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This is to certify that the
thesis entitled

Efficacy of Herbicide Safeners in
Corn (Zgg_maxs L.) and Grain Sorghum (Sorghum bicolor L.)

 

presented by

Marulak Simarmata

has been accepted towards fulfillment
of the requirements for

M.S. degree in C53

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Major professor

 

Date 03/06/92

 

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emana-pt

 

EFFICACY OF HERBICIDE SAFENERS
IN CORN [Zea mays L.] AND

GRAIN SORGHUM [Sorghum bicolor (L.) Moench]

By

Marulak Simarmata

A THESIS

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

MASTER OF SCIENCE

Department of Crop and Soil Sciences

1992

ABSTRACT
EFFICACY OP HERBICIDE BAFBNBRB

IN CORN [Zea mays 13.] AND
GRAIN SORGHUM [Sbrghum bicolor tn.) Noench]

BY

Narulak Binarmata

The efficacy of several commercial herbicide safeners was
evaluated in greenhouse and laboratory studies for protecting
corn and sorghum against injury from primisulfuron and
metolachlor. Protection by the safeners against corn injury
from primisulfuron plus terbufos insecticide and primisulfuron
plus antioxidants, piperonyl butoxide and metyrapone, was also
evaluated.

Significant protection against corn injury from
metolachlor and from primisulfuron interaction with terbufos
was observed with oxabetrinil, f lurazole , naphthalic
anhydride, dichlormid, and R-29148. Seed treatment with CGA-
133205 killed the corn. None of the safeners provided
protection against primisulfuron injury to sorghum, whereas
all provided protection against metolachlor injury to sorghum.

Antioxidants, piperonyl butoxide (PPO) and metyrapone,
increased primisulfuron injury to corn. Protection against
the increased corn injury from primisulfuron plus PPO was most
evident with naphthalic anhydride, f lurazole, and R-29148. No

protection was observed to the increased corn injury from

Marulak Simarmata
primisulfuron plus metyrapone. Naphthalic anhydride was also
an effective safener for PPO enhanced primisulfuron injury
when the PPO was applied foliarly 3 days prior to
primisulfuron application.

Absorption of 14C-primisulfuron by corn was not influenced
by PPO or naphthalic anhydride. Metabolism of 1“C-
primisulfuron was inhibited by PPO and.this inhibition was not
totally reversed by the safener, naphthalic anhydride.

These results suggest that safeners oxabetrinil,
flurazole, naphthalic anhydrid, dichlormid, and R-29148 were
effective in protecting corn against the injury from
metolachlor and the injury from primisulfuron interaction with
terbufos, and grain sorghum from metolachlor injury.
Naphthalic anhydride, flurazole, and R-29148 were effective
against the increased synergist primisulfuron injury induced
by PPO. The increased corn injury from primisulfuron plus PPO
was due to the inhibition of the primisulfuron metabolism.
The safening action of naphthalic anhydride did not totally
reverse this inhibition. Nomenclature: corn, [Zea mays L.];
sorghum, [Sorghum.bicolor (L.) Moench]; primisulfuron, 2-[3-
[4,6-bis(dif1uoromethoxy)-pyrimidin-2-yl]-ureidosulfonyl]-
benzoic acid methylester; metolachlor, 2-chloro-N-(2-ethy1-6-
methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide; terbufos,
S-[[(1,1-dimethylethyl)thio]methyl] 0,0-
diethylphosphorodithioate; piperonyl butoxide, a-[2-(2-

butoxyethoxyj ethoxy]-—4, 5-methylenedioxy-z-propyltoluene;

Marulak Simarmata
metyrapone, 2-methyl-1,2-di-3-pyridyl-1-propanone;
o:xaat>e‘t1:i.n ill, a- :( 1 ,23-d ic:x:o lair1-’2-yrl'-
methoxy) imino]benzeneacetonitrile; f lurazole, phenylmethyl 2-
chloro-4-(trifluoromethyl)-5-thiazolecarboxylate; naphthalic
anhydride, naphthalene-1,8-dicarboxylic acid anhydride;
dichlormid, 2 , 2-dichloro-N,N-di-Z-propenylacetamide,° R-29148 ,
3-(dichloroacetyl)-2,2,5-trimethyl-1,3-oxazolidine; CGA-
133205, 0-(1,3-dioxolan-2-yl-methyl)-2,2,2-trif1uoro-4’-

chloroacetophenone-oxime.

ACKNOWLEDGMENTS

I would like to express sincere gratitute to my major
professor, Dr. Donald Penner, for his patience, guidance, and
technical expertise throughout the entire project. I would
also like to express appreciation to my committee members, Dr.
Jim Kells and Dr. Bernard H. Zandstra, for their help in
reviewing this manuscript.

A special thanks is expressed to Frank C. Roggenbuck for
his assistance from the beginning of the research through the
completion of this thesis.

Finally, I extend appreciation to my wife, Estomihi, for

her patience and support through the completion of my study.

TABLE OF CONTENTS

PAGE

LISTOFTABLES..00.0000.00000000..00......0000000.00000Viii

INTRODUCTION

CHAPTER 1.

CHAPTER 2.

CHAPTER 3.

0.....000000...0. 0000000000 0000000....0... 1

REVIEW OF LITERATURE

Introduction .............................. 4
History of herbicide safeners ............. 5
Naphthalic Anhydride ....................... 6
Dichlormid ................................ 8
R-29148 .................................... 9
Cyometrinil, Oxabetrinil, CGA-133205 ...... 10
Flurazole ................................. 12
Summary ................................... 13
Bibliography .............................. 14

EVALUATION AND COMPARISON OF HERBICIDE
SAFENERS IN CORN [Zea mays L.) and GRAIN
SORGHUM [Sorghum bicolor (L.) Moench]

Abstract .................................. 19
Introduction ............................... 21
Materials and Methods ...................... 22
Results and Discussion ..................... 24
Summary .......... ........ .................. 25
Bibliography ............................... 26

INCREASED PRIMISULFURON INJURY TO CORN
(Zea mays L.) WITH ANTIOXIDANTS AND
PROTECTION WITH HERBICIDE SAFENERS

Abstract .................................. 31
Introduction ............................... 33
Materials and.Methods ..................... 35
Results and Discussion .................... 39
Summary ................................... 40
Bibliography ........... ...... ............. 41

vi

APPENDIX

A.

vii

Page

Chemical structure of naphthalic anhydride,
dichlormid, R-29148, oxabetrinil, CGA-133205,
andfluraZOIe000.000000000......000.0000000 45

Chemical structure of metolachlor and
primisulfuron ...................... ...... . 47

CHAPTER 2.

CHAPTER 3.

TABLE

10

LIST OF TABLES

PAGE

Protection of corn seedlings with
herbicide safeners from primisulfuron-
terbufos interaction injury.............. 27

Protection of corn seedlings with
herbicide safeners from metolachlor-
terbUfOSinjury00000.00000.0.00...0..... 28

Protection of sorghum seedlings with
herbicide safeners from primisulfuron-
terbufos injury .. ...... ............ ..... 29

Protection of sorghum seedlings with
herbicide safeners from metolachlor-
terbufos injury ... ....... ..... ......... . 30

Efficacy of herbicide safeners against
the increased injury from primisulfuron
plusantioxidants........................ 42

Efficacy of naphthalic anhydride against
increased primisulfuron injury to corn
fromPPOeeeoeoeeeooeeeeeoooooooeeooeeeee 43

Absorption and metabolism of
l4C-primisulfuron in corn seedlings in

the presence of the safeners, naphthalic
anhydride, and the antioxidant, PPO ..... 44

viii

INTRODUCTION

Application of herbicides in combination with other
pesticides is one of the management practices in crop
production that allows for control of a broad spectrum of
pests with a single application. Herbicides are commonly
applied before, simultaneously with, or after the use of
other pesticides. Unwanted interaction among pesticides,
such as an interaction between herbicides and insecticides
can occur. Certain interactions between herbicides and
insecticides may increase injury to crops and reduce
herbicide selectivity. One way to alleviate this problem is
to use chemicals, known as 'herbicide antidotes', ’herbicide
safeners' , 'herbicide protectants’ , or ’herbicide
antagonists’. These chemicals can selectively protect crops
from herbicide injury without protecting weeds.

The use of herbicide safeners has developed rapidly
since Hoffmann (21) introduced the concept of herbicide
safeners about three decades ago. 'There are numerous
currently marketed crop safeners for herbicides, such as

naphthalic anhydride (naphthalene-1,8-dicarboxylic acid

anhydride), dichlormid (2,2-dichloro-N,N-di-2-
propenylacetamide), R-29148 (3-[dich10roacetyl]-2,2,5-
trimetyl-l,3-oxazolidine), cyometrinil ([2]-

1

2

a[(cyanomethoxy)imino] benzeneacetonitrile), oxabetrini1.(a-
[(1,3-dioxolan-2-yl-methoxy)imino]benzeneacetonitrile),CGA-
133205 (O-[1,3-dioxolan-2-yl-methy1]-2,2,2-trifluoro-4’-
chloroacetophenone-oxime), GSA-154281 (4-[dichloroacetyl]-
3,4-dihydro-3-methyl-2H-1,4-benzoxazine), flurazole
(phenylmethyl 2-chloro-4-[trifluoromethle-S-
thiazolecarboxylate), fenclorim (4,6-dichloro-2-phenyl-
pyrimidine), MG-191 (2-dichloromethyl-2-methyl-1,3-
dioxolane), and others. The mechanism of action of the
herbicide safeners against herbicidal injury on crops is not
clearly understood.

The objectives of this research were: (a) to evaluate
and compare the potency of herbicide safeners in protecting
corn [Zea mays L.] and grain sorghum [Sorghum bicolor (L.)
Moench] from injuries caused by metolachlor (2-chloro-N-[2-
ethyl-G-methylphenyIJ-N-[2-methoxy-1-methylethyl]acetamide),
primisulfuron (2-[3-[4,6-bis(difluoromethoxy)-pyrimidin-2-
yl]-ureidosulfonyl]-benzoic acid methylester), and the
interaction with 'terbufos (S-[[(1,1-
dimethylethyl)thio]methyl]0,0-diethyl phosphorodithioate)
insecticide; (b) to determine whether the safening action of
herbicide safeners on corn was related to mixed function
oxidase (MFG) activity by’ using the MFO inhibitors or
antioxidants, piperonyl butoxide (a-[2-(2-
butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-propyltoluene) and

metyrapone (2-methyl-1,2-di-3-pyridy1-1-propanone); (c) to

3
study absorption and metabolism of 14C- primisulfuron
herbicide in the presence of the safener, naphthalic
anhydride, and the antioxidant, piperonyl butoxide (PPO), an

MFO inhibitor.

CHAPTER 1

REVIEW OF LITERATURE

0 on

The discovery and development of organic herbicides has
revolutionized weed management in crop production.
Selectivity obtained with organic herbicides has resulted in
their widespread use in modern agriculture. But, the limited
selectivity of certain herbicides has been a problem in
controlling weeds that are botanically related to the crops.

Several approaches to overcome this problem have been
reviewed by Hatzios (15). They involve development of new
herbicides that are more selective than those currently
available and enhancement of crop tolerance mechanically,
genetically, or chemically. The chemical approach is based
on the use of chemicals known as safeners, antidotes,
protectants, or herbicide antagonists. Hoffmann (21)
introduced this idea in 1962 and proposed that the use of
safeners could permit: the use of higher rates of
herbicides; the use of nonselective herbicides for selective
weed control; the use of herbicides under conditions where
crop damage is liable to occur; the protection of crops

accidentally treated with nonselective herbicides. In

5
addition, safeners could be a potential tool for elucidating

sites and mechanism of herbicidal action (15).

History of herbicide safeners

Activated carbon (activated charcoal) was the first
safener used directly on crop plants to protect them from
herbicide injury (2). It has great adsorptive capacity and
acts as a physical barrier to herbicide uptake. However,
activated charcoal has some limitations. Coating seed with
charcoal does not protect the emerging shoot from herbicide
injury (14), placing charcoal in rows results in ‘weed
protection (19), and large amounts of charcoal are required
to obtain the desired protection (1).

The use of chemicals to protect crops from herbicide
injury' is a ‘relatively’ new approach to selective ‘weed
control. It started in 1947 when tomatoes [Lycopersicon
esculentum Mill.] tested for their response.to 2,4-D analogs
were exposed accidentally to ‘the fumes of 2,4-D (2,4-
dichlorophenoxyacetic acid) and the epinastic responses were
absent (20). Observed antagonistic responses that led to
commercial products were 2,4,6-T (2,4,6-
trichlorophenoxyacetic acid) antagonism of 2,4-D on tomato
and the total inactivation of barban (4-chloro-2- butynyl-3-
chlorophenylcarbamate) by 2,4-D on wheat [Tritium aestivum
L.] (15) .

Based on this antagonistic herbicide interaction,

6

Hoffmann (21) evaluated several chemicals and introduced S-
449 (4-chloro-2-hydroxyiminoacetanilide) in 1962 as an
effective safener of wheat against injury from the herbicide
barban. Hoffmann (22) was also successful in the discovery
and development of naphthalic anhydride as an effective
safener in corn against injury from thiocarbamate herbicides.

The efforts to develop new safeners have continued and
resulted in several commercial safeners. Among them,
chloroacetamide safeners of corn against thiocarbamate and
chloroacetanilide herbicides have been most successful (31).
Other safener groups that are also important are oxime ether
derivatives (8, 10, 37) and disubstituted thiazole
carboxylates (38) . Both of them protect sorghum from
chloroacetanilide herbicide injury. More recently, fenclorim
was introduced as an effective safener of rice [Oriza sativa
L.] against injury from metolachlor and pretilachlor (2-
chloro-2’,6’-diethyl-N-[2-propoxyethyl]acetanilide) herbicide
(9). In 1988, GSA-154281 was also introduced as a new
safener to protect herbicidal injury of metolachlor to corn
(35). The following review will describe the uses, proposed
mode of action, and chemical structures of safeners that were

used in this research.

Naphthalic anhydride
Naphthalic anhydride was patented in 1971 as a herbicide

safener to ;protect corn, against EPTC (S-ethyl dipropyl

7
carbamothioate) injury (23). Naphthalic anhydride is
moderately effective as a seed treatment safener for a number
of chemical 1y unrelated herbicides , such as
phenylcarbamates, dithiocarbamates, chloroacetanilides,
sulfonylureas, imidazolinones, cyclohexenones, and
arylophenoxyalkanoic acids (15, 33) in primarily
monocotyledoneous crops such as corn, grain sorghum, oats
[Avena sativa L.], wheat, barley [Hordeum vulgare L.], and
rice (40).

The mechanism of action of naphthalic anhydride has not
been clearly defined. Naphthalic anhydride was found to
enhance the rate of EPTC metabolism and it was postulated
that naphthalic anhydride conferred protection by activating
enzymes involved in EPTC detoxication (13).

In both corn and sorghum, chloroacetanilide herbicides,
such as metolachlor, were detoxified by naphthalic anhydride
by conjugation with the tripeptide glutathione (GSH) (13).
Another report showed that the safener, naphthalic anhydride,
conferred protection against metolachlor injury by inducing
the de novo synthesis of GST-isozymes which catalyzed the
detoxication of the herbicides (7).

Seed treatment of corn with naphthalic anhydride did not
affect the activity of sulfonylurea and imazaquin (2-[4,5-
dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-
quinolinecarboxylic acid) herbicides which inhibit

acetolactate synthase (ALS) enzymes (3, 28). For imazethapyr

8
(2-[4,5-dihydro-4-methyl-4-(l-methylethyl)-5-oxo-1H-imidazol-
2-yl]-5-ethyl-3-pyridinecarboxylic acid), naphthalic
anhydride stimulated mixed function oxidase (MFO) activity
that can rapidly metabolize imazethapyr to hydroxy-
imazethapyr. The naphthalic anhydride safening effect can be
antagonized by the MFO inhibitor aminobenzotriazole (ABT)

(39).

Aisle—mid

The safener dichlormid was introduced commercially in
1973 as an antidote for EPTC injury to corn (32). Several
researchers reported that dichlormid also protected corn
seedlings from, injury’ caused. by (acetanilides (26, 43),
chlorsulfuron (2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-
triazin-2-yl)amino]carbonyl] benzenesulfonamide) (34), and
imazaquin.herbicides (4). In‘addition.to corn, grain sorghum
has also been protected by dichlormid from thiocarbamate and
acetanilide (12, 43) and from imazaquin herbicides (4).

Unlike naphthalic anhydride, which was ineffective at
low rates or nonselective at high rates as a soil applied
safener, dichlormid could be added to the EPTC formulations
and applied preplant incorporated. Dichlormid was
physiologically selective for preventing EPTC injury to corn
without reducing EPTC effectiveness for weed control (40).
Leavitt and Penner (26) reported that dichlormid was an

effective safener whether applied preemergence, preplant

9
incorporated, or as a tank mixture.

There has been, and still is, much controversy
surrounding the mode of action.of dichlormid.as tO‘whether it
acts at a site of herbicide action or protection is the
result of enhanced herbicide metabolism (11). Early studies
indicate that it did not act by inhibiting the uptake or
translocation of EPTC in plants (6). Lay and Casida (25)
suggested that the mechanism of action of dichlormid involved
an increase in the rate.of detoxication of the herbicide, via
conjugation to glutathione. They demonstrated that corn
pretreated with dichlormid had higher GSH levels, GST
activity, and metabolized EPTC sulfoxide faster than
nontreated plants. They proposed that dichlormid.protection
was the result of enhanced EPTC metabolism through elevation
in GSH levels and GST activity. Leavitt and Penner (27)
confirmed the EPTC sulfoxide conjugation to GSH, and proposed
that dichlormid acted by stimulating the formation of the

EPTC sulfoxide in corn.

R-29148
The safener R-29148 was first discovered in 1971 as a
safener for protecting corn from thiocarbamate injury (42).
It is normally incorporated into the soil applied as a tank
mixture with EPTC, butylate (S-ethyl bis[2-
methylpropyl]carbamothioate) or vernolate (S-propyl

dipropylcarbamothioate) . It is also effective as a seed

10
treatment. The safener R-29148 provided partial protection
to corn from the phytotoxic effects of chlorimuron (2-[ [ [ [ (4-
chloro-6-methoxy-2-pyrimidinyl) amino] carbonyl] amino]
sulfonyl] benzoic acid) and sulfometuron (2-[[[[(4,6-
dimethyl-Z-pyrimidinyl)amino]carbonyl] amino]sulfony1]benzoic
acid) (46). Currently, there is no published data concerning

the mode of action of R-29148.

Cyometrinil. Oxabetrinil, GOA-133305
Cyometrinil was marketed as CONCEP I in 1978,

oxabetrinil was marketed.as CONCEP II in 1982, and.CGA-133205
was marketed as CONCEP III in 1986. All of them are used as
herbicide safeners against injury from metolachlor and other
acetanilide herbicides in grain sorghum (17). They are
effective when applied as a seed treatment. When applied as
a tank mixture, they may also provide some protection to
several weeds of the genus sorghum. Turner et a1. (41)
reported the adverse effects caused by cyometrinil on the
seed germination of grain sorghum. As a result, CIBA-GEIGY
developed a chemical analog, oxabetrinil. But, there was an
undesirable interaction of oxabetrinil with downy mildew. To
solve this problem CIBA-GEIGY introduced GOA-133205 which is
expected to replace oxabetrinil in grain sorghum markets (8) .
Yenne and Hatzios (44) reported that metolachlor with seed-
applied CGA-133205 had no effect on sorghum germination,

while metolachlor with seed applied oxabetrinil significantly

11
reduced germination of grain sorghum.

The mechanism by which these safeners confer protection
are not clearly known, but several hypothesis have been
advanced. In an early study, Winkle et a1. (43) reported
that cyometrinil did not affect the metabolism of metolachlor
in sorghum seedlings. But, recent studies showed that
cyometrinil and oxabetrinil seed treatment caused accelerated
metolachlor metabolism in sorghum shoots, decreased
metolachlor content, and increased formation of the
glutathione conjugate (12). Zama and Hatzios (45) reported
that oxabetrinil enhanced metolachlor metabolism in grain
sorghum seedlings. It has also been proposed that
oxabetrinil promoted nonenzymatic GSH conjugation of
chloroacetanilides by a direct activation of the reaction.
However, Gronwald et a1. (13) were unable to confirm the
activation of nonenzymatic conjugation by oxabetrinil.

Unlike cyometrinil and oxabetrinil, the studies about
GOA-133205 are still limited. Moreland and Corbin (29)
proposed that grain sorghum tissue from CGA-133205 treated
seed.metabolized.metolachlor more extensively than untreated
seed. But, they have not yet separated the contribution to
the enhanced metabolism of metolachlor by the glutathione
system from those of the monooxygenase system in the safener

enhanced seedlings.

12
My;

Flurazole was discovered by MONSANTO in 1980 and
marketed under the trade name SCREEN. Flurazole is a seed
treatment protectant which safens grain sorghum and corn
seedlings from injury due to alachlor (2-chloro-N-[2,6-
diethylphenyl] -N- [methoxymethyl ] acetamide) , metolachlor , and
other chloroacetanilide herbicides (5, 38). Schafer (38)
reported that flurazole could be effective when applied in
furrow, but because it affords protection to weed species of
the genus sorghum, flurazole is marketed as a seed treatment
for sorghum seeds. Hatzios (16) applied flurazole as a
preplant incorporated treatment, but it was not very
effective in protecting corn against thiocarbamate and
chloroacetanilide herbicides.

The mechanism of the safening effect of flurazole is
poorly understood (5). Mozer et a1. (30) showed that
flurazole caused an increased in GST activity in corn
seedlings. Flurazole has also been reported to enhance the
level of GSH in grain sorghum (36). Breaux and his coworkers
(5) confirmed these two reports. They reported that
flurazole did not detectably change the metabolic pathway
that was responsible for detoxication of acetochlor (2-
chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-
phenyl]acetamide), but it did alter the rate of metabolism.
They also stated that acetochlor was converted to» GSH

conjugates in both safened and unsafened corn and grain

13
sorghum seedlings, but the ‘use of flurazole caused an
increase in both GSH levels and GST activity which catalyzed

the conjugation of acetachlor with GSH.

Summar

Currently, there are numerous herbicide safeners
available in the market. Research on the action of these
safeners can assist the process of discovering and developing
new herbicides. The herbicide safeners may allow cheaper
herbicides to replace the more expensive one for the control
of similar weed problem.

In a previous review (15) , several reports have proposed
mechanism of actions for herbicide safeners on various crops
and herbicides. But, their protective actions are not
totally understood. Further investigations to understand how
the herbicide safeners provide protection to crops have been

suggested by Hatzios and Hoagland in 1989 (18).

BIBLIOGRAPHY

Andersen, A.H. 1968. The inactivation of simazine and
linuron in soil by charcoal. Weed Res. 8:58-60.

Arle, H.F., O.A. Leonard, and V.C. Harris. 1948.
Inactivation of 2,4-D on sweet potato slips with
activated carbon. Science 107:247-248.

Barrett, M. 1989. Reduction of imazaquin injury to
corn (Zea mays) and sorghum (Sorghum bicolor) with
antidotes. Weed Sci. 37:34-41.

Barrett, M. and G.L. Olson. 1986. Protection from
imazaquin injury: studies on the mode of action of
herbicide protectants. IUPAC. Int. Cong. Pestic.
Chem., 6th. Abstr. No. 3D-14.

Breaux, E.J., M.A. Hobler, J.E. Patanella, and G.A.
Leyes. 1989. Mechanism of thiazole safeners, in: K.K.
Hatzios and R.E. Hoagland (eds.). Crop Safeners for
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pp. 163-175. Academic Press, New York.

Chang, F.Y., G.R. Stephenson, and J.D. Banden. 1974.
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seedlings. J. Agr. Food Chem. 22:245-248.

Dean, J.V., J.W. Gronwald, and C.V. Eberlein. 1990.
Induction of glutathione S-transferase isozymes in
sorghum by herbicide antidotes. Plant Physiol. 92:467-
473.

Dill, T.R. 1986. CGA 133205, A new seed applied
safener for sorghum. Proc. South. Weed Sci. Soc.
39:521.

Ebert, E., H.R. Gerber. 1989. Differential effects of
oxabetrinil and fenclorim against metolachlor and
pretilachlor injury on various grasses, in: K.K. Hatzios
and R.E. Hoagland (eds.). Crop Safeners for Herbicides:
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15

Ellis, J.F., J.W. Peek, J. Boehle, Jr., and G.
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Ezra, G. and G.R. Stephenson. 1989. Mechanism of
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Fuerst, E.P. and J.W. Gronwald. 1986. Induction of
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Sci. 34:354-361.

Gronwald, J.W., E.P. Fuerst, C.V. Eberlein, and M.A.
Egli. 1987. Effects of herbicide antidotes on
glutathione content and glutathione S-transferase
activity of sorghum shoots. Pest. Biochem. and Physiol.
29:66-76.

Gupta, O.P. and N.K. Niranwal. 1976. Increasing
herbicide selectivity in maize and cowpeas by seed
treatment with activated carbon and NA. PANS. 22:86-89.

Hatzios, K.K. 1983. Herbicide antidotes: development,
chemistry, and mode of action. .Adv. Agron. 36:265-316.

Hatzios, K.K. 1986. Interaction of the safener
flurazole with chloroacetanilide and thiocarbamate
herbicides on maize (Zea mays L.). Can. J. Plant. Sci.
66:353-359.

Hatzios, K.Kg 1989. Development of herbicide safeners:
Industrial and university perspective, in: K.K. Hatzios
and R.E. Hoagland (eds.). Crop Safeners for Herbicides:
Development, Uses, and Mechanism of Action. pp. 3-45.
Academic Press. New York.

Hatzios, K.K. and R.E. Hoagland. 1989. Herbicide
safeners: Progress and prospects, in: K.K. Hatzios and
R.E. Hoagland (eds.). Crop Safeners for Herbicides:
Development, Uses, and Mechanism of Action. pp. 355-
365. Academic Press. New York.

Henne, R.C. and R.T. Guest. 1974. Activated carbon as
a method of reducing metribuzin phytotoxicity to seeded
tomatoes. Proc. Northeast. Weed Sci. Soc. 28:242-248.

20.

21.

22.

23.

24.

25.

26.

27.

28.

290

30.

31.

16
Hoffmann, O.L. 1953. Inhibition of auxin effects by
2,4,6-trichlorophenoxyacetic acid. Plant Physiol.
28:622-628.

Hoffmann, O.L. 1962. Chemical seed treatments as
herbicidal antidotes. Weeds 10:322-323.

Hoffmann, O.L. 1969. Chemical antidotes for EPTC on
corn. Weed Sci. Soc. Am. Abstr. 9:12.

Hoffmann, O.L. 1971. U.S. Patent 3,564,768.

Hoffmann, 0.1” 1978. Herbicide antidotes: from concept

to practice, in: F.M. Pallos and J.E. Casida (eds.).
Chemistry and Action of Herbicide Antidotes. pp. 1-13.
Academic Press. New York.

Lay, M.M. and J.E. Casida. 1976. Dichloroacetamide
antidotes enhance thiocarbamate sulfoxide detoxification
by elevating corn root glutathione content and
glutathione S-transferase activity. Pestic. Biochem.
and Physiol. 6:422-456.

Leavitt, J.R.C. and D. Penner. 1978. Protection of
corn (Zea mays) from acetanilide herbicidal injury with
the antidote R-25788. Weed Sci. 26:653-659.

Leavitt, J.R.C. and D. Penner. 1979. In vitro
conjugation of glutathione and other thiols with
acetanilide herbicides and EPTC sulfoxide and the action
of the herbicide antidote R-25788. J. Agr. Food Chem.
27:533-536.

Milhomme, H. and J. Bastide. 1990. Uptake and
phytotoxicity of the herbicide metsulfuron methyl in
corn root tissue in the presence of the safener 1,8-
naphthalic anhydride. Plant Physiol. 93:730-738.

Moreland, D.E. and F.T. Corbin. 1990. Interaction
between safeners and the sorghum microsomal system.
International Conference on Herbicide Safeners,
Chemistry, Mode of Action, and Application. Abstr.
p.49.

Mozer, T.J., D.C. Tiemeir, and E.G. Jawoworski. 1983.
Purification and characterization of corn glutathione
S-transferase. Biochemistry 22:1068-1072.

Pallos, F.M., M.E. Brokke, and D.R. Arneklev. 1977.
U.S. Patent 4,021,224.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

420

17

Pallos, F.M., R.A. Gray, D.R. Arneklev, and.M.E. Brokke.

1978. Antidotes protect corn from thiocarbamate
herbicide injury, in: F.M. Pallos, and J.E. Casida
(eds.). Chemistry and Action of Herbicide Antidotes.
pp. 15-20. Academic Press, New York.

Parker, C. 1983. Herbicide antidotes: a review.
Pestic. Sci. 14:40-48.

Parker, C., W.G. Richarson, and T.M. West. 1980.
Potential for expanding the selectivity of DPX-4189 by
use of herbicide safeners. IProc. Br. Crop. Prot. Conf -
Weeds 1:15-21.

Peek, J.W., H.A. Collins, P.J. Propiglia, J.F. Ellis,
and W. Maurer. 1988. CGA-154281 - a new safener for
improved herbicide tolerance in corn. Weed Sci. Soc.
Am. Abstr. 28:35.

Rubin, B., O. Kirino, and J.E. Casida. 1985. Chemistry
and action of N-phenylmaleamic acids and their
progenitors as selective herbicide antidotes. J. Agric.
Food Chem. 33:489-494.

Rufener, J., A. Nyffeler, and J.W. Peek. 1982. CGA
92194, a new safener to protect sorghum from injurious
effects of metolachlor. Proc. Br. Crop Prot. Conf -
Weeds 2:461-467.

Schafer, D.E., R.J. Brinker, and R.O. Radke. 1981.
Evaluation of behavior and performance of MON-4606 for
alachlor in grain sorghum. Weed Sci. Soc. Am. Abstr.
21:26.

Shaner, D.L. 1990. Mode of action of naphthalic acid
as safener of imazethapyr on maize. International
Conference on Herbicide Safeners, Chemistry, Mode of
Action and Application. Abstr. p.52.

Stephenson, G.R. 1990. Milestones in the development
of herbicide safeners. International Conference on
Herbicide Safeners, Chemistry, Mode of Action, and
Application. Abstr. p. 15.

Turner, W.E., D.R. Clark, N. Helseth, T.R. Dill, J.
King, and E.B. Seifried. 1982. CGA-92194, a new
safener to protect sorghum from metolachlor injury.
Proc. South. Weed Sci. Soc. 35:388.

Weed Science Society of America. 1989. Herbicide
Handbook, 6th ed. Champaign, IL.

43.

44.

45.

46.

18

Winkle, M.E., J.R.C. Leavitt, and O.C. Burnside. 1980.
Acetanilide-antidote combinations for weed control in
corn (Zea mays) and sorghum (Sorghum bicolor). Weed
Sci. 28:699-704.

Yenne, S.P. and K.K. Hatzios. 1989. Influence of oxime
ether safeners and.metolachlor on acetate incorporation
into lipids and acetyl-CoA carboxylase of grain sorghum.
Pestic. Biochem. and Physiol. 35:146-154.

Zama, P. and K.K. Hatzios. 1986. Effects of CGA 92194
on the chemical activity of metolachlor with glutathione
and metabolism of metolachlor in grain sorghum (Sorghum
bicolor). Weed Sci. 34:834-841.

Zbiec, 1.1. and R.M. Devlin. 1990. Antidotal effects
of dichlormid and R-29148 on the herbicidal activity of
chlorimuron and sulfometuron. Proc. Northeast. Weed
Sci.Soc. 44:6-10.

CHAPTER 2
EVALUATION AND COMPARISON

OF HERBICIDE SAPENERS IN CORN [Zea mays L.]
AND GRAIN SORGHUM [sorghum bicolor (L.) Moench]

Abstract

Combinations of certain insecticides and.herbicides can
produce severe crop injury. Greenhouse studies were
conducted to evaluate and compare herbicide safeners for
protection of corn and grain sorghum from herbicidal injury
of metolachlor, primisulfuron, and the interaction with
terbufos insecticide.

Primisulfuron applied at 40 g ai/ha to corn not treated
with terbufos showed only slight injury, but corn tretated
with terbufos at 750 g/1000 m of row showed severe injury.
A high rate of metolachlor produced severe injury to corn
seedlings. The level of metolachlor injury was not affected
by terbufos. With the exception of CGA-133205 that killed
corn, the safeners oxabetrinil, flurazole, naphthalic
anhydride, dichlormid, and R-29148 provided significant
;protection. against injury' to corn from ‘the interaction
between primisulfuron and terbufos, and from metolachlor.

Primisulfuron applied at 10 g ai/ha and metolachlor

applied at 4.5 kg ai/ha caused severe injury to sorghum

19

20
seedlings with or without terbufos. Partial protection was
provided by the safeners, oxabetrinil, CGA-133205, flurazole,
naphthalic anhydride, dichlormid, and R-29148, against
metolachlor injury only. Nomenclature: corn, [Zea mays L. ];
sorghum, [Sorghum bicolor (L.) Moench]; metolachlor, 2-
chloro-N-(2-ethyl-6-methy1phenyl)-N-(2-methoxy-1-
methylethyl)acetamide; primisulfuron, 2-[3-[4,6-
bis(dif1uoromethoxy)-pyrimidin-2-yl]-ureidosulfonyl]benzoic
acid methylester; terbufos, S-[[(1,1-
dimethylethyl)thio]methyl]0,0-diethyl phosphorodithioate;
CGA-133205,0-(1,3,dioxolan-2-yl-methyl)-2,2,2-trifluoro-4’-
chloroacetophenone-oxime; oxabetrinil, a-[(1,3-dioxolan-2-
yl-methoxy)imino]benzeneacetonitrile); flurazole,
phenylmethyl 2-chloro-4-(trifluoromethyl)-5-
thiazolecarboxylate; naphthalic anhydride, naphthalene-1,8-
dicarboxylic acid anhydride; dichlormid, 2,2-dichloro-N,N-
di-2-propenylacetamide; R-29148, 3-(dichloroacety1)-2,2,5-

trimethyl-l,3-oxazolidine

21
IEEIQQEQEAQA

Many herbicides are available that will selectively
control grass weeds in corn and grain sorghum, but certain
herbicides may cause injury problems at the seedling stage.
Metolachlor is a preemergence herbicide for corn and grain
sorghum. Under certain condition, metolachlor may injure
corn seedlings (8) and grain sorghum seedlings (6).
Primisulfuron is a sulfonylurea herbicide that provides
postemergence control of several annual and perennial grasses
plus many broadleaf weeds in corn (7), but some varieties of
corn may be injured from the application of primisulfuron
(1) .

The use of multiple pesticides has become the usual
practice in crop production. Preemergence or postemergence
herbicides may follow the application of soil insecticides.
Although these two chemicals act independently, certain soil
insecticides may increase herbicidal injury (4). Of the 121
herbicide-insecticide interactions listed by Hatzios and
Penner (3), more than 93 percent were synergistic. Severe
corn injury from interactions between primisulfuron and
organophosphate insecticides have been reported (1, 2, 4, 5).

One way to reduce the injury problems is by using
chemicals known as herbicide safeners. The main purpose for
using safeners is to broaden the margin of herbicide
selectivity. Herbicide safeners may be applied as a seed

treatment, preplant incorporated, or as a tank mixture with

22
the herbicide. Commercial safeners evaluated in this study
include oxabetrinil, CGA-133205, naphthalic anhydride,
flurazole, dichlormid, and R-29148.

The objectives of this research were: a) to evaluate
the interaction between metolachlor and primisulfuron with
terbufos insecticide, and b) to evaluate and compare the
effectiveness of herbicide safeners against injury from
metolachlor, primisulfuron, and the interaction with terbufos

insecticide in corn and grain sorghum.

Materials and Methods

Three safener-treated or untreated corn seeds 'Northrup
King 9283' and.six safener-treated or untreated.sorghum seeds
'DK 46' were planted in 945-ml plastic pots containing an air
dried Spinks loamy sand soil with 1.0 percent organic matter
and pH 7.2. The safeners oxabetrinil (1.5 g ai/kg seed),
CGA-133205 (1.5 glai/kg corn seed only), naphthalic anhydride
(10 g ai/kg seed), and flurazole (2.5 g ai/kg seed) were
applied as seed treatment, dichlormid and R-29148 were
applied as a preplant incorporated treatment or tank mixture
with herbicide application at 0.6 kg ai/ha. Sorghum seed
treated or untreated with safener CGA-133205 (0.4 g ai/kg
seed) were provided by CIBA-GEIGY Corp.

Terbufos insecticide was preplant incorporated at a rate
equivalent to 750 g/1000 m of row in the top 3 cm of soil in

the pots.

23

Metolachlor was applied preemergence at the rates of 6.7
and 4.5 kg ai/ha for corn and grain sorghum, respectively.
Primisulfuron plus nonionic surfactant X-77 (0.1% v/v) was
applied postemergence at the three- to four-leaf stage
visible or with collars of corn and grain sorghum at the
rates 40 and 10 g ai/ha, respectively. The rates were lower
for sorghum because preliminary studies indicated sorghum was
more sensitive to metolachlor and primisulfuron injury than
corn. The herbicides were sprayed with a continuous link
belt sprayer at 193 kPa pressure and 230 L/ha volume.

Plants were grown in the greenhouse at 24 i 2 C with
supplemental lighting to provide a total mid-day light
intensity of 1200 uE m?s* from both supplemental and natural
light. Sorghum seedlings for the primisulfuron experiment
were thinned to two plants per pot. The plants were watered
as needed and fertilized weekly with water-soluble fertilizer
(20% N, 20% P205, 20% K20) at the rate of 1500 ppm.

All experiments were factorial combinations of
herbicides, terbufos and safeners in a completely randomized
design” Each treatment. had four replications and. all
experiments were repeated. Plant heights were measured 14
and 28 days after treatment (DAT) for primisulfuron and
metolachlor, respectively. The mean of the plants from each
pot was considered as one observation. Following the
analysis of variance the means were separated using Fisher’s

Protected LSD at the 5 percent level of significance.

24
Result and Discussion

Primisulfuron by itself reduced corn height slightly
(Table 1). The combination of primisulfuron and terbufos
resulted in severe injury to corn seedlings (Table 1) .
Metolachlor'at.high.rate of 6.7 kg ai/ha caused severe injury
to corn seedlings, but no interactions with terbufos
insecticide was evident (Table 2). Jk:a.previous study, corn
hybrid ’NK 9283' has been shown premierly sensitive to
metolachlor at a high rate (9).

Significant protection against primisulfuron-terbufos
interaction injury to corn was provided by oxabetrinil,
flurazole, naphthalic anhydride, dichlormid, and R-29148
(Table 1).

Corn ‘was protected from :metolachlor injury by ‘the
safeners oxabetrinil, flurazole, naphthalic anhydride,
dichlormid, and R-29148 whether or not it was treated with
terbufos (Table 2).

Seed treatment application of the safener CGA-133205
killed the corn (Tables 1 and 2). With the exception of CGA-
133205, there were no statistically significant effects of
safeners on the height of corn seedlings when applied by
themselves or in combination with terbufos (Tables 1 and 2).
There was no significant differences between tank mixtures
and preplant incorporated application of the safeners
dichlormid and R-29148 (Tables 1 and 2). Terbufos by itself

also did not reduced the height of corn seedlings (Tables 1

25
and 2).

Both primisulfuron and metolachlor caused severe injury
to sorghum seedlings (Tables 3 and 4) . No interaction
between terbufos and primisulfuron or terbufos and
metolachlor was evident on sorghum (Tables 3 and 4).

None of the tested safeners protected sorghum from
primisulfuron injury (Table 3). Protection from metolachlor
injury was provided by oxabetrinil, CGA-133205, flurazole,
naphthalic anhydride, dichlormid, and R-29148 (Table 4).
Preliminary studies showed. that terbufos by itself and
terbufos plus safeners did not reduce the height of sorghum
seedlings. Safeners by themselves did not influence the

growth of sorghum seedlings (Tables 3 and 4).

Summary

A Synergistic interaction between primisulfuron and
terbufos was observed in corn hybrid 'NK 9283’ . The safeners
oxabetrinil, flurazole, naphthalic anhydride, dichlormid, and
R-29148 provided protection against corn injury from
primisulfuron interaction with terbufos. Protection was also
provided by these safener against corn injury from
metolachlor.

Primisulfuron and metolachlor caused severe injury to
grain sorghum. Protection by the safeners, oxabetrinil, CGA-
133205, flurazole, naphthalic anhydride, dichlormid, and R-

29148 was only evident against metolachlor injury.

BIBLIOGRAPHY

Ahrens, W.H. 1990. Corn variety response to DPX-9360
and CGA-136872 with soil-applied insecticides. Proc.
North Cent. Weed Sci. Soc. Abstr. 45:34.

Diehl, R.E. and E.W. Stoller. 1990. Interaction of
organophosphate insecticides with nicosulfuron and
primisulfuron in corn. Proc. North Cent. Weed Sci. Soc.
Abstr. 45:31.

Hatzios, K.K. and D. Penner. 1985. Interaction of
herbicides with other agrichemicals in higher plants.
Rev. Weed Sci. 1:1-63.

Holshouser, D.L., J.M. Chandler, and H.R. Smith. 1991.
The influence of terbufos on the response of five corn
(Zea mays) hybrids to CGA-136872. Weed Tech. 5:165-168.

Ketchersid, M.L., J.M.Chandler, and M.G. Merkle. 1989.
Factors affecting the phytotoxicity of CGA-136872 to
corn. Proc. South. Weed Sci. Soc. 42:271.

Ketchersid, M.L., K. Norton, and M.G. Merkle. 1981.
Influence of soil moisture on the safening effect of CGA-
43089 in grain sorghum (Sorghum bicolor). Weed Sci.
29:281-287.

Propiglia, P.J., H.A.Collins, and J.W. Peek. 1988. CGA-
136872: a new corn herbicide. Weed Sci. Soc. Am. Abstr.
28:13.

Rowe, L. and D. Penner. 1987. Variability in corn
tolerance to acetanilide herbicides. Proc. North Cent.
Weed Sci. Soc. Abstr. 42:53.

Rowe, L. E. Rossman, and D. Penner. 1990. Differential
response of corn hybrids and inbrids to metolachlor.

26

27

TABLE 1. Protection of corn seedlings with herbicide
safeners from primisulfuron-terbufos interaction

 

 

injury.

Primi- Shoot height(l§DAT)

sulfuron Safeners‘ -Terbufos +Terbufos

(9/ha) ----- (cm/Plant) ------

0 None 51 50
Oxabetrinil (st) 49 51
CGA-133205 (st) 0 O
Flurazole (st) 51 50
NA (st) 48 47
Dichlormid (pre) 52 51
Dichlormid (ppi) 50 46
R-29148 (pre) 49 47
R-29148 (ppi) 47 45

40 None 42 33
Oxabetrinil (st) 45 43
CGA-133205 (st) 0 o
Flurazole (st) 46 39
NA (st) 43 4o
Dichlormid (tm) 45 38
Dichlormid (ppi) 45 39
R-29148 (tm) 47 43
R-29148 (ppi) 41 39

LSD (0.05) ------- 5 ----------

 

‘st=seed treatment; pre=preemergence; ppi= preplant incor-
porated; tm=tank mixture.

l'

28

TABLE 2. Protection of corn seedlings with herbicide
safeners from metolachlor-terbufos injury.

 

Shoot height (28 DAT)

 

Metolachlor Safener -Terbufos +Terbufos

(kg ai/ha) --- (cm/plant) -----

0.0 None 51 50
Oxabetrinil (st) 49 51
CGA-133205 (st) 0 o
Flurazole (st) 51 50
NA (st) 48 47
Dichlormid (pre) 52 51
Dichlormid (ppi) 50 46
R-29148 (pre) 49 47
R-29148 (ppi) 47 45

6.7 None 23 23
Oxabetrinil (st) 42 42
CGA-133205 (st) 0 O
Flurazole (st) 48 50
NA (st) 40 45
Dichlormid (tm) 45 47
Dichlormid (ppi) 46 46
R-29148 (tm) 45 48
R-29148 (ppi) 44 46

LSD (0.05) ...... 5 ...........

 

29

TABLE 3. Protection of sorghum seedlings with herbicide
safeners from primisulfuron-terbufos injury.

 

 

Primi-

sulfuron, Terbufos Safener' Shoot height (14 DAT)

(g ai/ha) (g/1000 m row) (cm/plant)

O 0 None 58
Oxabetrinil (st) 57
CGA-133205 (st) 55
Flurazole (st) 55
NA (st) 54
Dichlormid (post) 55
R-29148 (post) 51

10 0 None 36
Oxabetrinil (st) 33
CGA-133205 (st) 33
Flurazole (st) 29
NA (st) 27
Dichlormid (tm) 32
R-29148 (tm) 31

10 750 None 31
Oxabetrinil (st) 31
CGA-133205 (st) 32
Flurazole (st) 29
NA (st) 28
Dichlormid (tm) 30
R-29148 (tm) 32

LSD (0.05) 7

 

30

TABLE 4. Protection of sorghum seedlings with herbicide
safeners from metolachlor-terbufos injury.

 

 

Meto-

lachlor Terbufos Safener' Shoot height (28 DAT)

(kg ai/ha) (g/1000 m row) (cm/plant)

0.0 0 None 55
Oxabetrinil (st) 59
CGA-133205 (st) 60
Flurazole (st) 54
NA (st) 53
Dichlormid (pre) 54
R-29148 (pre) 50

4.5 0 None 19
Oxabetrinil (st) 47
CGA-133205 (st) 44
Flurazole (st) 46
NA (st) 46
Dichlormid (tm) 33
R-29l48 (tm) 50

4.5 750 None 17
Oxabetrinil (st) 45
CGA-133205 (st) 39
Flurazole (st) 45
NA (st) 46
Dichlormid (tm) 37
R-29l48 (tm) 47

LSD (0.05) 8

 

CHAPTER 3
INCREASED PRIHISULPURON INJURY TO CORN [Zea mays L.]
WITH ANTIOXIDANTS AND PROTECTION WITH HERBICIDE SAPENERS
M3233.

Greenhouse and laboratory studies were conducted to
evaluate the efficacy of herbicide safeners against corn
injury from primisulfuron and primisulfuron plus
antioxidants.

Antioxidants, piperonyl butoxide and metyrapone, applied
preemergence at 5000 ppm were observed to increase
primisulfuron injury to corn. Piperonyl butoxide (PPO) by
itself did not influence shoot height of corn seedlings, but
metyrapone reduced shoot height significantly. Naphthalic
anhydride, oxabetrinil, flurazole, dichlormid, and R-29148
provided protection against primisulfuron injury to corn.
The increased corn injury from primisulfuron plus PPO was
also protected by those safeners. Protection was most
evident with naphthalic anhydride, flurazole, and R-29148.
None of the safeners provided protection against increased
injury from metyrapone. Naphthalic anhydride was also an
effective safener against increased.corn injury'when 3000 ppm
PPO was applied foliarly 3 days prior to primisulfuron

application.

31

32
l4C-Primisulfuron absorption by corn was not influenced
by the antioxidant, PPO, or the safener, naphthalic
anhydride. However, metabolism of 1“C-primisulfuron was
inhibited by PPO. This inhibition was not totally reversed

by the safener, naphthalic anhydride. Nomenclature: corn,

[Zea .mays L.]; primisulfuron, 2-[3-[4,6-
bis(difluoromethoxy)-pyrimidine-2-yl-] ureidosulfonyl]-
benzoic acid methylester; piperonyl butoxide , a- [ 2- (2-

butoxyethoxy)ethony-4,5-methylenedioxy-2-propyltoluene;
metyrapone, 2-methyl-1,2-di-3-pyridyl-1-propanone;

naphthalic anhydride, naphthalene-1,8-dicarboxylic acid
anhydride; oxabetrinil, a-[(1,3-dioxolan-2-yl-
methoxy)imino]benzeneacetonitrile; flurazole, phenylmethyl
2-chloro-4-(trifluoromethyl)-5-thiazolecarboxylate;
dichlormid, 2,Z-dichloro-N,N-di-Z-propenylacetamide; R-

29148, 3-(dichloroacetyl)-2,2,5-trimethyl-1,3-oxazolidine.

33

I troduction

Primisulfuron is a new sulfonylurea herbicide for
postemergence weed control in corn. Ahrens (1) reported that
some varieties of corn may be injured by postemergence
application of primisulfuron. As with other sulfonylurea
herbicides, inhibition of acetolactate synthase (ALS) is the
site of action of primisulfuron (2). Selectivity of this
class of herbicide in cereal crops, corn, and soybean has
been correlated to the rapid metabolism of the sulfonylureas
to herbicidally inactive products (2).

Metabolism of primisulfuron in corn occurs by
hydroxylation and subsequent sugar conjugation of the phenyl
and pyrimidine rings, whereas the sulfonylurea bridge
cleavage constitutes a minor pathway (3). Hydroxylation of
primisulfuron was inhibited in vitro by tetcyclasis as well
as CO in the presence of oxygen. Fonne-Pfister et a1. (3)
concluded that hydroxylation of primisulfuron is catalyzed by
an inducible cytochrome P450 monooxygenase system.

Molecular oxygen is an obligatory substrate required for
the function of a group of hemoproteins known as mixed
function oxidase (MFO) enzymes (9). These enzymes are
actively involved in the metabolism of a number of herbicides
by plants (5). The activity of MFO is known to be inhibited

by antioxidant compounds or MFO inhibitors, such as piperonyl

34
butoxide (PPO), sesamex (a component of sesame oil), EDU (N-
[2-2-oxo-1-imidazolidinyl]ethyl-N-phenylurea), and n-propyl
gallate (8). Furthermore Rubin et a1. (8) observed that PPO
acted as an herbicide and insecticide synergists.

The use of chemicals to protect crops from herbicidal
injury has been an accepted practice in modern weed
management programs (7) . Hoffmann (6) introduced this
concept about three decades ago. Today, there are numerous
commercial crop safeners, such as oxabetrinil, flurazole,
naphthalic anhydride, dichlormid and R-29148. The mechanism
of action of these safeners is not clearly defined and
several theories have been proposed. According to Hatzios
(4), a safener-induced enhancement of herbicide detoxication
in protected plants seems to be the major mechanism involved
in the protective action of currently developed safeners.
The enhancement of oxidative reactions is currently viewed as
possibly involved in the protective action of herbicide
safeners.

The objectives of these research were: a) to determine
the effect of the antioxidants, PPO and metyrapone, on
primisulfuron activity, b) to study the efficacy of
herbicide safeners against primisulfuron injury to corn in
the presence of the antioxidants, PPO and metyrapone, and c)
to study the absorption and metabolism of 1“C-primisulfuron in
the presence of the safener, naphthalic anhydride, and the

antioxidant, PPO.

35

t n at o s

General greenhouse study: Corn ’NK 9283' treated or
untreated with herbicide safeners was planted three seeds per
pot in 945-ml plastic pots containing air-dried Spinks loamy
sand with 1.0 percent organic matter and pH 7.2. The
safeners naphthalic anhydride, oxabetrinil and f lurazole were
applied. as seed ‘treatment, dichlormid. and. R-29148 were
applied as tank mixtures with the herbicide. The rates for
the safeners were 10 g ai/kg seed, 1.5 g ai/kg seed, 2.5 q
ai/kg seed, 0.6 kg ai/ha, and 0.6 kg ai/ha for naphthalic
anhydride, oxabetrinil, flurazole, dichlormid and R-29148,
respectively.

The antioxidants, PPO and metyrapone, were applied as a
5000 ppm solution containing ethanol 0.5 % v/v 1.5 ml per pot
immediately after planting. Primisulfuron plus nonionic
surfactant X-77 (0.1 % v/v) was applied postemergence at the
rates 40 or 80 g ai/ha to corn at the three to four-leaf
stage.

The efficacy of the safener, naphthalic anhydride,
against the increased injury from primisulfuron at 40, 80 and
120 g ai/ha plus PPO to corn was evaluated in a greenhouse
study. In this study the corn was planted in a BACCTO
potting soil mixu [Naphthalic anhydride (10 g ai/kg seed) was
applied as a seed treatment, but PPO at 3000 ppm plus ethanol

(0.5 % v/v) and nonionic surfactant X-77 (0.1 % v/v) were

36
applied postemergence 3 days prior to primisulfuron
application. Primisulfuron and PPO were applied
postemergence using a continuous link belt sprayer at 193 kPa
pressure and 230 L/ha volume.

The plants were grown in the greenhouse at 24 1'2 C with
supplemental lighting to provide a total mid-day light
intensity of 1200 uE mfls“ from both supplemental and natural
light. The plants were watered as needed and fertilized
weekly with water-soluble fertilizer (20 % N, 20 % Pgfi, 20 %
190) at the rate of 1500 ppm.

These studies used a factorial treatment arrangement of
primisulfuron, antioxidants and safeners in a completely
randomized design. Each treatment combination had four
replications and all experiments were repeated. Shoot
heights were measured 14 days after treatment (DAT) and
calculated as a percent of control. The mean of three plants
from each pot was considered as one observation. Following
the analysis of variance the means were separated with
Fisher's Protected LSD at the 5 percent level of
significance.

“C-Primisulfuron absorption and metabolism: Corn 'NK
9283' treated or untreated with naphthalic anhydride was
planted 1 seed per pot in 945-ml plastic pots containing
BACCTO potting soil mix. Plants were grown in the greenhouse
for 1 week and then moved to the growth chamber. Temperature

in the growth chamber was maintained at 24 C during the day

37
and at 20 C at night, and lighting was provided for 16 h
period by high pressure sodium lamps.

PPO at 3000 ppm plus ethanol (0.5 % v/v) and nonionic
surfactant X-77 (0.1 % v/v) was applied postemergence 3 days
prior to primisulfuron application. Prior to treatment with
the l"C-primisulfuron, corn at the three- to four-leaf stage
was sprayed with primisulfuron plus x-77 (0.1 % v/v). While
spraying, leaves used for the l‘C-primisulfuron absorption and
metabolism studies were covered with plastic wrap.

A.2 ul drop of the l‘C-primisulfuron (activity 2.22 x 102
Bq, uniform ring labelled) and 18 1-ul drops (activity 4.44
x 103 Bq) for absorption and metabolism studies,
respectively, were applied to the second leaf of seedlings.
After the spots were dry, the seedlings were placed in growth
chamber until harvest. After 8 h the plants were harvested
for absorption study and after 10 h the plants were harvested
for the metabolism study. The leaves were rinsed for 45
seconds with 5 ml of 50 % MeOH (v/v). The leaves for the
metabolism study were placed in dry ice at -30 C until
extracted.

Absorption of 14C-primisulfuron was determined by
radioassaying the MeOH rinsate by liquid scintillation
spectrometry. Data are presented as a percent of total
applied.

For the metabolism study, the leaves were ground with a

Virtis grinder in 40 ml of 80 % acetone (v/v) for 6 minutes.

38

The extract was filtered under vacuum with a Whatman no 1
filter paper. The filtered solution was transferred to a
250-ml round bottom flask and concentrated to 3 ml in vacuo
at 35 C using a rotary evaporator» The aqueous plant extract
was placed in a 15-ml vial and the round bottom flask was
rinsed with 5 ml acetonitrile and then combined with the
aqueous plant extract.

The volume of the aqueous extract was reduced under a
stream of nitrogen to 2 ml. The aqueous plant extract was
then filtered under vacuum with a millipore-filter paper
(0.45 mM) and the vial was rinsed with 2 to 3 ml
acetonitrile. The volume was then reduced under a stream of
nitrogen to approximately 0.6 ml volume. Following the
addition of 0.4 ml of 80 % acetone (v/v) the extract was
placed on silica gel 60 FE“ TLC plates that were developed
with the solvent system of methanol : benzene (2:1 v/v).

The location of the 1“C-primisulfuron and its metabolites
on the TLC plates were determined with a scanner. The 14C-
labelled spots ‘were removed and. radioassayed. by liquid
scintillation spectrometry. Metabolism data are presented as
percent of 1“C absorbed. Both absorption and metabolism
studies were analyzed using a completely randomized design
with eight replications. Means were separated by Fisher's

Protected LSD at the 5 percent level of significance.

39
t n scuss 0

Effect of MPO inhibitors and herbicide safeners:
Primisulfuron applied at 40 and 80 g ai/ha injured the hybrid
’NK 9238' corn seedlings (Table 1). The level of injury
increased in the presence of the antioxidants, PPO and
metyrapone. PPO by itself did not injure corn seedlings, but
metyrapone caused a significant decrease in corn shoot height
(Table 1) . Corn injury from primisulfuron applied at 40 and
80 g ai/ha was reversed by the safeners naphthalic anhydride,
oxabetrinil, flurazole, dichlormid, and R-29148 (Table 1).
The increased corn injury from PPO plus primisulfuron at 40
and 80 g ai/ha was also protected by those safeners.
Protection against the incresed corn injury from
primisulfuron plus PPO was most evident from naphthalic
anhydride, flurazole, and R-29148 (Table 1). None of the
safeners provided protection against increased injury from
metyrapone (Table 1).

Further study to evaluate primisulfuron activity in the
presence of PPO and naphthalic anhydride is presented in
Table 2. Postemergence application of PPO also increased
corn injury from primisulfuron applied at 40, 80 and 120 g
ai/ha (Table 2) . The safener, naphthalic anhydride, reversed
the increased injury from the antioxidant, PPO, applied 3
days prior to primisulfuron application (Table 2).

"c-Primisulfuron absorption and metabolism: Absorption

of "C-primisulfuron was not influenced by the safener,

40
naphthalic anhydride, the antioxidant, PPO, or both the PPO
and the naphthalic anhydride (Table 3). The increased corn
injury from primisulfuron plus PPO appeared due to the
inhibition of the l4C-primisulfuron metabolism by the PPO
(Table 3) . The safener, naphthalic anhydride, partially
reversed this inhibition (Table 3). Naphthalic anhydride
itself did not influence the rates of l‘C-primisulfuron

metabolism (Table 3).

M
The antioxidants , piperonyl butoxide ( PPO) and

metyrapone, increased corn injury from primisulfuron. The
increased injury from PPO was reversed by the safeners,
flurazole and R-29148. However, protection by the safeners
against increased injury from metyrapone was not evident.
The increased injury from PPO was due to inhibition of
primisulfuron metabolism. The safener, naphthalic anhydride,

did not totally reverse this inhibition.

BIBLIOGRAPHY

Ahrens, W.H. 1990. Corn variety response to DPX-9360
and CGA-136872 with soil applied insecticides. Proc.
North Cent. Weed Sci. Soc. Abstr. 45:34.

Beyer Jr., E.M. M.J. Duffy, J.V. Hay, and D.D.
Schlueter. 1988. Sulfonylureas. In: P.C. Kearney and
D.D. Kaufman (eds.). Herbicides: Chemistry, Degradation
and.Mode of Action. ‘Vol. 3. Dekker, New York. pp. 117-
189

Fonne-Pfister, R. J. Gaudin, K. Kreuz, K. Ramsteiner,
and Ebert. 1990. Hydroxylation of primisulfuron by an
inducible cytochrome P450-dependent monooxygenase systems
from maize. Pest. Biochem. and Physiol. 37:165-173.

Hatzios, K.K. 1983. Herbicide antidotes: development,
chemistry, and mode of action. Adv. Agron. 36:165-316.

Hatzios, K.K. and D. Penner. 1982. Metabolism of
herbicides in higher plants. Burgess Publishing Co.,
Minneapolis. 142 pp.

Hoffmann, O.L. 1962. Chemical seed treatments as
herbicidal antidotes. Weeds 10:322-323.

Pallos, F.M. and J.E. Casida. 1978. Chemistry and
action of herbicide antidotes. Academic Press, New York.
171 pp.

Rubin, B. J.R.C. Leavitt, D. Penner, and A.W. Saettler.

1980. Interaction of antioxidants with ozone and
herbicide stress. Bull. Environm. Contam. Toxicol.
25:623-629.

Sato, R. and T. Omura. 1978. Cytochrome P-450.
Academic Press, New York. 233 pp.

41

42

TABLE 1. Efficacy of herbicide safeners against injury from
the interaction.of primisulfuron.and antioxidants.

 

 

 

Primi- Shoot height (14 DAT)
sulfuron Safener' None PPO Metyrapone
(g ai/ha) (percent of untreated)
0 None 100 98 92
40 None 85 64 81
NA (st) 94 89 80
Oxabetrinil (st) 94 81 84
Flurazole (st) 97 97 85
Dichlormid (tm) 93 81 87
R-29148 (tm) 98 95 86
80 None 83 70 74
NA (st) 89 84 81
Oxabetrinil (st) 91 85 82
Flurazole (st) 93 88 79
Dichlormid (tm) 91 78 76
R-29148 (tm) 94 92 83
LSD (0.05) --------- 8 ---------------

 

'st=seed treatment; tm=tank mixture.

43

TABLE 2. Efficacy of nahpthalic anhydride against increased
primisulfuron injury to corn with the PPO.

 

Shooth height (14 DAT)

 

Primisulfuron PPO -NA +NA
(g ai/ha) (ppm) (percent of control)
40 0 90 99
3,000 86 99
80 0 88 94
3,000 81 95
120 0 85 94
3,000 77 94

LSD (0.05) ........ 7 ........

 

44

TABLE 3. Absorption and metabolism of 1"C-primisulfuron in
corn seedlings in the presence of the safener,
naphthalic anhydride and the antioxidant, PPO.

 

Herbicide interaction Absorption Metabolism

 

(% of treated) (% of absorbed)

Primisulfuron 20.4 10.2
Primisulfuron + NA 21.4 11.1
Primisulfuron + PPO 21.4 5.7
Primisulfuron + NA + PPO 18.9 7.8
LSD (0.05)' 4.7 1.7

 

'Comparisons valid within columns.

APPENDIX A. Chemical structure of naphthalic anhydride,
dichlormid, R-29148, oxabetrinil, CGA-133205,
and flurazole.

45

Naphthalic Anhydride

)flQCH:£Ha
CL—4rF4k—NK
c 1 CHaCHEHa

Dichlormid

9-29148

46

Oxabetrinil

F

OT —c~a< “’3

N——0

CGA-133205

1°:
HAG
\c /

f3.
\c
I
c1
Flurazole

APPENDIX B. Chemical structure of metolachlor and
primisulfuron.

47

 

 

N
0
\dl
CHECH3
Metolachlor
0 F
\
\C—O—CH3 o—c:3——H
o H o H N
H I II I J —- F
S N " N
1| \5 / F
U N I
0—C—H

Primisulfuron F

"‘TillllllillMilli“