Chang, F.Y., J.D. Bandeen, and G.R. Stephenson. 1973. NJNfdial-
1y1-a,a-dichloroacetamide as an antidote for EPTC and other
herbicides in corn. Weed Res. 13:399-406.

Frear, D.S. and H.R. Swanson. 1970. Biosynthesis of §7(4-ethyl-
amino—6-isopropylamino-Zegytriazino) glutathione: partial
purification and properties of a glutathionee§rtransferase
from corn. Phytochemistry 9:2123.

Hubbell, J.P. and J.E. Casida. 1977. Metabolic fate of the NJNf
dialkyl—carbamoyl moiety of thiocarbamate herbicides in
rats and corn. J. Agric. Food Chem. 25:404-413.

Lamoreaux, G.L., L.E. Stafford, F.S. Tanaka. 1971. Metabolism
of 2-chloroegrisopropylacetamide (propachlor) in the leaves
of corn, sorghum, sugarcane and barley. J. Agric. Food Chem.
19:346-350.

Lay, M.M. and J.E. Casida. 1976. Dichloroacetamide antidotes
enhance thiocarbamate sulfoxide detoxification by elevating
corn root glutathione content and glutathionefiSftransferase
activity. Pestic. Biochem. Physiol. 6:442-456.

Lay, M.M., J.P. Hubbell, and J.E. Casida. 1975. Dichloroacetamide
antidotes for thiocarbamate herbicides: mode of action.
Science 189:287-289.

Meggitt, W.F., A.D. Kern, and T.F. Armstrong. 1972. Crop protec-
tants and the use of thiocarbamates in corn. Proc. North
Cent. weed Control Conf. 27:22.

Rains, L.J. and 0.H. Fletchall. 1971. The use of chemicals to
protect crops from herbicide injury. Proc. North Cent. Weed
Control Conf. 26:42.

28

ll. Stephenson, G.R., N.J. Bunce, R.I. Makowski, and J.C. Currie.
1978. Structure-activity relationship for §7ethy1fN,Nf
dipropylthiocarbamate (EPTC) antidotes in corn. J. Agric.
Food Chem. 26:137-140.

12. Stephenson, G.R. and F.Y. Chang. 1978. Comparative activity,
selectivity and field applications of herbicide antidotes.
IE_J.E. Casida and F.M. Pallos, eds., Chemistry and Action
of Herbicide Antidotes. Academic Press, New York. 192 pp.

Figure 1

29

Corn injury symptoms caused by pre-plant-incorporated
applications of EPTC and their prevention by R925788.

a) EPTC 0.0 kg/ha, b) EPTC 4.48 kg/ha, c) EPTC 13.44
kg/ha, d) EPTC 0.0 kg/ha plus R225788 1.12 kg/ha,

e) EPTC 4.48 kg/ha plus R225788 1.12 kg/ha, f) EPTC

13.44 kg/ha plus R-25788 1.12 kg/ha. Ruler scale is in
inches.

30

a

a', . " ' ‘. .- istiw- w- «we
'“‘n‘*v --‘~ ‘u‘, a”. 7 ' 0" g. .

We??? ----- - -

“ ' 1 ~ - .. ‘ -[ , ' i .._‘.‘s:- Vl—T‘;‘.

= a » . ,_ A“... “.1... .

.m6l97.12”90"..i.l V!.:v . -.-. H't'vu’sw-le-n ‘1 “'hhl‘hfig '~F!'
_..,_r b.‘ ... V ' .

      

. I» .1. .,~

Figure 2

31

Corn injury symptoms caused by pre-plant-incorporated
applications of metolachlor and their prevention by
R225788. a) metolachlor 0.0 kg/ha, b) metolachlor

4.48 kg/ha, c) metolachlor 13.44 kgfha, d) metolachlor
0.0 kg/ha plus R225788 1.12 kg/ha, e) metolachlor 4.48
kg/ha plus R-25788 1.12 kg/ha, f) metolachlor 13.44 kg/ha
plus R925788 1.12 kg/ha. Ruler scale is in inches.

32

 

 

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35

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37

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39

 

 

 

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40

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42

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CHAPTER 3

PREVENTION OF EPTC-INDUCED EPICUTICULAR.WAX
AGGREGAIION 0N CORN (ZEA MAYS) WITH R-25788

Abstract

Seanning electron micrographs showed that EPTC (§7ethyl-dipropyl-
thiocarbamate) caused an aggregation of the epicuticular wax layer of
corn (§g§;g§y§_L.). R925788 (2,Z-dichloroAEJEfdiallylacetamide) preven-
ted this aggregation when applied in combination with EPTC. Neither
EPTC, metolachlor (Z-chlorofigr(2-ethy1—6-methylphenyl)s§7(2-methoxy-l-
methyl)ethyacetamide), nor Rr25788 changed the weight of chloroform
extractable epicuticular wax on corn leaves. EPTC apparently does not
block lipid synthesis in corn as it does in other plant species. Thus
R225788 does not protect corn from.EPTC or metolachlor by overcoming
such a block, but EPTC did affect_wax arrangement on the leaf surface
and caused an increase in the cuticular transpiration of corn and pre-
disposed corn to injury from subsequent postemergence applications of
paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride). R-25788 protec-

ted corn against these deleterious effects.

43

44

Introduction

Thiocarbamate herbicides such as EPTC have been reported to
inhibit epicuticular wax deposition on peas (Pisum sativum L.) (8),
sicklepod (Cassia obtusifolia L.) (9), cabbage (Brassica oleracea var.
Capitata) (3, 4, 5) and navy bean (Phaseolus Vulgaris L.) (12). This
inhibition has been associated with EPTC-induced predisposition of navy
bean to root rot caused by Fusarium solani (Mart) Appel (12, 13). EPTC
has also been reported to inhibit lipid synthesis in isolated spinach
(Spinacia oleracea L.) chloroplasts (ll). R-25788, when applied in come
bination with EPTC, prevented this inhibition of lipid synthesis (10).
Since R925788 is an effective antidote to corn injury caused by EPTC
(2, 6, 7), it was suggested that the general mode of action of R925788
is to overcome an EPTC-induced inhibition of lipid synthesis (10). The
purpose of this research was to investigate the effects of EPTC on epi-
cuticular wax deposition on corn, to examine the interaction of R-25788
with any EPTC effect found, and to test whether any EPTC effect could
predispose corn to subsequent herbicide stress injury. Metolachlor was
included in the study since it has been reported that R—25788 protects

corn from metolachlor injury as effectively as it protects EPTC (6).

Materials and Methods
Plant culture and chemical application.
In all studies the plant material was corn (Zea mays L. 'Pioneer
3780') grown in a greenhouse soil (lzlzl, soil:sand:peat). All herbici-
des and R—25788 used were commercial formulations without any additional

surfactant. The plants for the epicuticular wax extraction, cuticular

45

transpiration, and scanning electron-micrograph (SEM) studies were grown
in 5 x 14.5 x 29 cm styrofoam trays placed outside (between the green-
houses) on a rough gravel bed (to prevent roots from growing out of the
trays). The soil in the styrofoam trays was treated with a commercial
N-P-K fertilizer (20:20:20) before herbicide application or planting.

Before planting the corn 2.0 cm deep, EPTC and/or R925788 in the
first study and metolachlor and/or R925788 in the second study were
sprayed on the surface of the soil on the trays with a link belt sprayer
(2.11 kg/cm; pressure; 935 L/ha spray volume). The chemicals were incor-
porated into the top 2.5 cm of soil. When Rr25788 was applied in combi-
nation with one of the herbicides, the herbicide was applied and incor-
porated first. The epicuticular wax extraction, cuticular transpiration,
and SEM studies were two-way factorial in design. EPTC at 0.0, 1.68,
3.36, or 6.72 kg/ha or metolachlor at 0.0, 1.68, or 3.36 kg/ha were ap-
plied in combination with 0 or 1.12 kg/ha R—25788. These studies consis-
ted of six replications per treatment, five of which were used for wax
extraction and cuticular transpiration measurements. The sixth was used
for Scanning Electron Microscopy (SEM). The experiment combining meto-
lachlor and R-25788 had five replications per treatment and was not
repeated since the results were of minor interest. The seeds were plan-
ted 2.0 cm deep in 946-ml waxed cups for the EPTC, Rr25788 paraquat
interaction study and the plants grown in a greenhouse with supplemental
lighting (16 hr day). The temperature ranged from 22 C at night to 30 C
during the day. This study was three-way factorial in design with four
replications combining EPTC at 0.0, 3.36, or 6.72 kg/ha, R-25788 at 0.0
or 1.12 kg/ha, and paraquat at 0.0 or 0.56 kg/ha. Immediately after

planting, the EPTC and Rr25788 were applied in 50 ml of solution soil

46

drench. When the corn was 12 days old (15 cm tall), the paraquat was
sprayed on the leaves with the link belt sprayer (2.11 kg/cm2 pressure;

935 L/ha volume). The non-absorbed paraquat was washed off the leaves

24 hr later. Forty-eight hr after paraquat treatment visual injury ratings
were taken, and five days later the fresh weight per plant was measured.
Except for the experiment with metolachlor, all data presented are the
means of two experiments.

Wax extraction, cuticular transpiration, SEM,4and GLC.

When the corn plants were in the fifth leaf stage, the leaf blades
from the third oldest leaves were removed for epicuticular wax extraction.
Upon removal, the leaf blade area was measured with an automatic area
meter (Lambda Instruments). Three hundred cm2 of blades were placed in
the bottom of a 1—L measuring cup and washed twice with glass-distilled
chloroform (once with 150 ml for 30 sec., once with 100 ml for 15 sec).
Both washes were combined. A 75-ml aliquot of the chloroform~wax mixtu-
re was filtered through Whatman #1 filter paper into predweighed 80-ml
aluminum pans. After the chloroform was evaporated for 18 hr, the pans
were re-weighed and the weight of epicuticular wax per cm2 leaf area
calculated. The cuticular transpiration was measured on the fourth olde
est leaf of the same plants. The leaf blades were harvested, their leaf
area measured with the automatic area meter and cut into three 10 cm.long'
sections. The incisions were covered with lanolin to prevent water loss.
Three leaves were placed into lO-cm-dia aluminum pans, weighed, allowed
to transpire for 45 hr in an exhaust hood, and then re—weighed. The water
loss per cm2 leaf area was calculated. Scanning electron micrographs
were taken of the adaxial surface of 4 by 8 mm leaf pieces of the leaf

blade of the third leaf (taken approximately 1/3 of the distance from

47

sheet to blade tips to the side of the mid vein) as previously described
(5). A sample of the chloroform wax solution was analyzed by gas-liquid
chromatography (GLC) for the effects of the chemical treatment upon the
major corn wax components. The solvent from a 10-ml aliquot of the solu-
tion of chloroform wax was evaporated under nitrogen and the wax subse-
quently redissolved in 1 ml fresh chloroform. Over 602 of the detected
area was in one peak with a retention time of 6.8 min and was identified
as l—dotriacontanol. This identification was made by comparison of the
retention time of the unknown to the retention times of a homologus
series of known standards. It has been previously reported that l-dotria-
contanol is the major constituent of the corn epicuticular waxes (l).

The GLC system used was as previously described except column temperature

was 280 C (5).

Results and Discussion

Neither EPTC nor metolachlor, applied alone or in combination with
R-25788, caused any significant deviation from the control in amount of
epicuticular wax extracted from corn leaf blade surfaces (Tables 1 and
2). However, the highest rate of EPTC plus R-25788 did result signifi-
cantly less epicuticular wax than did the lower rates of EPTC plus
Rr25788 (Table 1). Since EPTC did not decrease epicuticular wax deposi-
tion on corn, it may not decrease lipid synthesis in corn as it does in
other plant species. Since the antidote R-25788 protects corn from
injury by high rates of EPTC, the mode of action of R925788 apparently
is not to overcome an inhibition of lipid synthesis caused by either EPTC
or metolachlor. Cuticular transpiration was increased by EPTC when

applied alone but not by EPTC in combination with R-25788 (Table l).

48

Metolachlor had no effect on cuticular transpiration whether applied
alone or with R925788 (Table 2). The increase in cuticular transpira-
tion caused by EPTC, and its reversal by R925788, without concomitant
changes in the amount of epicuticular wax on the leaf surface can only
be explained by a change in either the chemical composition or the
distribution (fine structure) of the wax on the corn leaf surface.
Analysis of the epicuticular wax by GLC did not reveal any obvious
effects of EPTC or R925788 treatment on any of the unidentified minor
components. Analysis of the major wax component (l-dotriacontanol) by
GLC revealed a slight inhibition by EPTC that was not prevented by
R-25788 (Table 3), thus eliminating the possibility that R225788 reversal
of EPTC-induced changes in chemical wax composition could account for
the transpiration effect. However, scanning electron micrographs of corn
leaves (Fig. 1) showed that EPTC caused definite aggregation of the epi-
cuticular wax upon the surface. When R925788 was applied with EPTC, the
epicuticular wax layer appeared normal (Figure l). The increase in
cuticular transpiration caused by EPTC and its prevention by R925788 can
be explained by these observations. By inducing aggregation of the epi-
cuticular wax, it appears that EPTC causes areas of the underlying cuti-
cle layers to be relatively more exposed, leading to increased water
loss. Elimination of the aggregates by combining EPTC with R-25788 elimi-
nates the increase in transpiration. Though it is difficult to estimate
amounts of epicuticular wax from the micrographs, the treatments do not
appear to differ in amount of epicuticular wax despite the differences
in distribution.

EPTC treatments at planting predisposed corn to increased damage

from later application of paraquat (Table 4). Re25788 prevented this

49

predisposition. Apparently EPTC-induced epicuticular wax aggregation
caused increased uptake of paraquat. R-25788 eliminated the predispo-
sition effect by eliminating the aggregation effect.

In conclusion, EPTC caused a change in the distribution of epicu-
ticular wax on corn leaf surface wich R925788 prevented. R925788 preven-
ted EPTC-induced increases in cuticular transpiration and predisposition

to paraquat injury.

10.

ll.

12.

50

LITERATURE CITED

Bianchi, G. and F. Solamini. 1975. Glossy mutant of maize. IV.
Chemical composition of normal epicuticular waxes. Maydica
20(1):1-3.

Chang, F.Y., G.R. Stephenson, and J.D. Bandeen. 1973. Comparative
effects of three EPTC antidotes. Weed Sci. 21:292-295.

Flore, J.A. and M.J. Bukovac. 1974. Pesticide effects on the plant
cuticle: I. Response of Brassica oleracea L. to EPTC as
indexed by epicuticular wax production. J. Amer. Hort. Sci.
99(1):34—37.

Gentner, W.A. 1966. The influence of EPTC on external wax deposi-
tion. Weeds. 14:27-31.

Leavitt, J.R.C., D.N. Duncan, D. Penner, and W.F. Meggitt. 1978.
Inhibition of epicuticular wax deposition on cabbage by etho-
fumesate. Plant Physiol. (in press).

Leavitt, J.R.C. and D. Penner. 1977. Protecting corn against
alachlor, metolachlor, and H-22234 injury with the antidote
R925788. Proc. N. Cent. Weed Control Conf. 32: (In press).

Rains, L.J. and H.0. Fletchall. 1971. The use of chemicals to
protect crops from herbicide injury. Proc. N. Cent. Weed
Control Conf. 26:42.

Still, G.G., G.D. Davis, and G.L. lander. 1970. Plant epicuticular
lipids alteration by herbicidal carbamates. Plant Physiol.
46:307-314.

Wilkinson, R.E. 1974. Sicklepod surface wax response to photo-
period and §7(2,3-dichloroallyl) diisopropyl thiocarbamate
(Diallate). Plant Physiol. 53:269-275.

Wilkinson, R.E. and A.E. Smith. 1975. Reversal of EPTC induced
fatty acid synthesis inhibition. Weed Sci. 23:90-92.

Wilkinson, R.E. and A.E. Smith. 1975. Thiocarbamate inhibition
of fatty acid biosynthesis in isolated spinach chloroplasts.
weed Sci. 23:100-104.

wyse, D.L., W.F. Meggitt, and D. Penner. 1976. The interaction
of atrazine and EPTC on navy bean. Weed Sci. 24:5-10.

51

13. wyse, D.L., W.F. Meggitt, and D. Penner. 1976. Herbicide-root-
rot interaction in navy bean. weed Sci. 24:16-21.

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Table 3

Effect of EPTC and R-25788 on the major corn epicuticular
wax component, l-dotriacontanol.

 

 

 

EPTC rate Peak area/cm2

(ks/ha) (Z of control)
R925788
0.0 kg/ha 1.12 kg/ha

0.0 100 cda 106 d
1.68 108 c 84 b
3.36 89 be 78 ab
6.72 67 a 76 a

 

Values with the same letter or letters are not significantly different
at the 5% level using Duncan's Multiple Range Test.

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one on econommun mausmufiwfiowfim uos mum magnum; name some “one: muuuuoa no nouuoa 03mm ecu nous mosao>

 

55

 

 

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one m.H m m.H ~.H o.o NH.H + «n.w mmNmNIm + 09mm
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anxwx om.c a:\wx c.c m:\wx on.o ms\mx o.c
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magmas: enmum musfiofi shoe

 

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wmhmem use 09mm he unavoumm vmuaannImosmmuoBMomon aouu swans“ cu once we coauqnoomfipoum I e manna

56

Figure 1. Scanning electron micrographs of corn leaf surfaces showing
the epicuticular wax aggregation induced by EPTC and its pre-
vention by R-25788. (a) Control 2000x (b) R-25788 (1.12
kg/ha) 2000x (c) EPTC (6.72 kg/ha) 2000x Cd) EPTC (6.72 kg/
ha) + R-25788 (1.12 kg/ha) 2000x

CHAPTER 4

THE IN_VITRO CONJUGATION OF GLUTATHIONE AND OTHER THIOLS
WITH ACETANILIDE HERBICIDES AND EPTC SULFOXIDE
AND THE ACTION OF THE HERBICIDE ANTIDOTE R925788

Abstract

Non-enzymatic reaction in giggg of 3H-la‘beledoglutathione (GSH)
with 14C-alachlor, 14C-metolachlor, 14C-H—22234, and 14C-EPTC sulfoxide
formed dual labeled GSH-herbicide conjugates. GSH did not conjugate in
this system with the herbicides buthidazole, atrazine, EPTC or the herbi-
cide antidote Rr25788. Alachlor also conjugated with the thiol contai-
ning compounds cysteine, dithiothreitol, and coenzyme A but not with
methionine, acetyl CoA, mercaptoethanol, or ethanethiol. The alachlor-
GSH conjugation reaction yielded more product with increasing pH (over
pH 6.0) indicating that the reactive species of 683 is the GS- ion.
Through the GSH-acetanilide conjugation reaction had a low yield at
physiological pH it could be the basis for the protection of corn from
acetanilide herbicide injury by R-25788. Because R-25788 was required to
protect atrazine-susceptible corn from alachlor injury but not from
thiocarbamate injury it is suggested that R-25788 may protect corn from
EPTC injury by increasing the rate of EPTC sulfoxidation followed by
subsequent EPTC sulfoxide-GSH conjugation. R—25788 did not protect gene-

tically atrazine-susceptible corn from atrazine injury, indicating that

R—25788 does nor stimulate glutathionef§rtransferase activity or atrazine
GSH conjugation in corn.

58

59

Introduction

.EEHXEEEQ: non-enzymatic conjugation of glutathione (glutamylcyste-
inyl—glycine) (GSH) with three fungicides was reported by Seigel (17) in
1970. Atrazine (2—chloro-4(ethylamino)-6-(isopropylamino)fgftriazine)-
GSH conjugates have been isolated from sorghum (Sorghum vulgare Pers.)
leaf pieces by Lamoreaux 2E 51. (9), and a glutathioneޤrtransferase that
catalyzes SSH-atrazine conjugation has been identified in corn (Eggnmgzg
L.), sorghum and sugarcane (Saccharum officianarum L.) by Freer and
Swanson (6). GSHޤrtransferases that catalyze the conjugation of GSH
with fluorodifen (pynitrophenylfig:§:§7trifluoro-Z-nitrofiprtolyl ether)
have also been isolated from peas (Pisum sativum L.) and peanuts
(Arachis hypogaea L.) (7). Although Lay and Casida (11) reported a GSH-
Ԥ7transferase from corn roots that catalyzed the conjugation of GSH with
EPTC (Sfethyl dipropylthiocarbamate) sulfoxide, Carringer g£_§l, (2)
disputed the existence of this enzyme and reported that the GSH-EPTC
sulfoxide conjugation proceeded in gi££g_non-enzymatically in buffer only.
Both Lay and Casida (ll), Lay et a1. (12) and Carringer et a1. (3) repor-
ted that the herbicide antidote R925788 (§,§7diallyl-2,2-dichloroaceta-
mide) increased the GSH content of corn and hypothesized that this GSH
increase could cause an increased rate of EPTC detoxification by forming
increased GSH-EPTC sulfoxide conjugation (after initial EPTC sulfoxidation)
and thereby explain the mode of action of this antidote. Leavitt and
Penner (13) have recently reported that R925788 also protects corn from
five acetanilide herbicides as effectively as it protects corn from.EPTC.
The acetanilide herbicide analog, chloroacetamide, readily conjugates non-

enzymatically with certain thiol compounds, including GSH (14, 15).

60

The 633 conjugate of propachlor (2-chlor0fflrisopropylacetanilide) has
also been isolated from corn and a non-enZymatic GSH-propachlor conjur
gation reaction described (10). Preliminary experiments failed to find
a GSHe§7transferase responsible for SSH-acetanilide herbicide conju-
gation; therefore, the objective of this study were to a) characterize
the non-enzymatic conjugation of GSH and other thiols with the three
acetanilide herbicide alachlor (2-chloro-2',6'-diethylfN7(metoxymethyl)
acetanilide), metolachlor (Z-chlorofflr(2-ethyl-6-methylpheny1)-N-(2-
methoxy-l-methylethyl)acetamide), and H-22234'Qfi-cthroacetylfN7(2,6-
diethylphenyl)glycine ethyl ester) and the herbicide derivative EPTC
sulfoxide, and b) to determine whether the mechanism for the protective
action of R-25788 was the same for thiocarbamate and acetanilide herbi-

cide by using a GSHfiSrtransferase deficient inbred corn line.

Material and Methods

Reagents and Equipment.

L-(glycine-2-3H)—g1utahione (specific activity, s.a. 2500mCi/mM)
was purchased from New England Nuclear. Non-labeled GSH, L-cysteine, DL-
dithiothreitol, and Z-mercaptoethanol, were purchased from Sigma Chem
Company. Oxidized glutathione was prepared by bubbling 02 through a
solution of reduced GSH for 30 min. Coenzyme A (lypholized) was purcha-
sed from.Nutritional Biochemicals Company, acetyl CoA from Schwarz/Mann,
and ethanethiol from Eastman Organics. Formulated, technical, and uni-
formly l4--C ring labeled alachlor (s.a. 1.7 mCi/mM) were donated by
Mensanto Corp. Technical and uniformly 14C-ring labeled metolachlor (s.
a. 4.5 mCi/mM) as well as formulated and uniformly 14C-ring labeled atra-

zine (s.a. 2.1 mCi/mM) were donated by CibarGeigy Corp. Technical and

61

carbonyl 14C-labeled H-22234 (s.a. 1.2 mCi/mM) were donated by Hercules
Corp. Formulated butylate (§rethyl-diisobutyl thiocarbamate), formulated
and carbonyl labeled 140 EPTC (s.a. 1.33 mCi/mM) and formulated and tech-
nical R-25788 were donated by Stauffer Chemical Co. Labeled (14C labeled)
buthidazole (3-(5-(1,l-dimethylethyl)-l,3,4-thiadizol-2yl-4-hydroxy-l-
methyl-Z-imidazolidinone) (s.a. 12.7 mCi/mM) was donated by Velsicol
Chem. Corp. 14C-labeled EPTC sulfoxide (s.a. 1.33 mCi/mM) was synthesi-
zed from the 14C carbonyl labeled EPTC by the method of Lay and Casida
(11). All other chemicals used were reagent grade. Buffers were made

in sterilized, de-aerated, distilled water, to an ionic strength of

0.1 M by the method of Cherry (5). Liquid scintillation spectrometry
(lsc) was done by a Packard Tri—CarbR Model 3320 liquid scintillation

14C, and 233 Ra external

spectrometer with separate channels for 3H,
standard. The scintillation cocktail used was Aqueous Counting Scintil-
lantR from.Amersham. Mixtures were lypholized on a VertisR model lypho-
lizer. The thin-layer cromatograph (TLC) system used was: Silica gel
60 or 60 F pre-coated TLC plates (E. MerckR) developed in buthanol:
acetic acid: water 30:10:15, and visualized with either autoradiography
(Kodak No-ScreenR X-Ray film), ninhydrin spray reagent (l6), nitroprus-
side (sodium) spray reagent (20), or dividing the plate into 1 x 2 cm
blocks each block into scintillation vials for lsc.
Reaction of thiols with herbicides.

The reaction between 3H-GSH and 14C-acetanilide herbicide was
studied by adding the following, in the order given, to a 100 x 12 mm
screw top culture tube, mixed, and allowed to react for 3 h under a ni-

trogen atmosphere in a 30 C water bath: 1 m1 phosphate buffer pH 7.0,

100 nmoles 14C-acetanilide herbicide (alachlor, metolachlor, H-22234

62

each diluted to .07 uCi/lOO anwith non-labelled herbicide) in 10 ul
ethanol, 1300 nmoles GSH.in 0.2 m1 phosphate buffer, and 0.4 nmoles 3H-
GSH in 50 ul 0.05 N acetic acid (approx. 1.0 uCi). The reaction was
stopped by freezing the mixtures in a dry-ice acetone bath and then
lypholized. The residue was extracted with 0.5 ml methanol, and 100 ul
samples were applied to the TLC plates, developed, and visualized. These
experiments were also repeated without 3H—GSH. The specific activity of
the 14C-acetanilide herbicide and the 3H-GSH in the reaction mixtures was
approximately equal so that any conjugate formed containing 1 mole of GSH
residue per mole of herbicide residue would have near equal amounts of

3H and 140 dpm. The pH experiments contained 1 m1 of the following buf-
fers: acetate pH 4.6, phosphate pH 6.0, phosphate pH 7.0, phosphate 8.0
or tris-HCl pH 8.6, plus 100 nm 14C-alachlor and 1,000 nm.GSH in one
experiment and 10,000 nm GSH in another. The reaction mixture for the
alachlorbthiol experiments was identical to the acetanilide-GSH experi-
ment except only non-labeled thiols were used at a concentration of 1000
nm per reaction mixture. The reaction between GSH and other herbicides
was studied by substituting the following for the acetanilide herbicides
in the standard reaction mixture: 100 nmoles technical R225788, 100 nm

14 146-, 38 nmoles 14C-EPTC sulfoxide,

C-buthidazole (1.3 u Ci), 38 nmoles
and 23 nm.14C atrazine, all in 10 ul ethanol (except the buthidazol which
was in 20 ul ethanol). Results from all experiments were expressed as
the percent of 140 recovered from the TLC plate as conjugate as compared
to the total amount of 14C recovered per spot.

Plant culture for herbicide-antidote response study.

Atrazine susceptible, glutathioneޤftransferase.deficient corn

inbred GTLLZ (18) was grown in a greenhouse mix soil (1:1:1 sand:peat:

63

soil) in 946 ml waxed cups in a greenhouse supplemented with artificial
lighting (16 h day) with a maximum temperature of 38 C and a minimum.of
30 C. The response of this inbred to the herbicides EPTC, butylate,
alachlor, and atrazine, alone or in combination with R—25788, was measu-
red in three experiments. All herbicides and R925788 were applied pre-
plant-incorporated with 2.1 kg/cm2 pressure in 935 1/ha spray volume
with a link belt sprayer. When the herbicides were applied in combina-
tion with R925788, the herbicides were applied and incorporated first
and the R-25788 applied and incorporated 15 min later. After 4 weeks,
corn heights and dry weights were measured. Only plant heights are re-

ported. The dry weight results were similar.

Results and Discussion

The GSH conjugates of alachlor, metolachlor, and H-22234 were
identified on TLC plates as spots with both 3H and 14C co-chromatograr
phing in near equal relative abundance (Table 1). These spots reacted
‘with ninhydrin (wich reacts with the free amino group in the GSH and
therefore visualize both conjugated and non-conjugated GSH), did not
react with nitroprusside (which reacts with free thiol groups and there-
fore visualizes unconjugates GSH only), and did not co-chromatograph with
any of the original reactants (Table 1). Failure of the dual-labeled
conjugate to react with nitroprusside indicates that the site of conju-
gation was the sulfur of the GSH. The presence of 14C in all three
conjugates indicates that the phenyl ring of the herbicides was mantained
in the conjugate since both alachlor and metolachlor were phenyl labeled.

The carbonyl carbon was also mantained in the conjugate since the

H-22234 was carbonyl labeled. Based on this evidence the proposed

64

structure of the GSH-acetanilide herbicide conjugate was formulated
(Figure 1). Although there were no significant differences between the
amounts of conjugate formed by the three acetanilide herbicides (Table

1), the trend in amounts formed is the opposite of their relative toxi-
city to corn (13) (i.e., alachlor less than metolachlor less than H-22234).

The pH dependence of the alachlor-GSH conjugation reaction can be
seen in Table 2. Except for acetate buffer at pH 4.6, the amount of
conjugate formed inugi££g_increased with increasing pH to almost 1002.
when 10,000 nmoles of GSH were used in tris-HCl buffer pH 8.6. The pK
of the sulfhydryl group of GSH has been reported as 8.66 (I). This means
that the reactive species of GSH is the GS-ion as previously reported
for GSHschloroacetamide conjugation (15). The anamalous behavior in
acetate buffer at pH 4.6 could be the result of a different reaction
mechanism, or the alachlor may be suitable at the low pH.

Alachlor also conjugated with cysteine, dithiothreitol, and co-
enzyme A (Table 3). No appreciable conjugate formation was detected
with methionine, acetyl CoA, mercaptoethanol, or ethanethiol. Although
R-25788 has been reported to increase GSH content of corn, (2, ll, 12),
the autors are unaware of any reports on the effect of R-25788 on the
concentration in corn of other thiols such as cysteine or coenzyme A.

No conjugation product of GSH with R-25788 could be detected
(Table 4). GSH also did not conjugate in yitgngith other chemicals
buthidazole, EPTC, or atrazine. However, GSH conjugated with the EPTC-
sulfoxide with 60.3% of the recoverable 14C found in the conjugate
(Table 4). These results support the conclusion of Carringer et a1.

(2) that EPTC sulfoxide conjugates non-enzymatically with GSH.

Although the physiological significance of the GSH-acetanilide

65

herbicide conjugation is unknown in yigg, it occurs at physiological

pH. The reported stimulation of GSH synthesis by Rr25788 (3, ll, 12),
coupled with GSH-acetanilide conjugation, could explain the protective
action of R-25788 against the acetanilide herbicides in corn. Similar
rationale has been used to explain the protection of corn from EPTC injur
ry (3). The possibility that R225788~cou1d have the same mode of action
in preventing acetanilide and thiocarbamate herbicide injury was investi-
gate by examining the response of inbred corn line, GT112, to both herbi-
cide classes. This inbred is glutathionee§7transferase deficient and
atrazine susceptible (18). As shown in Table 5, the two thiocarbamate
herbicides EPTC and butylate caused no inhibition of growth in this corn
genotype, whereas alachlor and atrazine did. The growth inhibition by
alachlor was prevented by R-25788 but the inhibition by atrazine was not.
In two other genotypes of corn, normal (DeKalb XL 316) and thiocarbamate
susceptible (DeKalb XL 306), atrazine at 6.72 kg/ha did not inhibite
growth (data not presented). Butylate at 3.36 kg/ha inhibited the growth
of the thiocarbamate susceptible corn. EPTC at 6.76 kg/ha and alachlor
at 4.48 and 6.72 kglha inhibited the growth of both genotypes. R925788
prevented injury from all butylate, EPTC and alachlor to both genotypes.
Since the inbred corn line, GT112, responded differently to thiocarbama-
tes and alachlor, the metabolism of these two herbicide groups must
differ. Whatever rendered this genotype thiocarbamate tolerant did not
protect it from alachlor injury, and furthermore the action of R-25788 to
prevent alachlor injury was not required for the prevention of EPTC
injury. Therefore, not only is the metabolism of the two herbicide
classes different, but the protective effect of R-25788 must have a dif-

ferent basis. Differences in GSH conjugation could result from the

66

requirement that EPTC be converted to its sulfoxide prior to GSH
conjugation which is not required for alachlor. Furthermore, EPTC
sulfoxide formed three times as much GSH conjugate in vitae in these
experiments as alachlor, indicating that the GSH content in corn could
be relatively more important for acetanilide detoxication than for

EPTC detoxication. Casida et a1. (4) reported that corn was injured by
EPTC at 3.4 kg/ha but could tolerate EPTC sulfoxide applications of 27
kg/ha without damage. EPTC sulfoxide was more toxic to other plants
than EPTC, however. Thus corn can readily detoxify EPTC sulfoxide
without prior R-25788 treatments to raise the GSH content. Rr25788
could therefore protect corn from EPTC injury only by increasing the
rate of EPTC sulfoxidation. Increased rate of EPTC sulfoxide metabolism
by increased GSH conjugation would only be secondary. Since the aceta-
nilide herbicides do not react as readily with GSH as EPTC sulfoxide nor
require prior activation in order to react with GSH, R-25788 may prevent
acetanilide herbicide injury by simply increasing the GSH content of
corn. The differential response of the GT112 inbred corn line to both
herbicides could be explained by ease of GT112 sulfoxidation of EPTC,
which then reacts with the natural GSH levels; GSH levels, however, not
high enough to protect the genotype from alachlor unless R925788 raises
them. Failure of R925788 to protect this inbred corn line from atrazine
injury indicates that R925788 does not stimulate glutathione-§7transfe-

rase activity or the rate of atrazine-GSH conjugation in corn.

10.

ll.

67

LITERATURE CITED

Boyland, E. and L.E. Chasseaud. 1969. The role of glutathione
and glutathionee§7transferases in mercapturic acid biosyn-
thesis. Adv. in Enzymology 32:173-219.

Carringer, R.D., C.E. Rieck and L.P. Bush. 1978. Effect of
R225788 on EPTC metabolism in corn (Zea mays L.). Weed Sci.
26:167-171.

Carringer, R.D., C.E. Rieck, and L.P. Bush. 1978. Metabolism of
EPTC in corn (Zea mays L.). weed Sci. 26:157-160.

Casida, J.P., R.A. Gray, and H. Tilles. 1974. Thiocarbamate sul-
foxides: potent, selective, and biodegradable herbicides.
Science 184:503-574.

Cherry, J.E. 1973. Molecular biology of plants. Columbia Univ.
Press. New York and London.

Frear, D.S. and H.R. Swanson. 1970. Biosynthesis of S-(4-ethyl—
amino-6-isopropylamino-Zigftriazino) glutathione: partial
purification and properties of a glutathioneegrtransferase
from corn. Phytochem. 9:2123-2132.

Frear, D.S. and H.R. Swanson. 1973. Metabolism of substituted
diphenyl ether herbicides in plants. L. Enzymatic cleavage
of fluorodifen in peas (Pisum sativum.L.) Pestic. Biochem.
Physiol. 3:473-482.

Jaworski, E.G. 1956. Biochemical action of CDAA, a new herbicide.
Science 123:847-848.

Lamoreux, G.L., R.E. Shimabukuro, H.R. Swanson, and D.S. Frear.
1970. Metabolism of 2-chloro-4-ethylamino-6-isopropylamino-
§7triazine (atrazine) in excised sorghum leaf sections. J.
Agr. Food Chem. 18:81-86.

Lamoreux, G.L., L.E. Stafford, and F.S. Tanaka. 1971. Metabolism
of 2-chlor05N7isopropylacetanilide (propachlor) in the leaves
of corn, sorghum, sugarcane, and barley. J. Agr. Food Chem.
19:346-350.

Lay, M.M. and J.E. Casida. 1976. Dichloroacetamide antidotes
enhance thiocarbamate sulfoxide detoxification by elevating
corn root glutathione content and glutathioneޤftransferase
activity. Pest. Biochem. Physiol. 6:442-456.

12.

l3.

14.

16.

17.

18.

19.

20.

68

Lay, M.M., J.P. Hubbell, and J.E. Casida. 1975. Dichloroacetamide
antidotes for thiocarbamate herbicides: mode of action.
Science 189:287-289.

Leavitt, J.R.C. and D.Penner. 1978. Protection of corn (Zea mays
L.) from acetalinide herbicide injury with the antidote
R-25788. weed Sci. (In press).

Lindley, H. 1960. A study of the kinetics of the reaction between
thiol compounds and chloroacetamide. Biochem. J. 74:577-584.

Lindley, H. 1962. The reaction of thiol compounds and chloroaceta-
mide. Biochem. J. 82:418-425.

Patton, A.R. and P. Chism. 1951. Anal. Chem. 23:1863.

Siegel, M.R. 1970. Reactions of certain trichloromethyl sulfenyl
fungicides with low molecular weigh thiols in vitro studies
with glutathione. J. Agr. Food Chem. 18:819-822.

Shimabukuro, R.E., D.S. Frear, H.R. Swanson, and W.C. welsh.
1971. Glutathione conjugation, an enzymatic basis for atra-
zine resistance in corn. Plan Physiol. 47:10-14.

Shimabukuro, R.E., G.L. Lamoreux, H.R. Swanson, W.C. Welsh, L.E.
Stafford, and D.S. Frear. 1973. Metabolism of substituted
diphenylether herbicides in plants. II. Identification of a
new fluorodifen metabolite, S-(2-nitro-4-trifluoromethylphe-
nyl)-glutathione in peanut. Pestic. Biochem. Physiol.
3:483-494.

Toennies, G. and J.J. Kolb. 1951. Anal. Chem. 23:823.

69

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cmmumIm I I oom.H~ IIIIII on. I «m
.maoo smNNNImImu IIII I + ooH.H ooN.H as. I we
emu + + IIIIII ooo.a~ on. I «a
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Emulmm + «MNNNISIQQH
.numaouws I I con.na IIIIII on. I ma
.nsoo.aoomsImo IIII I + cow ooa.H he. I am
mmo + + IIIIII oom.mH cm. I mm
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I HO Um Oqul
emu mm + a; H use
HOHSUNHM I l OO¢¢¢ llllll who " MM
.ncou.auIHmImu IIII I + ooa.a ooA.H as. I «a
:mo + + IIIIII ooH.mH cm. I mm
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emu mm + H: H usH
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swocHN .umaoucoIoo :uHB zxm 02mm m

:mo mo :OHuommm I H wanna

70 .

Figure l - Structure of GS-acetanilide herbicide conjugates.

71

O O O

0

2

II II II II
HO-C-CH-CHZ-C-NH-CH-C-NH-CH -C-OH
I I

NH2 932

8

C-0

I
N-
, R1
CH3-CH2-.—R2
for GS-alachlor conjugate

for GS—metolachlor conjugate

for GS-H-22234 conjugate

Rl- ca3-o-cnz-

R2- 033-ca2- CH3

I

Rl- CH3-0-CH2-CH--

R2" CH3" 0
II

Rl- CH3-CH2-C-CH2--

Rz- CH3-CH2--

72

 

Table 2 - The pH dependence of the GSH-alachlor conjugation reaction.
pH 1,000 nm GSH 10,000 nm.GSH
4.6 18.5 b1 43.6 b
6.0 7.4 a ' 8.9 a
7.0 17.0 ab 38.5 b
8.0 ' 26.4 b 85.5 c
8.6 76.5 c 99.1 d

 

1Means within columns followed by a common letter or letters are not
significantly different at the 52 level as judged by the Duncan's
Multiple Range Test.

73

Table 3 - Reaction of alachlor with other thiols.

 

Z of extractible 14C

Reactants Rf of conjugate in conjugate
14C-alachlor + Cysteine 0.51 11.72 a2

+ Dithiothreitol 0.35 9.12 a

+ Coenzyme A 0.36 3.01 a

+ Methionine N.R.3 --

+ Acetyl CoA N.R. --

+ Mercaptoethanol N.R. --

+ Ethane thiol N.R. --

 

lTLC system used: Silica gel 60F, E. Merck, in BuOh, AcOh, H20 30:10:15
2Means followed by the same letter or letters are not significantly dif-
ferent at the 52 level as judged by Duncan's Multiple Range Test.

3N.R. - no reaction

74

Table 4 - Reaction of GSH with Various herbicides.

l4

Reactions Rf of Conjugate1 Z of extractable C

in conjugate

 

3

H-GSH + R-25788 N.R. -
+ 14C-Buthidazole N.R. I --
+ l4C-EPTC N.R. --
14
+ C-EPTC s=0 0.38 60.3:
+ lac-Atrazine N.R. --

 

lTLC system: Silica gel 60F, E. Merck: BuOh, AcOh, 320 30:10:15

75

Table 5 - Response of atrazine suscrptible, GSH-S-transferase deficient
corn inbred line GT-112 to butylate, EPTC, alachlor, and
atrazine in three experiments.

 

 

 

Exp. Herbicide (rate) Corn Height
(kg/ha) R—25788
0.0fikglha 1.12 kg/ha
(mm) (cm)
(1) Control 21.7 c1 23.7 c
Butylate (3.36) 23.4 c 22.1 c
Alachlor (4.48) 13.9 b 23.5 c
Atrazine (6.72) 6.9 a 8.9 a
(2) Control 41.0 b 34.5 b
EPTC (6.72) 39.0 b 36.6 b
Atrazine (6.72) 14.0 a 17.4 a
(3) Control 26.2 b 24.2 ab
Alachlor (6.72) 20.8 a 26.3 b
1

Means within experiments followed by a common letter or letters are not
significantly different at the 52 level according to Duncan's Multiple
Range Test.

CHAPTER 5

Summary

The herbicide antidote R-25788 protects corn from herbicide
injury by either acting as a competitive inhibitor to the herbicide, by
increasing the rate of herbicide detoxification by glutathione conjuga-
tion, by increasing the rate of thiocarbamate sulfoxidation, or by some
combination of these hypothesis. R-25788 does not protect corn by
preventing herbicide induced inhibitions of gibberellin synthesis or
lipid synthesis in corn. The competitive inhibitor hypothesis is sup-
ported by the observation that the antidote is similar in structure to
both the acetanilide and thiocarbamate herbicides and protects corn from
both. The hypothesis that R-25788 could protect corn by increasing the
rate of glutathione-herbicide conjugation is supported by the rapid
conjugation reaction that occures between glutathione and aCetanilide
herbicides or thiocarbamate sulfoxides in 31552,
However, since R-25788 was required to protect atrazine-susceptible corn
from alachlor injury but not from thiocarbamate injury, the mode of action
of R-25788 could differ between the acetanilide and the thiocarbamate
herbicides. It is therefore suggested that R—25788 might increase the
rate of sulfoxidation of thiocarbamates to their non-phytotoxic sulfo-
xide derivates. The driving force responsible for increased thiocarbama-
te sulfoxidation, however, could be increased thiocarbamate sulfoxide-

glutathione conjugation.

76

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10.

11.

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APPENDICES

81

Appendix A - Structures of one thiocarbamate and five acetanilide
herbicides referred to in the text.

82

'0'
CH
EPTC °"3""°"2 -S- C - N ’ 2
\CH2 —CH2—CI‘I3

'0' NC" — o—CI'I3
ALACl-ILOR CI—cu2 — c— N<
cuzcu3
.. e?
“3
II ’:u -CI-I2— O III-CH3
METOLACHLOR CI -CH2- C — N \ CH3

CH26H3’ 0

fl /cu2cH —CII2 — o—CH3
ACETOCHLOR CI—cuz—c—I N\

N’Cflz — IC' IIIICI-l2 III-CH

H “22234 Cll-ICI'I2 - '6' _N H 23c" 3
CH2CH3
CH
3
II WCHZ :0; 0

CH3
CH26H3

83

Appendix B - Structures of five compounds screened as potential
antidotes to acetanilide herbicide injury to corn.

R- 25788

R- 29148

CDAA

CARBOXIN

NA

84

H o

I ll ’Cflz—CH2=CH2
Cl—C-C —N

“Ma-cu2 =cu

Cl 2
OH
H 0 / 3
I “ }"2‘ c .4,
Cl— C - C - N I
I. \ 0
Cl (0"
° 3 CH3
H

0
Cl- C - C - N .-
.I' \Cflz-CHZ - CH2

”'I’I'I'ITIIIII

 

TATE

293

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