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LIBRARY L
Michigan State

University

This is to certify that the

thesis entitled

ABSORPTION, TRANSLOCATION, METABOLISM, AND MODE OF ACTION
OF BUTHIDAZOLE(3-[5-(1,1-DIMETHYLETHYL)-l,3,4-THIADIAZOL-
2-YL]-4-HYDROXY-l-METHYL-Z-IMIDAZOLIDINONE) AS RELATED
TO ITS SELECTIVITY BETWEEN CORN AND REDROOT PIGWEED AND

BETWEEN ALFALFA AND QUACKGRASS
presented by

KRITON KLEANTHIS HATZIOS

has been accepted towards fulfillment
of the requirements for

Ph.D Weed Science

degree in

 

 

EvQQ Ly? vQ-y
/

Mflm1mfiuuu

Date 347/]?
/ / '

0-7639

 

 

 

OVERDUE FINES:

25¢ per day per item

RETURMIG LIBRARY MATERIALS:
Place in book return to remove
charge from circulation records

 

1e.
. ‘ 1 _¢

t .Ilullifl.]

\I“I:l.ll..\nl It‘ll}! lllxll l[ll

ABSORPTION, TRANSLOCATION, METABOLISM, AND MODE OF ACTION OF
BUTHIDAZOLE (3-[5-(1,l-DIMETHYLETHYL)-l,3,4-THIADIAZOL-2-YL1-4-
HYDROXY-l-METHYL-2-IMEDAZOLIDINONE) AS RELATED TO ITS SELECTIVITY

BETWEEN CORN (Egg mazs L.) AND REDROOT PIGWEED

(Amaranthus retroflexus L.) AND BETWEEN ALFALFA (Medicago

sativa L.) AND QUACKGRASS [Agropyron repens (L.) Beauv.]

 

 

By

Kriton Kleanthis Hatzios

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1979

ABSTRACT

ABSORPTION, TRANSLOCATION, METABOLISM, AND MODE OF ACTION OF
BUTHIDAZOLE (3-[5-(1,l-DIMETHYLETHYL)-l,3,4-THIADIAZOL-2-YL]-4-
HYDROXY-l-METHYL-Z-IMIDAZOLIDINONE) AS RELATED TO ITS SELECTIVITY

BETWEEN CORN (37.3.3 mazs L.) AND REDROOT PIGWEED

(Amaranthus retroflexus L.) AND BETWEEN ALFALFA (Medicago

sativa L.) AND QUACKGRASS [Agropyron repens (L.) Beauv.]

 

By
Kriton Kleanthis Hatzios

Buthidazole (3-[5-(1,l-dimethylethyl)-l,3,4-thiadiazol-2-yl]-4-
hydroxy-l-methyl-Z-imidazolidinone) is a promising new herbicide for
selective weed control in corn (Egg EéZ§.L') following preemergence or
early postemergence applications and in established alfalfa (Medicago
sativa L.) applied in fall or spring.

Absorption, translocation, metabolism, and mode of action were
studied as potential factors contributing to buthidazole selectivity
between tolerant corn and susceptible redroot pigweed (Amaranthus retro-
flexus L.) and between tolerant alfalfa and susceptible quackgrass
[Agropyron repens (L.) Beauv.]
14C-buthidazole was absorbed by both leaves and roots of all species
and moved acropetally. Basipetal movement was evident only in redroot pig-
weed following foliar application. However, rapid uptake by the roots and
rapid movement to the leaves via the xylem appeared to be the main pathway
of uptake and translocation of 1‘.C-buthidazole in all species. Differential
absorption and translocation did not appear to contribute to buthidazole
selectivity between alfalfa and quackgrass. There were no differences in
buthidazole absorption between corn and redroot pigweed. However, 140-
buthidazole supplied to the roots was translocated more quickly to the

redroot pigweed shoots and leaves. This may have contributed to the sele-

ctivity between corn and redroot pigweed.

Kriton Kleanthis Hatzios

Metabolism of 14C-buthidazole was studied in the leaves and stems of
all four plant species following root application and in corn following
application to the emerging coleoptile. Alfalfa metabolized 14C-buthidazole
very rapidly yielding five metabolites beside the unmetabolized parent
buthidazole. An unidentified metabolite with an Rf value of 0.21 (developing
system Chloroform 80% : Methanol 20%) appeared to be the major metabolite
in alfalfa accounting for 40% of the total radioactivity present in the
leaves of treated plants 6 days after treatment. Metabolism of 14C-buthida-
zole was very slow in quackgrass. Unmetabolized 14C-buthidazole was present
as 87% of the total radioactivity even after 6 days. Two metabolites with
Rf values similar to those of the amine and dihydroxy derivatives of buthi-
dazole were present in higher amounts in alfalfa as compared to quackgrass,
whereas a metabolite with an Rf value similar to that of the methyl urea
derivative of buthidazole was present only in alfalfa. Thus, differential
rate of metabolism appeared to be a major factor contributing to buthidazole
selectivity between alfalfa and quackgrass. Corn and redroot pigweed meta-
bolized l4C-buthidazole in a similar manner but at a different rate yielding
as major metabolite an unidentified buthidazole derivative with Rf value of
0.24 to 0.26 in the chloroform:methanol (4:1) developing system. This meta-
bolite appeared to be similar to the one detected in alfalfa. Corn formed
this metabolite very rapidly even in roots, whereas the formation of this
buthidazole derivative in redroot pigweed leaves and stems was slow. Minor
metabolites detected in both corn and redroot pigweed appeared to be similar
to the urea and dihydroxy derivatives by Thin Layer Chromatography (TLC). A
metabolite with Rf value similar to the amine derivative of buthidazole was
present only in corn leaves following application of labeled buthidazole
to the emerging coleoptile. Thus, a differential rate of metabolism, combined

with the faster movement of unmetabolized buthidazole from the roots to the

Kriton Kleanthis Hatzios

shoots and leaves of redroot pigweed appeared important in the selectivity
of buthidazole between corn and redroot pigweed.

Time-course and concentration studies on the localization of the meta-
bolic site of action of buthidazole were conducted with enzymatically iso-

lated leaf cells from navy beans (Phaseolus vulgaris L.) and appropriate

 

radioactive substrates. These studies revealed that photosynthesis was the
most sensitive and first metabolic process inhibited by buthidazole at
concentrations as low as 0.1 pH and as early as 30 min of incubation. Buthi-
dazole at concentrations 10 and 1004uM also inhibited RNA and lipid synthe-
ses, whereas protein synthesis was not affected by buthidazole at any con-
centration examined. Furhter studies with isolated spinach (Spinacea
oleracea L.) chloroplasts indicated that inhibition of photosynthetic
electron transport by buthidazole was primarily at the reducing side of
photosystem II (between the unknown quencher Q and plastoquinone). A less
inhibitory effect of buthidazole on the mechanism of water oxidation (oxidi-
zing side of photosystem II) was also evident from the data. 13 gi!g_measu-
rements of total photosynthesis and dark respiration of corn, redroot pig-
weed, alfalfa, and quackgrass plants treated with various rates of buthida-
zole applied postemergence were conducted by means of an infrared C02 ana-
lyzer at different time periods after application. The results confirmed
the observations of the in 23533 studies, suggesting that buthidazole was a
strong and rapid inhibitor of photosynthesis of the sensitive redroot pig-
weed and quackgrass plants, with less effect on corn and alfalfa. Corn
appeared to be tolerant to low rates of buthidazole following preemergence
application. Postemergence application of buthidazole at rates as low as
0.28 kg/ha inhibited total photosynthesis of corn as early as 4 hours after

spraying. Inhibition of anthocyanin formation in corn did not appear to be

connected to the effect of this herbicide on photosynthesis since low rates

Kriton Kleanthis Hatzios

that inhibited anthocyanin biosynthesis did not inhibit total photosynthesis
of corn following preemergence application of buthidazole. Buthidazole did
not affect respiration of corn and redroot pigweed seedlings except for a
transitory increase in corn 12 days after preemergence or 4 hours after post-
emergence treatment with buthidazole at 0.56 or 0.84 and 1.12 kg/ha, respe-
ctively. Respiration in alfalfa was also showed a transitory increase 4 hours
after treatment with buthidazole at 1.12 and 2.24 kg/ha following post-
emergence application. A long-term inhibition of quackgrass respiration 96
hours after treatment with buthidazole at 1.12 and 2.24 kg/ha was evident.

Transmission electron microscopy studies showed that postemergence
application of buthidazole at 0.28 and 1.12 kg/ha reduced or prevented
starch accumulation in corn bundle sheath chloroplasts of treated plants
as early as 4 hours after treatment, indicating thus interference with
photosynthesis. Ultrastructural disruptions in some mesophyll chloroplasts
of treated corn plants were also evident.

Germination studies with seeds of corn, redroot pigweed, alfalfa, and
quackgrass indicated that buthidazole is not a germination inhibitor.

The mode of action studies with buthidazole indicate that a diffe-
rential mode of buthidazole action between tolerant and susceptible species
did not appear to contribute to crop selectivity.

Attempts to increase corn tolerance to higher rates of buthidazole by
using herbicide antidotes found that CDAA (2-chlorofN,Nfdiallylacetamide)
and NA (1,8-naphthalic anhydride) offered partial protection, with R-25788
(2,2-dichlorofiN,N7diallylacetamide) being ineffective. CDAA appeared to be
more effective than NA and a ratio 1:3 (buthidazole:CDAA) was optimal for
the protection effect. Although this ratio, 1:3, may not be practical, it

showed that buthidazole safety to corn could be chemically enhanced.

zrfi uvfiun 100 narépa uou Kkedven, rfis unrépas uou Ataudvrms,

Kai toO ddsloofl uou Hérpou

In memory of my father Kleanthis, my mother Diamando, and

my brother Petros

ii

Afyo éKoua

ed 600m: rfs duuydahés v’ dvefcow
rd udpuapa v6 Adunouv 016v filto

rfi Balaooa vd Kuuarfcet

Afyo drdua,

vd onKmGOOue Afyo wnlorepa

Pimpyos Xeoépns , dné 16 Muetordpnuo

Just a little more
and we shall see the almond trees in blossom
the marbles shining in the sun
the sea, the curling waves
Just a little more
let us rise just a little higher
George Seferis, From Mythistorema

Greek poet, Nobel laureate, 1963

iii

ACKNOWLEDGEMENTS

The enthusiasm, patience, and knowledge of Dr. Donald Penner, my
academic advisor, have provided the framework for my education as a
scientist. I thank him for all that he has contributed during my graduate
studies and for his trusted friendship. I also wish to thank Dr. G.A.
Mourkides, Aristotelian University of Thessaloniki, Greece, for encou-
raging me to pursue my M.S. and Ph.D. studies in the United States.

Drs. H. Johnson, F.W. Meggit, A. Putnam, and M. Zabik, are appre-
ciated for their service in the guidance committee and their constructive
criticism of my work. Drs. N.E. Good and D. Bell, Dept. of Botany and
Plant Pathology, Michigan State University, are acnowledged for their
technical advise and support in the electron transport studies with iso-
lated spinach chloroplasts. The technical advice of Dr. F.M. Ashton, and
Mr. R.K. Glenn, Dept. of Botany, University of California, Davis, on the
details of their method for isolating active leaf cells form beans, is
also appreciated. Dr. K. Baker is acknowledged for her assistance with the
electron microscopy studies.

The moral support of my fianchée Maria and my family in Greece, have
contributed greatly to the success of my academic studies. Special thanks
are also offered to all my Greek and American friends and fellow graduate
students for making my stay in East Lansing a pleasant experience full of
good memories.

I am grateful to Velsicol Chemical Corporation for granting this
study and for supplying 14C-buthidazole and analytical reference standards
of buthidazole and its derivatives. Thanks are also expressed to the North
Central Regional Office of the Pesticide Impact Assessment for the finan-
cial support for part of my research.

iv

LIST OF TABLES . . .
LIST OF FIGURES . . .

INTRODUCTION . . . .

CHAPTER 1: Absorption, Translocation, and Metabolism of
dazole in Alfalfa (Medicago sativa L.) and Quackgrass
[AgropL ron repens (L. ) Beauv. ].

TABLE OF CONTENTS

 

ABSTRACT . . .

INTRODUCTION . .

0

MATERIALS AND METHODS .

RESULTS AND DISCUSSION

LITERATURE CITED

CHAPTER 2: Site of Uptake and Translocation of
Corn (Zea mays L.) and Redroot Pigweed (Amaranthus

retroflexus L.)

ABSTRACT . . .

INTRODUCTION .

MATERIALS AND METHODS

RESULTS AND DISCUSSION

LITERATURE CITED

 

l4

C-Buthidazole in

14

C-Buthi-

 

CHAPTER 3: The Role of Metabolism in Buthidazole Selectivity

Between Corn (Zea mays L.) and Redroot Pigweed
(Amaranthus retroflexus L.)

 

ABSTRACT . . .

INTRODUCTION .

MATERIALS AND METHODS .

RESULTS AND DISCUSSION

LITERATURE CITED

29

30

30

. 30

. 31

34

57

58

59
59
62

73

CHAPTER 4: Localizing the Metabolic Site of Action of Two Thia-

diazolyl Herbicidal Derivatives . . . . . . . . . .
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . .
MATERIALSANDMETHODS..............
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . . . . . . . . . . .

CHAPTER 5: Inhibition of Photosynthetic Electron Transport in

Isolated Spinach Chloroplasts by Two 1,3,4-thiadia-

zolyl Derivatives . . . . . . . . . . . . . . . .
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . .

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

LITERATURE CITED . . . . . . . . . . . . . . . . . . . .

CHAPTER 6: Physiological Effects of Buthidazole on Corn (Zea ma

L.), Redroot Pigweed (Amaranthus retroflexus L.),

 

Alfalfa (Medicago sativa L.), and Quackgrass [Agropyron

repens (L.) Beauv.] . . . . . . . . . . . . . . . .
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . .
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . . . . . . . . .
CHAPTER 7: Some Effects of Buthidazole on Corn (532 male L.)
Photosynthesis, Respiration, Anthocyanin Formation,
and Leaf Ultrastructure . . . . . . . . . . . . .
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . . . . . . . .

MATERIALS AND METHODS .'. . . . . . . . . . . . . .

vi

Page

74
74
74
75
79
87

89
89
90
91
93

100

102

102

. 102

103

106

119

120

. 120

. 121

. 121

RESULTS AND DISCUSSION . . . . .
LITERATURE CITED . . . . . . . .

CHAPTER 8: Potential Antidotes Against
(Zea mays L.) . . . . . . .

ABSTRACT . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . .
MATERIALS AND METHODS . . . . . .
RESULTS AND DISCUSSION . . . . .
LITERATURE CITED . . . . . . . .
CHAPTER 9: Summary and Conclusions . .

LIST OF REFERENCES . . . . . . . .

vii

Page
123

138

140
140
141
142
143
156
158

161

LIST OF TABLES

CHAPTER 1

1.

14C-Buthidazole absorption by alfalfa and quackgrass _ _

plants harvested 1, 3, and 14 days after leaf treatment. .
14C-Buthidazole absorption by alfalfa and quackgrass

plants harvested 1, 3, and 6 days after root application .
Distribution of total 14C found in various parts of alfalfa
plants harvested at 1, 3, and 14 days after leaf application
of l4C-buthidazole . . . . . . . . . . . . . . . . . . . . .

Distribution of total 14C found in various parts of
quackgrass plantz harvested at 1, 3, and 14 days after leaf
application of l C-buthidazole . . . . . . . . . . . .

Distribution of total 14C found in various parts of alfalfa
plants harvested 1, 3, and 6 days after root application of
C-buthidaZOJ-e O C O O O O O O O O O I I O O O I 0 O O O 0

Distribution of total 14C found in various parts of
quackgrass plants harvested at 1, 3, and 6 days after root
application Of 14C-buthida2018 o o o o o o o o o o o o

Methanol-soluble metabolites of l4C-buthidazole in
alfalfa leaves and stems l, 3, and 6 days after root
application . . . . . . . . . . . . . . . . . . . .

Methanol-soluble metabolites of 14C-buthidazole in quack-
grass leaves and stems l, 3, and 6 days after root appli-
cation 0 O O O O O O O O O O O O O O I O O O 0

CHAPTER 2

l.

2.

14C-Buthidazole absorption by corn and redroot pigweed seed-

lings harvested 1, 3, and 14 days after leaf application . .
14C-Buthidazole absorption by corn and redroot pigweed seed-
lings harvested 1 and 3 days after root application

Distribution of total 14C found in various parts of corn
plants harvested 1, 3, and 14 days after leaf application
of l4C-buthidazole . . . . . . . . . . . . . . . . . . .

Distribution of total 14C found in various parts of redroot

pigweed plants harvested 1, 3, and 14 days after leaf appli-
cation of 14C-buthidazole . . . . . . . . . . . . . .

viii

Page

21

22

23

24

25

26

27

28

48

49

50

51

Page

Distribution of total 140 found in various parts of corn

5.
planzs harvested 1,3, and 14 days after root application
C- buthidazole . . . . . . . . . . . . . . . . . . . . . 52
6. Distribution of total 14C found in various parts of redroot
pigweed plants harvested 1, 3, and 6 days after root appli-
cation of l4C-buthidazole . . . . . . . . . . . . . 53
7. The effect of buthidazole on 'Pioneer 3780' corn height and
weight 30 days after applying the herbicide on the root zone
vszspraying on the soil surface . . . . . . . . . . . ... . 54
8. The effect of buthidazole application method on 'Pioneer 3780'
corn height and fresh weight after 30 days . . . . . . . . . 55
9. Distribution of total 14C found in various parts of corn
plants harvested 3, 8, and 16 days after treatment with 14C-
buthidazole at the coleoptile stage . . . . . . . . . . . . 56
CHAPTER 3
1. Rf values of analytical reference standards used for identi-
fication of unknown buthidazole metabolites . . . . . . . . 71
2. Methanol-soluble metabolites of 14C-buthidazole in corn
seedlings 1, 3, and 14 days after root application . . . . . 71
3. Methanol-soluble metabolites of 14C-buthidazole in the first
leaf of corn plants treated with buthidazole applied to the
emerging coleoptile 3, 8, and 16 days after treatment . . . 72
4. Methanol-soluble metabolites of 14C-buthidazole in redroot
pigweed leaves 1, 3, and 6 days after root application . . . 72
CHAPTER.4
1. The effect of buthidazole and tebuthiuron on photosynthesis
of isolated bean cells . . . . . . . . . . . . . . . . . . 83
2. The effect of buthidazole and tebuthiuron on RNA synthesis
of isolated bean cells . . . . . . . . . . . . . . . . . . . 84
3. The effect of buthidazole and tebuthiuron on protein synthe-
sis of isolated bean cells . . . ... . . . . . . . . . . . . 85
4. The effect of buthidazole and tebuthiuron on lipid synthesis

of isolated bean cells . . . . . . . . . . . . . . . . . . . 86

ix

CHAPTER 5

I.

II.

III.

IV.

Effects of buthidazole and tebuthiuron on uncoupled electron
transport in illuminated spinach chloroplasts . . . . . . .

Effects of buthidazole and tebuthiuron on photosystem II-
mediated electron transport in illuminated spinach chloro-
plasts O C O O O O O O O O O C O O O O O O O O O O O O I O 0

Effects of buthidazole and tebuthiuron on photosystem I-
mediated electron transport in illuminated spinach chloro-
plasts O O O I I O O O O O I O O O O O O O O O O I O O O O 0

Effects of buthidazole and tebuthiuron on electron transport

in hydroxyiamine—treated and illuminated spinach chloroplasts

CHAPTER 6

1.

The effect of buthidazole on germination of four plant
species .'. . . . . . . . . . . . . . . . . . . . . . . .

Viability and vigor of 'Pioneer 3780' corn seeds obtained
from plots treated with preemergence applied buthidazole . .

Viability and vigor of 'Pioneer 3780' corn seeds obtained
from plots treatd with preplant incorporated buthidazole . .

The effect of buthidazole on corn total photosynthesis and
dark respiration 12 days after preemergence application . .

The effect of buthidazole on redroot pigweed total photo-
synthesis and dark respiration at various time intervals
after postemergence application . . . . . . . . . . . . . .

The effect of buthidazole on 'Vernal' alfalfa total photo-
synthesis at various time intervals after postemergence
application 0 O O O O I O O O I I O O O O O O O O O O O O O

The effect of buthidazole on quackgrass total photosynthesis
at various time intervals after postemergence application .

The effect of buthidazole on dark respiration of 'Vernal'
alfalfa at various time intervals after postemergence
application . . . . . . . . . . . . . . . . . . . . . . . .

The effect of buthidazole on quackgrass dark respiration
at various time intervals after postemergence application .

CHAPTER 7

1.

Effect of buthidazole on 'Pioneer 3780' corn total photo-
synthesis at various time intervals after preemergence
application . . . . . . . . . . . . . . . . . . . .

Page

96

97

98

. 110

111

112

113

114

115

116

117

118

133

Effect of buthidazole on 'Pioneer 3780' corn total photo-
synthesis at various time intervals after postemergence
application . . . . . . . . . . . . . . . . . . . . . . .

Effect of buthidazole on 'Pioneer 3780' corn dark respi-
ration at various time intervals after preemergence
application . . . . . . . . . . . . . . . . . . . . . . . .

Effect of buthidazole on 'Pioneer 3780' corn dark respi-
ration at various time intervals after postemergence
application I O O O O O O O O O O O O O O O O I O O O O O O

The effect of buthidazole on anthocyanin formation in the
sheaths of 'Pioneer 3780' corn leaves 14 days after pre—
emergence application . . . . . . . . . . . . . . . . . . .

CHAPTER 8

1.

2.

Effects of R-25788, R-29l48, and GA3 on corn injury from
buthidazole, 30 days after preemergence treatment . . .

Effect of CDAA on corn injury from buthidazole, 30 days
after preemergence treatment . . . . . . . . . . . . .

Effect of CDAA on corn injury from buthidazole, 30 days
after preemergence treatment . . . . . . . . . . . . . .

Effects of NA and Carboxin on corn injury from buthidazole,
30 days after preemergence application . . . . . . . . . .

Effects of six herbicides on corn injury from buthidazole,
20 days after preemergence treatment . . . . . . . . . . .

Effects of R-25788, Alachlor, and Diethatyl at 5.60 kg/ha
on corn injury from buthidazole, 30 days after preemergence
treatment 0 O I O O O O O O I O O I O O O O O O O O O O I 0

Effects of alachlor, metolachlor, and butylate + R-25788

at 2.24 kg/ha on corn injury from buthidazole, 30 days
after preemergence treatment . . . . . . . . . . . . . . .

xi

Page

. 134

. 135

136

137

. 148

. 149

150

. 151

152

. 154

. 155

LIST OF FIGURES

Page

CHAPTER 1

1.

Chemical structure and chemical name of the herbicide buthi-
dazole. The aigerisk (*) indicates the radioactive labeled
carbon atom ( C) . . . . . . . . . . . . . . . . . . . . . . 12

Translocation of 14C-buthidazole in alfalfa plants. Treated
plants harvested (A) 1 day, (B) 3 days, and (C) 14 days after
foliar application. Corresponding radioautographs below

( a,b,c ) . . . . . . . . . . . . . . . . . . . . . . . . . . l4

Translocation of 14C-buthidazole in quackgrass plants. Treated
plants above harvested (A) 1 day, (B) 3 days, and (C) 14 days
after foliar application. Corresponding radioautographs

below ( a,b,c ) . . . . . . . . . . . . . . . . . . . . . . . l6
Translocation of l4C-buthidazole in alfalfa plants. Treated
plants above harvested (A) 1 day, (B) 3 days and (C) 6 days

after root application. Corresponding radioautogrpahs below

( a,b,c ) . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Translocation of 14C-buthidazole in quackgrass plants. Treated
plants above harvested (A) 1 day, (B) 3 days, and (C) 6 days
after root application. Corresponding radioautographs below

( a,b,c ) . . . . . . . . . . . . . . . . . . . . . . . . . . 20

CHAPTER 2

1.

Translocation of l4C-buthidazole in corn plants. Treated

plants on the left harvested (A) 1 day, (B) 3 days, and (C)

14 days after foliar application. Corresponding radioautographs
to the right ( a,b,c ) . . . . . . . . . . . . . . . . . . . . 39

Translocation of l4C-buthidazole in redroot pigweed plants.
Treated plants on the left harvested (A) 1 day, (B) 3 days,

and (C) 14 days after foliar application. Corresponding radio-
autographs to the right ( a,b,c ) . . . . . . . . . . . . . . 41

Translocation of 14C-buthidazole in corn plants. Treated

plants above harvested (A) 1 day, (B) 3 days, and (C) 14

days after root application. Corresponding radioauto—

graphs below ( a,b,c ) . . . . . . . . . . . . . . . . . . . . 43

Translocation of l4C-buthidazole in redroot pigweed plants.
Treated plants above harvested (A) 1 day, (B) 3 days, and

(C) 6 days after root application. Corresponding radio-
autographs below ( a,b,c ) . . . . . . . . . . . . . . . . . 45

xii

Page

Translocation of 14C-buthidazole in corn plants. Treated

plants above harvested (A) 3 days, (B) 8 days, and (C)

16 days after application to the emerging coleoptile.
Corresponding radioautographs below ( a,b,c ) . . . . . . . . 47

CHAPTER 3

1.

Chemical structure and chemical name of the herbicide buthi-
dazole. The asterisk (*) indicates the radioactive labeled
carbon atom (14C) 0 O O O I O O O I O O I O O O O O O O O O O 66

Radioscans of thin-layer chromatograms of extracts of redroot
pigweed and corn treated with 14C-btuhidazole, 1 day after
application to the roots. The developing system was

Chloroform: Methanol (4:1) . . . . . . . . . . . . . . . . . . 68

Radioscans of thin-layer chromatograms of extracts of redroot
pigweed and corn treated with 14C-buthidazole, 3 days after
application to the roots. The developing systen was Chloro-

form: Methanol (4:1) . . . . . . . . . . . . . . . . . . . . . 70

CHAPTER 4

1.

Chemical structures of the herbicides buthidazole and tebu-
thiuron O O O O I I O O O O I O O O O I O C O O O O O O O O O 82

CHAPTER 5

1.

Chemical structures of the herbicides buthidazole and tebu-
thiuron O O O O O O O O O O O O O O O O O O I O O O O O O O O 95

CHAPTER 7

1.

Chemical structure and chemical name of the herbicide buthi-
dazole . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Electron micrographs of corn bundle sheath chloroplasts from
a) Control 96 h after treatment with water and from buthida-
zole treated plants; b) and c) 24 and 96 h after treatment
with 0.28 kg/ha and d) and e) 24 and 96 h after treatment

at 1.12 kg/ha. Bar represents 0.5 pm in a, b, e, 0.2 um in c,
and 0.25 um in d . . . . . . . . . . . . . . . . . . . . . . 130.

Electron micrographs of corn mesophyll chloroplasts and mito-
chondria from a) Control 96 h after treatment with water and

from buthidazole-treated plants; b) mitochondria 96 h after
treatment at 1.12 kg/ha, c) and d) 24 and 96 h after treat-

ment at 0.28 kg/ha, and e) and f) 24 and 96 h after treatment

at 1.12 kg/ha. Bar represents 0.5 um in a, f, 0.1 um in b, and
0.25 um in c, d, and e . . . . . . . . . . . . . . . . . . . 132

xiii

Page
CHAPTER 8

1. Chemical structures and chemical names of the herbicide
antidotes, R-25788, R-29148, CDAA, carboxin, and NA . . . . 146

xiv

INTRODUCTION
Extensive use of airelatively persistent herbicide like atrazine
[2-chloro-4-(ethylamino)-6-(isopropylamino)fgftriazine] applied repeatedly
to the same area for many years may create conditions for ecological popu-
lation expansion of resistant strains of weed species. Biotypes of common

groundsel (§enecio vulgaris L.), redroot pigweed (Amaranthus retroflexus L.),

 

 

and common lambsquarters (Chengpodium album L.) resistant to atrazine have

 

been reported (10,71,73). In a continuous corn (Egg mazg_L.) cropping system,
rotation of herbicides applied from year to year may provide an effective
means of preventing an ecological buildup of resistant strains. Therefore,
apart from the economical reasons, development of new selective herbicides
for weed control in corn is still a challenge to the herbicide industry.
Thiadiazole derivatives are chemical compounds containing the 1,3,4-
thiadiazolyl ring and they have been reported to possess phytotoxicity (49,75).
Original work in Japan revealed that l,1-dimethyl-3-(SfEEEEfbutyI-l,3,4-
thiadiazol-Z-y1)urea showed the strongest herbicidal activity among the
thiadiazolyl urea derivatives tested (49). Later on, tebuthiuron (N-[S-(1,1-
dimethylethy1)-1,3,4-thiadiazol-2-yl]f§,§7dimethylurea) and buthidazole
(3-[5-(1,1-dimethylethy1)-1,3,4-thiadiazol-2-y1]-4-hydroxy-l-methyl-Z-imi-
dazolidinone) were synthesized and developed as industrial herbicides in
united States (2,3). These two herbicides have also shown promise for
agricultural uses. Thus, buthidazole has shown potential for selective weed
control in corn following preemergence or early postemergence application at

low rates and in established alfalfa (Medicagg sativa L.) applied in fall or

 

spring. Among the plant species susceptible to buthidazole are redroot pig-
weed and quackgrass [Agropyron repens (L.) Beauv.], serious weed problems
interfering with the production of corn and alfalfa, respectively. The reason

for this selectivity of buthidazole between corn and redroot pigweed and between

alfalfa and quackgrass is not known. Differential rate of metabolism by N-
demethylation has been proposed as the main factor contributing to selecti-

vity of other thiadiazolyl urea herbicides between barley (Hordeum vulgare

 

L.) and wheat (Triticum aestivum L.) (57). However, differential absorption,
translocation, and mode of action have also been recognized as bases for
selective chemical weed control (9,27).

The margin of buthidazole selectivity between tolerant and susceptible
plant species is very narrow. Use of herbicide antidotes or protectants
offers a potential alternative for increasing the margin of selectivity (11,
68). Three compounds have received commercial interest as herbicide antido-
tes. These are NA (1,8-naphthalic anhydride), R-25788 (2,2-dichlorofi§,§f
diallylacetamide) and concep or CGAr43089 [a-(cyanomethoximino)-benzonitri-
le]. However, the concept of using herbicide antidotes has been successful
only in protecting grass species, primarily corn, against injury from
specific herbicidal groups such as the thiocarbamates and the acetanilides.
Protection of broadleaf crop plants against injury from any herbicidal group
or protection of any plant species against injury from herbicides known to
act as photosynthetic inhibitors, like the triazines and the substituted
ureas, has been unsuccessful with chemical compounds evaluated as herbicidal
antidotes (43). However, antagonistic interactions leading to protection of
broadleaf species from herbicide injury or antidoting the effect of photo-
synthetic inhibitors with other herbicides have been reported (30,51).

The objectives of this research were: a) to understand the basis for
buthidazole selectivity by studying herbicide absorption, translocation, meta-
bolism, and mode of action in corn, redroot pigweed, alfalfa, and quackgrass
and b) to broaden the margin of buthidazole selectivity in corn by using
antidotes or antagonistic interactions with other herbicides. In some of the
mode of action studies the effects of buthidazole on plant physiological

processes were compared to those of its analog tebuthiuron.

CHAPTER 1

Absorption, Translocation, and Metabolism of 14C-Buthidazole in Alfalfa

(Medicago sativa L.) and Quackgrass [Agropyron repens (L.) Beauv.]

 

 

ABSTRACT
The pattern of buthidazole (3-[5-(l,ldimethylethyl)-l,3,4-thiadiazol-
2-yl]-4-hydroxy-l-methyl-Z-imidazolidinone) uptake, translocation, and
metabolism and their potential contribution to crop selectivity were

studied in tolerant alfalfa (Medicago sativa L.) and susceptible quackgrass

 

[Agrgpyron repens (L.) Beauv.]. 14C-buthidazole at 0.1 uCi was applied to

 

a single leaf of established alfalfa and quackgrass plants and the plants
harvested 1, 3, and 14 days after application. In the root uptake study,
plants were placed for one day in Hoagland's no. 1 solution containing

10 uCi/L of 14C-buthidazole and were harvested 1, 3, and 6 days after treat-
ment. l4C-buthidazole was absorbed by leaves of both alfalfa and quackgrass
plants but did not move basipetally to the roots or nontreated leaves.
14C-buthidazole was taken up very rapidly by the roots of both species and
translocated to the leaves through the xylem. No differences in absorption
and tranlocation of buthidazole between alfalfa and quackgrass were evident,
indicating that these processes did not contribute to crop selectivity.
Apart from unmetabolized ll’C-buthidazole, five metabolites were present in
alfalfa leaf extracts 6 days after root treatment. A major unidentified
metabolite had an Rf value of 0.21 (developing system; Chloroform 80%:
Methanol 20%), and accounted for 40% of the detected radioactivity 6 days
after root application. Metabolism of buthidazole was very slow in quack-
grass. Unmetabolized 14C-buthidazole was present as 87% of the total radio-
activity even after 6 days. Two metabolites with Rf values similar to those

of the amine and dihydroxy derivatives of buthidazole were present in

higher amounts in alfalfa as compared to quackgrass, whereas a metabolite

similar to the methyl urea derivative of buthidazole was present only in

alfalfa. Thus, differential rate of metabolism appeared to be a major

factor contibuting to buthidazole selectivity between alfalfa and quackgrass.
INTRODUCTION

Buthidazole, marketed under the trade name RavageTM, is a relatively
new herbicide used for industrial and non-cropland weed control (1). Use of
buthidazole as selective herbicide for weed control in established alfalfa
is under investigation (2). Buthidazole applied in Spring of fall at rates
0.28 to 4.48 kg/ha has been found promising in controlling quackgrass, a
tough perennial weed interfering with alfalfa production (2). The basis
for this selectivity of buthidazole between alfalfa and quackgrass is not
known.

Differential absorption, translocation, and metabolism have long been
recognized as bases for selective chemical weed control (3,8). Most of the
work on uptake, translocation, and metabolism of herbicides has been done
with radioactive labeled compounds (5,7).

The purpose of this study was to determine the contribution of foliar
and root uptake and translocation, and of metabolism following root uptake
to buthidazole selectivity between alfalfa and quackgrass plants.

MATERIALS AND METHODS
Plant material. Seeds of 'Vernal' alfalfa were planted into wooden boxes
54 by 36 by 18 cm filled with a volume mixture of soil, sand, and peat
(1:1:1). The plants were grown to maturity under greenhouse conditions of
33 C day and 20 C night temperature without additional artificial light.
At flowering the plants were cut to 6 cm height, allowed to regrow to 12

cm, and placed outdoors to undergo dormancy during winter of 1978. The

plants were then transferred back to the greenhouse for acclimation, thin-
ned to one plant per 946-ml cup, and when they reached 15 to 20 cm in height,
they were used for the foliar application study. Alfalfa seedlings grown
under greenhouse conditions to a height of 8 to 10 cm, were used for the

root application study. Quackgrass plants were produced from rhizomes col-
lected after dormancy from a field in East Lansing, Michigan. The rhizomes
were placed in 946-ml waxed cups and grown under the same environmental
conditions described earlier. When they reached the height of 18 to 25 cm,
the plants were used in both the foliar and root application studies.

Foliar and root application of 14C-buthidazole. A 5 ul drop containing 0.1

 

uCi of 14C-buthidazole, labeled at the 5 carbon atom of the thiadiazole

ring (Figure l), was placed inside a lanolin ring enclosure on the second
oldest leaf of quackgrass and in the middle leaf of one of the upper trifo-
liolate leaves of alfalfa. The specific activity of 14C-buthidazole was

12.7 mCi/mmole. Following treatment, the plants were placed in a greenhouse
with the same conditions as described earlier. Treated plants were harvested
1, 3, and 14 days after foliar application of 14C-buthidazole.

For the root uptake study, alfalfa and quackgrass plants were placed
into modified Hoagland's no. 1 solution (6) containing 10 uCi/L of 14C-
buthidazole for one day and then transferred to non-radioactive Hoagland's
no. 1 solution. The growing conditions were the same as thoSe of the leaf
application study. The plants were harvested 1, 3, and 6 days after root
application.

Following both the foliar and root applications of 14C-buthidazole,
the harvested plants were divided into treated leaf, remaining shoot, and
roots. Radioactivity was determined by combustion and is expressed as dpm/

mg of leaf or root dry tissue. Translocation was determined both qualita-

tively by radioautography and quantitatively by radioassay of the various

portions of the treated plants. For the radioautography, the harvested
plants were freeze-dried, rehydrated, mounted on blotter paper, pressed,

and radioautographed. Figures are representative of two experiments with

two replications per each experiment. For the quantitative measurement of
translocation, harvested plants were freezeedried and dissected into various
sections according to the type of buthidazole application. Thus, in the
foliar application, plants were sectioned into the treated leaf, shoot and
non-treated leaves, and roots. In the roots application, the plants were
divided into shoots and leaves and into the roots. These plant portions

were homogenized in an Sorval-Omni mixer for 5 minutes in 20 ml of 100%
methanol, and each homogenate was filtered through Whatman #1 filter paper
under vacuum. Aliquots of 0.5 ml from the methanol-soluble extracts were
used for radioactivity determinations by liquid scintillation spectrometry.
The methanol-insoluble residues were combusted under 02 in a biological
oxidizer (OX-200; P. J. Harvey Instr.) and their radioactivity was again
determined by liquid scintillation radioassay (Beckman LS 8100). The results
of the radioactivity measurements were expressed as percent (Z) of the

total 14C found and as dpm/mg of plant tissue. Data presented are the means
of two experiments with two replications per experiment. Percent values less
than 15% or greater than 85% were transformed to arcsine values for analysis
of variance. Duncan's multiple range test was used for mean separation.
Extraction, Separation, and Quantitation of Buthidazole and its Metabolites.
Since uptake by roots and rapid translocation to the shoots and leaves
appeared to be the predominant pattern of buthidazole in the previous studies
with both plant species, the methanol-soluble extracts from the shoots and
leaves of the root-treated plants, obtained as described in the translocation

study, were used for the metabolism studies. Partitioning of 15 ml of the

methanol-soluble leaf extracts against 15 ml of hexane still left 95% of

the radioactivity in the aqueous fraction. One hundred ul samples from the
aqueous phase of each extract were spotted on Thin Layer Chromatography (TLC)
plates precoated with silica gel G layer 250 um thick. The plates were deve-
loped in a solvent system containing 80% chloroform and 20% methanol (4:1),
and the Rf values were determined following application of radioautography
and TLC-scanning with a Berthold LB 76 scanner with a slit width 2 mm.
Quantitation of the radioactivity present in each metabolite was done by
integration of the peaks obtained for each spot using the TLC scanner. The
results are expressed as percent of the total radioactivity found in the 100
pl sample of each extract used.

Identification of Metabolites. The TLC absorbent containing the 14C labeled
methanol-extractable spots was removed from the plate, extracted with 2 ml
of methanol, centrifuged to 500 x g, and filtered through a glass fiber
filter under vacuum. The filtrates were evaporated down to 100 pl under
nitrogen. Then the separated metabolites were co-chromatographed with known
compounds on 20 by 20 cm plates (Redi/Plate, Analtech, Inc.) coated with
silica gel CF (250 pm) in the same solvent system of chloroform:methanol
(4:1). Localization of the metabolites was achieved by exposure to ultra-
violet light (UV lamp 254 nm). Presence of small amounts of unknown l4C"
metabolites was confirmed by TLC scanning. The following buthidazole deri-
vatives used for identification were kindly provided by Velsicol Chemical
Corporation: a) Analytical grade buthidazole 98.7% pure by Infrared Spec-
troscopy (IR). b) Buthidazole-DiOH (3-[5-(l,ldimethylethyl)-l,3,4-thia-
diazol-2-yl]-4,5-dihydroxy-2-imidazolidinone), 100% pure by TLC.

c) Buthidazole methylurea (3-[5-(1,l-dimethylethyl)-l,3,4-thiadiazol-2-yl]-
l-methyl)urea, 99% pure by TLC. d) Buthidazole urea (3-[5-(l,l-dimethyl-

ethyl)-l,3,4-thiadiazol-2—yl]-urea), 99% pure by TLC and e) Buthidazole

amine [5-amino-2-(l,l-dimethylethyl)-l,3,4-thiadiazole], 99.8% pure by
Liquid chromatography.
RESULTS AND DISCUSSION

Following foliar 14C-buthidazole application, alfalfa and quackgrass
absorbed almost equal amounts of 14C-buthidazole expressed either as dme
plant or dpm/mg of dry treated leaf tissue (Table 1). When 14C-buthidazole
was supplied to the roots, quackgrass absorbed five times more herbicide
than alfalfa seedlings when the radioactivity was expressed as dpm/plant
(Table 2). However, when the results were expressed as dpm/mg of leaves or
roots, alfalfa seedlings concentrated two times more 14C-buthidazole in the
leaves andfseven times as muchlAC-buthidazole in the roots as quackgrass
did (Table 2). Therefore, the observed results suggest that the plant size
was the reason for the differences in absorption since alflafa plants used
were seedlings while the quackgrass used were older plants. Furthermore,
these results show that alfalfa seedlings rapidly take up and concentrate
buthidazole. The difference in depth of rooting between established alfalfa
and quackgrass may also contribute to selectivity of buthidazole under
field conditions.

Buthidazole did not translocate appreciably in both alfalfa and quack—
grass plants following foliar application at any harvesting period after the
treatment (Figures 2 and 3). Acropetal movement toward the tip of the treated
leaf was evident but no signs of basipetal translocation were present. Radio-
assay of various plant parts of the treated plants revealed the same pattern
of acropetal movement in both plant species (Tables 3 and 4).

Rapid translocation of 14C-buthidazole from the roots to the leaves
of both alfalfa and quackgrass plants was evident in the radioautographs
shown in Figures 4 and 5. Quantitative measurement of translocation indi—

cated that 1 day after root application of buthidazole 90 and 66% of the

radioactivity detected in both the methanol-soluble and insoluble parts
was translocated to the leaves of alfalfa and quackgrass, respectively
(Tables 5 and 6). Examining the results obtained from both the foliar and
root application studies, it appears that rapid uptake by the roots and
rapid translocation to the leaves where the herbicide exerts its action '
by inhibiting photosynthesis (4) seems to be the pattern of uptake and
translocation of buthidazole with little difference between species.

One day after root application alfalfa metabolized 14C-buthidazole
very rapidly, yielding at least five metabolites apart from the unmeta-
bolized parent compound (Table 7). The distribution of the radioactivity
detected in each metabolite revealed that an unidentified metabolite with
Rf value of 0.21 seems to be the major metabolite in alfalfa with the others
being minor. Data presented in Table 7 indicates that the amount of radio-
activity associated with this metabolite increased as a function of time,
whereas the radioactivity in the parent compound decreased along with time.
For comparison reasons, the Rf values of the analytical reference buthida-
zole derivatives used for identification are included in Tables 7 and 8,
below the unknown metabolites with Rf values similar to the respective
standard. Thus, metabolites #3, 4, and 5 with Rf values of 0.51, 0.60, and
0.64, respectively, appeared to be similar to the amine, dihydroxy, and
methylurea derivatives of buthidazole (Table 7). One more unidentified meta-
bolite with Rf value of 0.42 accounted for 17% of the total radioactivity
after 6 days (Table 7).

Quackgrass does not metabolize 14C-buthidazole rapidly (Table 8).
unmetabolized l4C-buthidazole was present as 87% of the total radioactivity
even after 6 days. An unknown metabolite with Rf value of 0.25, believed to
be similar to the major metabolite found in alfalfa, was present only in

small amounts (Table 8). Two minor metabolites with Rf values resembling those

10

of the amine and dihydroxy derivatives of buthidazole were also present in
quackgrass but in smaller amounts than in alfalfa (Tables 7 and 8). It is
clear, therefore, from the data obtained in the metabolism studies that
metabolism appears to be a major factor contributing to buthidazole selectivi-
ty' between alfalfa and quackgrass. The results of buthidazole metabolism

in alfalfa seem to be in agreement with those reported elsewhere (9). Buthi-‘
dazole metabolites in alfalfa previously reported included 5—amino-(l,l-
dimethylethyl)-l,3,4-thiadiazole, 3-[5-(1,l-dimethylethyl)-l,3,4-thiadiazol-
2-yl]-hydroxy-2-imidazolidinone and 5-(l,l-dimethylethyl)-l,3,4-thiadiazol-
2-yl)urea (9). Whether the major unidentified metabolite in alfalfa resembles
the desmethyl derivative, which has been reported as a major metabolite

in the aforementioned study (9), or another unknown buthidazole derivative

is not known yet.

In summary then , we conclude that buthidazole is absorbed by the
leaves and roots of both alfalfa and quackgrass in a similar manner. Trans-
location followed the apoplastic pathway in the xylem driven by the trans-
piration stream in both alfalfa and quackgrass plants. Rapid metabolism
of buthidazole by alfalfa seedlings appears to be the basis for the observed
selectivity of this herbicide between alfalfa and quackgrass. An unidenti-
fied metabolite with Rf value of 0.21 accounted for 40% of the total radio-
activity in alfalfa leaves 6 days after root application of 14C-buthidazole
and is believed to be very important for the observed selectivity. Quack-

grass only slightly metabolized buthidazole.

11

Figure 1. Chemical structure and chemical name of the herbicide buthidazole.

The asterisk (*) indicates the radioactive labeled carbon atom (14C).

12

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Figure 2. Translocation of l C-buthidazole in alfalfa plants. Treated plants
harvested (A) 1 day, (B) 3 days, and (C) 14 days after foliar appli-

cation. Corresponding radioautographs below ( a,b,c ).

15

Figure 3. Translocation of 14C-buthidazole in quackgrass plants. Treated plants
above harvested (A) 1 day, (B) 3 days and (C) 14 days after foliar

application. Corresponding radioautographs below ( a,b,c ).

 

l7

4
Figure 4. Translocation of 1 C-buthidazole in alfalfa plants. Treated plants
above harvested (A) 1 day, (B) 3 days, and (C) 6 days after root

application. Corresponding radioautographs below ( a,b, c ).

18

 

19

Figure 5. Translocation of l4C-buthidazole in quackgrass plants. Treated plants
above harvested (A) 1 day, (B) 3 days, and (C) 6 days after root

application. Corresponding radioautographs below ( a, b, c ).

21

 

 

 

Table l. 14C-Buthidazole absorption by alfalfa and quackgrass plants
harvested 1, 3, and 14 days after leaf treatmenta.
Harvesting l4C-uptake
Species Time Plant Treated leaf
(daYS) (dpm/ plant) (dpm/mg)
Alfalfa 1 32563 a 5617 a
3 17980 a 4448 a
14 16875 a 2954 a
Mean 22473 4340
Quackgrass 1 31937 a 4916 a
3 31915 a 2787 a
14 27652 a 3586 a
Mean 30501 3763

a Means within columns with similar letters are not significantly
different at the 5% level by Duncan's multiple range test.

22

 

 

 

Table 2. 14C-Buthidazole absorption by alfalfa and quackgrass plants
harvested 1, 3, and 6 days after root applicationa.
Harvesting 14C-uptake
Species Time Plant leaves roots
(daYS) (dpm/pl;nt) (dpm/mg) (dpm/mg)
Alfalfa 1 36787 a 15183 bc 5066 b
3 37597 a 20567 c 2213 a
6 37699 a 19054 c 1331 a
Mean 37361 18268 2870
Quackgrass 1 184124 c 6352 a 852 a
3 112352 b 11189 ab 283 a
6 157482 bc 5726 a 262 a
Mean 151319 7756 466

 

a Means within columns with similar letters are not significantly
different at the 5% level by Duncan's multiple range test.

23

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27

Table 7. Methanol-soluble metabolites of l4C-buthidazole in alfalfa

leaves and stems l, 3, and 6 days after root applicationa.

 

Days after treatment

 

Metabolite ‘ Rf value* 1 3 6

 

 

( % of total radioactivity )

Unknown #1 0.21 13.2 de 31.5 g 39.8 1

unknown #2 ' 0.42 6.6 b 9.8 c _l7.0 f

unknown #3 0.51 2.4 a 16.0 ef 12.5 cd
Buthidazole amine 0.54

Unknown #4 0.60 4.7 ab 3.8 ab 13.6 de
Buthidazole DiOH 0.62

Unknown #5 0.64 1.9 a 1.9 a 2.3 a

Buthidazole methyl urea 0.65

Buthidazole 0.71 71.2 j 37.0 h 14.8 def

 

* Deve10ping system; Chloroform : Methanol (4:1).

a Means within rows and columns with similar letters are not significantly
different at the 5% level by Duncan's multiple range test.

Table 8. Methanol-soluble metabolites of 14

28

C-buthidazole in quackgrass

leaves and stems l, 3, and 6 days after root applicationa.

 

 

Metabolite Rf value*
Unknown #1 0.25
Unknown #2 0.50
Buthidazole amine 0.54
Unknown #3 0.61
Buthidazole DiOH 0.62
Buthidazole 0.73

Days after treatment

 

 

1 3 6

( % of total radioactivity )
1.9 a 4.8 a 6.5 a
1.5 a 2.5 a 1.9 a
6.1 a 5.6 a 4.8 a
90.5 b 87.1 b 86.8 b

 

Developing system; Chloroform :

Methanol (4:1)

a
Means within rows and columns with similar letters are not significantly
different at the 5% level by Duncan's multiple range test.

29

LITERATURE CITED

Anonymous. 1976. RavageTM, herbicide for experimental use as an
industrial and non-cropland herbicide. Tech. Inf. Bulletin issued
by Velsicol Chemical Corp., Chicago, IL 6 pp.

. Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural

use. Tech. Inf. Bulletin issued by Velsicol Vhemical Corp., Chicago,
IL 5 pp.

. Ashton, F.M. and W.A. Harvey. 1971. Selective chemical weed control.

California Agr. Exp. Sta. Circ. 558. 17 pp.

Hatzios, R.K. and D. Penner. 1979. Mode of action of buthidazole.
Abstr. Weed Sci. Soc. Amer. 19:104-105.

. Hay, J.R. 1976. Herbicide transport in plants. Pages 365-396 in

L.J. Audus (ed.) Herbicides: Physiology, Biochemistry, Ecology,
Vol. 1. Academic Press, London. 608 pp.

. Hoagland, D.R. and D.J. Arnon. 1960. The water culture method for

growing plants without soil. California Agr. Exp. Sta. Circ.
347. 32 pp.

Naylor, A.W. 1976. Herbicide metabolism in plants. Pages 397‘426.$E
L.J. Audus (ed.) Herbicides: Physiology, Biochemistry, Ecology,
Vol. 1. Academic Press, London. 608 pp.

Wain, R.L. and M.S. Smith. 1976. Selectivity in relation to metabolism.
Pages 279-302 in L.J. Audus (ed.) Herbicides: Physiology, Biochemistry,
Ecology, Vol. 1. Academic Press, London. 608 pp.

Weed Science Society of America. 1979. Herbicide Handbook, 4th Edition.
p. 82.

CHAPTER 2
. 14
Site of Uptake and Translocation of C-Buthidazole in

Corn (Zea mays L.) and Redroot Pigweed (Amaranthus retroflexus L.)

 

ABSTRACT
Uptake and translocation of l4C-buthidazole (3-[5-(l,1-dimethylethyl)-
1,3,4-thiadiazol-2-y1]-4-hydroxy-l-methyl-Z-imidazolidinone) in corn (Zea

mays L.) and redroot pigweed (Amaranthus retroflexus L.) were studied

 

following both foliar and root treatments under greenhouse and growth chamber
environments. Following foliar application, 14C-buthidazole was absorbed
by the leaves of corn and redroot pigweed seedlings in similar amounts.
Translocation occured only toward the tip of the treated leaves in corn,
whereas in redroot pigweed thel4C moved both acropetally and basipetally.
Rapid uptake by the roots and rapid movement to the leaves via the xylem
seems to be the main pathway of uptake and translocation of 14C-buthidazole
supplied to the roots of redroot pigweed plants. Uptake by both the roots
and the emerging coleoptile and transport to the foliage seems to be the
pattern of absorption and translocation of buthidazole in corn following
preemergence application. Difference in absorption did not appear to be
an important factor contributing to selectivity of 14C-buthidazole between
corn and redroot pigweed. However, translocation of 14C-buthidazole supplied
to the roots was faster to the redroot pigweed shoots than to corn shoots.
INTRODUCTION

Extensive use of a relatively persistent herbicide like atrazine
[2—chloro-4-(ethylamino)-6-(isopropylamino)figftriazine] applied repeatedly
to the same area for many years may create conditions for the growth of

resistant strains of weed species. Thus, common groundsel (Senecio vulgaris

 

L.), redroot pigweed, and common lambsquarters (Chenopodium album L.) have

 

been reported to have strains resistant to atrazine (3,7,8). In a continuous
corn cropping system, rotation of herbicides applied from year to year may

30

31

provide an effective means of preventing an ecological buildup of resistant
strains. Therefore, apart from the economical reasons, development of new
selective herbicides for weed control in corn is still a challenge to the
herbicide industry.

Buthidazole (3-[5-(l,l-dimethylethyl)-l,3,4-thiadiazol-2-y1]-4-hydroxy-
1- methyl-2-imidazolidinone) is a new promissing herbicide for selective weed
control in corn applied preemergence or early postemergence (1). For herbi-
cides applied preemergence or preplant incorporated, the roots and emerging
coleoptile of grass species are important pathways of herbicide entry. In
postemergence-applied herbicides, the leaves and occasionally the roots are
important routes of entry of the herbicide.

The objectives of this study were a) to examine 14C-buthidazole absorp-
tion and translocation in tolerant corn and susceptible redroot pigweed
following foliar and root applications, b) to determine the site of buthi-
dazole uptake in corn following preemergence application and c) to determine
whether differential absorption or translocation of buthidazole plays a role
in crop selectivity.

MATERIALS AND METHODS

Foliar and Root Applications of 14C-buthidazole.

 

a. Plant material. Five 'Pioneer 3780' corn and ten redroot pigweed

 

seeds were planted 4.0 and 0.5 cm deep, respectively, into greenhouse soil
(1:1:1 soil, sand, peat) in 946-ml waxed cups. After planting, the cups
were placed in a greenhouse with temperature ranging from 20 C at night to
33 C during the day without additional artificial light. After emergence, the
plants were thinned to one plant per cup. Corn seedlings 15 cm tall and
redroot pigweed seedlings 10 cm tall were used for the uptake and trans-
location studies.

b. Application of l4C-buthidazole. Radioactive buthidazole was labeled

at the 5 carbon atom of the thiadiazole ring and had a specific activity

32

f 14C-buthidazole was

of 12.7 mCi/mM. A 5 ul drop containing 0.1 uCi o
placed on the center area of the second older leaf of corn and redroot pig-
weed seedlings. Following treatment, the plants were moved to a growth
chamber with a 16-h day and a light intensity of 19 Klux. Day and night
temperature was maintained at 25 i 1 C. Treated plants were harvested 1,

3, and 14 days after treatment. In the root uptake study the roots of corn
and redroot pigweed seedlings were placed in a modified Hoagland's (6) No.

1 solution containing 10 uCi/L of 14C-buthidazole. The plants remained

in the radioactive Hoagland's solution for only 1 day. Then they were trans-
ferred and continued to grow in cups containing Hoagland's No. 1 solution
without 14C-buthidazole. The environmental conditions throughout this study
were the same as those in the foliar application. The plants were harvested
after 1, 3, and 6 days for redroot pigweed and l, 3, and 14 days for corn.

The susceptible redroot pigweed plants were near death 6 days after treatment.
Following both foliar and root applications of l4C-buthidazole, the
treated plants were analyzed for radioactivity in the specific plant portions.

Radioactivity measurements were expressed as dpm/plant or dpm/mg of leaf

or root tissue. Translocation was determined both qualitatively by
radioautography and quantitatively by radioassay with liquid scintillation
spectrometry of the radioactivity found in various parts of the treated
plants following combustion of the plant parts. The treated plants were
divided into treated leaf, shoot and non-treated leaves, and roots follow-
ing the foliar application and into shoot and leaves and roots following

the root treatment. Photographs presented are representative of two experi-
ments each with two replications. Radioactivity determinations are expressed
as percent (%) of the total or they are calculated as dpm/mg of l4C-buthidazole.
Data presented are the means of two experiments each with two replications.

Percent data involving values less than 15% or greater than 85% were

33

transformed to arcsine values for analysis of variance. Duncan's multiple
range test was used for mean separation.
Site of Buthidazole Uptake Following Preemergence Application to Corn.
Determination of the site of buthidazole uptake by corn included three
separate studies. First, the effect of buthidazole on corn growth was ex-
amined after selective application of the herbicide to various corn root
and shoot regions. Comparison of corn injury caused by buthidazole, applied
either preemergence or preplant incorporated into the soil, was the subject
of the second study. Finally, placement of l"C-buthidazole on the emerging
coleoPtile of corn seedlings was used in the third study to determine the
contribution of absorption by the coleOptile in buthidazole uptake by corn.
In the first study a slight modification of the method reported by
Armstrong et a1. (2) was used. An activated charcoal layer separated the
root and shoot regions of corn and prevented vertical movement of the her-
bicide in the soil. Buthidazole was applied preemergence separately to
the root zone (below the charcoal layer) and on the soil surface at rates
of 0, 0.56, 1.12, and 2.24 kg/ha. Five 'Pioneer 3780' corn seeds were planted
into greenhouse soil in 946~ml waxed cups. After planting and treatment
with buthidazole, the cups were placed in a greenhouse with the same con-
ditions as in the foliar and root uptake studies. Water was applied to
provide moisture for growth of the plants. Shoot height and fresh weight
were recorded 30 days after planting and treatment with the herbicide.
In the second study buthidazole was applied either preemergence on
the soil surface or preplant incorporated into the top 6.0 cm of soil at
rates of 0, 0.56, 1.12, and 2.24 kg/ha. Five 'Pioneer 3780' corn seeds
were planted 5.0 cm deep and the cups were placed in the same greenhouse
as in the previous study. Thirty days after treatment, shoot height and

shoot fresh weight were recorded. In both the first and second studies

34

two experiments each with two replications were performed and the data
analyzed for variance followed by Duncan's multiple range test.

In the last study a 5 pl drop containing 0.2 uCi of 14C-buthidazole
was placed on the emerging coleoptile of 'Pioneer 3780' corn seedlings
as soon as they penetrated the soil surface. Following treatment, the plants
were placed in a growth chamber with the same environmental conditions
as those described in the foliar and root application studies. Treated
plants were harvested at 3, 8, and 16 days after treatment, radioautographed,
and analyzed for translocation by radioassay after dissection into four
parts. These parts were the first leaf, shoot and untreated leaves, the
primary roots, and the adventitious roots. Radioactivity determinations
were again expressed as percent (%) of the total or dpm/mg of the plant
part. Photographs and data presented are from two experiments with two
replications per experiment.

RESULTS AND DISCUSSION

Three days following foliar application, corn and redroot pigweed
had absorbed similar amounts of radioactivity, expressed as dpm/plant (Table
1). However, when the results are expressed as dpm/mg in the treated leaf,
pigweed absorbed more l4C-buthidazole than did corn (Table 1). When l4C-
buthidazole was supplied to the root system, both corn and pigweed absorbed
the same amount of radioactivity on a per plant basis (Table 2). Dividing
the plant into leaves and roots and expressing the results as dpm/mg of
leaf or root tissue indicates that pigweed concentrated more 14C-buthidazole
into the roots after 3 days than did corn (Table 2). These results indicate
that there were no substantial differences in buthidazole uptake by the
roots of the two plant species and suggest that the role of absorption by
roots was not important for cr0p selectivity following preemergence appli-

cation of buthidazole.

35

Translocation of l4C-buthidazole in corn was limited to acropetal
movement toward the tip of the treated leaf following foliar application
(Figure 1). Quantitative measurement of translocation shows also that
the bulk of radioactivity was present in the treated leaf even 14 days
after application (Table 3). On the contrary, both acropetal and basipetal

translocation of 14

C-buthidazole was evident in radioautographs from red-
root pigweed plants (Figure 2). Quantitative measurements indicate that
there were no differences in the distribution pattern of 1['C found in various
plant parts as a function of time (Table 4). Thus distribution was rapid

and occurred within 1 day. Fourteen days after treatment 65% of the total
radioactivity was present in the treated leaf, 33% was translocated to the
shoots and other leaves, and only traces were found in the roots (Table

4). Rapid uptake from the roots and translocation of 14C-buthidazole to

the leaves was observed to occur in both corn and redroot pigweed plants
following root application of the herbicide (Figures 3 and 4). Therefore,
movement of the herbicide from the roots to the leaves seems to follow pri-
marily the apOplastic route, through the xylem in the transpiration stream.
Furthermore, 14 days after treatment, the new leaves of corn plants formed
after the treatment with the l4C-labeled herbicide contained very little

or insignificant amounts of radioactivity (Figure 3). Data presented in
Tables 5 and 6 show that the percent of 14C-buthidazole or of its metabolites
increased in leaves and decreased in the roots as a function of time in

corn (Table 5), whereas in the redroot pigweed plants 97% of the radioactivity
moved to the leaves duing the first day after the treatment (Table 6).

Thus, while the pattern of buthidazole uptake and translocation seems to

be the same in corn and pigweed following root application, redroot pigweed
seems to translocate the herbicide to the shoot faster than corn. These

results are consistent with the observed selectivity.

36

Site of Buthidazole Uptake binorn Followinngreemepgence Application.
Preemergence placement of buthidazole on the soil surface reduced both
plant height and fresh weight of the new seedlings (Table 7). Placement of
buthidazole in the root zone of corn (below the charcoal layer) did not
affect corn seedlings even at rates up to 2.24 kg/ha. In grasses like corn,
the primary root lives a relatively short time and the root system is formed
by adventitious roots arising from the shoot (5). Therefore, buthidazole
absorption by the adventitious roots could have been very important in the
previous study. Buthidazole uptake by the emerging coleoptile of corn seed-
lings could also have been important in this study. However, since primary
roots were found capable of absorbing buthidazole in the root application
studies described earlier, inactivation of buthidazole by the activated
charcoal layer appears to be the most logical explanation for the abscence
of injury observed in corn seedlings following application of buthidazole
to the root zone (Table 7). Incorporation of herbicides into the upper
6.0 to 10.0 cm of soil decreased the herbicide concentration at the soil
surface, and one might expect decreased absorption by the emerging shoots.
However, comparison of preemergence application versus preplant incorporation
of buthidazole showed that preplant incorporated treatment was more active
than the preemergence treatment (Table 8) indicating that uptake by both
the primary and adventitious roots may be important. This supports the
previous conclusion that buthidazole was inactivated by the activated char-
coal layer used to separate the root and shoot zones. A study with acetanili4
de herbicides, which are absorbed primarily by newly emerging yellow nutsedge
shoots, showed that preplant incorporated treatment provided greater control
than the preemergence treatment (4). Information shown in Figure 5 and Table
9 indicates that uptake by the emerging coleoptile played a role in buthi-

dazole absorption but the bulk of radioactivity, even 16 days after treatment,

37

remained in the first leaf with the remainder in the older leaves (Table 9).
In summary, then, buthidazole was absorbed by the leaves of both
corn and redroot pigweed seedlings following foliar application but moved
only acropetally in corn in contrast to redroot pigweed in which both
acropetal and basipetal movement was observed. Rapid uptake by the roots
and rapid movement to the leaves via the xylem occured in redroot pigweed.
Uptake by both the roots and the emerging coleoptile of corn, followed by
translocation to the leaves, was the pattern of buthidazole absorption and
translocation in corn following preemergence application. Differences in
absorption did not appear to be an important factor contributing to sele-
ctivity of buthidazole between corn and redroot pigweed. However, translo-
cation of 14C-buthidazole supplied to the roots was faster to the shoots
of redroot pigweed than to corn shoots. Thus, faster translocation of
buthidazole in redroot pigweed appears to be important for the observed

selectivity.

38

14
Figure 1. Translocation of C-buthidazole in corn plants. Treated plants on

the left harvested (A) 1 day, (B) 3 days, and (C) 14 days after

foliar application. Corresponding radioautographs to the right

(a,b,c).

39

 

 

40

Figure 2. Translocation of 14C-buthidazole in redroot pigweed plants.
Treated plants on the left harvested (A) 1 day, (B) 3 days, and
(C) 14 days after foliar application. Corresponding radioautographs

on the right (a,b,c).

 

42

Figure 3. Translocation of 14C-buthidazole in corn plants. Treated plants
above harvested (A) 1 day, (B) 3 days, and (C) 14 days after root

application. Corresponding radioautographs below (a,b,c).

44

Figure 4. Translocation of 14C-buthidazole in redroot pigweed plants.
Treated plants above harvested (A) 1 day, (B) 3 days, and
(C) 6 days after root application. Corresponding radioautographs

below (a,b,c).

 

46

Figure 5. Translocation of 14C-buthidazole in corn plants. Treated
plants above harvested (A) 3 days, (B) 8 days, and (C) 16 days
after application to the emerging coleoptile. Corresponding

radioautographs below (a,b,c).

47

 

48

14

Table 1. C-Buthidazole absorption by corn and redroot pigweed seedlings

harvested 1, 3, and 14 days after leaf applicationa.

 

 

 

l4C uptake
Harvesting
time Plant Treated leaf

Species (days) (dpm/plant) (dpm/mg)
Corn 1 20079 ab 1316 a
3 24077 b 663 a
14 12720 a 397 a

Mean 18959 792
Redroot pigweed 1 17191 ab 3091 b
3 22824 b 3324 b
14 23165 b 1235 a

Mean 21060 2550

 

a Means within columns with similar letters are not significantly different

at the 5% level by Duncan's multiple range test.

49

14
Table 2. C-Buthidazole absorption by corn and redroot pigweed seedlings

harvested 1 and 3 days after root applicationa.

 

 

 

1"C uptake
Harvesting

time Plant Leaves Roots
Species (days) (dpm/plant) (dpm/mg) (dpm/mg)
Corn 1 161395 a 6116 ab 1081 a
3 194146 a 4513 a 855 a

Mean 177770 5315 968
Redroot pigweed 1 195059 a 7089 ab 1655 ab
3 177272 a 6984 ab 2373 b

186165 7037 2014

 

a Means within columns with similar letters are not significantly different

at the 5% level by Duncan's multiple range test.

50

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54

Table 7. The effect of buthidazole on 'Pioneer 3780' corn height and fresh

weight 30 days after applying the herbicide on the root zone vs spraying on the

soil surface.a

 

 

 

 

Main effects Buthidazole Zone of application Plant ht Plant fresh wt
(kg/ha) (cm/plant) (g/plant)
Root zone 52.00 b 4.25 b
1) Zone of
application Soil surface 47.73 a 3.29 a
0 53.01 c 4.52 b
2) Buthidazole 0.56 50.78 bc 4.03 b
1.12 47.95 ab 3.22 a
2.24 47.71 a 3.31 a
Interactions
0 Root zone 53.03 c 4.49 cd
0.56 Root zone 50.5 c 4.05 cd
1.12 Root zone 50.33 c 3.73 c
2.24 Root zone 54.15 c 4.72 d
0 Soil surface 53.00 c 4.55 d
0.56 Soil surface 51.07 c 4.01 cd
1.12 Soil surface 45.58 b 2.71 b
2.24 Soil surface 41.26 a 1.91 a

a
Means within the same column with similar letters are not significantly

difdferent at the 5% level by Duncan's multiple range test.

55

Table 8. The effect of buthidazole application method on 'Pioneer 3780' corn height

and fresh weight after 30 days.8

 

 

 

 

Main effects Buthidazole Method of Plant ht Plant fresh wt
(kg/ha) application (cm/plant) (g/plant)
Method of PREb 48.97 b 3.27
application PPIc 34.56 a 1.62
0 52.00 c 4.32
Buthidazole 0.56 50.76 c 3.33
1.12 36.71 b 1.68
2.24 27.59 a 0.44
0 PRE 52.00 d 4.32
Interactions 0.56 PRE 58.85 e 4.71
1.12 PRE 50.29 d 3.26
2.24 PRE 34.72 b 0.78
0 PPI 52.00 d 4.32
0.56 PPI 42.67 c 1.94
1.12 PPI 23.13 a 0.12
2.24 PPI 20.46 a 0.10

 

a Means within columns with the same letters are not significantly different at the
5% level by Duncan's multiple range test.
b PRE = preemergence.

C PPI = preplant incorporated.

56

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57

LITERATURE CITED

. Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural use.

Tech. Inf. Bulletin issued by Velsicol Chemical Corp., Chicago, IL. Spp.

Armstrong, T.F., W.F. Meggit, and D. Penner. 1973. Absorption, trans-
location, and metabolism of alachlor by yellow nutsedge. Weed
Sci. 21: 357-360.

. Bandeen, J.D. and R.D. McLaren. 1976. Resistance of Chenopodium album

 

to triazine herbicides. Can. J. Plant Sci. 56: 411-412.

. Cornelius, A.J., W.F. Meggit, and D. Penner. 1978. Yellow nutsedge

(Cyperus esculentus L.) control with acetanilide herbicides. Abstr.
Weed Sci. Soc. Amer. 18: 13-14.

 

. Esau, K. 1977. Anatomy of Seed Plants, 2nd ed. John Wiley and Sons, Inc.

N.Y. 550 pp.

. Hoagland, D.R. and D.J. Arnon. 1950. The water culture method for

growing plants without soil. Calif. Agr. Exp. Sta. Circ. 347. 32 pp.

. Peabody, D. 1973. Aatrex tolerant pigweed found in Washington. Weeds

Today. 4: 17.

. Ryan, 0.1. 1970. Resistance of common groundsel to simazine and atra-

zine. Weed Sci. 18:614-616.

CHAPTER 3
The Role of Metabolism in Buthidazole Selectivity Between

Corn (Zea mays L.) and Redroot Pigweed (Amaranthus retroflexus L.)

 

ABSTRACT
The metabolism of l4C-buthidazole (3-[5-(1,1-dimethylethyl)-l,3,4-
thiaidiazol-Z-yl]-4-hydroxy-1-methyl-2-imidazolidinone) was studied in

corn (Zea mays L.) and redroot pigweed (Amaranthus retroflexus L.) fol-

 

lowing root application. Corn and redroot pigweed seedlings were placed
for 1 day in Hoagland's no. 1 solution containing 10 uCi/L of 14C-buthi-
dazole then transferred into non-labeled nutrient solution and harvested
1, 3, and 6 or 14 days after treatment with buthidazole. In corn, metabo-
lism was also studied following application of 0.2 uCi of 14C-buthidazole
to the emerging coleoptile. The first leaf of the emerged treated seed-
lings was harvested 3, 8, and 16 days after treatment and analyzed for
buthidazole and metabolites. After partitioning of methanol-soluble
extracts from leaf tiisues against hexane, the bulk of the radioactivity
remained in the aqueous phase. Both corn and redroot pigweed metabolized
buthidazole in a similar manner but at different rates, yielding as a
major metabolite an unknown buthidazole derivative with an Rf value of
0.24 to 0.26 (developing system; chloroform:methanol, 4:1). Corn formed
this metabolite very rapidly even in the roots, whereas the buildup of
this metabolite in redroot pigweed was very slow, following root appli-
cation. Minor metabolites with Rf values similar to those of the urea and
dihydroxy derivatives of buthidazole were present in both plant species.
A metabolite with Rf value corresponding to the amine derivative of buthi-

dazole was detected in corn plants but only after application to the

emerging coleoptile. The dihydroxy derivative of buthidazole formed in

58

59

redroot pigweed seedlings appeared to be futher metabolized since the

radioactivity associated with it decreased as a function of time. A dif—

ferential rate of buthidazole metabolism in corn and redroot pigweed seems

to be very important for the observed selectivity of this herbicide.
INTRODUCTION

Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]—4-'
hydroxy-l-methyl-Z-imidazolidinone) is a new herbicide with potential for
selective weed control in corn, following preemergecne or early post—
emergence application (1).

A previous study on the pattern of l4C-buthidazole uptake and trans-
location in corn and redroot pigweed showed that redroot pigweed translo-
cated l4C-buthidazole both acropetally and basipetally whereas corn trans-
located buthidazole only acropetally into the apoplast (2). Thus, faster
herbicide movement from the redroot pigweed roots, the main site of entry,
to the leaves, the site of action on photosynthesis (3), was considered to
be an important factor contributing to buthidazole selectivity between
corn and redroot pigweed (2).

The purpose of this study was to examine the potential contribution
of metabolism in the selectivity of buhtidazole between corn and redroot
pigweed folowing root application.

MATERIALS AND METHODS

Plant material. Five 'Pioneer 3780' corn and ten redroot pigweed seeds were

 

planted 4.0 and 0.5 cm depp, respectively, into greenhouse soil mixture (1:
1:1 soil, sand, peat) in 946-ml waxed cups. After planting, the cups were
placed in a greenhouse with temperature ranging from 20 C at night to 33 C
during the day without additional artificial light. After emergence, the

plants were thinned to 1 plant per cup. Corn seedlings 15 cm tall and

60

redroot pigweed seedlings 10 cm tall were used for the metabolism studies
following root application of 14C-buthidazole. 'Pioneer 3780' corn seeds
planted into waxed cups, 1 seed per cup were used for the metabolism
study in corn, following application of l4C-buthidazole to the emerging
coleoptile.

Application of l4C-buthidazole. Radioactive buthidazole was labeled at the

 

5 carbén atom of the thiadiazole ring (Figure 1) and had a specific activi-
ty of 12.7 mCi/mM. Corn and redroot pigweed seedlings, grown as described,
were placed into cups with Hoagland's no. 1 solution (5) containing 10 uCi/

L of 14C-buthidazole. The plants remained in the radioactive solution for
only 1 day. Then they were transferred and continued to grow in cups contain-
ing Hoagland's no. 1 solution without l4C-buthidazole. After treatment, the
plants were grown in a growth chamber with a 16-h day and a light intensity
of 19 Klux. Day and night temperature was maintained at 25 i 1 C. Corn plants
were harvested 1, 3, and 14 days after treatment and redroot pigweed plants
were harvested 1, 3, and 6 days after treatment. 14C-buthidazole metabolism
was also studied in corn plants treated with a 5 ul drop containing 0.2 uCi
of 14C-buthidazole applied to the emerging coleoptile of corn seedlings as
soon as they penetrated the soil surface. The treated corn seedlings
continued to grow under the same conditions as those described for the

root application study and were harvested 3, 8, and 16 days after the
treatemnt with 14C-buthidazole. Since the results of the previous study

had revealed that following application of labeled buthidazole to the
emerging coleoptile of corn, the bulk of radioactivity was associated with
the first leaf (2), only the first leaf of the treated corn seedlings was
used for this metabolism study.

Extraction, Separation, and Quantitation of Buthidazole and its Metabolites.

Following the 14C-buthidazole treatment the plant tissues were homogenized

61

in a Sorval-Omni mixer for 5 min in 20 ml of 100% methanol. The homegenates
were filtered through Whatman #1 filter paper under vacuum and 15 m1 of

the filtrate were partitioned against 15 m1 of hexane. After partitioning,
the bulk of radioactivity (more than 95%) remained in the aqueous phase.
The roots of harvested corn and redroot pigweed seedlings were not used

for the metabolism studies because the amount of radioactivity associated
with them was very low, as reported earlier (2). Only the roots of corn
plants harvested 1 day after root application were examined for metabolism
since in the aforementioned study the amount of radioactivity associated
with them was relatively high, accounting for 40% of the total radioacti-
vity detected (2). The procedure for extracting buthidazole and metabolites
form these roots, was similar to the one used for the leaves and stems of
the treated plants. One hundred ul samples from the aqueous phase of each
extract were spotted on Thin Layer Chromatography (TLC) paltes precoated
with a silica gel G layer 250 um thick. The plates were developed in a
solvent system containing 80% chloroform and 20% methanol (4:1) and loca-
lization of the separated metabolites was determined by both radioauto-
graphy and TLC scanning by using a Berthold LB scanner with a slit width
of 2 mm. Quantitation of the radioactivity present in each metabolite was
done by integration of the area under the peak obtained for each spot,
using the TLC scanner. The results of quantitation were expressed as percent
of the total radioactivity found in the 100 pl samples of each extract.

Identification of Metabolites. The TLC absorbent containing the 14C labeled

 

methanol-extractable spot was removed from the TLC plate and extracted
with 2 ml of methanol, centrifuged to 500 g, and filtered through glass
fiber filter under vacuum. The filtrates were evaporated down to 100 pl

under nitrogen. Then the separated metabolites were co-chromatographed

62

with known compounds on 20 x 20 cm plates (Hedi/Plate, Analtech, Inc.)
coated with silica gel CF (250 um) in the same solvent system of chloro-
form to methanol in a 4:1 ratio. Localization of the metabolites was
achieved by exposure to ultraviolet light (UV 254 nm). Presence of
small amounts of unknown 14C-metabolites was confirmed by TLC scanning.
The following known buthidazole derivatives used for identification were
kindly provided by Velsicol Chemical Corporation: a) Analytical grade
buthidazole 98.7% pure by infrared spectroscopy (IR). b) Buthidazole
DiOH (3-[5-(1,1-dimethylethyl)-l,3,4-thiadiazol-2-y1]-4,5-dihydroxy-
2-imidazolidinone, 100% pure by TLC. c) Buthidazole methylurea (3-[5-
(1,l-dimehtylethyl)-l,3,4-thiadiazol-2—yl]-1-methy1)urea, 99% pure by
TLC. d) Buthidazole urea (3-[5-(1,l-dimethylethy1)-1,3,4-thiadiazol-2-
y1]-urea, 99% pure by TLC. and e) Buthidazole amine [5-amino-2-(1,1-
dimethylethy1)-l,3,4—thiadiazole], 99.8% pure by Liquid chromatography.
RESULTS AND DISCUSSION

The Rf values of the analytical reference standards for identifica—
tion of the unknown metabolites are shown in Table l. The methanol-solu-
ble metabolites detected in corn plants, 1, 3, and 14 days after root
application are shown in Table 2. Corn metabolized buthidazole very fast,
even in the roots, yielding a major metabolite with Rf value of 0.26 and
two minor metabolites with Rf values of 0.57 and 0.62, beside the unmeta-
bolized 14C-buthidazole. From the data presented in Table 2 and in Figures
2 and 3, it is clear that as the amount of radioactivity in unknown #1
increases, the radioactivity in the parent buthidazole decreases. Fourteen
days after the treatment 53% of the total radioactivity was present in the
major metabolite #1, whereas the level of the unmetabolized buthidazole
had dropped to 27% of the total radioactivity (Table 2). A two-fold increa-

se of the radioactivity in unknown #3 was also noticeable 3 days after

63

treatment and then remained constant to 14 days (Table 2). Following
application of 14C—buthidazole to the emerging coleoptile, the number

of detected metabolites of buthidazole in corn appeared to be higher,
with two new metabolites present (Table 3). The metabolite with Rf

value of 0.25 appeared again to be the major metabolite, accounting for
52% of the total radioactivity 3 days after application of l4C-buthida-
zole (Table 3). However, the amount of radioactivity associated with the
unmetabolized buthidazole and the five metabolites remained more or'less
constant with time. This may be related to the apoplastic translocation
paterrn of buthidazole in corn. The two new metabolites had Rf values of
0.46 811d 0.53, respectively. Comparison of the Rf values of the standard
reference compounds to those of the buthidazole metabolites detected in
corn (Tables 1,2,3) indicates that unknown #3 (Table 3) may be the amine
derivative of btuhidazole whereas unknowns #2 and 3 (Table 2) or unknowns
#4 and 5 (Table 3) may be the urea and dihydroxy derivatives of buthidazole.
Urea and dihydroxy derivatives of buthidazole have been reported as minor
metabolites of buthidazole in metabolism studies with sugarcane (4). Data
presented in Table 4 indicates that metabolites with Rf values similar to
those of the urea and dihydroxy buthidazole derivatives were also present
in redroot pigweed. However, the amount of radioactivity associated with
the dihydroxy derivative of buthidazole decreases with time indicating

that this metabolite might be further metabolized in redroot pigweed (Table
4). The metabolite with Rf value 0.25 appears to be the major buthidazole
derivative but its formation is very slow as compared to its formation

in corn (Tables 2,3,4 and Figures 2 and 3). Thus, 1 day after root appli-
cation unknown #1 is present only as 4% of the total radioactivity, but

this increased to 32.6% of the total at 6 days (Table 4). The unknown #2

64

with an Rf value of 0.47 (Table 4) appeared to be similar to unknown #2
formed in corn (Table 3).

The major metabolite of buthidazole in corn and redroot pigweed with
Rf values ranging from 0.24 to 0.26 (Tables 2,3,4) did not appear to have
chromatographic properties similar to any of the buthidazole derivatives
shown in Table l. Attempts to identify this metabolite have been unsuccess-
ful so far. The desmethyl derivative of buthidazole has been reported as
the major metabolite of buthidazole in sugarcane (4). N-demethylation of
other thiadiazolyl herbicides has also reported and it is considered very
important for the selective action of these herbicides in grass species
(6,7). Whether the unknown metabolite with Rf value of 0.24 to 0.26 cor-
responds to the N-demethylated derivative or to an other unknown metaboli-
te of buthidazole is not known at present.

In summary we concluded that corn and redroot pigweed metabolized
buthidazole similarly but at different rates. An unknown metabolite with
an Rf value of 0.26 to 0.26 appeared to be the major metabolite of buthida- ‘
zole in corn following both root and coleoptile applications. Formation
of this metabolite in corn was very fast, occuring even in the roots, 1
day after root application of 14C-buthidazole. The buildup of this meta-
bolite in redroot pigweed was slow apparently contributing to the observed
selectivity of buthidazole between these two species. Minor metabolites
with Rf values similar to those of the urea and dihydroxy derivatives of
buthidazole were present in both corn and redroot pigweed. A minor meta-
bolite in corn following application to the emerging coleoptile, appeared
to be the amine derivative of buthidazole. A differential rate of buthidazo-
le metabolism in corn and redroot pigweed, combined with the differential
rate of translocation, reported earlier, seem to be two very important

factors contibuting to buthidazole selectivity between these two species.

65

Figure 1. Chemical structure and chemical name of the herbicide buthidazole.

The asterisk(*) indicates the radioactive labeled carbon atom (14C).

66

BON<O_I.50

2050:3025 INJEEE I _
556»; IVA—5|? .3050}. Iv.0.— IA .>;_o_5£oE_.uI—.— VIA—I0

O
1U

= m
u m _
«5 I z\ /z I o“ /ulu lmxo

_
__ __

_
:o.

67

Figure 2. Radioscans of thin-layer chromatograms of extracts of redroot pigweed
and corn treated with 14C-buthidazole, 1 day after application to the

roots. The developing system was Chloroform : Methanol (4:1).

cpm x100

cpm x 200

68

I redroot pigweed (I clay)

 

V"

b

I
INLWWML IIIIIVII- ML!

04"! O“ 0.61 0.13

com extract ( I day ) I

 

LInLLMI . II IJ n Inw'I‘u III/LII III I#

0.0 0.26 0.51 on. o .73

Retention value ( Rf)

67

Figure 2. Radioscans of thin-layer chromatograms of extracts of redroot pigweed

and corn treated with 14C-buthidazole, 1 day after application to the

roots. The developing system was Chloroform : Methanol (4:1).

 

 

68

(N redroot pigweed (1 day)

.5
“““~

cpm x 100

U3
“'\

 

“ LILQILLLILLLL III/ILL LLL

I O 2‘! ' 64"! o“ 061 0.1

:1, corn extract ( 1 day) I

. . IA
' l
i l

I I
IIMIM WWII III L IL
051 05?.

0.73

 

I LMLLMVI

0.

 

o. as .
Retention value ( Rf)

69

Figure 3. Radioscans of thin-layer chromatograms of extracts of redroot pigweed
and corn treated with 14C-buthidazole, 3 days after application to the

roots. The developing system was Chloroform : Methanol (4:1).

 

 

 

7O

 

 

 

7, w redroot pigweed (3 days) 1‘.
7 u
(,0
3 . 3' 1
X 5 f I.
E 1
u 1., I .' i
3 .1 \
N i ' ‘
J '.
L . "i 1
2 L {M ‘ ‘ 1 r ‘
3' \ l‘ H i
1 1 ' 9“- .91 [-1 ' ' i
13%., m. A; i“ ,s‘ H iv a.“
, (‘3 '\ -i'\ l, :1 ' \ Q
g IN ‘4 \1& ‘V' 51 e VLML ; ¢ Xi. 5 y“ ‘4le
0-0 0.1% 0.41 0.55 0.0 0.13
7 i corn extract(3doys)
8‘” 1,5 I!
i:
7 I- } L
6 ’ .1 I
1
8 5 _ .' 1
N ‘ ;
x y
E 4 _ 5
D.
” i
3 b l
v. I
2 M" ,IH'F". f
i‘ p" i '
. I
i > i I i“ '
A v" ' t. i .
o n . c o ’ -. l I I H tiff I I |L l
.l._l___.l imhl_.:_____4_._.__.L__L._.LL,M_L --- --4-.- _- -._;—
o. o 0 26

35;; 0.69. o. 73
Retention value( Rt

71

Table l. Rf values of analytical reference standards used for identification

of unknown buthidazole metabolites.

 

Analytical Standard Rf value*

 

 

.Buthidazole amine 0.54
Buthidazole urea 0.56
Buthidazole DiOH 0.62
Buthidazole Methylurea‘ 0.65
Buthidazole 0.71 to 0.73

 

Developing system; Chloroform: Methanol (4:1).

Table 2. Methanol-soluble metabolites of 14C-buthidazole in corn plants 1, 3,

and 14 days after root applicationa.

 

 

Days after treatment

1 3 l4

 

 

Metabolite Rf value* Leaves Roots Leaves Leaves

 

( Z of total radioactivity )

Unknown #1 0.26 30.4 e 28.2 e 43.5 f 53.0 g
Unknown #2 0.57 2.4 ab 2.5 ab 1.2 a 5.7 bc
Unknown #3 0.62 6.9 c 3.3 b 13.3 d 13.9 d
Buthidazole 0.73 60.3 h 66.0 h 42.0 f 27.4 e

 

-- “--_ _--——

 

Means within rows and columns with similar letters are not significantly
different at the 5% level by Duncan's multiple range test.

72

Table 3. Methanol-soluble metabolites of 14C-buthidazole in the first leaf
of corn plants treated with buthidazole applied to the emerging coleo-

ptile 3, 8, and 16 days after treatmenta.

 

 

Days after treatment

 

 

 

 

Metabolite Rf value* 3 8 l6
( Z of total radioactivity )

Unknown #1 0.25 52.0 h 38.4 d 44.6 ef
Unknown #2 0.46 0.8 a 1.4 a 0.8 a
Unknown #3 0.53 0.4 a 1.9 a 0.4 a
Unknown #4 0.57 0.4 a 1.4 a 0.4 a
Unknown #5 0.63 3.6 ab 9.4 c 5.2 b
Buthidazole 0.74 42.8 e 47.5 fg 48.6 g
a Means within rows and columns with similar letters are not significantly

different at the 5% level by Duncan's multiple range test.
*

Developing system ; Chloroform : Methanol (4:1).

Table 4. Methanol-soluble metabolites of l4C-buthidazole in redroot pigweed

leaves 1, 3, and 6 days after root applicationa.

 

Days after treatment

 

Metabolite Rf value* 1 3 6

( Z of total radioactivity )

Unknown #1 0.24 4.0 a 22.4 de 32.6 f
Unknown #2 0.47 ‘ 3.7 a 6.5 abc 11.2 c
Unknown #3 0.55 1.8 a 2.4 a 5.0 ab
Unknown #4 0.61 24.7 e 18.3 d 10.6 bc

Buthidazole 0.73 65.8 i 50.4 h 40.6 g

 

 

 

73

LITERATURE CITED

. Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural use.

Tech. Inf. Brochure issued by Velsicol Chemical Corp., Chicago, IL. Spp.
l4

. Hatzios, R.K. and D. Penner. 1979. Site of uptake and translocation of C-

buthidazole in corn (Zea mays L.) and redroot pigweed (Amaranthus retro-
flexus L.). weed Sci. (In review).

 

. Hatzios, R.K. and D. Penner. 1979. Mode of action of buthidazole. Abstr.

Weed Sci. Soc. Amer. 19: 104-105. N0. 221.

Hilton, H.W. and N.S. Nomura. 1979. Metabolism of 14C-VEL-5026 in sugarcane
plants via root and foliar application. Abstr. Papers Am. Chem. Soc. 176th
meeting, PEST 106.

. Hoagland, D.R. and D.J. Arnon. 1950. The water culture method for growing

plants without soil. California Agr. Exp. Sta. Circ. 347. 32 pp.

. Lee, I.N. and K. Ishizuka. 1976. A mode of selective action of thiadiazolyl

urea herbicides. Arch. Environ. Toxicol. 4: 155-165.

. Lab, A., S.D. west, and T.D. Macy. 1978. Gas chromatographic analysis of

tebuthiuron and its metabolites in grass, sugarcane and sugarcane by-
products. J. Agric. Food Chem. 26: 410-413.

CHAPTER 4
Localizing the Metabolic Site of Action
of Two Thiadiazolyl Herbicidal Derivatives
ABSTRACT
Enzymatically isolated leaf cells from navy beans (Phaseolus vulgaris L.,
cv. 'Tuscola') were used to study the effect of buthidazole (3-[5-(1,l- '
dimethylethyl)-1,3,4-thiadiazol-2—yl]-4-hydroxy-l-methyl-2-imidazolidinone)
and tebuthiuron (N-[5-(1,l-dimethylethyl)-l,3,4-thiadiazol—2-yl]-N,N'-dimethy1-
urea) on photosynthesis, ribonucleic acid (RNA), protein, and lipid synthesis.

14

The incorporation of NaH C0 14C-uracil, 14C-leucine, and 14C-acetic acid as

3:
substrates for the respective metabolic process were measured. Time-course and‘
concentration studies included incubation periods of 30, 60, and 120 minutes and
concentrations of 0.1, 1, 10, and 100 uM of both herbicides. Photosynthesis
was very sensitive to both buthidazole and tebuthiuron and was inhibited in
30 min by 0.1 uM of both herbicides. RNA and lipid syntheses were inhibited
50 and 87%, respectively, by buthidazole and 42 and 64%, respectively, by tebu-
thiuron after 120 min at 100 uM concentration. Protein synthesis was not affe-
cted by any herbicide at any concentration or any exposure time period. The
inhibitory effects of buthidazole and tebuthiuron on RNA and lipid syntheses
may be involved in the ultimate herbicidal action of these herbicidal chemicals.
INTRODUCTION

Following the original description of a procedure for the preparation
of physiologically active tobacco mesophyll cells by Takebe §£_§1,(1), Jensen
and coworkers (2,3) developed techniques for the isolation of mesophyll cells
that could photosynthesize or absorb and incorporate protein and ribonucleic
acid precursors. Since suspensions of separated leaf cells can be handled like
bacteria or unicellular algae, they offer several advantages over whole plants

in studying the mode of action of herbicides. Thus, use of single cells in

“I I.

75

studies on the mode of action of several herbicides has already been
reported (4,5,6,7).

Compounds containing 1,3,4-thiadiazoly1 group in their molecular
structures have been reported to be phytotoxic (8,9). Original work
in Japan revealed that 1,l-dimethyl-3-(SfEEEEfbutyl-l,3,4-thiadiazol-
2-yl)urea showed the strongest herbicidal activity among the thiadiazolyl
urea derivatives tested (8). Later on, tebuthiuron (N-[S-(l,l-dimethyl-'
ethyl)-l,3,4-thiadiazol-2-yl]-N,N'-dimethylurea) and buthidazole (3-[5-
(l,l-dimethylethyl)-l,3,4-thiadiazol-2-y1]—4-hydroxy-1-methyl-2-imida204
lidinone) were synthesized and developed as industrial herbicides in the
United States (10,11). These two herbicides have also shown promise for
agricultural uses. Thus, buthidazole has shown potential for selective
weed control in corn (Egg mgy§_L.) following preemergence or early post-
emergence application at low rates and in dormant alfalfa (Medicago
sativa L.) following postemergence application (11). Tebuthiuron has been
reported promising for broad spectrum weed control in sugarcane (Saccharum

officinarum L.) following preemergence application (12).

 

The chemical structures of buthidazole and tebuthiuron are shown in Figure l.
The purpose of this research was to determine the primary metabolic
site of the herbicidal action of buthidazole and tebuthiuron by examining
the effects of these herbicides on photosynthesis, RNA, protein, and lipid
syntheses of isolated navy bean cells under various time-course and con-
centration conditions.
MATERIALS AND METHODS

Plant material. Navy bean (Phaseolus vulgaris L., cv. 'Tuscola') seeds were

 

 

grown in a mixture of vermiculite and greenhouse soil (1:1) in waxed cups
at a temperature 23 i 1 C. One day after emergence the cups were transferred

to a growth chamber with 25 i 1 C temperature and light intensity of 16 Klux

76

at the level of the primary leaves. Light was supplied by a combination
of fluorescent and incandescent lamps for a l6-hr period which was
followed by an 8-hr dark period.

.§E§$E° The maceration, wash, and incubation media were the ones used by
Ashton gt a1.(6), modified in some cases as suggested by Porter and
Bartels (5). The maceration medium for all cell preparations contained

2% macerase (Calbiochem) with 0.3% potassium dextran sulfate (Calbiochem)
and 0.7 M sorbitol at pH 5.8. The maceration medium was made up daily
prior to use from a 0.7 M stock sorbitol solution, adding the appropriate
amounts of the maceroenzyme and dextran sulfate. The wash medium con-

tained 0.65 M sorbitol, 1 mM KNO 0.2 mM KH P0 0.1 mM mgsoa, 1 mM CaCl

3’ 2 4’
1 uM KI, and 1 uM CuSO4, adjusted to pH 5.8. The incubation medium was

2’

identical to wash medium but contained 0.625 M instead of 0.65 M sorbitol.
The incubation medium was buffered with 0.05 M HEPES adjusted to

pH 7.2 with 0.1 M ROM for the photosynthesis studies. For protein, RNA,

and lipid synthesis studies, the incubation medium was buffered with

50 mM MES and adjusted to pH 5.8 with KOH. The wash and incubation stock

media were made up weekly.

Isolation of cells. Cells were isolated according to the method of

 

Jensen gt al,(2) as modified by Ashton gt al.(6) and by Porter and

Bartels (5). Primary leaves from 7- to lO-day-old bean plants wer har-
vested 4 to 5 hr after initiation of the light period, rinsed in distilled
water, blotted, deveined, and cut into 2 x 3 mm pieces. Five grams of
tissue were then vacuum infiltrated with 30 ml of maceration medium untill
they were fully infiltrated. After vacuum infiltration the leaf tissue

was filtered through a 242 um nylon net, transferred to another 30 ml

of maceration medium, and slowly stirred on a magnetic stirrer for 10 min.

The solution was again filtered through the same nylon net and the filtrate

77

was discarded. The leaf tissue was transferred again to 30 ml of macera-
tion medium and it was stirred for 50 min. The cells released during this
period were filtered again through the same net and the tissue was washed
with 20 ml of maceration medium. The released cells were centrifuged for

3 min at 60 x g at room temperature. The supernatant fraction was removed
by suction and the cells were washed three times with 10 ml aliquots of
wash medium by centrifugation at 60 x g for 3 min at room temperature.

The supernatant solution was removed by suction and the cells were made

up to a desired volume with incubation medium so that the cell prepara-
tions uséd for the assays contained 0.04 to 0.06 mg of chlorophyll per

ml, or 0.08 to 0.1 mg of chlorophyll per assay. In each assay the assaying
mixture contained 2 m1 of the cell preparation in a 25 ml Erlenmyer flask,
0.1 ml of the radioactive substrate and 0.05 ml of the herbicide solution,
making a volume of 2.15 ml. For the chlorophyll determination 1.0 m1 of the
cell suspension was added to 4 ml of 80% acetone and centrifuged. Then the
chlorophyll content was determined by the method described by Arnon (13).

Metabolic studies

 

Photosynthesis. Photosynthesis was assayed according to the method of

 

Jensen st 31. (2) as modified by Ashton gt al.(6). The cells were incubated
14 12

with 5 uCi NaH CO3 (sp. act. 10 mCi/mmole) containing 6.0 uM of NaH C03.
The erlenmyer flasks with the assay mixtures were sealed and placed in

a shaking waterbath at 25 C. The flasks were illuminated from above with

a combination of fluorescent and incandescent lamps with an intensity of
4.5 Klux at the level of the flasks. After the specific incubation periods
used, a 100 pl sample was removed with a pipet and placed on a 2.3 cm
Whatmann 3 uM filter paper disc. The discs were dried under a hair dryer,

acidified with 100 pl of 88% formic acid and dried again for 1 hr. Radio-

activity was determined by radioassaying the discs by liquid scintillation

78

spectrometry. Photosynthesis was calculated as cpm of 14002 fixed per mg

of chlorophyll.

Protein and RNA synthesis. Incorporation of 14C-leucine and l4C—uracil
was determined by the method of Francki gt a1. (3) as modified by Ashton
35 a1. (6). One uCi of L-[U-IACI-leucine (sp. act. 70 mCi/mmole) and 5 uCi
of [Z-IACJ-uracil (sp. act. 65 mCi/mmole) were added to the cells.
Incubation conditions were the same as described for photosynthesis.
Five hundred ul samples were collected and added to 1.9 m1 of ice-cold
12% trichloroacetic acid (TCA) containing 50 mM L-leucine for the protein
synthesis and 30 mM uracil for the RNA synthesis study and left overnight
at 4 C. The protein and ribonucleic acid precipitates were then collected
by filtering through 2.1 cm glass fiber filter discs (Arthur Thomas),
washed three times with ice-cold 10% TCA, three times with 80% ethanol,
once with acetone, and twice with diethyl ether. The discs were then

put in vials, dried in an oven for 30 min and the radioactivity determined
by liquid scintillation spectrometry. Protein and RNA syntheses were
calculated as cpm per mg of chlorophyll.

Lipid synthesis. Lipid synthesis was determined by the method of Ashton
33 a1. (6). One uCi of [1,2-14C]acetic acid, sodium salt (sp. act. 56.2
mCi/mmole) was added to the cells. Incubation conditions were the same as
in photosynthesis. Five hundred-pl samples were collected in 2 m1 of

0.35 M H2804 and 0.05 M CHBCOOH in conical centrifuge tubes. The samples
were allowed to sit in the acid for at least 15 min and they were centri-
fuged for 10 min at 160 x g at room temperature. The supernatant fraction
was removed by auction and 4 ml of CHClB/CH3OH (2:1) was added and mixed.
The tubes were stoppered and left overnight at room temperature. Two m1

of distilled water was added and the mixture was centrifuged for 5 min

at 160 x g at room temperature. The top layer was removed by suction.

79

This procedure was repeated three times. The chloroform solution was
filtered through glass fiber filter discs into vials and the discs were
washed two times with CHClB/CHBOH (2:1). The filtered solution was dried
under a current 6f air, and the radioactivity in the lipid fraction was
determined by liquid scintillation spectrometry. Lipid synthesis was
calculated as cpm per mg of chlorophyll.

Radioactivity determination. Radioactivity was determined by adding 10 ml

 

of Aqueous Counting Scintillant (ACS, Amersham) to samples for radioassay
with a Beckman LS 8100 Liquid Scintillation Spectrometer.
Time-course and concentration studies with buthidazole and tebuthiuron.
Analytical grade (more than 99% pure) buthidazole and tebuthiuron were
diluted in 5 m1 of ethanol and made up to volume with distilled water.
Herbicide concentrations of 0.1, l, 10, and 100 uM were used in all assays.
The assay mixtures in all studies were incubated for time periods of 30,
60, and 120 minutes. All experiments were repeated three times. Data
presented are the means of these three experiments analyzed for analysis
of variance in a two-way factorial design. Duncan's multiple range test
was used to separate the means.
RESULTS AND DISCUSSION

The effects of buthidazole and tebuthiuron on four metabolic pro-
cesses of isolated bean cells were examined (Tables 1 through 4). Inhibition~
of photosynthesis was very rapid, reaching maximum levels in 30 min
incubation time with the high concentrations of both herbicides. However,
in the case of the low concentration 0.1 uM, the inhibition rate increased
from 27 and 30% after 30 min to 44 and 46% after 60 min of incubation
with buthidazole and tebuthiuron, respectively (Table 1). Although the
results clearly suggest that both herbicides are strong photosynthetic

inhibitors at high concentrations, the inhibition rates obtained are

80

somewhat lower than those reported by Ashton gt a1.(6) for other well-
known photosynthetic inhibitors such as atrazine, bromacil, and monuron
used at the same concentrations. However, buthidazole and tebuthiuron
caused greater inhibition of photosynthesis at the low concentrations

of 0.1 uM compared to the aforementioned herbicides in the study by
Ashton EE.21° (6). Differences in absorption or subcellular transport,
‘although not documented, may account for the observations. In another
study, both buthidazole and tebuthiuron were found comparable to atrazine
and diuron as inhibitors of photosynthetic electron transport in isolated
spinach chloroplasts (14).

Significant inhibition of RNA synthesis was found to be caused by
buthidazole at l, 10, and 100 uM and tebuthiuron at 10 and 100 uM (Table
2). Inhibition of RNA by buthidazole and tebuthiuron did not appear to be
a function of the incubation time period since the inhibition percentages
remained unchanged for all incubation times (Table 2).

Protein synthesis was not affected by any herbicide even at the
maximum concentration and maximum exposure time (Table 3). This appears
to be an exception to the behavior of photosynthetic inhibitors used as
herbicides as reported by Ashton 33 a1. (6) where they found significant
inhibitions of protein synthesis by atrazine, bromacil, and monuron at
high concentrations.

Lipid synthesis was inhibited significantly by the high concentrations
(10 and 100 uM) of both herbicides reaching levels of 84 and 64% inhibition
for buthidazole and tebuthiuron at 100 uM, respectively (Table 4). A slight
stimulation of lipid synthesis by the lower concentration of 0.111M, which
inhibited photosynthesis, was found with tebuthiuron at any incubation
time and with buthidazole at the maximum exposure time. This agrees with

the results of Ashton_g£.al. (6) for other herbicidal photosynthetic

81

inhibitors.

The lowest concentration of buthidazole and tebuthiuron that inhi-
bited any of the four metabolic processes studied was 0.1 uM which at
120 min inhibited photosynthesis 43 and 35% respectively (Table 1).
Protein and RNA syntheses are essentially unaffected by this concentration
at any exposure time (Tables 2 and 3). At the highest concentration of 100
uM and maximum exposure time of 120 min, photosynthesis was inhibited 87 and
81% , RNA synthesis 50 and 42%, protein synthesis 11 and 10%, and lipid
synthesis 84 and 64% by buthidazole and tebuthiuron, respectively.

The results of this study indicate that both buthidazole and tebuthiu-
ron act in a similar manner. Photosynthesis was the most sensitive and first
metabolic process inhibited. The inhibitory effects on RNA and lipid syntheses
caused by both herbicides at high concentrations may be involved in the ulti-
mate herbicidal action of buthidazole and tebuthiuron. Protein synthesis was

not affected by any of these herbicides.

82

3-[s-m-dimchylochyl)-1,3,4-:hiadiazoI-2-yl1-4-hydroxy-
l-methyI-2-imidozolidinone

buthidazole

N—[5-( l,l-dimethylethyl )- 1,3,4- thiadiazoI—Z- yIJ- N, N'-
dimeihylu reo
tebuthiuron

Figure]. Chemical structures of ihe herbicides buthidazole
and tebuthiuron

83

Ho>ma Nm onu um ucoHQMMHm maucmofimacmwm uo: mum muouuma umHHEHm :uH3

.umou owamu onHuHoE m.:mocoo an

mcssaoo casuwa mono: m

 

 

HO on OOOH OOH HO OH «Ohm OOH

we Ou OONO OH OO on OOOH OH

HH w OOmON H OH O HOHH H ONH

mm ; OHOHN H.O OH O ONOOH H.O

O H HHHOO O O H OHOHH O

an on HOOO OOH OO OH OOON OOH

HO OO OHOHH OH OO Om HOOH OH

HO HO OOHOH H an Oo Omem H OH

OH H HOOOH H.O HH H OOOOH H.O

O H OONOO O O O HHHHN O

HO O HONN OOH OO O OOOH OOH

me an HOOO OH HO O ONOH OH

OH Oo OHOO H Ne OH NOON H Om

Om Ou HNOO H.O NH O HHOO H.O

O HO HHOHH O O o HHOO O

HNV HHOO Os\sOoO Haze HNO HHOO ws\eOoO Hznv HOHeO
OOHHHOHOOH OOHumxHO OOHOHHHOOOO OOHOHOHOOH OOHquHO OHONHOHHOOO msHau-

Nomad NOOHH cowumnsooH

 

 

.mmaaoo coon moumaomfi mo mammcuozm0uona co cousfinuonou can maonmofinuon mo uomwwo one .H manme

84

HO>OH NO OOH um HOOOOOOHO

.ummu owcmu vanguaoa m.:mo:=a xn

haucmuawfiawwm uo: mum muouuma umHHBHm suds mcsoaoo casufia memo: m

 

 

 

OH mO ONOOH OOH Om Om OOOOH OOH

OH O HOOOH OH Om OOO OOOOH OH

O O OHOOO H OH HO OOOHO H OOH

O O NOOHH H.O O OH OOOOO H.O

O O ONNON O O H OOOOO O

HO OOO OOOOH OOH Om OOH OOOOH OOH

OO Ou OOHNH OH HO OOOO HOHOH OH

O OO OOHOH H ON OOO HOOOH H OO

OH HO HHHOH H.O HH OOO OOOOO H.O

O O OOOHH O O HO OOHOO O

OO O HOOO OOH OH O HOOOH OOH

OH HO OOOO OH Om Om OHOHH OH

HH on OOOO H OH OOO OOOOH H OO

O on OOOOH H.O OH OOO OHOHO H.O

O OO OOHHH O O HO OHOOO O

HOV HHOO Oa\eOuO Hznv HOV HHOO Oe\aOuO Hzav HOHsO
I coauunwnaH woumuomuooca :onownusnoh cowuanfion woumuoauoocH maonmofisuom oEHHII

afiomuoloca Hwomuoloqa coaumnoucH

II .mmHHmu coon omumHomH mo mammnuahm <zm co couofisusamu mam oaowmmfinusn mo uoomwo one .N manna

85

HO>OH NO OOH OO

.ummu owomu oamauaoa m.:moosn he
acouowmao haucmoHMHowfim uoc mum muouuoa HOHHEHO Saga mqasaoo afieuws memo: m

 

 

OH OO OOOHO OOH HH O OOOOO OOH

H OO OOOOO OH O u OOHOO OH

O OO OOHOO H O OO OHOOO H OOH

O OO OOHOO H.O O OO OOHOO H.O

O O HOOOO O O O OOOOO O

H OO OOOHO OOH O O OOHOH OOH

O OO OOOHO OH O O ONOOH OH

O OO OOOHN H H O HOOOH H OO

O OO OOOOO H.O H O HOOOH H.O

O OO OOOHN O O O HHHOH O

OH O HOHOH OOH HH O OHOOH OOH

O O ONOOH OH O O HHOHH OH

O O OOOOH H O O OHOHH H on

O O HOOOH H.O H O OOHNH H.O

O O OOOOH O O O OOOOH O

HOV AHOO Oa\aOOO Hznv HOV HHOO Oa\sOOO Hznv . HOHaO
It coauwnwsaH moumuoauooaH cognanuonoh coeufinasaH woumuoahoocH oaoumvwzusm oawhli

mawosoaloca ocwosoaloea coaumnsoaH

 

.mmHHou coon vmumHomH mo mamonuchm cfiououm co aouowsuon0u poo oaoumvwnuon mo uoommo 059 .m manna

86

.umou omcmu oaowuaoa m.omuoan me

 

 

 

Ho>oH Mm onu um ucouowMHQ hfiucmowmacwfim no: mum mumuuma umawaam saga moeoaou ofisufia ammo: m
HO OO HOOO OOH HO O OONH OOH
NH OOO OOOO OH HN OO OHON OH
HH OO NOHOH H OO O OHON H ONH
O O HOHNH H.O O O HHNHH H.O
O O HHOOH O O O NOHHH O
OH OO HOHH OOH OO O OONH OOH
ON OO NONO OH HO O OOON OH
N OOO ONOO H NO OOO ONOH H OO
O OOO OONHH H.O N OO OONO H.O
O OOO HOHO O O OO OHOO O
HH O OOOO OOH OO O ONON OOH
ON OO HOHH OH NO OOO ONOO OH
O OO NOHO H ON OOO HNNH H OO
O OOO NOOw H.O N OO OOOH H.O
O OO ONOO O O OO OONO O
HNO HHOO Oa\aOOO HOOO HNO HHOO OOHOOOO HOOO HOHOO
coauwnanoH woumuwauoucm cou=H£u=nmh cofiuanancH voumuoouooaH «Houmvwsuam mafia
oumuousiru.H mumuoomloqa coaumnousH
.omHHou some voumaomw mo mumonuchm vHoHH so consanusnmu pom oaonmpHSusn mo uomumo one .q manna

10.

11.

12.

87

REFERENCES

. Takebe,I., Y. Otsuki, and S. Aoki. 1968. Isolation of tobacco meso-

phyll cells in intact and active state. Plant Cell Physiol. 9:115-
124.

Jensen,R.G., R.I.B. Francki, and M. Zaitlin. 1971. Metabolism of
separated leaf cells I. Preaparation of photosynthetically active
cells from tobacco. Plant Physiol. 48: 9-13.

Francki,R.I.B., M. Zaitlin, and R.G. Jensen. 1971. Metabolism of
separated leaf cells II. Uptake and incorporation of protein and
ribonucleic acid precursors by tobacco cells. Plant Physiol. 48:
14-18 e

. Kulandaivelu,G., and A. Cynanam. 1975. Effect of growth regulators

and herbicides on photosynthetic partial reactions in isolated
leaf cells. Physiol. Plant. 33: 234-240.

. Porter,E.M.,and P.G. Bartels. 1977. Use of single leaf cells to

study mode of action of SAN 6706 on soybean and cotton. weed
Sci. 25: 60-65.

Ashton,F.M., O.T. DeVilliers, R.K. Glenn, and W.B. Duke. 1977.
Localization of metabolic sites of action of herbicides. Pestic.
Biochem. Physiol. 7: 122-141.

Malakondaiah,N., and S.C. Fang.1&979. Differential effects of phenoxy
herbicides on light-dependent CO fixation in isolated cells of

C3 and C4 plants. Pestic. Biochem. Physiol. 10: 268-274.

Kubo,H. R. Sato, I. Hamura, and T. Ohi. 1970. Herbicidal activity of
1,3,4-thiadiazole derivatives. J. Agr. Food Chem. 18:60-65.

Schafer,G., A. Trebst, and K.H. Buchel. 1975. 2-anilino-1,3,4-thia-
diazoles, Inhibitors of oxidative and photosynthetic phosphorylation.
Z. Naturforsch. 30c : 183-189.

Anonymous. 1975. Technical report on Spike, experimental herbicide.
Brochure issued by Lilly Research Laboratories, Indianapolis, IN, p.6.

Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural use.
Technical Inf. Brochure issued by Velsicol Chemical Corp. Chicago, IL.
p. 5.

Pafford,J.L. and C.D. Hobbs. 1974. Tebuthiuron: A new herbicide for
premergence weed control in sugarcane. Abstr. weed Sci. Soc. Amer.
14: 114. Abstr. No 266.

88

13. Arnon, D.J. 1949. Copper enzymes in isolated chloroplasts. Polyphenol-
oxidase in Beta vulgaris L. Plant Physiol. 24: 1-15

 

l4. Hatzios, R.K., D. Penner, and D. Bell. 1979. Inhibition of photosynthe-
tic electron transport in isolated spinach chloroplasts by two 1,3,4-
thiadiazolyl derivatives. Plant Physiol. Suppl. 63(5): 41.

CHAPTER 5
Inhibition of Photosynthetic Electron Transport in Isolated
Spinach Chloroplasts by Two 1,3,4-Thiadiazolyl Derivatives
ABSTRACT
Buthidazole (3-[5-(1,1-dimethylethy1)-l,3,4-thiadiazol-2-y1]-4-
hydroxy-l-methyl-Z-imidazolidinone) and tebuthiuron (N-[S-(l,l-dimethyl-
ethyl)-l,3,4-thiadiazol-2-yl]-N,N'-dimethylurea), are two new promiéing
herbicides for selective weed control in corn (223 gays L.) and sugar-

cane (Saccharum officinarum L.), respectively. The effects of these two

 

compounds on various photochemical reactions of isolated spinach (Spinacea
oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5,
5, and 500 uM. Buthidazole and tebuthiuron at concentrations higher than
0.5 uM inhibited uncoupled electron transport from.water to ferricyanide
or to methylviologen very strongly. Photosystem II-mediated transfer of
electrons from water to oxidized diaminodurene, with 2,5-dibromo-3-methyl-
6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited

34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 uM.
Inhibition of photosystem I-mediated transfer of electrons from diamino-
durene to methylviologen, with 3,4-dichlorophenyl-l,1-dimethylurea (DCMU)
blocking photosystem II, was insignificant with both herbicides at any
concentration tested. This suggests that both buthidazole and tebuthiuron
do not inhibit electron transport through photosystem I. Transfer of ele-
ctrons from catechol to methylviologen in hydroxylamine-washed chloroplasts
was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at
0.5 uM. The data indicate that the inhibition of electron transport by both
herbicides is primarily at the reducing side of photosystem II. However,
since catechol is an electrbn donor at the oxidizing side of photosystem

II, between water and chlorophyll 3680’ and lower inhibition levels were

89

90

observed in the last study (catechol to methylviologen), it may be that
there is also a small inhibition of the mechanism of water oxidation by
both herbicides.
INTRODUCTION

Substituted 1,2,4- and 1,3,4-thiadiazoles have been reported to
possess herbicidal activity (5,10). In the case of 1,3,4-thiadiazoles,
this activity was found to be strongly associated with the 5-(l,l-dimethyl-
ethyl)-l,3,4-thiadiazole nucleus of the molecule (10). At present two
5-(1,l-dimethylethyl)-l,3,4-thiadiazolyl derivatives are marketed com-
mercially as herbicides for industrial weed control under the common names
buthidazole and tebuthiuron (Figure 1). These two herbicides have also
shown promise for agricultural uses. Thus buthidazole (3-[5-(1,1-dimethyl—
ethyl)-l,3,4-thiadiazol-2-yl]-4-hydroxy-l-methyl-2-imidazolidinone) has
shown promise for selective weed control following preemergence application
in corn (Egg gays L.) and postemergence application in established alfalfa

(Medicagg sativa L.) (l). Tebuthiuron (N-[5-(1,l-dimethylethyl)-1,3,4-

 

thiadiazol-Z-yl]-N,N'-dimethylurea) has exhibited potential for rangeland
brush control (4) and for broad spectrum weed control in sugarcane (Saccha-

rum officinarum L.) (14).

 

Inhibition of photosynthesis appears to be involved in the action of
1,3,4-thiadiazolyl herbicides. Thus buthidazole inhibited corn photosynthesis
in zigg following either pre- or post-emergence application (8). Prevention
of starch accumulation in bundle sheath chloroplasts and some ultrastru-
ctrural disruption of mesophyll chloroplasts of corn plants treated with
buthidazole applied postemergence were also observed in the previous study
(8). Phytotoxicity symptoms suggested that inhibition of photosynthesis is
also the mode of action of tebuthiuron (2).

Interference with photo-induced electron transport and coupled

91

phosphorylation reactions mediated by isolated chloroplasts has been studied
extensively (11) and used as a means to explain the mechanism of action of
many structurally diverse herbicides known to act as photosynthetic inhi-
bitors (7). Electron transport and photophosphorylation were found to be
inhibited by 1,2,4- and 1,3,4-thiadiazole derivatives in assays with iso-
lated chloroplasts (5,15,17). The purpose of this study was to examine the
effects of buthidazole and tebuthiuron on the electron transport chain of
isolated spinach chlorOplasts and to locate the site of the inhibition by
segmenting the photosynthetic electron transport pathawy.

MATERIALS AND METHODS

Chloroplast isolation. Chloroplasts were isolated from commercial spinach

 

(Spinacea oleracea L.) obtained from a local market. Leaves were washed

 

with cold distilled water and ground in a Wering blendor for 5 sec in a
medium containing 0.3 M.NaC1, 30 mM tricine-NaOH buffer (pH 7.8), 3 mM
M3012, and 0.5 mM EDTA. The homogenate was filtered through eight layers

of cheesecloth and the chloroplasts were sedimented at 2500 g for 2 min.
The chloroplast pellet was then resuspended in a medium consisting of 0.2 M
sucrose, 5 mM HEPES-NaOH buffer (pH 7.4) and 2 mM MgC12. After a 60-sec
centrifugation at 2000 g to remove cell debris, the chloroplasts were
centrifuged again (3000 g for 3 min) and finally suspended in a few milli-
liters of the above suspending medium. Chlorophyll content was determined
spectrophotometrically by the method of Arnon (3). All operations were
conducted at 0 to 5 C temperature.

Hydroxylamine-treatment of isolated chloroplasts. In the assay of electron
transport from catechol to methylviologen the chloroplast suspensions used,
were washed with hydroxylamine in order to eliminate flow of electrons from
water to methylviologen. Hydroxylamine treatment of chloroplasts was per-

formed according to the method of Izawa and Ort (9). Two m1 of chloroplast

92

stock suspension prepared as described in the previous paragraph were added
to 20 ml of a freshly prepared medium containing 0.2 M sucrose, 5 mM HEPES-
2, 5 mM NHZOH and 1 mM EDTA. The mixture was
allowed to stand at room temperature (22 C) for 20 min, then diluted with

NaOH buffer (pH 7.4), 2 mM MgCl

cold, NHZOH-free suspending medium, and centrifuged at 4000 g for 5 min at
O C. The chloroplasts were washed twice by centrifugation (4000 g for 5 min,
0 C) with a large volume of the suspending medium to remove NHZOH and EDTA,

and finally suspended in NH OH-free suspending medium.

2

Electron transport assays. Artificial or unnatural electron acceptors and

 

donors have been frequently used in studies of partial reactions of the
photosynthetic electron transport mediated by isolated chloroplasts (12,16).
In this study uncoupled electron transport from water to ferricyanide and
photosystem II-mediated electron transport were assayed spectrophotometrical-
ly..;by recording the rate of ferricyanide reduction at 420 nm. Uncoupled
electron transport from water to methylviologen, photosystem.I-mediated
electron transport and whole chain electron transport from catechol to
methylviologen were assayed as 02 uptake resulting from aerobic reoxidation
of reduced methylviologen. A membrane-covered Clark-type electrode was

used for these 02 assays. In all assays light for illumination of the
chloroplast preparations was provided by the 500-watt incandescent lamp of
a slide projector with a l-liter round bottomed flask with diluted CuSO4
acting as condenser and heat filter. The light was then passed through a
broad band red glass filter (transmission greater than 600 nm)‘before it
impinged on the reaction cuvette. The reaction conditions in each of the
assays were as described in Tables I through IV. In all assays the
reaction volume was made up to 2.0 ml with distilled water and the reaction
temperature was 18 C. Analytical grade buthidazole (100% pure) and tebuthi-

ron (99% pure) were used at concentrations 0, 0.05, 0.5, 5, and 500 uM.

93

RESULTS AND DISCUSSION

The effects of buthidazole and tebuthiuron on uncoupled electron
transport assayed in two ways are shown in Table I. Both compounds at
concentrations 0.5 uM and higher inhibited electron transport from water
to ferricyanide or methylviologen very strongly (Table I). The inhibitions
by the concentrations of 0.05 and 0.5 uM of both herbicides were progressive,
indicating a dependence of the ractions on time. Thus preincubation of the
reaction mixture with the herbicide in the dark was necessary before
initiation of the light reaction. Therefore, electron transport rates for
the reactions containing 0.05 and 0.5 uM herbicidal concentrations cor-
respond to reactions preincubated for 5 min in the dark before exposure
of the chloroplast preparations to the light.

Photosystem II-mediated transfer of electrons from.water to oxidized
diaminodurene, with DBMIB acting as block of photosystem I, was also '
inhibited very strongly by both herbicides at concentrations 0.5 uM or
higher (Table II). The inhibition levels obtained with buthidazole and
tebuthiuron at 0.05 uM were 34 and 37% respectively.

Inhibition of photosystem I-mediated electron transport from ascorbic
acid Idiaminodurene to methylviologen, with DCMU acting as block of
photosystem II, was insignificant with both herbicides at any concentration
examined (Table III). This suggests that both buthidazole and tebuthiuron
do not inhibit electron transport through photosystem I.

Data presented in Tables I, II, and III indicate that the inhibition
of electron transport by both buthidazole and tebuthiuron is primarily at
the reducing side of photosystem II, between Q, the unknown primary electron
acceptor for photosystem II, and plastoquinone. Thus the site of buthi-
dazole and tebuthiuron inhibition of photosynthesis appears to be the

same or very near the site of action of diuron and atrazine (7). This is

94

not altogether suprising in that the two herbicides tested here are,
like diuron, substituted ureas.

Finally, examination of whole chain of electron transport from
ascorbate/catechol to methylviologen in NHZOH-treated chloroplasts indi-
cated that again electron transport was strongly inhibited by both herbi-
cides (Table IV). However, the inhibition levels caused by both herbicides
in this assay were somewhatLIess than those obtained in the uncoupled
electron transport from water to methylviologen (Tables I and IV). Thus
the inhibition levels by buthidazole and tebuthiuron at 0.5 uM were 50
and 47%, respectively, as compared to the 89% inhibition levels of the
uncoupled electron transport by the same concentration of both herbicides.
Since catechol is an electron donor at the oxidizing side of photosystem
II, between water and chlorophyll 3680’ these last results indicate that
there might be a small inhibition of the mechanism of water oxidation by
both herbicides. This possibility of a secondary site of inhibition on the
oxidizing side of photosystem II has also been reported by York and
Arntzen (17) who came to the same conclusion on the basis of fluorescence
measurements of the effect of buthidazole on electron transport reactions
of isolated pea chloroplasts. However, it is also possible that the
lower levels of inhibition obtained in the last study might be a consequence
of some nonbiological photo-oxidation of catechol or ascorbate, which is
not inhibited by the herbicides.

In conclusion, both buthidazole and tebuthiuron inhibited photo-
synthetic electron transport in 33552. This inhibition was primarily at
the reducing side of photosystem II with a small inhibition of the mecha-

nism of water oxidation by both herbicides.

95

OH

I

2

CH- N- CH3
3-C

3C\ SC/ -N\Cl/
3-[5-i1,1-dimethylethyl)-1,3,4-thiodiozol-2-yl1-4-hydroxy-
I-methyl-2-imidozolidinone
buthidazole

N—N

‘1“3“ II 1'3

CH -C|-C -N-C— N-CH
3_ (:S\ /C II 3

CH3

N—[5-( 1,1-dimethylethyl )-1,3,4- thiadiazol— 2- yl ]- N, N'-
dimethylu reo
tebu thi uron

Figure]. Chemical structures of the herbicides buthidazole
and tebuthiuron

96

Table I. Effects of buthidazole and tebuthiuron on uncoupled electron
transport in illuminated spinach chloroplasts.
Reaction conditions:

50 mM tricine-NaOH buffer (pH 8.0), 2 mM MgCl 0.2 M sorbitol,

2!
0.5 mM ferricyanide or 0.5 mM methylviologen, 2 ug/ml gramicidin,

and 15 or 7.5 pg of chlorophyll per reaction mixture.

 

 

 

 

 

Herbicide Photosynthetic Photosynthetic methyl-
concentration reduction of % viologen-mediated O2 %
(HM) .. ferricyanide1 Inhibition uptake2 Inhibition
Control 1177 0 275 0
Buthidazole
0.05 941 20 130 53
0.5 102 91 29 89
5 18 98 ll 96
500 10 99 7 98
Tebuthiuron
0.05 906 23 117 58
0.5 112 90 30 89
5 32 97 13 95
500 14 99 7 98
1

Data expressed as u moles of ferricyanide reduced/hr/mg of chlorOphyll.
Data expressed as u moles of 02 consumed/hr/mg of chlorophyll. To
compare with electron transport rates in ferricyanide reduction

values must be multiplied by 4.

Table 11. Effects of buthidazole and tebuthiuron on photosystem II-mediated

electron transport in illuminated spinach chlorOplasts.

Reaction conditions:

50 mM tricine-NaOH buffer (pH 8.0), 2 mM MgCl

2’

0.2 M sorbitol,

2 mM ferricyanide, 0.5 mM diaminodurene, 0.5 uM DBMIB, and 15 pg

of chlorophyll per ml of reaction mixture.

 

 

 

 

Herbicide Buthidazole-treated Tebuthiuron-treated
concentration Electron trans- % Electron trans— %
(uM) port rate1 Inhibition port rate1 Inhibition
Control 798 0 798 0
0.05 525 34 500 37
0.5 148 82 176 78
5 18 98 37 95
500 0 100 0 100

 

Data expressed as u moles of ferricyanide reduced/hr/mg of chlorophyll.

98

Table III. Effects of buthidazole and tebuthiuron on photosystem I-mediated
electron transport in illuminated spinach chlorOplasts.
Reaction conditions:

50 mM tricine-NaOH buffer (pH 8.0), 2 mM MgCl 0.2 M sorbitol,

2’
0.1 mM.methylviologen, 2.5 mM diaminodurene, 2.5 mM ascorbate,

1.5 uM DCMU, and 7.5 ug of chlorophyll per ml or reaction mixture.

 

 

 

 

 

Herbicide Buthidazole-treated Tebuthiuron-treated
concentration Electron trans- 2 Electron trans- 2
(uM) port rate1 Inhibition port rate1 Inhibition
Control 1165 O 1165 0
0.05 1101 5 1065 9
0.5 1225 -5 1191 -2
5 1264 -8 1176 -l
500 1230 -6 1187 -2
1

Data expressed as u moles of O consumed/hr/mg of chlorOphyll. The values

2
given must be multiplied by a factor of between 1 and 2, depending on the
endogenous superoxide dismutase and catalase activities to obtain a mea-

sure of the true electron transport in the chlorOplasts (13).

99

Table IV. Effects of buthidazole and tebuthiuron on electron transport
in hydroxylamine-treated and illuminated spinach chlorOplasts.
Reaction conditions:

50 mM tricine-NaOH buffer (pH 8.0), 2 mMngCl 0.2 M sorbitol,

2’
0.5 mM ascorbate, 0.5 mM catechol, 0.5 mM methylviologen, 2 ug/ml

gramicidin, and 7.5 ug of chlorophyll per ml of reaction mixture.

 

 

 

 

 

“Herbicide Buthidazole-treated Tebuthiuron-treated
Electron concentration Electron trans- % Electron trans- %
donor (uM) port rate: Inhibition port rate Inhibition
H20 -- 2821 -- 2821 --
H20
(NHZOH tmt) - 30 - 301 H-
Asc/Cat.
(NHZOH tmt) Control 1372 0 1372 0
" 0.05 116 15 120 12
" 0.5 69 50 73 47
" 5 43 69 44 68
" 500 25 82 27 80
1 Data expressed as u moles of 02 consumed/hr/mg of chlorOphyll where
values must be multiplied by 4 to give electron transport rates.
2

Data also expressed as u moles of O2 consumed/hr/mg of chlorophyll

but values must be multiplied by 2 to give electron transport rates.

100

LITERATURE CITED

1. Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural

10.

11.

12.

l3.

14.

use. Tech. Inf. Brochure issued by Velsicol Chemical Corp., Chicago,
IL, p. 5.

. Anonymous, 1975. Technical report on Spike, experimental herbicide.

Brochure issued by Lilly Research Laboratories, Indianapolis, IN, p. 6.

. Arnon, D.J. 1949. Copper enzymes in isolated chloroplasts. Polyphenol-

oxidase in Beta vulgaris L. Plant Physiol. 24: 1-15.

 

Bovey, R.W., R.E. Meyer, and J.R. Baur. 1975. Evaluation of tebuthiuron
for woody plant control. Abstr. Weed Sci. Soc. Amer. 15: 22-23.
Abstr. No. 54.

. Bracha, P., M. Luwisch, and N. Shavit. 1972. Thiadiazoles of herbicidal

activity. IE_A.S. Tahori, ed., Proceedings Second International IUPAC
Congress of Pesticide Chemistry, Vol. 5. Gordon and Breach, New York,

. Good, N.E. and S. Izawa. 1973. Inhibitors of photosynthesis. In P.M.

Hochstes, M. Kates, J.H. Quartel, eds., Metabolic Inhibitors: Vbl. 4.
Academic Press, New York, pp. 179-214.

Goss, R.J., E.P. Richard, C.J. Arntzen, and F.W. Slife. 1978. The site
of atrazine and diuron electron transport inhibition in isolated pea
chloroplasts. Abstr. weed Sci. Soc. Amer. 18: 74. Abstr. No. 163.

Hatzios,K.K. and D. Penner. 1978. The effect of buthidazole on corn
photosynthesis, respiration, and leaf ultrastructure. North Central
weed Control Conf. Abstr. 33: 48-49.

. Izawa, S. and D.R. Ort. 1974. Photooxidation of ferricyanide and iodide

ions and associated phosphorylation in NHZOH-treated chloroplasts.
Biochim. Biophys. Acta. 357: 127-143.

Kubo, H., R. Sato, I. Hamura, and T. Ohi. 1970. Herbicidal activity of
1,3,4-thiadiazole derivatives. J. Agric. Food Chem. 18: 60-65.

Moreland, D.E. and J.L Hilton. 1976. Actions on photosynthetic systems.
IE_L.J. Audus, ed., Herbicides: Physiology, Biochemistry, Ecology,
Vol. 1, Academic Press, London, pp. 493-523.

Moreland, D.E. 1977. Measurements of reactions mediated by isolated
chloroplasts. In B. Truelove, ed., Research Methods in Weed Science,
Ed. 2, Southern Weed Science Society, Auburn, Alabama, pp. 141-149.

Ort, D.R. 1975. Quantitative relationship between photosystem I electron
transport and ATP formation. Arch. Biochem. Biophys. 166: 629-638.

Pafford, J.L. and C.D. Hobbs. 1974. Tebuthiuron: A new herbicide for
preemergence weed control in sugarcane. Abstr. Weed Sci. Soc. Amer.
14: 114. Abstr. No. 266.

101

15. Schafer, G., A. Trebst, and K.H. Buchel. 1975. 2-anilino-1,3,4-thiadia-
zoles,Inhibitors of oxidative and photosynthetic phosphorylation.
Z. Naturforsch. 30c: 183-189.

16. Trebst, A. 1974. Energy conservation in photosynthetic electron
transport of chloroplasts. Ann. Rev. Plant Physiol. 25: 423-458.

17. York, A.C. and C.J. Arntzen. 1979. Photosynthetic electron transport
inhibition with buthidazole. Abstr. weed Sci. Soc. Amer. 19: 103.
Abstr. No. 218.

CHAPTER 6
Physiological Effects of Buthidazole on Corn (239 m§y§_L.)
Redroot Pigweed (Amaranthus retroflexus L.), Alfalfa (Medicago
sativa L.), and Quackgrass [Agropyron repens (L.) Beauv.]
ABSTRACT
Buthidazole (3-[S-(l,l-dimethylethyl)-l,3,4-thiadiazol-2-yIJ-4-

6 to 10'“ M

hydroxy-l-methyl-Z-imidazolidinone) at concentrations of 10-
did not affect germination of corn (Zea mays L., 'Pioneer 3780'), redroot
pigweed (Amaranthus retroflexus L.), alfalfa (Medicago sativa L.,‘Verna1'),

and quackgrass [Agropyron repens (L.) Beauv.] seeds. Stressing the seeds

 

obtained from mature corn plants treated either preemergence or preplant
incorporated with buthidazole at several rates by accelerated aging and
cold treatments further indicated that this herbicide did not affect germi-
nation. Total photosynthesis and dark respiration of corn plants 12 days
after preemergence application and of redroot pigweed, alfalfa, and quack-
grass plants after postemergence application of buthidazole at several
rates were measured with an infrared CO2 analyzer. The results suggested
that buthidazole was a rapid inhibitor of photosynthesis of the sensitive
redroot pigweed and quackgrass plants, with less effect on corn and alfalfa.
Buthidazole did not affect respiration of the examined species except for
a transitory increase in corn and alfalfa 12 days after preemergence or 4 h
after postemergence treatment with buthidazole at 0.56 or 1.12 and 2.24
kg/ha, respectively. A long-term inhibition of quackgrass respiration 96 h
after treatment with buthidazole at 1.12 and 2.24 kg/ha was also evident.
INTRODUCTION

Buthidazole (3-[5-(1,l-dimethylethyl)-l,3,4-thiadiazol-2-yl]-4-

hydroxy-l-methyl-2-imidazolidinone) has shown potential as a preemergence

herbicide for selective weed control in corn (1). Applied at low rates

102

103

ranging from 0.28 to 0.56 kg/ha, it controls a wide spectrum of weeds.
In established alfalfa, buthidazole applied postemergence at 1.12 kg/ha
during dormancy effectively controls quackgrass, a serious weed problem
in alfalfa (1).

Inhibition of germination, photosynthesis, and respiration have
frequently been cited as potential modes of herbicidal action (2,4,6).

The action and selectivity of this herbicide were studied using
seedling corn, a crop tolerant to low preemergence application rates of
buthidazole, and seedling redroot pigweed, a susceptible weed in the
first study. In the second study, buthidazole was applied to alfalfa
as the tolerant crop plant and to quackgrass as the susceptible weed as
postemergence application to dormant plants.

The purpose of this study was to examine a) effects of buthidazole
on germination of corn, pigweed, alfalfa, and quackgrass seeds; b) effects
on viability and vigor of corn seeds obtained from plants treated with
buthidazole; and c) effects of buthidazole on total photosynthesis and
dark respiration of corn and pigweed, following preemergence application,
and of alfalfa and quackgrass, at various time periods after postemergence
application.

MATERIALS AND METHODS

Germination studies. Twenty seeds of corn (Zea mays L., 'Pioneer 3780')

 

and quackgrass [Agropyron repens (L.) Beauv.] and fifty seeds of alfalfa

 

(Medicago sativa L., 'Vernal') and redroot pigweed (Amaranthus retroflexus
L.) were placed in 'Petri' dishes whose bottoms were covered with Whatmann
#1 filter paper. Ten milliliters of herbicide solution, pH 6.8, containing.
0, 10-6, 10-5, and 10.4 M of technical buthidazole (95% purity) were placed

in the 'Petri' dishes and the seeds germinated in an incubator at 25 C in

the dark. An additional 10 ml of the respective herbicide solution was

104

added 4 days later to keep the paper moist. After 7 days, germinated
seeds were counted and the results expressed as percent germination. Data
presented are the means of two experiment with three replications per
experiment. The data were analyzed for variance followed by Duncan's
multiple range test to separate the means.

Viability and vigor studies of corn seeds obtained from buthidazole-

treated corn fields. Seeds harvested from 'Pioneer 3780' corn plants

 

treated in the field with 0, 0.28, 0.56, 0.84, and 1.12 kg/ha of buthi-
dazole applied preemergence or preplant incorporated were obtained from
velsicol Chemical Corporation, Chicago, Illinois. These seeds were
analyzed for percent germination and vigor under various environmental
conditions as follows:

a) Standard germination test. A standard, warm germination test (5) was

 

run on two loo-seed lots from each sample. The seeds were placed on
moist paper towels covered with waxed paper at 25 C for 7 days. Only normal
seedlings were recorded. The results were expressed as percent germination.

b) Accelerated aging test. In the accelerated aging stress test (5), two

 

lOO-seed lots from each sample were subjected to 42 C for 3 days of 100%
relative humidity. They were then transferred to conditions of the standard
germination test (25 C, 7 days) and the number of normal seedlings reported
as percent germination.

c) Cold treatment. The cold test (5) was performed by placing the seeds

 

on moist unsterilized soil mixture (2/3 greenhouse soil, 1/3 vermiculite),
50 seeds per 473-ml waxed cups, under growth chamber conditions at 10 C

for 5 days. The conditions of the growth chamber were then changed to l6-h
day at 30 C and 8-h night at 20 C for 6 days. Percent germination, emergence,
and seedling height were recorded. In all cases the data were analyzed for

variance followed by Duncan's multiple range test to separate the means.

105

Photosynthesis and respiration studies

Plant material. Five 'Pioneer 3780' corn seeds and ten pigweed seeds were

 

planted into greenhouse soil (1:1:1 soil, sand, peat) in 946-ml waxed cups.
Buthidazole was applied preemergence at rates 0, 0.56 and 1.12 kg/ha. After
planting, the cups were placed in a greenhouse with temperature ranging
from 20 C at night to 33 C during the day. After 12 days photosynthesis

and respiration were measured. However, the sensitive pigweed plants died
so quickly after emerging from the soil surface that to examine the effect
of buthidazole on pigweed photosynthesis we applied buthidazole post-
emergence on pigweed plants 25 cm tall.

'Vernal' alfalfa seeds were planted into greenhouse soil in 54 x 36 cm
wooden boxes and grown to maturity under greenhouse conditions. Then they
were cut to 6 cm, allowed to regrow to 12 cm and placed outdoors during the
winter of 1978. Then they were transplanted one plant per 946-ml cup and
placed in a greenhouse for acclimation and growth. When the plants reached
the height of 25 cm, they were treated with buthidazole and used for the
photosynthesis and respiration measurements.

Twenty quackgrass seeds were planted 2.0 cm deep into greenhouse soil
in 946-ml waxed food cups and were allowed to grow to maturiy under green-
house conditions. Then they were cut to‘6 cm in height, allowed to regrow
to 12 cm, and placed in a controlled environment chamber at 0-5 C for 2
weeks of acclimation. Then the cups were returned to greenhouse conditions
and the plants allowed to attain a height of 25-30 cm for use in this study.
Measurement of total photosynthesis and dark respiration. Total photo-
synthesis and dark respiration of all plant species were measured with an
infrared CO2 analyzer (3,7) in an open air flow system at a slow rate of
500 cm3/min. The plants, after reaching the aforementioned heights, were

placed inside a clear cylinder located in the interior of a growth chamber.

106

The cylinder was sealed and the lights were turned on and off to create’
appropriate conditions for measuring total photosynthesis and dark respi-
ration, respectively. The environmental conditions of the growth chamber
were 25 i 1‘C day and night temperature and light intensity of 21 Klux or
280 microeinsteims/mzlsec energy. The measurements were recorded as C02
uptake (total photosynthesis) and CO2 evolution (dark respiration).
Following preemergence application of buthidazole in corn, photosynthesis
and respiration of corn plants were measured 12 days after treatment. The
leaf area of the measured plants was also recorded and the results were
expressed as mg C02/dm2/h as reported by Sestak gt El. (7). For the post-
emergence treatment of buthidazole, photosynthesis and respiration were

made prior to treatment (original measurements) and then 4 and 24 hours
later for redroot pigweed and 4, 24, 48, and 96 hours after treatment for
alfalfa and quackgrass plants. The results were expressed as the percentage
of the original photosynthetic and respiratory measurements. Redroot pig-
weed plants were treated with 0, 0.28, 0.56, 0.84, and 1.12 kg/ha of buthi-
dazole, and alfalfa and quackgrass plants were treated with 0, 1.12 and

2.24 kg/ha of the herbicide. In all cases, buthidazole was formulated as

a 50% wettable powder and was applied with a link belt sprayer at 2.1 kg/cm2
pressure in 935 L/ha spray volume.

All data presented are the means of two experiments with two repli-
cations per experiment. The data were analyzed for variance followed by
Duncan's multiple range test to separate the means.

RESULTS AND DISCUSSION
Buthidazole at concentrations of 10-6, 10-5, and 10-4 M did not

have any apparent effects on the germination of seeds of corn, redroot

pigweed, alfalfa, and quackgrass (Table l). The redroot pigweed had a low

107

germination percentage, but this was not the result of treatment with
buthidazole since non-treated seeds also germinated poorly.

Extensive testing involving the use of accelerated aging and cold
treatment to stress corn seeds during germination might possibly reveal
effects of the herbicide that would not show up under normal conditions.
However, these tests did not indicate any buthidazole effect on germina-
tion (Tables 2 and 3). Seeds for the experiments had been harvested from
corn plants treated either preemergence or preplant incorporated with
buthidazole at rates 0, 0.28, 0.56, 0.84, and 1.12 kg/ha. In both cases,
exposure of seeds to heat stress did not give percent germination values
different from those of the regular test. However, exposure of seeds to
cold treatment often gave lower germination percentages. There were no
significant differences between the germination values of seeds obtained
from treated and non-treated plants under the same tests (regular, accele-
rated aging, and cold germination tests). Percent emergence and seedling
height did not show any significant differences between control and herbi-
cide-treatment (Tables 2 and 3).

The influence of buthidazole on total photosynthesis and dark respi-
ration of corn, receiving preemergence application, and redroot pigweed,
alfalfa, and quackgrass, all receiving postemergence application, is
shown on Tables 4 through 9. Buthidazole appeared to be a rapid photo-
synthetic inhibitor, acting as early as 4 hours after postemergence appli-
cation (Tables 5,6,7). However, photosynthesis of corn, 12 days after pre-
emergence application, was not affected by buthidazole at the rate of 0.56
kg/ha, whereas buthidazole at 1.12 kg/ha decreased the photosynthetic
rate significantly (Table 4). Following postemergence application, total

photosynthesis of redroot pigweed was markedly inhibited by any rate of

108

buthidazole examined as early as 4 hours after treatment (Table 5).

In the second study, total photosynthesis of alfalfa and quackgrass was
significantly inhibited very early following postemergence application of
buthidazole even at the rate of 1.12 kg/ha (Tables 6 and 7). The time

of application may be important for the selective performance of buthi-
dazole in the alfalfa-quackgrass system. For technical reasons in mea-
suring photosynthesis, the plants were allowed to grow to a height of

25 to 30 cm, providing a leaf area greater than in established alfalfa
during or right after dormancy. The time of application may be of great
significance under field conditions.

The influence of buthidazole on respiration of corn (Table 4),
redroot pigweed (Table 5), alfalfa (Table 8), and quackgrass (Table 9)
did not appear to be related to the main herbicidal action of the sub-
stance. However, it is of interest to note the significant increase in
corn respiration 12 days after preemergence treatment with 0.56 kg/ha
and in alfalfa respiration 4 hours after postemergence treatment with
1.12 and 2.24 kg/ha (Tables 4 and 8). This burst of C02 release was
transitory, and levels dropped to normal very rapidly 24 hours after
treatment in the case of alfalfa (Table 8). This transitory increase
of respiration, observed only in corn and alfalfa, might be an indication
of rapid metabolism of buthidazole in these tolerant crop plants. A
significant long-term inhibitory effect of buthidazole on quackgrass res-
piration was detected 96 hours after treatment with 1.12 and 2.24 kg/ha
(Table 9). Since this effect was observed only at 96 hours after treat-
ment and not earlier, and furthermore it was observed only in quackgrass,
inhibition of respiration did not seem to be a means by which buthidazole

exerted its primary mode of action.

109

In conclusion, the mode of action of buthidazole appears to be
a strong and rapid inhibition of photosynthesis with germination of

all tested species unaffected.

110

*
Table 1. The effect of buthidazole on germination of four plant species

 

 

 

Redroot
Buthidazole Corn Pigweed Alfalfa Quackgrass
( Molar ) ( percent germination)
Concentration
0 100 a 45.0 a 78.3 a 73.3 a
10’6 100 a 41.8 a 83.3 a 76.6 a
10"5 100 a 41.6 a 80.0 a 76.6 a
10‘4 100 a 43.6 a 85.0 a 68.3 a
*

Means within columns with similar letters are not significantly dif-
ferent at the 5% level by Duncan's multiple range test. Percent values
less than 15% or greater than 85% were transformed to arcsine values

for analysis of variance.

111

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113

Table 4. The effect of buthidazole on corn total photosynthesis and dark

*
respiration 12 days after preemergence application .

 

12 days after treatment

 

 

 

Buthidazole Photosynthesis+ Respiration:
2
( kg/ha ) ( mg COZ/dm /hr )
O 41 b 9 a
0.56 44 b 17 b
1.12 33 a 11 a
*

Means within columns with similar letters are not significantly dif-
ferent at the 5% level by Duncan's multiple range test.

CO2 uptake

C02 evolution

H-

114

Table 5. The effect of buthidazole on redroot pigweed total photosynthesis

and dark respiration at various time intervals after postemergence

 

 

 

 

*
application .
Hours after postemergence treatment
4 24
... 1'
Buthidazole Photosynthesis Respiration Photosynthesis Respiration
( kg/ha ) ( ppm of CO2 as percent of the original values )
0 121 d 100 a 150 b 124 a
0.28 48 c 103 a 0 a 80 a
0.56 16 b 109 a 0 a 125 a
0.84 12 ab 98 a 0 a 92 a
1.12 2 a 87 a O a 95 a

*
Means within columns with similar letters are not significantly different
at the 5% level by Duncan's multiple range test.

+ CO2 uptake

+

- CO2 evolution

115

Table 6. The effect of buthidazole on 'Vernal' alfalfa total photosynthesis

*
at various time intervals after postemergence application .

 

Hours after treatment

 

 

 

Buthidazole
4 24 48 96
( kg/ha ) ( ppm of CO2 uptake as % of the original values )
0 117 b 1491: 185 c 190 c
1.12 7 a 9 a 10 a 21 a
2.24 3 o a 0 a o a 0 a
*

Means whithin rows and columns with similar letters are not significantly

different at the 5% level by Duncan's multiple range test.

116

Table 7. The effect of buthidazole on quackgrass total photosynthesis at

*
various time intervals after postemergence application .

 

Hours after treatment

 

 

Buthidazole
4 24 48 96
( kg/ha ) ( ppm of CO2 uptake as % of the original values )
0 p 94 b 117 c 124 c 99 b
1.12 9 a 0 a 0 a- 0 a
2.24 8 a 0 a 0 a 0 a

 

Means within rows and columns with similar letters are not significantly

different at the 5% level by Duncan's multiple range test.

117

Table 8. The effect of buthidazole on dark respiration of 'Vernal' alfalfa

*
at various time intervals after postemergence application .

 

Hours after treatment

 

 

Buthidazole =
4 24 48 96
( kg/ha ) ( ppm of CO2 evelution as Z of the original values )
0 115 a 108 a 112 a 110 a
1.12 169 b 140 a 132 a 137 a
2.24 189 b 136 a 115 a 117 a

 

Means within rows and columns with similar letters are not significantly

different at the 5% level by Duncan's multiple range test.

118

Table 9. The effect of buthidazole on quackgrass dark respiration at various

*
time intervals after postemergence application .

Hours after treatment

 

 

 

Buthidazole
4 24 48 96
( kg/ha ) ( ppm of CO2 evelution as % of the original values )
0 88 abc 114 cd 113 cd 126 d
1.12 ’99 bcd 111 bed 95 abcd 82 ab
2.24 91 abc 92 abc 83 abc 68 a
*

Means within rows and columns with similar letters are not significantly

different at the 5% level by Duncan's multiple range test.

119

LITERATURE CITED

Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural use.
Tech. Inf. Brochure issued by Velsicol Chemical Corp., Chicago, IL,
p. 5.

Ashton, F.M and A.S. Craftes. 1973. Mode of Action of Herbicides. John
Wiley, New York, pp. 69-99.

Davis, D.E. and B. Truelove. 1977. The meaSurement of photosynthesis
and respiration using whole plants or plant organs. Pages 119-129
in B. Truelove, ed., Research Methods in Weed Science. Southern weed
Science Society, Auburn, Alabama.

Kirkwood, R. C. 1976. Action on respiration and intermediary metabolism.
Pages 444-492 in L. J. Audus, ed., Herbicides: Physiology, Biochemistry,
Ecology, Vol. 1, Academic Press, London.

McDonald, M.B., Jr. 1975. A review and evaluation of seed vigor tests.
Proc. AOSA 65, 109-139.

Moreland, D.E. and J.L. Hilton. 1976. Actions on photosynthetic systems.
Pages 493-523 in L.J. Audus, ed., Herbicides: Physiology, Biochemistry,
Ecology, Vol. 1, Academic Press, London.

Sestak, Z., J. Catsky, and P.G. Jarvis. 1971. Plant Photosynthetic Pro-
duction: Manual of Methods. Dr. W. Junk N. V. Publishers, The Hague,
pp. 162-166.

CHAPTER 7
Some Effects of Buthidazole on Corn (Egg mayg L.) Photosynthesis,
Respiration, Anthocyanin Formation, and Leaf Ultrastructure
ABSTRACT

The effect of the herbicide buthidazole (3-[5-(l,l-dimethylethyl)-
1,3,4-thiadiazol-2-yl]-4-hydroxy-l-methyl-Z-imidazolidinone) on photo-
synthesis, respiration, anthocyanin formation, and leaf ultrastructure
of corn (Eggnmgyg L., var. Pioneer 3780) was studied following pre- or
post- emergence applications. Total photosynthesis and dark respiration
were measured with an infrared CO2 analyzer in an open air flow system
12, 18, and 24 days after preemergence treatment with 0, 0.56, 1.12, and
2.24 kg/ha of buthidazole. The 0.56 and 1.12 kg/ha preemergence treatments
had no effect on total corn photosynthesis even 24 days after treatment,
whereas buthidazole at 2.24 kg/ha inhibited photosynthesis as early as 12
days. Total photosynthesis and dark respiration were also measured in whole
plants, 30 cm tall, before herbicide application and 4, 24, 48, and 96 h
after postemergence treatment with buthidazole at 0, 0.28, 0.56, 0.84, and
1.12 kg/ha. Following postemergence treatment, buthidazole inhibited total
photosynthesis at any rate examined as early as 4 h after treatment. Neither
pre- or postemergence buthidazole applications influenced respiration with
the exception of a transitory increase caused by 0.56 kg/ha 12 days after
preemergence treatment and by 0.84 and 1.12 kg/ha 4 h after postemergence
treatment. Transmission electron micrographs revealed that buthidazole ap-
plied postemergence at 0.28 and 1.12 kg/ha reduced or prevented the accumu-
lation of starch in bundle sheath chloroplasts as early as 24 h after treat-
ment. Ultrastructural disruptions in some mesophyll chloroplasts of treated
corn plants were also evident. Preemergence application of buthidazole at

rates of 0.28, 0.42, 0.56, and 1.12 kg/ha inhibited anthocyanin formation

120

121

indicating an alteration in corn metabolism.
INTRODUCTION

Photosynthesis and respiration have been repeatedly reported to be
affected by many herbicides (3,13,15). Electron microscope studies have
served as a useful tool in studying the mode of action of herbicides (l).

Derivatives of 1,3,4-thiadiazoles exhibit insecticidal (4) and
herbicidal (14) activities. Buthidazole, a derivative of 1,3,4-thiadiazole
(Figure l), is currently marketed as a herbicide for industrial weed control.
Its potential for selective weed control in corn, using preemergence or
early postemergence applications at low rates (0.28 to 0.56 kg/ha), is the
object of current research (2). High rates are phytotoxic even to corn,
making corn a suitable species for the study of buthidazole selectivity and
mode of action. Our objectives were to study the mode of buthidazole action
by examining the effect of buthidazole on photosynthesis, dark respiration,
anthocyanin formation, and leaf ultrastructure in corn.

MATERIALS AND METHODS

Plant material and herbicide application. 'Pioneer 3780' corn was seeded
five seeds per 946-m1 pot in a 1:1:1 volume mixture of soil, sand, and peat
and placed in the greenhouse at 25 i 3 C. Buthidazole was applied preemerge-
nce at 0, 0.56, 1.12, and 2.24 kg/ha for the photosynthesis and respiration
studies and at 0, 0.28, 0.42, 0.56, and 1.12 kg/ha for the anthocyanin study.
In separate experiments when corn plants were 30 cm tall, at 20 days of age,
buthidazole was applied postemergence at rates 0, 0.28, 0.56, 0.84, and 1.12
kg/ha for photosynthesis and respiration measurements. In all cases the herbi-
cide was formulated as a 50% wettable powder, and it was sprayed with a
link belt sprayer at 2.1 kg/cm2 pressure in 935 L/ha spray volume.
Photosynthesis and respiration measurements. Total photosynthesis and dark

respiration were measured with an infrared CO analyzer in an open gas flow
2

122

system operated at 500 cm3/min. After acclimation to insure open stomata
the plants were placed in a sealed cylinder inside a growth chamber with
conditions of 25 i l C day and night temperature and light intensity of

21 Klux or 280 uE/mzlsec energy. The measurements were over the period of

1 hour as ppm of CO2 uptake (total photosynthesis) and as ppm of CO2 evo-
lution (dark respiration). Following the preemergence application of buthi-
dazole, total photosynthesis, dark respiration, and leaf area were measured
12, 18, and 24 days after treatment. The results were expressed as mg 002/
dm2/h by means of the following formula (17).

Flow rate ACO 273 K 44gC02/mole

 

 

 

x 2x x X1039-S-X6O—1nin
(L/min) (ppm) 298 K 22.4 L/mole
mgCO /dm2/h-
2 2 dm2 6 1
Leaf area (m ) X 100-—§ X‘lO -3— (ppm)
m L

In this equation AC02 refers to the difference in CO2 content of the inlet
and outlet gas streams.

For the postemergence treatment of buthidazole, photosynthesis and
respiration measurements were made before treatment and then 4, 24, 48, and
96 h after treatment. The results are expressed as the percentage of the
pretreatment photosynthetic and respiratory measurements.

Data presented are the means of two experiments with two replications
per experiment for all studies. The data were analyzed by analysis of
variance in a two-way factorial design with factor A as the herbicide rate
and factor B as the time period after treatment with the herbicide. Mean
separation was by Duncan's multiple range test. A student's t-test was
also used to compare the values obtained for the treated and non-treated

plants at the various time intervals after the postemergence application

to those corresponding to the pretreatment measurements.

123

Transmission electron microscopyg(TEM) study. Tissue samples from corn
leaves were obtained from an area 6 cm from the tip of the third leaf
24 and 96 h after treatment with buthidazole at rates of O, 0.28, and
1.12 kg/ha. These tissues were fixed for 2 h at 25 C in 5% (v/v)
glutaraldehyde and Sorensen's phosphate buffer, pH 7.2 (10). The material
was then washed in the same buffer and fixed for 1 h in 1% (v/v) osmium
tetroxide. After washing, the tissues were stained in 0.5% (w/v) aqueous
uranyl acetate for 2 h. The material was then dehydrated in ethanol and
embedded in Epon-Araldite (6). Thin sections were obtained in an ultra- O
microtome equipped with a diamond knife. These sections were stained with
lead citrate and examined with a Philips 201 TEM at 60 kV.
Anthocyanin extraction. Plants used for this study were grown in a growth
chamber with l6-h day and 8-h night under the same conditions as in the
photosynthesis studies. Fourteen days after preemergence treatment with
buthidazole, the plants were harvested and anthocyanin was extracted as
described by Duke gt gl.(9). The sheaths from the first leaves were
ground with 10 m1 of cold methanolic HCl (1% HCl) in a mortar and pestle.
The extract was centrifuged at 1700 g for 10 min, and the absorbance of
the supernatant was measured spectrophotometrically at 525 nm. The
values presented are the means of three experiments with five repications
per experiment.
RESULTS AND DISCUSSION

Preemergence application of buthidazole at 0.56 and 1.12 kg/ha
had no effect on total photosynthesis of corn even 24 days after treatment
(Table 1). However, buthidazole at 2.24 kg/ha caused significant inhibition
of photosynthesis as early as 12 days after treatment (Table 1). The

significant difference observed between the photosynthetic rates at 12

124

days and 18 or 24 days, even for the control plants, was due to senescence
of part of the lower leaves of the older plants 18 and 24 days after treat-
ment.

Following postemergence treatment buthidazole inhibited total corn'
photosynthesis at any rate examined as early as 4 h after treatment (Table
2). The data indicate that buthidazole applied either pre- or postemergence
is a photosynthetic inhibitor and the effect appears dose dependent.
Greater time was required to inhibit photosynthesis following preemergence
than postemergence buthidazole application due to the time required for
germination and development of roots and the emerging shoot (coleoptile)
which appear to be important for the uptake of soil applied buthidazole (11).

No effect of buthidazole on dark respiration of corn was evident fol-
lowing either pre- or postemergence applications, with the exception of
an increase of the respiratory rates caused by 0.56 kg/ha 12 days after
preemergence treatment and by 0.84 and 1.12 kg/ha after postemergence
treatment (Tables 3 and 4). This burst of CO2 release was transitory and
levels dropped to normal rapidly (Tables 3 and 4).

Electron micrographs of leaf sections obtained from corn plants 24
and 96 h after postemergence treatment with buthidazole at 0, 0.28, and
1.12 kg/ha are shown in figures 2 and 3. Reduction of the amount of starch
synthesized or prevention of its accumulation in bundle sheath chloro-
plasts of treated corn plants was noticeable 24 h after treatment with
1.12 kg/ha of the herbicide (Figure 2d). Mesophyll chloroplasts appeared
swollen 24 h after treatment with 0.28 kg/ha of buthidazole, and ultra-
structural disruprions of chloroplast membranes were present 96 h after
treatment with 0.28 and 1.12 kg/ha of the herbicide (Figures 3c, 3d, and

3f). However, normal mesophyll chloroplasts were also observed in some

125

sections obtained 96 h after treatment with this compound (Figure 3e).
Swelling of chloroplast thylakoids has been reported to be caused by
other herbicides and is often considered as an early stage of chloroplast
breakdown (1). No obvious abnormalities due to treatment with this herbi-
cide were observed in mitochondria of treated corn plants (Figure 3b).
Thus, the ultrastructural studies support the results obtained from the
photosynthesis and respiration measurements, indicating again that buthi-
dazole is a photosynthetic inhibitor. This conclusion is also supported
-from another study in which buthidazole was found to be a very strong inhi-
bitor of photosynthetic electron transport in isolated spinach chloro-
palsts, comparable to atrazine and diuron (12).

Preemergence application of buthidazole at rates as low as 0.28 kg/ha
inhibited anthocyanin formation in the sheaths of the first leaves in corn,
indicating another effect on metabolism (Table 5). Anthocyanins are fla-
vonoid compounds that are nearly always present as glycosides containing
most commonly one or two glucose or galactose units attached to the hydroxyl
group in the central ring of their molecule. These sugars can be formed
from degradation of starch or fat in storage organs during seedling
development or from photosynthesis in chlorophyll-containing cells (5,16).
Therefore, this effect of buthidazole on anthocyanin formation in corn
seems to be a consequence of the effect of buthidazole on corn photosynthesis
discussed earlier. Diuron [3-(3,4-dichlorophenyl)-l,l-dimethylurea] and
monuron [3-(p-chlorophenyl)-l,l-dimethylurea], two well-known powerful
photosynthesis inhibitors, have also been demonstrated to reduce photo-
induced levels of anthocyanin (5,7,8) and activity of phenylalanine
ammonia lyase (18), respectively. However, light-induced formation of antho-

cyanin in corn seedlings was found to -be independent of photosynthesis in

126

one study (9), and in our study inhibition of photosynthesis was not
evident at rates causing inhibition of anthocyanin formation in the sheaths
of corn leaves (Tables 1 and 5). In such a case a possible effect of buthi-
dazole on the activity of phenylalanine ammonia lyase, or other key
enzymes in anthocyanin biosynthesis, could serve as a basis to explain
the aforementioned effect of buthidazole on anthocyanin formation in corn.
Further work is needed to elucidate this point.

In conclusion, buthidazole appears to be a strong inhibitor of
photosynthesis and anthocyanin biosynthesis in corn. Buthidazole at low

rates stimulated respiration in corn, but the effect was transitory.

127

Figt_1re 1. Chemical structure and chemical name of the herbicide buthidazole.

128

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129

Figure 2. Electron micrographs of corn bundle sheath chloroplasts from
a) Control 96 h after treatment with water and from buthidazole-
treated plants; b) and c) 24 and 96 h after treatment at 0.28
kg/ha and d) and e) 24 and 96 h after treatement at 1.12 kg/ha.

Bar represents 0.5 um in a, b, e, 0.2 um in c, and 0.25 um in d.

 

130

 

131

Figure 3. Electron micrographs of corn mesophyll chloroplasts and mito-
chondria from a) Control 96 h after treatment with water and
from buthidazole-treated plants; b) Mitochondria 96 h after
treatment at 1.12 kg/ha, c) and d) 24 and 96 h after treatment
at 0.28 kg/ha, and e) and f) 24 and 96 h after treatment at
1.12 kg/ha. Bar represents 0.5 um in a, f, 0.1 um in b, and

0.25 um in c,d, and e.

 

132

 

133

Table 1. Effect of buthidazole on 'Pioneer 3780' corn total photosynthesis

at various time intervals after preemergence application.a

 

Days after treatment

 

 

Buthidazole
12 18 24
(kg/ha) (C02 uptake as mg COZIdm2/hr)
0 40 fg 26 de 26 de
0.56 45 g 25 cde 23 cde
1.12 33 ef 24 cde 17 bcd
2.24 15 bc 10 ab 2 a

 

a Means within rows and columns with similar letters are not significantly

different at the 5% level by Duncan's Multiple Range Test.

134

Table 2. Effect of buthidazole on 'Pioneer 3780' corn total photosynthesis

at various time intervals after postemergence application.a

 

Hours after treatment

 

 

Buthidazole
4 24 48 96
(ppm of C02 uptake as % of the original values)b
(kg/ha)

0 118 f 133** g 158** h 152** h

0.28 84* e 53** cd 58** de 55** cd

0.56 71** de 33** b 33** b 29** b

0.84 41** be 10** a 9** a 9** a

1.12 30** b 7** a 7** a 4** a

 

a Means within rows and columns with similar letters are not significantly
different at the 5% level by Duncan's multiple range test.

b Asterisks (* or **) within rows indicate significant differences at the

5% and 1% levels between the means of the measurements conducted at 4,

24, 48, and 96 h after treatment and the original values by student's

t-test.

135

Table 3. Effect of buthidazole on 'Pioneer 3780' corn dark respiration

at various time intervals after preemergence application.a

 

Days after treatment

 

 

Buthidazole
12 18 24
(kg/ha) (C02 evolution as mg COzldmzlhr)
0 8.3 ab 6.7 ab 8.9 ab
0.56 16.0 c 6.5 ab 9.1 b
1.12 8.5 ab 7.3 ab 6.5 ab
2.24 8.7 ab 5.5 a 7.6 ab

 

a Means within rows and columns with similar letters are not significantly

different at the 5% level by Duncan's Multiple Range Test.

136

Table 4. Effect of buthidazole on 'Pioneer 3780' corn dark respiration

at various time intervals after postemergence application.a

 

. Hours after treatment
Buthidazole

 

 

4 4:4 48 96
(kg/ha) (ppm of C02 evolution as % of the original values)b
0 94 ab 91 ab 102 b 97 ab
0-28 87 ab 78 a 89 ab 76 a
0056 92 ab 79 a 82 ab 87 ab
0-84 134** c 84 ab 85 ab 88 ab
1-12 144** c 83 ab 94 ab 78 a

 

a Means within rows and columns with similar letters are not significantly
different at the 5% level by Duncan's Multiple Range Test.

b Asterisks (**) within rows indicate significant differences at the 1%
level,between the means of the measurements conducted at 4 h after treat-

ment and the original values by student's t-test.

137

Table 5. The effect of buthidazole on anthocyanin formation in the

sheaths of 'Pioneer 3780'

corn leaves 14 days after preemergence

 

 

applicationa.

Buthidazole Anthocyanin formation
(kg/ha) (Absorbance at 525 nm)

0 0.559 c

0.28 0.287 b

0.42 0.205 ab

0.56 0.082 a

1.12 0.126 ab

 

a

Means within columns with similar letters are not significantly different

at the 5% level by Duncan's multiple range test.

10.

ll.

12.

l3.

14.

138

LITERATURE CITED

. Anderson, J.L. and W.W. Thomson. 1973. The effects of herbicides on

the ultrastructure of plant cells. Residue Rev. 47: 167-189.

Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural
use. Tech. Inf. Brochure issued by Velsicol Chemical Corp., Chicago,
IL, 5 pp.

Ashton, F.M. and A.S. Crafts. 1973. Mode of Action of Herbicides, John
Wiley, NY, 504 pp.

. Bracha, P., M. Luwish, and N. Shavit. 1972. Thiadiazoles of herbicidal

activity. Pages 444-492 in A. S. Tahori, ed., Proceedings Second Inter-
national IUPAC Congress of Pesticide Chemistry, Vbl. 5. Gordon and
Breach, NY.

Creasy, L.L. 1968. The significance of carbohydrate metabolism in fla-
vonoid synthesis in strawberry leaf discs. Phytochemistry 7: 1743-1749.

Dawes, C.J. 1971. Biological Techniques in Electron Microscopy. Barnes
and Noble, NY, 193pp.

DeNicola, M., M. Piatelli, V. Castrogiovanni, and V. Amico. 1972.
The effects of light and kinetin on amaranthin synthesis in relation
to phytochrome. Phytochemistry 11: 1011-1018.

. Downs, R.J., H.W. Siegelman, W.I. Butler, and S.B. Hendricks. 1965.

Photoreceptive pigments for anthocyanin synthesis in apple skin.
Nature (London) 205: 909-910.

Duke, O.S., S.B. Fox, and A.W. Naylor. 1976. Photosynthetic indepen-
sence of light-induced anthocyanin formation in Zea seedlings.
Plant Physiol. 57: 192-196.

Gomori,G. 1955. Preparation of buffers for use in enzyme studies. Page
143 $3.3-P- Colowick and N.O. Kaplan, eds., Methods in Enzymology,
Vol. 1, Academic Press, NY.

Hatzig s, K. K. and D. Penner. 1978. Role of absorption and translocation
of QC-buthidazole in crop selectivity. Proceedings North Central
Weed Control Conf. 33:113.

Hatzios, R.K. and D. Penner. 1979. Inhibition of photosynthetic ele-
ctron transport in isolated spinach chloroplasts by two 1,3,4-
thiadiazolyl derivatives.P1ant Physiol. Suppl. 63(5): 41.

Kirkwood, R. C. 1976. Action on respiration and intermediary metabolism.
Pages 444- 492 in L. J. Audus, ed., Herbicides: Physiology, Biochemistry,
Ecology, Vol. 1. Academic Press, London.

Kubo, H., R. Sato, I. Hamura, and T. Ohi. 1970. Herbicidal activity
of 1,3,4-thiadiazole derivatives. J. Agric. Food Chem. 18: 60-65.

139

15. Moreland, D.E. and J.L. Hilton. 1976. Actions on photosynthetic
systems. Pages 493-523'ig_L.J. Audus, ed., Herbicides: Physiology,
Biochemistry, Ecology, Vol. 1. Academic Press, London.

16. Salisbury, F.B. and C.W. Ross. 1978. Plant Physiology, 2nd edition.
Wadsworth Publishing Co., Inc., Belmont, 422 pp.

17. Catsky, J., J. Janac, and P.G. Jarvis. 1971. General principles of
using IRGA for measuring CO exchange rates. Pages 161-166 in
Z. Sestak, J. Catsky, and P.G. Jarvis, eds., Plant Photosynthetic
Production: Manual of Methods, Dr. Junk N. V. Publishers, The
Hague.

18. Zucker, M. 1969. Induction of phenylalanine ammonia-lyase in
Xanthium leaf discs: photosynthetic requirement and effect of
daylength. Plant Physiol. 44: 912-922.

CHAPTER 8
Potential Antidotes Against Buthidazole Injury
to Corn (_Z_e_ar_n_ay_§ L.)
ABSTRACT
Buthidazole (3-[5-(1,l-dimethylethyl)-l,3,4-thiadiazol-2-le-4-
hydroxy-l-methyl-Z-imidazolidinone) has shown promise for selective
weed control in corn (Egg m§y§_L.). However, at high rates, injury
symptoms were evident in corn seedlings. Greenhouse studies were initiated
to test potential antidotes for buthidazole injury to corn seedlings. NA
(1,8-naphthalic anhydride) and CDAA (2-chloro-N,N-diallylacetamide) were
the most promising of six chemicals evaluated. The other chemicals tested
were R-25788 (2,2-dichloro-N,N-diallylacetamide), R-29l48 (2,2-dimethyl-
5-methyl-dichloroacetyloxazolidine), carboxin (2,3-dihydro-5-carboxanilido-
6-methy1-l,4-oxanthiin) and gibberellin (GA3). Seven herbicides were also
tested for their antagonistic interactions but none offered protection.
The herbicides were alachlor [2-chloro-2',6'-diethyl-Nf(methoxymethyl)aceta-
mide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-
methylethy1)acetamide], diethatyl [N-(chloroacetyl)-N-(2,6-diethy1phenyl)-
glycine ethyl ester], H-26910 [N-(chloroacetyl)-N-(2-methyl-6-ethylphenyl)-
glycine isopropyl ester], EPTC (S-ethyl dipropylthiocarbamate), butylate
(§-ethyl diisobutylthiocarbamate) plus the antidote R-25788, and trifluralin
(g,g,g-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine). A ratio 1:3 (buthi-
dazole:CDAA) appeared to be optimal for the protection effect. CDAA appeared
to be more effective than NA. Since both CDAA ahd NA offered limited
protection but R-25788 did not, this action appears to be through a dif-
ferent mechanism than the one proposed against the thiocarbamate or aceta-

nilide herbicides.

140

141

INTRODUCTION

The concept of using herbicide antidotes, introduced by Hoffman in
1962 (12), offers a potential alternative for increasing the margin of
selectivity of the currently available herbicides. Three compounds have
received commercial interest, NA (1,8-naphthalic anhydride) for protecting
corn against thiocarbamate, dithiocarbamate, and acetanilide herbicides
(13) and for protecting rice, grain sorghum, and oats against other herbicides
(6,15,24). R-25788 (2,2—dichloro-N,N-diallylacetamide) was effective against
thiocarbamate injury to corn (23) and has been found effective against
perfluidone (l,l,l-trifluoro-N-[2—methyl-4-(phenylsulfonyl)phenyllmethane-
sulfonamide) (24), barban (4-chloro-2-butynyl m-chlorocarbanilate) (4),
and acetanilide (18) injury to corn. Protection offered by R-25788 appears
very specific to corn (25). CGA 43089 [g-(cyanomethoximino)-benzonitrile]
has shown promise in protecting sorghum against metolachlor (8,22). Numerous
other compounds have been tested including R-29148 (2,2-dimethyl-5-methyl-
dichloro-acetyloxazolidine) and CDAA (2-chloro-N,N-diallylacetamide), an
analog of R-25788 (Figure l), which were less effective as antidotes against
thiocarbamate and acetanilide injury to corn than R-25788 (5,16,19). Gib-
berellin (GA3) and carboxin (2,3-dihydro-5-carboxanilido-6-methy1-l,4-oxan-
thiin) also have been reported to decrease injury from herbicides (7,21).

Buthidazole (3-[5-(l,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-
l-methyl-2-imidazolidinone) is a promising herbicide for selective weed
control in corn. However, the margin of selectivity is narrow and buthi-
dazole at rates higher than 0.56 kg/ha may cause injury to corn seedlings
following preemergence application (1).

The purpose of this study was to determine whether corn tolerance
to buthidazole could be increased using chemicals or herbicides previously

reported to have antidote activity.

142

MATERIALS AND METHODS
Plant material and chemical treatment evaluation. 'Pioneer 3780' corn
plants for all studies were grown in greenhouse soil (1:1:1 soil, sand,
peat), 5 seeds per 946-ml waxed cups. Four acetanilide (alachlor, metol-
achlor, diethatyl, H-26910), two thiocarbamate (EPTC and butylate+R-25788)
and one dinitroaniline (trifluralin) herbicides were evaluated for their
efficacy in protecting corn from buthidazole when applied in combination
with buthidazole. These herbicides and the potential antidotes R-25788,
CDAA, and GA3, were sprayed on the soil as formulated emulsifiable concen-
trates in an oil-in-water emulsion. The formulated emulsifiable concentrate
of R-29l48 was sprayed in a 50% water-50% ethanol mixture. Buthidazole
formulated as a 50% wettable powder and the herbicides and antidotes were
sprayed sequentially with a link belt sprayer at 2.1 kg/cm2 pressure in
935 L/ha spray volume. Buthidazole was applied at 0, 1.12, 2.24 kg/ha in
all studies and, in addition, at 0.42, 0.56 and 0.84 kg/ha in two studies.
The six antidotes used throughout this study were: R-25788 at 0, 1.12,
2.24, and 5.60 kg/ha, R-29148 at 0, 1.12, and 2.24 kg/ha, gibberellin (GAB)
as 3.91% liquid concentrate at 0 and 1.12 kg/ha, CDAA at 0, 1.12, 2.24,
3.36, 4.48, 5.6 and 6.72 kg/ha, NA and Carboxin at 0 and 0.5% (w/w) seed
treatments. The herbicides tested for antagonistic interactions with buthi-
dazole were alachlor at 0, 1.12, 2.24, 3.36, and 5.6 kg/ha, metolachlor
at 0, 1.12, 2.24, and 3.36 kg/ha, diethatyl at 0, 0.56, 3.36 and 5.6 kg/ha,
H-26910 at 0, 0.56, and 3.36 kg/ha, EPTC at O, 1.12, and 3.36 kg/ha, buty-
late+R-25788 at 0, and 2.24 kg/ha, and trifluralin at 0, 0.28 and 0.56 kg/ha.
After planting and preemergence application of the chemicals, the cups were
placed in a greenhouse with temperature ranging from 20 C at night to 33 C
during the day. Twenty or 30 days after planting, the plants were harvested

and plant heights and fresh weights were measured. The data are expressed

143

as the average shoot height in cm per plant per cup and the average shoot

fresh weight in g per plant per cup. Data presented are the means of two

experiments with five replications per experiment. These data were analyzed

for variance and Duncan's multiple range test was used to separate the means.
RESULTS AND DISCUSSION

The antidote R-29148 at 2.24 kg/ha offered limited protection against
2.24 kg/ha of buthidazole (Table 1). R-25788 was ineffective at that rate
or at 1.12 kg/ha. GA3 applied at 1.12 kg/ha increased shoot height but
not shoot fresh weight, indicating that the plants were taller but still
injured (Table l). CDAA at 1.12 and 2.24 kg/ha offered limited protection
to corn injury from buthidazole at 1.12 kg/ha (Table 2). Increasing the
ratio of buthidazole to CDAA to 1:3 provided greater protection from CDAA
(Table 2).

Increasing the rates of CDAA to 4.48, 5.6, and 6.72 kg/ha did not
increase the protection (Table 3). Although the ratio of 1:3 (buthidazole:
CDAA) may not be practical, it shows that buthidazole safety to corn can
be enhanced chemically.

Data in Table 4 indicate that NA at 0.5% (w/w) also offered limited
protection to corn from buthidazole injury at rates of 0.84 and 1.12 kg/ha,
whereas carboxin failed to give any protection. Thus, NA, a chemical anti-
dote against a spectrum of herbicides in corn and other grasses (2), also
appeared promising against buthidazole injury to corn. CDAA differs from
R-25788 only in that it has one less chlorine (Figure 1). This difference
appears important in protecting thiocarbamate and acetanilide injury to
corn, with R-25788 being superior to CDAA (16,19). A 12:1 ratio of EPTC
or butylate to R-25788 was very effective for corn protection (18). R-
25788 applied at a 5:1 ratio of antidote to buthidazole, which corresponds

to amount of R-25788 60 times greater than that needed to work against

144

thiocarbamates, failed to protect corn from buthidazole injury (Table 6).
The difference in chemistry of R-25788 and CDAA.was again evident, however,
this time CDAA.was active and R-25788 inactive.

Protection of corn from EPTC and other thiocarbamate injury is believed
to be the result of an R-25788-mediated increase of the rate of EPTC sul-
foxidation followed by subsequent EPTC sulfoxide-glutathione conjugation
(3,16,17,20). The moderate antidotal activity of CDAA against EPTC injury
to corn may also result from.increased EPTC metabolism (16). A similar
mode of action has also been proposed for NA which was reported to stimulate
EPTC (9) and cisanilide (l4) metabolism in corn. To protect corn against
buthidazole injury, CDAA and NA may act through a different mechanism than
the one proposed for the thiocarbamates or acetanilide herbicides. This
hypothesis is partially supported by the finding that 14C buthidazole did
not conjugate with 3H-labeled blutathione (GSH) $2 ZiE£2.(20)- Although
this observation does not exclude an enzymatic GSH conjugation of buthi-
dazole ip gigg, it does suggest that the activity of the responsible gluta-
thione-g-transferase was not stimulated by R—25788, CDAA, and NA.

Buthidazole has been reported to act as a strong inhibitor of photo-
synthesis in corn and other plants (10) blocking photosynthetic electron
transport primarily at the reducing site of photosystem II (11). Therefore,
the protection of corn injury from buthidazole obtained with the use of
NA and CDAA appears to be a successful attempt in antidoting a herbicide
affecting photosystem 11. Thus far, no one has reported success in anti-
doting diuron or other herbicides affecting photosystem II (13).

The acetanilide, thiocarbamate, and dinitroaniline herbicides, tested
for antagonistic interactions with buthidazole, were not effective in pre-
venting corn injury from buthidazole (Tables 5,6,7). At a herbicide to

antidote ratio of 2:3, diethatyl offered slight protection (Table 5).

145

However, this does not appear promising, as higher rates of diethatyl were
phytotoxic to corn (Table 6). York and Slife (26) reported promising an-
tagonistic interactions between buthidazole and acetanilide herbicides

on corn, however, the rates of buthidazole and acetanilide herbicides used
in their studies were not reported. The data in Table 7 indicate that ala-
chlor at 2.24 kg/ha offered limited protection against 0.56 kg/h a of buthi-
dazole, but no protection was evident against higher rates of buthidazole.
Metolachlor and butylate+ R-25788 also failed to offer any protection

(Table 7).

In summary, CDAA.and NA, although they offered only partial protection,
were the most effective antidotes tested against buthidazole injury to corn.
The mechanism of CDAA and NA action appeared different from that proposed
for these antidotes and R-25788 against thiocarbamate or acetanilide injuries
in corn. Perhaps analogs of CDAA, other than R-25788 or R-29l48, may offer

greater protection against buthidazole.

146

Figpre 1. Chemical structures and chemical names of the herbicide anti-

dotes, R-25788, R-29l48, CDAA, carboxin, and NA.

147

I a [m 2_¢H2=CI-l2~

R- 25788 cI-c-c-k
(I. (NZ-cu2 :0"2

2, 2 - dichloro -N,N -dio|Ioncetomide

" 1'1 ,<=’H.~ ’6"
I C—
R-29l48 CI-c-c-N< | "
.1 °
\
.12;

2,2 -dimethyl-5-methyl-dichloroocetyloxozolidine

It 0 —
I II [Caz-CH2 -cuz
CDAA Cl- C - c - N‘ —

2-chloro-N,N-diol|ylocetomide

/°\
CARBOXIN I ll

CH2 C — C — N *::>
2V5 II I
0 III
2,3-dihydre-corboxonilido-b-methyI-l,4- exonrhiin

I’D

c’:
NA
c0

I ,8- naphtholic onhydride

148

Tgblg 1. Effects of R-25788, R-29148, and GA3 on corn injury from buthidazole,

30 days after preemergence treatmenta.

 

 

 

 

Buthidazole Antidotes Rate Shoot ht Shoot fresh wt
(ks/ ha) (kg/ ha) (cm/ plant) (8/ plant)
Main effects
(1) Antidotes none 0.0 44.0 a 2.7 a
R-25788 2.24 46.1 a 3.0 ab
R-29148 2.24 43.2 a 2.8 ab
GA3 1.12 51.5 b 3.2 b
(ii) Buthidazole 0.0 57.1 c 5.0 c
1.12 47.3 b 2.9 b
2.24 34.3 a 0.8 a
Intera°t1°ns 0.0 none 0.0 56.7 f 5.2 f
1.12 0.0 45.2 cd 2.4 cd
2.24 0.0 30.1 a 0.4 a
0.0 R-25788 2.24 57.8 f .3 f
1.12 2.24 47.5 cde 3 de
2.24 2.24 33.2 ab .4 a
0.0 R—29148 2.24 49.8 de 4.
1.12 2.24 43.1 c
2.24 2.24 36.7 b 1.6 bc
0.0 GA3 1.12 63.9 g 5. f
1.12 1.12 53.4 ef 2 de
2.24 1.12 37.1 b 0.8 ab

 

Means within columns for any given effect comparison followed by similar
letters are not significantly different at the 5% level according to
Duncan's multiple range test.

149

Table 2. Effect of CDAA on corn injury from buthidazole, 30 days after pre-

a
emergence treatment .

 

Main effggtg

Interactions

 

Buthidazole CDAA Shoot ht Shoot fresh wt
( kg/ha ) (ks/ha) (cm/ plant) (8/ plant)
(1) CDAA 0.0 44.0 a 2.7 a
1.12 49.6 b 3.1 ab
2.24 53.3 c 3.4 bc
3.36 53.9 c 3.6 c
(ii) Buthidazole 0.0 59.4 c 5.1 c
1.12 53.3 b 3.5 b
2.24 37.9 a _ 1.0 a
0.0 56.8 de 5.2 e
1.12 none 45.0 c 2.4 c
2.24 30.1 a 0.4 a
0.0 1.12 58.3 d 4.9 e
1.12 1.12 52.7 d 3.4 d
2.24 1.12 37.8 b 0.9 ab
0.0 2.24 60.9 e 5.1 e
1.12 2.24 57.5 de 3.9 d
2.24 2.24 41.4 bc 1.2 ab
0.0 3.36 61.5 e 5.1 e
1.12 3.36 57.8 de 4.2 de
2.24 3.36 42.3 bc 1.6 bc

 

Means within columns for any given effect comparison followed by similar

letters are not significantly different at the 5% level according to
Duncan's multiple range test.

150

Table 3. Effect of CDAA on corn injury from buthidazole, 30 days after pre-

a
emergence treatment .

 

 

 

 

 

Buthidazole CDAA Shoot ht Shoot fresh wt
(kg/ha) (kg/ha) (cm/plant) (s/plant)
Main effects
(1) CDAA 0.0 47.7 a 2.7 a
4.48 52.7 b 3.0 ab
5.6 55.7 b 3.5 c
6.72 55.3 b 3.2 bc
(ii) Buthidazole 0.0 74.2 c 7.1 c
1.12 53.4 b 2.0 b
2.24 30.9 a 0.2
lfltera°t1°ns 0.0 73.2 e 7.2 d
1.12 none 41.4 c 0.6 a
2.24 28.4 ab 0.2 a
0.0 4.48 74.8 e 7.1 d
1.12 4.48 55.8 d 1.6 b
2.24 4.48 27.5 a 0.2 a
0.0 5.6 75.6 e 7.4 d
1.12 5.6 59.3 d 2.9 c
2.24 5.6 32.0 ab 0.2 a
0.0 6.72 73.1 e 6.6 d
1.12 6.72 57.2 d 2.8 c
2.24 6.72 35.5 be 0.3 a

 

Means within columns for any given effect comparison followed by similar
letters are not significantly different at the 5% level according to

Duncan's multiple range test.

151

Tablg 4. Effects of NA and Carboxin on corn injury form buthidazole, 30 days

a
after preemergence treatment .

A

Buthidazole Antidotes Rate Shoot ht Shoot fresh wt

 

 

 

(kg/ha) (% w/w) (cm/plant) (g/plant)
Main effects

(1) Antidotes none 0.0 54.7 ab 3.0 a
NA 0.5 57.8 b 4.1 b

Carboxin 0.5 53.7 a 1 2.8 a

(ii) Buthidazole 0.0 68.8 c 6.1 c
0.56 61.9 b 4.0 b

0.84 45.8 a 1.5 a

1.12 45.1 a 1.5 a

Intera°t1°ns 0.0 none 0.0 72.1 d 6.3 d
0.56 0.0 63.0 c 4.3 c

0.84 0.0 42.8 a 0.6 a

1.12 - 0.0 41.0 a 0.7 a

0.0 NA 0.5 61.0 c 5.5 d

0.56 0.5 59.6 be 4.3 c
0.84 0.5 56.6 be 3.4 bc

1.12 0.5 54.0 b 3.1 b

0.0 Carboxin 0.5 73.5 d 6.5 d
0.56 0.5 63.0 c 3.4 be

0.84 0.5 37.9 a 0.6 a

1.12 0.5 40.4 a 0.7 a

 

Means within columns for any given effect comparison followed by similar
letters are not significantly different at the 5% level according to
Duncan's multiple range test.

Table 5. Effects of six herbicides on corn injury from buthidazole, 20 days

152

a
after preemergence treatment .

 

 

Buthidazole Herbicides Rate Shoot ht Shoot fresh wt
(ks/ha) (kg/ha) (cm/plant) (g/plant)
Neimsfifissss
(i) Herbicides none 0.0 31.2 b 1.6 a
Alachlor 3.36 31.0 b 1.8 abc
Metolachlor 3.36 32.1 bc 1.9 bc
Diethatyl 3.36 31.1 b 2.0 c
H-26910 3.36 31.7 bc 1.9 bc
EPTC 3.36 28.6 a 1.7 ab
Trifluralin 3.36 33.9 c 2.0 c
(ii) Buthidazole 0.0 37.5 c 3.1 c
1.12 31.9 b ,l.7 b
2.24 24.7 a 0.8 a
Interactions 0.0 36.0 f8 2.7 d
1.12 none 32.5 ef 1.6 c
2.24 25.0 bc 0.7 a
0.0 Alachlor 3.36 34.5 fg 2.6 d
1.12 3.36 33.6 ef 1.9 c
2.24 3.36 24.9 bc 0.9 ab
0.0 Metolachlor 3.36 38.5 gh 3.2 e
1.12 3.36 32.9 ef 1.8 c
2.24 3.36 25.0 be 0.7 a
0.0 Diethatyl 3.36 35.1 fg 3.1 e
1.12 3.36 30.2 de 1.7 c
2.24 3.36 28.0 cd 1.1 b
0.0 H-26910 3.36 40.0 hi 3.3 e
1.12 3.36 32.3 ef 1.8 c
2.24 3.36 22.8 ab 0.7 a

153

Table 5. (continued)

 

Buthidazole Herbicides Rate Shoot ht Shoot fresh wt

 

(kg/ha) (kg/ha) (cm/plant) (g/plant)
InteractiQEE 0.0 EPTC 3.36 35.5 fg 3.1 e
1.12 3.36 29.8 de 1.5 c
2.24 3.36 20.4 a 0.6 a
0.0 Trifluralin 0.56 42.7 1 3.4 e
1.12 0.56 32.3 ef 1.6 c
2.24 0.56 26.6 bcd 0.9 ab

 

Means within columns for any given effect comparison followed by similar
letters are not significantly different at the 5% level according to
Duncan's multiple range test.

154

Table 6. Effects of R-25788, Alachlor, and Diethatyl at 5.60 kg/ha on corn

injury from buthidazole, 30 days after preemergence treatmenta.

 

 

 

 

Buthidazole Antidotes Rate Shoot ht Shoot fresh wt
(kg/ha) (kg/ha) (cm/plant) (s/Plant)
Main effects

(1) Antidotes none 0.0 47.0 b 2.6 ab
R-25788 5.6 42.6 b 2.5 a

Alachlor 5.6 43.7 b 3.1 b

Diethatyl 5.6 36.0 a 2.5 a

(ii) Buthidazole 0.0 64.7 c 6.9 c
1.12 36.3 b 1.0 b

2.24 25.9 a 0.2 a

lgtera°t1°ns 0.0 none 0.0 73.2 e 7.2 e
1.12 0.0 41.4 c 0.6 ab

2.24 0.0 26.3 ab 0.2 ab
0.0 R-25788 5.6 65.4 e 6.8 de

1.12 5.6 33.4 be 0.5 ab

2.24 5.6 29.1 ab 0.1 a

0.0 Alachlor 5.6 67.1 e 7.5 e

1.12 40.7 c .5 c

2.24 23.2 a 0.4 ab

0.0 Diethatyl 5.6 53.2 d 6.1 d
1.12 5.6 29.8 ab 1 bc

2.24 5.6 24.8 ab 0.3 ab

 

Means within columns for any given effect comparison followed by similar
letters are not significantly different at the 5% level according to
Duncan's multiple range test.

155

Table 7. Effects of alachlor, metolachlor, and butylate + R-25788 at 2.24

kg/ha on corn injury from buthidazole, 30 days after preemergence
treatment4;

 

 

Main effects
(i) Herbicides

 

(ii) Buthidazole

Interactions

 

 

Buthidazole Herbicides 'Rate Shoot ht Shoot fresh wt
(ks/ha) (ks/ha)(cm/plant) (slplant)
none 0.0 49.6 b 2.7 a
alachlor 2.24 48.6 b 3.1 b
metolachlor 2.24 43.4 a 2.6 a
butylate +
R-25788 2.24 49.9 b 2.6 a
0.0 65.9 e 6.1 d
0.42 61.4 d 4.6 c
0.56 51.2 c 2.0 b
1.12 34.5 b 0.3 a
2.24 26.4 a 0.2 a
0.0 none 67.9 g 6.5 h
0.42 65.5 g 4.6 de
0.56 51.4 f 2.0 b
1.12 34.5 cd 0.3 a
2.24 29.0 be 0.2 a
0.0 alachlor 2.24 65.1 g 6.2 gh
0.42 2.24 64.1 g 5.1 ef
0.56 2.24 54.9 f 3.4 c
1.12 2.24 33.5 cd 0.5 a
2.24 2.24 25.3 ab 0.2 a
0.0 metolachlor 2.24 64.8 g 5.8 gh
0.42 2.24 52.9 f 4.1 d
0.56 2.24 43.6 e 2.5 b
1.12 2.24 33.2 cd 0.3 a
2.24 2.24 22.6 a 0.2 a
0.0 butylate + 2.24 66.0 g 5.7 fg
0.42 R-25788 2.24 63.2 g 4.6 de
0.56 2.24 54.9 f 2.4 b
1.12 2.24 36.9 d 0.3 a
2.24 2.24 28.8 be 0.2 a

 

a Means within columns for any given effect comparison followed by similar
letters are not significantly different at the 5% level by Duncan's

multiple range test.

1.

10.

ll.

12.

13.

14.

15.

156
LITERATURE CITED
Anonymous. 1977. Experimental herbicide VEL-5026 for agricultural use.

Tech. Inf. Brochure issued by Velsicol Chemical Corp., Chicago, IL,
5 pp.

. Blair, A.M., C. Parker, and L. Kasasian. 1976. Herbicide protectants

and antidotes-- A review. PANS 22: 65-74.

. Carringer, R.D., C.E. Rieck, and L.P. Bush. 1978. Effect of R-25788

on EPTC metabolism in corn (Zea mays L.). Weed Sci. 26: 167-171.

. Chang, F.Y., J.D. Bandeen, and G.R. Stephenson. 1973. N,N-diallyl-a,a-

dichloroacetamid (R-25788) as an antidote for EPTC and other herbi-
cides in corn. weed Res. 13: 399-406.

. Chang, F.Y., G.R. Stephenson, and J.D. Bandeen. 1973. Comparative

effects of three EPTC antidotes. Weed Sci. 21: 292-295.

. Chang, F.Y., G.R. Stephenson, G.W. Anderson, and J.D. Bandeen. 1974.

Control of wild oats in oats with barban plus antidote. weed Sci.
22: 546-548.

. Chang, F.Y., H.V. March, Jr., and P.H. Jennings. 1975. Effect of

alachlor on Avena seedlings: inhibition of growth and interaction
with gibberellic acid and indoloacetic acid. Pestic. Biochem.
Physiol. 5: 323-329.

. Ellis, J.F., J.W. Peek, J. Boechle, Jr., and G. Muller. 1978. A new

herbicide safener which permits effective grass control in sorghum.
Abstr. Weed Sci. Soc. Amer. 18: 21-22. Abstr. No. 44.

. Guneyli, E. 1971. Factors affecting the action of 1,8-naphthalic

anhydride in corn treated with S-ehtyl-dipropylthiocarbamate (EPTC).
Dissert. Abstr. International (B) 32: 1957-1958.

Hatzios, R.K. and D. Penner. 1979. Mede of action of buthidazole.
Abstr. Weed Sci. Soc. Amer. 19: 104-105. Abstr. No. 221.

Hatzios, R.K., D. Penner, and D. Bell. 1979. Inhibition of photosynthe-
tic electron transport in isolated spinach chloroplasts by two
1,3,4-thiadiazoly1 derivatives. Plant Physiol. Suppl.63(5): 41.

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

Hoffman, O.L. 1978. Herbicide antidotes: from concept to practice.
Pages l-13 ig_F.M. Pallos and J.E. Casida, eds., Chemistry and
Action of Herbicide Antidotes. Academic Press, NY.

Holm, R.E. and 8.8. Szabo. 1974. Increased metabolism of a pyrroli-
dine urea herbicide in corn by a herbicide antidote. weed Res.
14: 119-122.

Jordan, L.S. and V.A. Jolliffe. 1971. Protection of plants from herbi-
cides with 1,8-naphthalic anhydride as illustrated with sorghum.
Bull Envir. Cont. Toxicol. 6: 417-421.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

157

Lay, MOM. and J.E. Casida. 1976. Dichoroacetamide antidotes enhance
thiocarbamate sulfoxide detoxication by elevating corn root gluta-
thione.content and glutathione-S-transferase activity. Pestic.
Biochem. Physiol. 6: 442-456.

Lay, M.M. and J.E. Casida. 1978. Involvenent of glutathione and
glutathione-S7transferases in the action of dichloroacetamide
antidotes for thiocarbamate herbicides. Pages 151-160 ig_F.M.
Pallos and J.E. Casida, eds., Chemistry and Action of Herbicide
Antidotes. Academic Press, NY.

Leavitt, J.R.C. and D. Penner. Protection of corn (Zgg mays L.)
from acetanilide herbicidal injury with the antidote R-25788.

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

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

Miller, S.D. and J.D. Nalewaja. 1976. Herbicide antidotes. Proc.
North Central Weed Control Conf. 31: 175.

Nyffeler, A., H.R. Berger, and J. Hensley. 1978. Laboratory studies
on the behavior of the safener CGA-43089. Abstr. weed Sci. Soc.
Amer. 18: 22. Abstr. No. 45.

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

Parker, C. and M.L. Dean. 1976. Control of wild rice in rice.
Pest. Sci. 7: 403-416.

Stephenson, G.R. and F.Y. Chang. 1978. Comparative activity,
selectivity, and field applications of herbicide antidotes. Pages
36-6l,ig,F.M. Pallos and J.E. Casida, eds., Chemistry and Action

of Herbicide Antidotes. Academic Press, NY.

York, A.C. and F.W. Slife. 1979. Antagonism of btuhidazole injury
to corn by acetanilide herbicides. Abstr. weed Sci. Soc. Amer.
19: 24. Abstr. No. 48.

CHAPTER 9
Summary and Conclusions

Buthidazole was absorbed by both leaves and roots of alfalfa, quack-
grass, corn, and redroot pigweed and moved acropetally following both foliar
and root applications of 14C-buthidazole. Redroot pigweed was the only
plant species in which basipetal movement of 14C-buthidazole was evident.
However, rapid uptake by the roots and rapid movement to the shoots and
leaves, via the xylem, appeared to be the main pathway of 14C-buthidazole
uptake and translocation in all four plant species. Differential absorption
and translocation did not appear to be a factor contributing to buthidazole
selectivity between alfalfa and quackgrass. There was no difference in
buthidazole absorption between corn and redroot pigweed but the faster
translocation of buthidazole from the roots to the shoots and leaves of
redroot pigweed seedlings may play a role in the selective action of this
herbicide between corn and redroot pigweed.

A differential rate of metabolism appeared to be the primary factor
contributing to buthidazole selectivity both between alfalfa and quack-
grass and between corn and redroot pigweed. A rapidly formed unidentified
metabolite with Rf values ranging from 0.21 to 0.26 seems to be very
important for the observed alfalfa and corn tolerance as its increase with
time appeared coupled to a proportional decrease of the parent 14C-buthi-
dazole. Formation of this buthidazole derivative was very slow in redroot
pigweed. Quackgrass formed only very small amounts of this metabolite at
any time period. Other metabolites of buthidazole detected in the plant
species examined, had Rf values corresponding to the dihydroxy, urea,
methylurea, and amine derivatives of buthidazole. Further confirmation
of the identity of these metabolites is needed prior to positive identi-

fication. Howevr, at present, this is technically difficult since the

158

159

obtained metabolites were present in small quantities, far below the
resolution limitations of the existing analytical procedures.

Data obtained from the mode of action studies revealed that buthida-
zole and its analog, tebuthiuron, are very strong photosynthetic inhibi-
tors, comparable to atrazine and diuron. The inhibitory effect was asso-
ciated with the reducing side of photosystem II of the photosynthetic
electron transport. A possible minor inhibition on the oxidizing side of
photosystem II was also evident. Strong inhibition of total photosynthesis
of all four plant species examined was also evident from 1.3 v_i_\_r_o_
measurements with an infrared C02 analyzer. However, corn photosynthetic
rates were not affected by low rates of buthidazole applied preemergence.
Postemergence application of buthidazole at rates as low as 0.28 kg/ha
inhibited total photosynthesis of corn very rapidly. Inhibition of antho-
cyanin formation in corn did not appear to be connected to the effect of
this herbicide on photosynthesis since low rates that inhibited anthocyanin
biosynthesis did not inhibit total photosynthesis of corn following pre-
emergence application of buthidazole. Reduction or prevention of starch
accumulation in bundle sheath chloroplasts of corn Plants treated with
buthidazole and ultrastructural disruptions of some meSOphyll chlorOplasts
of the same corn plants observed after postemergence application of buthi-
dazole, further indicated that the herbicidal action of this compound is
strongly associated with photosynthesis.

The transitory increase of corn and alfalfa respiration, observed only
in plants treated with low buthidazole rates, may be indicative of the
faster buthidazole metabolism in these tolerant species.

Buthidazole did not inhibit seed germination of all plant species

examined in this research.

160

CDAA and NA were the most promising of six chemicals evaluated as
antidotes to increase the tolerance of corn to higher rates of buthidazole.
CDAA was more effective than NA, whereas R-25788 was totally ineffective.
A ratio 1:3 (buthidazole:CDAA) appeared to be optimal for the protection
effect. Even though this ratio (1:3) may not be practical, it indicates

that buthidazole safety to corn can be enhanced chemically.

10.

ll.

12.

13.

14.

15.

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