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This is to certify that the
dissertation entitled

Potential Interactions of Clomazone with
Metribuzin, Linuron, and Atrazine in

Soybean (Glycine max) and Common Cocklebur
(Xanthium Strumarium)

presented by

Frederick Paul Salzman

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Crop and Soil Sciences

Major pro ssor

Date W

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

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MSU II An Affltmattvo Action/Equal Opportunity Institution
cMma-p.‘

 

 

 

 

 

 

 

 

POTENTIAL INTERACTIONS OF
CLOMAZONE WITH METRIBUZIN, LINURON, AND ATRAZINE IN
SOYBEAN (GLYCINE fléfi) AND COMMON COCKLEBUR (XANTHIUM SIRUMARIUM)

By

Frederick Paul Salzman

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

1991

ABSTRACT

POTENTIAL INTERACTIONS OF
CLOMAZONE WITH METRIBUZIN, LINURON, AND ATRAZINE IN

SOYBEAN (swim; MAX.) AND COMMON COCKLEBUR (W W)
By

Frederick Paul Salzman

Observations in the field indicated, a synergistic interaction
between clomazone plus metribuzin and clomazone plus linuron in soybean.
Experiments were conducted to determine if these herbicide combinations
injured soybean and resulted in yield reduction compared to combinations
of alachlor plus metribuzin and alachlor plus linuron, and if atrazine
residues influenced these interactions. Further experiments were
conducted to determine if temperature and rainfall after 'herbicide
application were influencing these interactions and what rates of
clomazone plus metribuzin ‘would cause a synergistic interaction in
soybean, common cocklebur, and redroot pigweed. The effect of clomazone
plus metribuzin and clomazone plus linuron was also studied to determine
if one herbicide was affecting the uptake, partitioning, and/or metabolism
of the other. Field experiments indicated that combinations of clomazone
plus metribuzin and clomazone plus linuron increased soybean injury, and
reduced leaf area, shoot weight, and root weight. Yield was reduced 19%
by combinations of clomazone plus linuron in one year. Injury was most
severe in soils with low organic matter and clay content, which reduced
the amount of metribuzin absorbed, allowing for increased plant uptake.

The interactions of clomazone plus linuron and clomazone plus metribuzin

were not changed. when soybean. was germinated under cool and. warm
temperature regimes. Soybean shoot dry weight was reduced an average of
89% from combinations of clomazone plus metribuzin. Placement of
herbicide-treated soil in the same zone as the soybean seed increased
injury from clomazone plus metribuzin 31% but did not increase from
clomazone plus metribuzin, compared to when treated soil was placed above
or below the seed. Combinations of clomazone plus metribuzin were
synergistic and reduced soybean and common cocklebur shoot weights in the
greenhouse. Equivalent rates of clomazone and metribuzin reduced soybean
shoot dry weight more on a 2.5% organic matter loam soil compared to a
4.4% organic matter loam soil. Studies with “C-herbicides indicated that
parent metribuzin levels averaged 9% higher in soybean roots and shoots,
and averaged 15% higher in common cocklebur roots and shoots when
clomazone was present. Parent linuron levels were 19% higher in soybean
roots. There were no differences in the uptake or partitioning of
clomazone, metribuzin, or linuron by the addition of metribuzin or linuron

to clomazone, or clomazone to metribuzin or linuron.

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation to his major
professor, Dr. Karen Renner, for her guidance, support, and friendship in
completing this dissertation” iAppreciation is also extended to Dr. Donald
Penner for his guidance in the studies conducted in the laboratory anthis
friendship. The willingness of Dr. James Flore and Dr. Frank Ewers to
assist as Guidance Committee members is gratefully appreciated” .A special
thanks is extended to Dr. Bruce Branham for agreeing to serve on the
Dissertation Defense Committee. A sincere thank you is extended to
Patrick Svec for assisting with all aspects of this research, especially
in the laboratory. Teresa Petersen, Jeff Petersen, and Kelly Veit also
assisted in data collection. Gary Powell expertly coordinated all field
operations. His technical assistance in the field studies is gratefully
acknowledged. Appreciation is extended to Gary Powell and.Harold Webster
for allowing studies to be conducted on their land in 1990. Special
thanks are due to fellow students Mark VanGessel, Teresa Crook, Jason
Woods, and Troy Bauer for assistance with field experiments and for making
the experience as a student enjoyable. Finally, the love and support of
Paul and Doris Salzman, without which this dissertation would not have

been completed, is acknowledged.

iv

TABLE OF CONTENTS

LIST OF TABLES
INTRODUCTION

CHAPTER 1 . . . . . . . . . . . . . . . . . . . . . . . . .
INTERACTIONS AND HERBICIDAL EFFECTS ON PLANT PROCESSES
INTERACTIONS
LITERATURE CITED . . . . . . .
HERBICIDAL EFFECTS ON PLANT PROCESSES
LITERATURE CITED

CHAPTER 2 .

INTERACTION IN SOYBEAN OF CLOMAZONE, METRIBUZIN, LINURON,

ALACHLOR, AND ATRAZINE
ABSTRACT . .
INTRODUCTION . .
MATERIALS AND METHODS .
RESULTS AND DISCUSSION
LITERATURE CITED

CHAPTER 3 .

INTERACTION OF CLOMAZONE AND METRIBUZIN IN SOYBEAN, COMMON

COCKLEBUR, AND REDROOT PIGWEED
ABSTRACT

INTRODUCTION . .
MATERIALS AND METHODS .
RESULTS AND DISCUSSION
LITERATURE CITED

CHAPTER 4 .

ABSORPTION, TRANSLOCATION, AND METABOLISM OF CLOMAZONE,

METRIBUZIN, AND LINURON IN SOYBEAN AND COMMON
COCKLEBUR . . .

ABSTRACT

INTRODUCTION . .

MATERIALS AND METHODS .

RESULTS AND DISCUSSION

LITERATURE CITED

SUMMARY .

17

19
21
28
45

47

47
47
48
49
51
56

58

58
58
60
61
65
77

79

CHAPTER 2

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

1.

10.

11.

LIST OF TABLES

Soil characteristics at East Lansing and
Hickory Corners in 1988, 1989, and 1990

Herbicide treatments, application methods, and rates in
1988, 1989, and 1990

Residual atrazine in soil 1 year after
application

Response of soybean to herbicides applied alone
and in combination at Hickory Corners and East
Lansing in 1988

Total rainfall recorded for 7 weeks after planting
soybeans at East Lansing and Hickory Corners in 1988,
1989, and 1990 . . . . .

Response of soybean to herbicides applied alone and
in combination at Hickory Corners in 1989

Response of soybean to herbicides applied alone and
in combination at East Lansing in 1989

Response of soybean to herbicides applied alone and
in combination at Hickory Corners in 1990

Response of soybean to herbicides applied alone and
in combination at atrazine rates of 1.1 kg ha‘1 and
3.4 kg ha“ at East Lansing in 1990

Response of soybean shoot weight to applications of
herbicides applied alone and in combination averaged
over two temperature regimes

Response of soybean to placement of soil treated with

clomazone plus metribuzin, clomazone plus linuron, or
atrazine plus metribuzin in relation to the seed

vi

22

24

29

31

32

34

35

37

39

4O

42

CHAPTER 3

Table 1.

Table 2.

CHAPTER 4

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Dry shoot weight of soybean treated with clomazone
and/or metribuzin grown in Capac silt loam soil
with 2.5% or 4.4% organic matter contents

Dry shoot weight of common cocklebur grown in soil
treated with clomazone and metribuzin

Uptake and partitioning of clomazone, metribuzin,
and linuron alone and as influenced by another
herbicide in soybean and common cocklebur
Unextracted radioactivity in plant residues

Distribution of clomazone and major clomazone
metabolites in soybean and common cocklebur

Distribution of metribuzin and major metribuzin
metabolites in soybean and common cocklebur

Distribution of linuron and major linuron
metabolites in soybean and common cocklebur

vii

52

54

66

68

70

72

75

INTRODUCTION

Clomazone, a soil-applied herbicide for weed control in soybean, has
a limited weed control spectrum making it desirable to apply it with other
herbicides to increase the species of weeds controlled. In 1986, it was
noted that combinations of clomazone plus metribuzin and clomazone plus
linuron interacted synergistically in soybeans resulting in severe injury,
stand and yield reductions. Research on these potential synergistic
interactions could provide several benefits. Environmental influences on
these interactions could be documented and potential explanations
formulated. Research on the rates of clomazone and metribuzin that
interact synergistically in soybean and common cocklebur could lead to
changes in recommendations that would provide increased control of common
cocklebur, yet still be safe to soybean. An understanding of the uptake,
translocation, and uwmabolism of each herbicide would not only supply
answers to why the synergism was occurring, but could also provide
information on the degradation of clomazone in soybean and common
cocklebur. .At this time, the degradation pathway of clomazone is unknown,
though hypotheses have been formulated. A broad overview of the problem
of synergism would allow for an opportunity to tie field observations to
experiments in more controlled environments or to laboratory studies in
order to better understand the synergism of clomazone plus metribuzin and
clomazone plus linuron.

This research was conducted to determine: (a) if combinations
clomazone plus metribuzin and clomazone plus linuron were synergistic and
to compare the effects of these combinations to combinations of alachlor

plus metribuzin and alachlor plus linuron; (b) if atrazine residues were

2

influencing the synergism of clomazone plus metribuzin and clomazone plus
linuron; (c) if either temperature or herbicide placement in the soil
influenced soybean response to these herbicide combinations; (d) if the
range of application rates at which soybean demonstrated the synergistic
interaction of clomazone plus metribuzin and determine if the synergistic
interaction could be exploited in common cocklebur; and (d) if the basis
for the synergism in soybean and common cocklebur to clomazone plus
metribuzin and clomazone plus linuron is due to differences in uptake,

partitioning, and/or metabolism.

CHAPTER 1
INTERACTIONS AND HERBICIDAL EFFECTS ON PLANT PROCESSES
A REVIEW OF THE LITERATURE
INTERACTIONS

With the realization that chemicals could be used to selectively
control unwanted plants growing alongside desirable plants, scientists
began to combine two or more herbicides that had limited weed spectrums
individually in an attempt to control a wider range of weed species.
However, the results of' these herbicide combinations 'was sometimes
unexpected. In some instances two chemicals that when applied separately
resulted in death and/or severe injury to individual weeds did not appear
to have the same effect when applied together. Alternatively, some
combinations of chemicals resulted in more severe injury to the weed
and/or crop than was anticipated from the action of each individually.

The most accepted term to describe an unespected plant response to
a herbicide combination is interaction. Research involving herbicide
interactions has been conducted for some time, but there has not been
general agreement on describing chemical interactions in terms of plant
response. It should be noted that chemical interactions are not the same
as statistical interactions. Interaction in a strict statistical sense in
an analysis of variance has been defined as a measure of the departure of
the simple effects from an additive law or model based on main effects
only (10). In a factorial experiment this means the difference in
response between the levels of one factor are not the same at all levels
of the other factor (6). Drury (3) noted that a statistical interaction

is actually rooted in calculus and is the action of y on the action x on

4

f(x,y) or the second partial derivative, d2f(x,y)/dxdy. Alternatively, the
term interaction may be used statistically to describe responses that have
been shown to be interactions by proper use of Fisher's analysis of
variance (5).

Most weed scientists view an interaction in plant physiology terms,
specifically phytotoxicity. Interaction is defined as the total response
to a combination of individual toxicants (8). This implies that each
herbicide or compound in an interaction has a response of its own. Many
papers have been published that report plant responses to mixtures of
chemicals and describe the response without using the term interaction.
In this review the term interaction shall be defined as a phytotoxic
interaction.

Plant responses to herbicide mixtures are described as synergistic,
antagonistic, additive, or enhanced effect. Scientists have defined a
synergistic response as ”if over a range of rates and ratios, the response
is greater than that obtained when one chemical is substituted for the
other at rates based on the activity of each chemical used singly” (1).
There has been no agreement among researchers as to the definition of
antagonism. Akobundo et al. (1) defined antagonism as the opposite of
synergism or "if over a range of rates and ratios the response is less
than that obtained when one chemical is substituted for the other at rates
based on the activity of each chemical used singly". Nash (8) used a
similar definition although he emphasized the comparison of the action of
the mixture to the sum of the individual chemicals.

There is disagreement.as tO‘whether additive and enhancement effects

can be considered interactions. An additive effect is what is normally

5

expected from the mixture of two chemicals; the response is the same when
one chemical is substituted for another. Nash (8) did not consider an
additive effect an interaction. An enhancement is the "effect of a
herbicide and.a nontoxic adjuvant applied in.combination.on.a plant...(if)
the response is greater than that obtained when the herbicide is used at
the same rate without the adjuvant (l). Enhancement is not usually
included in discussions of interaction because the adjuvant alone is
usually not active.

A great deal of the difficulty in.defining an interaction comes from
attempts to quantify the expected responses of a plant to a herbicide
mixture. A formula devised by Colby (2) has been used frequently by weed
scientists in describing the effect of'a herbicide mixture on plants. The
formula is:

E-XY/lOO

where E - the expected percent inhibition of growth by herbicides A and B
at p and q rates; X - the percent inhibition of growth by herbicide A at
p rate; and'Y - the percent inhibition of growth by herbicide B at q rate.
If the actual value of E is greater than the expected value the
interaction is synergistic; if the actual value is less than the expected
value the interaction is antagonistic. Statistical significance between
the actual value and the expected value can be determined by using the
modified Least Significance Difference equation developed by Hamill and
Penner (4). Rummens (9) used the formula developed by Colby yet modified
it to calculate inhibition (X and Y in Colby's formula). He gave this
formula as:

X or Y-A/(x/c)3+l

6

where X or Y - measured response; x - concentration of herbicide used; A
- response at x - 0; c - concentration of herbicide for which the response
is reduced to 50% of A; and B - a dimensionless parameter defining the
sharpness of the 'bend' in the response curve. Synergism was then defined
as any significant deviation from E according to Colby's formula and could
be either positive or negative. The negative response was defined as
antagonism. These methods of dealing with ‘herbicide mixtures are
considered to be multiplicative survival models and are useful when
studying a mixture containing herbicides that have dissimilar action (7).

Another method to describe chemical interactions was developed by
Tammes (11). This method involves transforming the data through probit
analysis and developing a new curve, or isobole, from constant inhibition
values (usually I050). Drury (3) used calculus to calculate a multiple
regression equation followed by differentiation with respect to each
herbicide and then differentiating with respect to both herbicides
combined. The second derivative values were then graphed to indicate the
areas of interaction. Nash (8) noted that this method was no better than
the multiple regression equation that is calculated and instead proposed
a regression estimate method. In this method, best fit equations are
obtained for separate herbicides and the response values were calculated
as functions of pesticide dosage. This provided estimated value of
reductions obtained from the product of the individual regression
equations. This method also gave statistical significance to the
deviation from the expected values, something that is lacking in some

other methods of determining interactions.

Herbicide interactions remain an area that merits research. More
time will need to be spent understanding and refining methods of
calculating interactions and measuring or quantifying the interaction.
With increased use of herbicide combinations to broaden weed control
spectrums, there will continue to a demand for research on specific

interactions.

10.

11.

LITERATURE CITED

Akobundo, I. 0., R. D. Sweet, and W. 8. Duke. 1975. A method of
evaluating herbicide combinations and determining herbicide
synergism. Weed Sci. 23:20-25.

Colby, S. R. 1967. Calculating synergistic and antagonistic
responses of herbicide combinations. Weeds 15:20-22.

Drury, R. E. 1980. Physiological interaction, its mathematical
expression. Weed Sci. 28:575-579.

Hamill, A. S. and D. Penner. 1973. Interaction of alachlor and
carbofuran. Weed Sci. 21:330-335.

Lockhart, J. A. 1965. The analysis of interactions of physical and
chemical factors on plant growth. An. Rev. Plant Phys. 16:37-52.

Montgomery, D. C. 1984. Design and Analysis of Experiments. John
Wiley and Sons, New York. p. 190.

Morse, P. M. 1978. Some comments on the assessment of joint action
in herbicide mixtures. Weed Sci. 26:58-71.

Nash, R. G. 1981. Phytotoxic interaction studies-techniques for
evaluation and presentation of results. Weed Sci. 29:147-155.

Rummens, F. H. A. 1975. An improved definition of synergistic
effects. Weed Sci. 23:4-6.

Steel, R. G. D. and J. H. Torrie. 1980. Principles and Procedures
of Statistics: A Biometrical Approach. McGraw-Hill Book Co., New
York. p. 340-342.

Tammes, P. M. I“ 1964. Isoboles, a graphic representation of
synergism in pesticides. Neth. J. Plant Path. 70:73-80.

HERBICIDAL EFFECTS ON PLANT PROCESSES

An understanding of the plant process affected by herbicides is

essential in studying herbicide interactions. Traditionally, the nature
of herbicidal uptake, translocation, and mode of action are usually not
e1ucidated.until herbicidal properties have'been.demonstrated, though this
has changed in recent years. Therefore it has been possible for an
interaction to be noted without an understanding of the basis for the
interaction. An understanding of the chemical and/or physiological
reasons for an interaction may allow for the prediction of interactions by
similar chemicals, an opportunity to better understand the mode of action
of'a herbicide, and may offer a starting point in altering, overcoming, or
preventing interactions.
Atrazine. Atrazine is in the triazine family of herbicides and is used
for controlling annual broadleaf and grass weeds in corn (Zea mays L.).
Atrazine has been noted for its carryover potential, especially in the
Midwest and Great Plains, where a common rotation is corn followed by an
atrazine-sensitive crop such as soybeans [Glycine max (L.) Merr.]. In
Missouri, residual levels of atrazine of 0.19 kg ha‘1 have been found one
year after an application of 2.24 kg ai ha‘1 (27).

Uptake of atrazine by the soybean seed is believed to be a physical
process, such as diffusion, since there was no difference in uptake
between living and dead seeds (23). The rate of absorption was rapid for
the first few hours, but then decreased steadily until germination (24).
Absorption by plant roots grown in an aqueous solution was also in two
phases with initial rapid uptake followed by a slower continuous uptake.

Rates of absorption.and translocation‘were found to be proportional to the

10

amount of water absorbed and/or the translocation rate (24). These
results are indicative of apoplastic movement.

Once in the plant, atrazine inhibits photosynthesis with an
associated decrease in transpiration. Atrazine blocks the electron
transport chain in photosynthesis between the primary electron acceptor in
photosystem II, a plastoquinone with special properties termed Q, and the
plastoquinone (PQ) pool (1). This results in decreased photosynthesis and
inhibition of carbon dioxide (C02) that has been noted by researchers
(8,15) . However, atrazine does not affect nonphotosynthetic C02 fixation
(8). While the effects of atrazine on Chlorella vulgaris could be
countered by the addition of glucose (1), it has been noted that the
symptoms of atrazine injury are not consistent with a slow starvation of
a plant and may be due to a secondary factor (1,5,25). However, the
effects of the secondary factors have been found to be reversible if
carbohydrate is not limiting (25).

Atrazine injury is indicated by chlorosis of plant tissue in
susceptible species. The blocking of electron transport leads to the
formation of excited chlorophyll that can only dissipate the excess energy
by fluorescence or free radical formation. Free radical formation
ultimately leads to formation of hydroxyl free radicals which attack cell
membranes resulting in their peroxidation and eventual desiccation of the
plant.

Metribuzin. Metribuzin, like atrazine, is also in the triazine family ‘of
herbicides. It is applied to control annual grass and broadleaf weeds in
many crops including soybeans. Metribuzin has not been studied as

extensively as atrazine and discussions of metribuzin activity have been

11

dependent on findings of the action of other triazine herbicides.
Movement in the plant is thought to be apoplastic. Metribuzin was more
mobile in a sensitive species, hemp sesbania (Sesbania exultata L.), than
in soybean (16). Metribuzin inhibits photosynthesis by blocking electron
transport in photosystem II in the same manner as atrazine and was also
found to inhibit plant respiration (4,10).

Soybean cultivars have displayed differential tolerance to
metribuzin (13,26). Metribuzin has been found to be readily absorbed by
the roots and translocated to the shoot in both tolerant and susceptible
cultivars (14,26). Therefore, differences in tolerance are believed to be
due to the metabolism of metribuzin (14,26), with tolerant cultivars
detoxifying metribuzin more rapidly (l8). Oswald and coworkers (21) have
proposed that tolerance in soybeans is related to the presence of an
unknown enzyme found in both tolerant and susceptible cultivars.
Linuron. linuron is a substituted urea herbicide used for control of
annual grass and broadleaf weeds in soybeans and other crops. Rapid
initial uptake of linuron occurred in soybean roots followed by slower
uptake, suggesting that the uptake process was passive (l9). Absorption
of linuron by soybean roots from nutrient solution appeared to be passive
and governed by the entrance of water (20). Uptake by a susceptible
species, giant foxtail (Setaria faberii L.), was greater by the plant
shoot than by the root (17). However, Walker (30) reported that more
linuron was taken up by the roots of turnip (Brassica rapa L.), lettuce
(Lactuca sativa L.), and ryegrass (Lolium perenne L.) than by shoots. In
tolerant species, linuron did not translocate out of the plant roots to

the same degree observed in sensitive species (1).

12

The primary action of linuron, like most of the substituted urea
herbicides, is thought to be inhibition of photosynthesis by blocking
electron transfer between Q and PO in photosystem II. Diuron, another
substituted urea herbicide, is often used in research to block photosystem
II to enable isolation of photosystem I.

Alachlor. Alachlor, a chloroacidanilide herbicide, controls grasses and
some broadleaf weeds in many crops, including soybeans. Alachlor is
absorbed by soybean roots and translocated to the shoots (7), although
among other species there is considerable shoot uptake. In the plant,
alachlor does not affect the Hill reaction or photosystems I or II (6).
In barley (Hordeum vulgare L.), alachlor has been shown to inhibit
gibberellic acid-induced alpha-amylase synthesis by repressing the genes
that code for alpha amylase (9). Alachlor may also act as an alkylating
agent.

Clomazone. Clomazone has been developed for grass and broadleaf weed
control in soybeans. Information has only recently been published about
the mode of action or uptake of clomazone. Susceptible plants in the
field turn white, become chlorotic, and are shortened. Duke and coworkers
(12), suggested that clomazone blocks both diterpene and tetraterpene
synthesis in pitted morningglory (Ipomoea lacunosa L.). Growth of
etiolated pitted morningglory plants treated with clomazone was inhibited,
indicating a reduction in gibberellin levels. Cotyledons of treated
plants had levels of protochlorophyllide equal to cotyledons of untreated
plants; however, the Shibata shift, a shift in the absorbance of precursor
of chlorophyll a because of the addition of phytol, was reduced.

Carotenoid levels were also greatly reduced in treated plants. Both

13

phytol and gibberillins are diterpenoids while carotenoids are
tetraterpenoids. In similar research with cowpea (Vigna unguicula L.), a
less susceptible species, only the reduction in growth of etiolated
seedlings and elimination of the Shibata shift was observed (11). Uptake
of clomazone by the susceptible species redroot pigweed (Amaranthus
retroflexus L.) and livid amaranth (Amaranthus lividus L.) was greater
then uptake by the tolerant species soybean and smooth pigweed (Amaranthus

hybridus L.) (28)

10.

11.

12.

13.

14

LITERATURE CITED

Ashton, F. M., T. Bisalputra, and E. B. Risley. 1966. Effect of
atrazine on Chorella vulgaris. Am. J. Bot. 53:217-219.

Ashton, F. M. and R. K. Glenn. 1979. Influence of chloro-,
methoxy-, and. methylthio-substitutions of 'bis(isopropy1amino-s-

triazine) on selected metabolic processes. Pestic. Biochem.
Physiol. 11:201-207.

Ashton, F. M. and A. S. Crafts. 1981. Mode of Action of
Herbicides. J. Wiley & Sons. New York. 525pp.

Boger, P. and U. Schlue. 1976. Long term effects of herbicides on
the photosynthetic apparatus: I. Influence of diuron, triazines and
pyridazinones. Weed Res. 16:149-154.

Bush, P. B. and S. K. Ries. 1974. Effect of atrazine on elongation
of the embryonic axis of red kidney bean. Weed Sci. 22:227-229.

Chandler, J. M., L. I. Croy, and P. W. Santelmann. 1972. Alachlor
effects on plant nitrogen metabolism and Hill reaction. J. Agric.
Food Chem. 20:661-664.

Chandler, J. M., E. Basler, and P. W. Santlemann. 1974. Uptake and
translocation of alachlor in soybean and wheat. Weed Sci. 22:253-
258.

Couch, R. W. and D. E. Davis. 1966. Effect of atrazine, bromacil,
and diquat on 1“Goa-fixation in corn, cotton, and soybeans. Weeds
14:251-255.

Devlin, R. M. and R. P. Cunningham. 1970. The inhibition of
gibberellic acid induction of a-amylase activity in barley endosperm
by certain herbicides. Weed Res. 19:316-320.

Draber, K., K. H. Buchel, K. Dickore, A. Trebst, and E. Pistorius.
1969. Structure-activity correlation of 1,2,4-triazinones, a new
group of phototsynthetic inhibitors. pp. 1789-1795 in H. Metzner ed.
Prog. Photosynthesis Research. Vol. III.

Duke, 8. O. and W. H. Kenyon. 1986. Effects of dimethazone (FMC
57020) on chloroplast development: II. Pigment synthesis and
photosynthetic function in cowpea (Vigna unguiculata L.) primary
leaves. Pestic. Biochem. Physiol. 25:11-18.

Duke, 8. 0., W. H. Kenyon, and R. N. Paul. 1985. FMC 57020 effects
on chloroplast development on pitted morningglory (Ipomoea lacunosa)
cotyledons. Weed Sci. 33:786-794. '

Eastin, E. F., J. W. Sij, and.J. P. Craigmiles. 1980. Tolerance of
soybean genotypes to metribuzin. Agron. J. 72:167-168.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

15

Falb, L. N. and A. E. Smith, Jr. 1984. Metribuzin metabolism in
soybeans: Characterization of the intraspecific differential
tolerance. J. Agric. Food Chem. 32:1425-1428.

Funderburk, H. H. and M. C. Darter. 1965. The effect of amitrole,
atrazine, dichlobenil, and paraquat on the fixation and distribution
of‘“’CO2 in beans. Proc. 18th Southern Weed Control Conf. P. 607.

Hargroder, T. G. and R. L. Rogers. 1974. Behavior and fate of
metribuzin in soybean and hemp sesbania. Weed Sci. 22:238-245.

Knake, E. L. and L. M. Wax. 1968. The importance of the shoot of
giant foxtail for uptake of preemergence herbicides. Weed Sci.
16:393-395.

Mangeot, B. L., F. E. Slife, and C. E. Rieck. 1979. Differential
metabolism by two soybean (Glycine max) cultivars. Weed Sci.
27:267-269.

Moody, K., C. D. Kurst, and K. P. Buchholtz. 1970. Release of
herbicides by soybean roots in culture solutions. ‘Weed Sci. 18:214-
218.

Nashed, R. B., and R. D. Ilnicki. 1970. Absorption, distribution,
and metabolism of linuron in corn, soybean, and crabgrass. Weed
Sci. 18:25-28.

Oswald, T. H., A. E. Smith, and D. V. Phillips. 1978.
Phytotoxicity and detoxification of metribuzin in dark-grown
suspension cultures of soybeans. Pestic. Biochem. Physiol. 8:73-83.

Rao, V. S. and W. B. Duke. 1976. Effect of alachlor, propachlor,
and prynachlor of GAP-induced production of protease and.a-amylase.
Weed Sci. 24:616-618.

Rieder, G., K. P. Buchholtz, and C. A. Kurst. 1970. Uptake of
herbicides by soybean seed. Weed Sci. 18:101-105.

Scott, H. D. and R. Phillips. 1973. Absorption of herbicides by
soybean seed. Weed Sci. 21:71-76.

Shimabukuro, R. H., V. J. Masteller, and W. C. Walsh. 1976.
Atrazine injury: Relationship to metabolism, substrate level, and
secondary factors. Weed Sci. 24:336-340.

Smith, A. E., and R. E. Wilkinson. 1974. Differential absorption,
translocation, and metabolism of metribuzin (4-amino-6-tert-buty1-3-
(methylthio)-as-triazine-5(4H)one) by soybean cultivars. Physiol.
Plant. 32:253-257.

Talbert, R. E. and.O. H. Fletchall. 1964. Inactivation of simazine
and atrazine in the soil. Weeds 12:33-37.

28.

29.

30.

16

Vencill, W} K., K. K. Hatzios, and H. P. Wilson. 1990. Absorption,
translocation, and metabolism of 1‘C-clomazone in soybean (Glycine

max) and three Amaranthus weed species. .J. Plant Growth Regul.
9:127-132.

Vostral, J. J., K. P. Buchholtz, and C. A. Kust. 1970. Effect of

root temperature on absorption and translocation of atrazine in
soybeans. Weed Sci. 18:115-117.

Walker, A. 1973. Vertical distribution of herbicides in soil and

their availability to plants: Shoot compared with root uptake.
Weed Res. 13:407-415.

CHAPTER 2

INTERACTION IN SOYBEAN OF CLOMAZONE, METRIBUZIN, LINURON, ALACHLOR, AND
ATRAZINE

ABSTRACT

Field observations in 1986 indicated that a synergistic interaction
in soybean could occur from combinations of clomazone plus metribuzin and
clomazone plus linuron. Field experiments were conducted in 1988, 1989,
and 1990 at two locations in Michigan. Atrazine treatment consisted of
rates of 0, 1.1, 2.2, and 3.4 kg ai ha‘1 to determine if residues in soil
would influence the soybean herbicide interactions. Atrazine was applied
the year previous to the plots. Herbicide treatments in soybean included
clomazone, metribuzin, linuron, and. alachlor alone, and in 'various
combinations. A synergistic response in soybean from clomazone plus
linuron and clomazone plus metribuzin occurred, however, soil
characteristics and weather conditions impacted soybean response.
Experiments were conducted in the growth chamber to measure these
synergistic interactions and to determine if they were influenced by
ambient air and/or soil temperature and.placement of the herbicide in the
soil. The temperature regime did not affect the response of soybean to
these herbicides applied alone or in combination. Under both cool and
warm temperature regimes there was a synergistic interaction from a
combination of clomazone plus metribuzin. Leaf area and shoot dry weight
were equally reduced from a combination of clomazone plus metribuzin when
placed in any soil zone. There was a decrease in leaf area and shoot dry
weight only when soil treated with clomazone plus linuron or atrazine plus
metribuzin was placed in the same zone as the soybean seed and not when

the herbicide-treated soil was either above or below the germinating seed.

17

18

Nomenclature: alachlor, 2-chloro-N-(2,6-diethylpheny1)-N-
(methoxymethy1)acetamide; atrazine, 6-chloro-N-ethy1-N’-(1-
methylethyl)-1,3,5-triazine-2,4-diamine; clomazone, 2-[(2-
chloropheny1)methyl]-4,4-dimethy1-3-isoxazolidinone; linuron, N’-(3,4-
dichlorophenyl)-N-methoxy-N-methylurea; metribuzin, 4-amino-6-(l,1-
dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one; soybean, Glycine

max (L.) Merr. 'Century'.

19

INTRODUCTION

Increased phytotoxicity and stand reduction to soybean have occurred
following clomazone plus metribuzin and clomazone plus linuron
applications when compared to alachlor or metolachlor combined with either
metribuzin or linuron (personal observations). Increased injury to
soybeans was noted in eastern Arkansas from combinations of clomazone plus
metribuzin applied preemergence (PRE) compared to metribuzin applied PRE
alone (17). Soybean yield was reduced by applications of clomazone plus
metribuzin compared to untreated soybeans. Werling and Buhler (16)
observed increased injury to no-till soybeans from applications of
clomazone plus metribuzin compared to clomazone applied alone in
Wisconsin. Yields were reduced when clomazone and metribuzin were applied
together early preplant (EPP) or PRE compared to when clomazone was
applied EPP followed by metribuzin applied PRE. In addition to the
synergistic interactions of clomazone plus metribuzin and clomazone plus
linuron there exists the potential for synergistic interactions among
other soybean herbicides and residual atrazine. Atrazine applied for weed
control in corn may persist in the soil and injure soybeans (5), and a
synergistic injury response in soybean to residual atrazine and metribuzin
in soil has been reported (8).

Rainfall patterns, soil moisture, and soil and/or air temperature
during the initial stages of soybean development may influence soybean
response to herbicides. Increased soybean injury from applications of
either metribuzin alone or clomazone plus metribuzin on soils with organic
matter contents of 1.3% was attributed to rainfall totals of 7.7 cm 1 week

after herbicide application making the herbicide readily available and

20

rapidly moving the herbicide to the soybean roots (17) . Significant
injury had not been noted the year previously, a year with normal rainfall
patterns. In both years, however, applications of metribuzin at 560 g‘ha‘1
alone or combined with clomazone resulted in soybean yield reduction.
Conversely, in studies conducted on a soil with an organic matter content
of 5.5%, decreased soybean injury occurred from metribuzin applied alone
when 8.5 cm of rain fell 10 days after treatment, compared to soybean in
plots that received 0.6 cm of rainfall during that same time period (15).
Yields were not reported in the study. The affinity of metribuzin for
organic matter (11, 13), and planting depth may have influenced these
differences in soybean response to metribuzin following heavy rainfall
within 10 days of application.

Early season injury from metribuzin is not always reflected in yield
reductions. Wax observed yield reductions only from metribuzin applied
preplant incorporated (PPI) at 1.1 kgfiha“, while significant visual injury
also occurred from metribuzin applied PPI or PRE at 0.6 and 0.8 kg ha”.
Hagood et a1. (6) studied the relationship between early-season soybean
injury and yield response under weed-free conditions. Significant injury
from metribuzin at the one to two trifoliolate-leaf growth stage was not
an adequate indicator of yield response unless there was a concomitant
reduction in soybean stand.

The objectives of these studies were to: (a) determine if
combinations of clomazone plus metribuzin and clomazone plus linuron.were
synergistic, (b) determine if soybean.injury increased and yield decreased
from combinations of clomazone plus metribuzin and clomazone plus linuron

compared to combinations of alachlor plus metribuzin and alachlor plus

21

linuron, and (c) determine if either temperature or herbicide placement in

the soil influenced soybean response to these herbicide combinations.

MATERIALS AND METHODS

Field studies. Field experiments were conducted in 1988, 1989, and
1990 at two locations in Michigan. Soil characteristics for each location
and year are summarized in Table 1.

The experimental design.was a split plot with atrazine levels as the
main plot treatments and soybean herbicides as the subplot treatments.
Treatments of atrazine consisted of rates of 0, 1.1, 2.2, and 3.4 kg ha”.
The atrazine was applied to the plots 1 year before soybean planting.
Corn was grown using standard crop production practices. The following
spring the main plots were moldboard plowed and disked in one direction,
perpendicular to the corn rows to evenly distribute the atrazine residues
and prepare the soil for planting. Two soil samples consisting of five
cores each to a depth of 15 cm were taken from each atrazine rate in each
replication. The atrazine was extracted from the soil for quantification
following the method developed by Smith (14). The procedure in brief is
as follows. Twenty grams of wet soil were extracted for 23 hr in a
Soxhlet tube containing 150 ml of methanol:water (9:1). The methanol was
removed by rotoevaporation, the residue suspended in methylene chloride,
and extracted in methylene chloride with distilled water. The methylene

chloride was removed by rotoevaporation and the sample resuspended in

22

Table 1. Soil characteristics at East Lansing and Hickory Corners in
1988, 1989, and 1990‘.

 

------------------------------ Location------------------------------
Year East Lansing Hickory Corners
1988 Capac loam Oshtemo sandy loam
Mixed, mesic Aerie Ochraqualf Mixed, mesic Typic Hapludalf
46% sand, 40% silt, 14% clay 71% sand, 15% silt, 14% clay
pH-6.5, 2.5% OM pH-6.3, 1.6% OM
1989 Capac loam ' Oshtemo sandy loam
Mixed, mesic Aerie Ochraqualf Mixed, mesic Typic Hapludalf
48% sand, 39% silt, 13% clay 65% sand, 23% silt, 12% clay
pH-6.0, 4.4% OM pH-6.3, 1.6% OM
1990 Celina loam Kalamazoo loam
Mixed, mesic Aquic Hapludalf Mixed, mesic Typic Hapludalf
25% sand, 35% silt, 40% clay 39% sand, 30% silt, 31% clay
pH-6.1, 2.2% OM pH-5.9, 2.6% OM

 

aOM-organic matter

23

isooctane. The atrazine residues were quantitated. by' using a gas
chromatograph1 equipped with a nitrogen/phosphorous detector. The column
temperature was set on a gradient program with an initial temperature of
100 C and a final temperature of 240 C. The temperature was increased 20
C min‘. Quantification was accomplished by injecting samples containing
known levels of atrazine to make a standard curve. Samples were then
compared.against the standard curve to determine the total atrazine in the
isooctane. Total amount in the soil was based on the soil dry weight.
Soybean herbicides were applied on May 11, 1988, May 18, 1989, and
May 9, 1990 at East Lansing and.May 6, 1988, May 16, 1989, and May 2, 1990
at Hickory Corners (Table 2). Soybean herbicide treatments were applied
with a tractor-mounted, compressed-air sprayer in 206 L ha” of water at
207 kPau Herbicides were incorporated. with. a. Danish S-tine field
cultivatorz set to a depth of 7 cm. 'Century' soybean, intermediate in

metribuzin tolerancea

, were planted in 76-cm rows. Plots were four rows
wide and 9 m in length. Plots were kept weed free all season with a
tractor-mounted cultivator and hand hoeing.

Injury ratings and stand counts were recorded.3 weeks after planting

(WAP) from observations made on soybean plants in the middle 6 m of the

two center rows in each plot. Injury ratings were based on soybean

1HP 5890A. Hewlett-Packard Co. Palo Alto, CA 94304.

2Kongskilde. Kongskilde Corp. Bowling Green, OH 43402.

3Field sheet, "Soybean tolerance to metribuzin-northern edition.",
January, 1991. Mobay Corporation, Kansas City, MO 64120.

24

Table 2. Herbicide treatments, application methods, and rates in 1988,
1989, and 1990.

 

Application

Herbicide method? Rate
kg ha’1

Clomazoneb PPI 0.8
Clomazone PPI 1.1
Metribuzinc PPI 0.3
Metribuzin PPI 0.4
Linuron PRE 0.6
Alachlor PPI 2.2
Clomazone + PPI + 1.1 +
metribuzin PPI 0.4
Clomazone + PPI + 1.1 +
metribuzin° PPI 0.3
Clomazone + PPI + 0.8 +
metribuzinc PPI 0.4
Clomazone + PPI + 1.1 +
linuron PRE 0.6
Alachlor + PPI + 2.2 +
linuron PRE 0.6
Alachlor + PPI + 2.2 +
metribuzin PPI 0.4

Untreated --

 

aPPI-preplant incorporated; PRE-preemergence.
0Treatment at MSU in 1989 and KBS in 1990 only.
°Treatments at KBS and MSU in 1989 and 1990 only.

25

stunting, leaf discoloration, chlorosis, and necrosis. An injury rating
of 0 indicated no visible injury while a rating of 100 indicated all
plants in the observation area were dead. At 7 WAP, six plants were
harvested from each plot outside of the area to be harvested for yield.
These six plants were evaluated for number of nodules, leaf area, dry root
weight, and dry shoot weight. Nodulation counts were included because
linuron has been implicated in reducing soybean nodulation (4). Plant
response to the herbicide treatments was more pronounced 3 weeks after
planting, but the later harvest date accounted for plant recovery from
earlier injury, thus, it may be a better indicator of season-long
response. At maturity the center 6 m of the middle two rows was harvested
for yield with a small plot combine. Yields were adjusted to 13.5%
moisture.

In 1988, all data collected was subjected to analysis of variance
and means separated by Least Significant Differences (LSD) at the 5%
level. The data collected in 1989 and 1990 was converted to the percent
of untreated plants, with the exception of visual injury data, before
analysis of variance and mean separation. Data were not combined over
years because of significant year by location by treatment interactions.
For the herbicide combinations the expected mean was also calculated
following the method of Colby (2). The modified LSD test developed by
Hamill and Penner (7) was used to determine if the response of soybean to
two herbicides was additive or synergistic. Data is averaged over three
replications at each location each year with the exception of Hickory

Corners in 1989 which is averaged over four replications.

26

Temperature effects. Experiments were conducted in the growth chamber4
to determine if the ambient air temperature at the time of soybean
emergence would influence soybean response to herbicides applied alone and
in combination“ .Air-dried.Capac loam soil (fine-loamy, mixed” mesic.Aeric
Ochraqualf, 52% sand, 25% silt, 23% clay, pH-6.4, 2.5% organic matter
(OM)) was placed into 473-ml plastic pots prior to herbicide application.
After the herbicide was incorporated.with a soil mixer, it was placed into
946-ml plastic cups, already containing 473 m1 of untreated soil.
Herbicide treatments included clomazone PPI at 1.1 kg ai ha”, metribuzin
PPI or PRE at 0.4 kg ai ha”, linuron PRE at 0.6 kg ai ha”, alachlor PPI at
2.2 kg ai ha”, clomazone PPI at 1.1 kg ha'1 plus metribuzin PPI at 0.4 kg
ha“, clomazone PPI at 1.1 kg ha’1 plus metribuzin PRE at 0.4 kg ha",
clomazone PPI at 1.1 kg ha'1 plus linuron PRE at 0.6 kg ha”, and clomazone
plus alachlor PPI and PPI at 1.1 plus 2.2 g ha”. In addition, untreated
plants were maintained for use as comparisons. All herbicides were
applied with a stationary, air-pressurized, greenhouse pot sprayer in 206
L ha’1 of water at 207 kPa. Three Century soybean seeds were planted in
each pot and thinned to one plant per pot following emergence. Pots did
not have any drainage holes to prevent herbicides from leaching out the
bottom from watering. To prevent overwatering, pots were weighed after
planting and herbicide application, and soil moisture levels based on 18%
by weight were calculated and recorded. When the soil surface was

observed to be dry, soil was irrigated to 18% moisture.

‘Conviron CMP 3244. Controlled Environments, Ltd” Winnipeg,
Manitoba, Canada.

27

Pots were subjected to one of two temperature regimes. The warm
regime had temperatures of 18 C for the daily low and 28 C for the daily
high. The cool regime had temperature settings of 8 C for the daily low
and 18 C for the daily high for 0 to 10 days after planting and 13 C and
23 C for daily high and low, respectively, for 10 to 21 days after
planting. Lights in the growth chambers had an intensity of 320 DE m"s'2
and were set for a 15 hour day. Plant shoots were harvested at soil level
when the third trifoliolate leaf was fully expanded, cotyledons removed,
air dried, and weighed.

The experimental design was a split plot with temperature regime as
the main plot and herbicide treatments as the subplot. The experiment was
repeated twice in time with four replications each time. Means were
subjected to analysis of variance and significant treatment means
separated by LSD at the 5% level. Expected response from herbicide
combinations and the Hamill-Penner LSD were calculated as described in the
field studies.

Herbicide placement. Experiments were conducted in the greenhouse to
determine if the placement of herbicide-treated soil in relation to the
soybean seed would influence soybean shoot weight or leaf area. Air-dried
Capac loam soil (fine-loamy, mixed, mesic Aeric Ochraqualf, 52% sand, 25%
silt, 23% clay, pH-6.4, 2.5% OM) received applications of clomazone PPI at
840 g ha’1 plus metribuzin PPI 280 g ha“, clomazone PPI at 840 g ha" plus
linuron PRE at 560 g ha“, and atrazine PPI at 5 g ha'1 plus metribuzin PPI
at 280 g ha". Herbicide applications were made with a stationary,
compressed air, pot sprayer in 206 L ha'1 of water at 207 kPa. All

treatments were thoroughly mixed with a soil mixer. Pots with a volume of

28

946 ml were filled to the 473-m1 level with untreated soil; this was
measured to be 7 cm below the final soil surface. A 2.5 cm layer of
treated soil was positioned either below, above, or in the same zone as
the soybean seed. The treated soil was separated from the untreated soil
by placing a 0.5 cm layer of activated charcoal above and below the
treated soil layer. When the treated soil was placed above the seed,
charcoal was only placed below the layer as the top was also the soil
surface. In all placement regimes, three soybean seeds were planted 3.5
cm below the soil surface. The soybean seedlings were thinned to one per
pot. Soil was maintained at 18% soil moisture by surface irrigation as
described in the previous experiment. Natural sunlight was supplemented
by sodium lamps with an intensity of 300 “E m434 set for a 15 hour day.
Pots were arranged in a completely randomized design and the experiment
was repeated twice in time with four replications each time. Plants were
harvested at soil level at the V3 growth stage, cotyledons removed, leaf
area measured, and shoots dried and weighed. All data was subjected to
analysis of variance. Mean comparisons were made by LSD at the 5% level

for soil placement within each herbicide treatment.

RESULTS AND DISCUSSION
Field studies. Atrazine. Extractable atrazine remaining in.the soil one
year after application was below 200 parts per billion, a level considered
harmful to soybean5 (Table 3). Atrazine levels varied depending on
location and year. At five of six plot locations, atrazine was detected

5Field sheet, "General guidelines to crop sensitivity to popular
herbicides”, Grower Service, Lansing, MI 48906.

29

Table 3. Residual atrazine in soil 1 year after application..

 

---------------- Location----------------
Year Application
sampled rate East Lansing Hickory Corners
kg ha4 ------------------- ppbb -----------------
1988 0 6 i 8 28 i 27
1.1 17 i 72 33 i 35
2.2 24 i 32 58 i 50
3.4 25 i 43 70 i 62
1989 0 0 8 i 2
1.1 7 i 5 9 i 17
2.2 11 i 9 13 i 11
3.4 28 i 16 15 i 10
1990 O 7 i 1 6 i 1
1.1 18 i 79c 33 i 20
2.2 34 i 10 49 i 18
3.4 39 i 7 88 i 34

 

“ppb-parts per billion. Lower detection limit was 0.5 ppb in the soil.
bi standard deviation.
0Mean of three replications was 17 ppb. Mean of one replication was 221

ppb.

30

in all main plots, including those that had not received an atrazine
application the year previouslyu Atrazine is a commonly used.herbicide in
corn as part of a rotation with soybean in Michigan and occasionally has
been found to persist in the soil up to 9 years after application (1).
1988. Nodulation, leaf area, dry shoot weight, and dry root weight at
Hickory Corners and East Lansing in 1988 were not influenced by atrazine
residues or by soybean herbicide treatments (data.not presented). Soybean
injury and stand count were not affected by atrazine residues at Hickory
Corners or East Lansing, but differences were noted among soybean
herbicide treatments (Table 4). Visual injury from the combination of
clomazone plus metribuzin was greater than that from each herbicide
applied alone at East Lansing but not at Hickory Corners, while clomazone
plus linuron reduced soybean stand compared to clomazone applied alone at
Hickory Corners only. However, yield differences amongst treatments were
not significant at East Lansing and Hickory Corners in 1988 (data not
presented).

For 7 weeks after planting, rainfall was 6 and 29% of the following
2 years at Hickory Corners and East Lansing, respectively (Table 5). This
lack of rainfall may have reduced herbicide availability to soybeans.
1989. Despite the low moisture levels of the 1988 growing season,
atrazine residues were below 28 ppb at East Lansing and 15 ppb at Hickory
Corners when measured at soybean planting in 1989 (Table 3). These low
residual atrazine levels did not influence soybean response to herbicide

treatments, and all results in 1989 are averaged over atrazine levels.

31

Table 4. Response of soybean to herbicides applied alone and in
combination at Hickory Corners and East Lansing in 1988.

 

 

Herbicide Soybean
Treatment rate Visual injury stand
--------------- Location----------------
East ----- Hickory Corners -----
Lansing
------------------ 3 WAP‘---------------
Plants
kg ha'1 ------------ % ------------ 12 m“1
Clomazone 1.1 7 13 84
Metribuzin 0.4 3 26 82
Linuron 0.6 0 37 78
Alachlor 2.2 1 16 77
Clomazone + 1.1 +
metribuzin 0.4 14 21 68
Clomazone + 1.1 +
linuron 0.6 5 29 68
Alachlor + 2.2 +
linuron 0.6 0 23 66
Alachlor + 2.2 +
metribuzin 0.4 - 16 68
LSD(0.05) - 6 12 ll

 

aWAP-weeks after planting.

32

Table 5. Total rainfall recorded for 7 weeks after planting soybeans at
East Lansing and Hickory Corners in 1988, 1989, and 1990.

 

------------------ Location-------------------
Year East Lansing Hickory Corners
uuuuuuuuuuuuuuuuuuuuu cm----------------------
1988 1 6
1989 22 21

1990 19 18

33

Combinations of clomazone plus metribuzin.and.clomazone plus linuron
resulted in more injury than did combinations of alachlor plus metribuzin
and alachlor plus linuron, respectively at Hickory Corners (Table 6).
Nodule number was reduced by metribuzin at 0.4 kg ha'1 and combinations of
clomazone plus metribuzin and clomazone plus linuron. Reductions in leaf
area, shoot 'weight, and root weight occurred from combinations of
clomazone plus metribuzin. Reductions in leaf area, shoot weight, and
root weight occurred from clomazone plus linuron compared to alachlor plus
linuron or untreated plants; this response was also synergistic. Yield
was reduced, compared to untreated plants, by applications of clomazone
plus linuron at Hickory Corners and the response was synergistic.
Rainfall of 13 cm within 16 days after planting may have moved herbicides
in the soil to where they were readily taken up by the emerging soybeans.
The sandy loam soil had 1.6% OM, which would be conducive to herbicide
movement and increased availability for uptake.

At East Lansing, applications of alachlor plus metribuzin and
alachlor plus linuron reduced root weight compared to applications of
clomazone plus metribuzin and clomazone plus linuron (Table 7).
Reductions in leaf area, shoot weight, and root weight occurred from
combinations of clomazone plus metribuzin. At East Lansing, yield was
reduced only by metribuzin plus clomazone applied at the highest rate,
compared to untreated plants. No interactions at East Lansing were

synergistic. High OM (4.4%) in the loam soil may have made herbicide

.rm: w CummlpHon ”w N.Hlu£wH03 uoou "w ¢.¢lu£wH03

 

34

 

 

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.meuoume an nuHs pouocec one mQOHuomuousH OHOmHmuocmm .wcHuceHa Mauve exoozlm<§a
mH «H «H NH cu m Amo.ovamH
mm me we on on H ¢.o :HNdnHuuoa
+ N.~ + MOHnomH<
ea mm am an om m o.o souchH
+ ~.~ + H0H£omH<
.H« .mm .mm .Hm mm mm w.o cousaHH
+ H.H + encumEOHo
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+ H.H + encumEOHo
mm mm me He mo wH ¢.o :Huanuuoe
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noH mm mm mm OOH o ~.~ H0H£omH<
mOH mm mm mm oo o m.o couacHH
mm mm on mm on m ¢.o cHuanuuoz
oOH mm mm om mm H m.o cHnanuuoz
mOH No mm mm mm oH H.H eacNeEOHo
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vHon uanos uanoa wows vmucmHa husficH ouch uaoaumouh
uoom uoonm mmoH me moHspoz HmsmH> opHoHnum:
................... m<3 n------------------- --m<3 m--
.ammmH

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.rmn m oaemluHeHh ”w e.HIu£mHo3 uccu “w m.mluano3
uoosm ”=:.Hwnlmoum mmeH umNlemuceHQ me moHSUOC we “open: "ouo3 mucaHn peumuuucs you meaHe> emcue><a
.wcHucmHm Houmw axeoslmdza

 

35

 

0H HH NH wH NH m Amo.ovamH

moH mm «a Ho mu oH ¢.o :HuanHuuea
+ N.~ + MOHnode

no em HHH HoH mm o o.o couscHH
+ N.N + uOHnomH<

em Hm mm mm on NH c.o couscHH
+ H.H + ocONmEOHU

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+ H.H + oGONmEOHo

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+ H.H + encumEOHo

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+ m.o + oGONmEOHo
HoH mo NHH qHH mm N N.~ MOHcomH<
MOH ma NHH AOH em 0 w.o CouscHH
mm mm mm om we mH ¢.o :Huanuuoz
mm mm mm mm on «H m.o :HndnHuuoz
00H mm on moH «m N H.H econmaoHo
mm an «m cm as o w.o macnmaoHo

.................... apoumouucs mo w--------------------- m rm: wx
pHon uanoB uanwB some chucmHQ xuaan oumu unoaumous
uoom uoofim mmoH _me moHdpoz HmzmH> opHoHnumm
................... m4: a------------------- --m<3 m--

 

.omme CH mchGMH ummm um fioHumaHnEoo CH paw oCOHm poHHmnm mopHOHnuen cu cmonmom mo uncommom .n oHan

36

unavailable to plants, even though 15 cm of rain fell within 14 days of
planting. This may explain that reductions in leaf area, shoot weight,
and root weight were not as great as at Hickory Corners, despite similar
rainfall totals after planting.

1990. Clomazone at 1.1 kg ha'1 injured soybean at Hickory Corners in 1990
and addition of metribuzin did not elevate injury levels (Table 8). Leaf
area and shoot weight measured 7 WAP were reduced by clomazone plus
metribuzin at 1.1 plus 0.4 kg ha”, clomazone plus linuron, alachlor plus
linuron, and alachlor plus metribuzin. In addition, shoot weight was
reduced by clomazone alone at 1.1 kg ha4. Root weight was reduced by
clomazone alone at 1.1 kg ha”, clomazone plus metribuzin at 1.1 plus 0.4
kg ha” and at 1.1 plus 0.3 kg ha”, clomazone plus linuron, and alachlor
plus metribuzin. There was no decrease in leaf area, shoot weight, and
root weight from combinations of clomazone plus metribuzin or clomazone
plus linuron compared to alachlor plus metribuzin or alachlor plus
linuron. Yield, as compared to untreated plants, was not affected by any
of the herbicide treatments.

Neither visual injury, soybean stand, or yield were reduced from any
of the soybean herbicide treatments at any atrazine level at East Lansing
in 1990 (data not presented). There was an interaction between atrazine
levels and soybean herbicide treatments for leaf area, dry shoot weight,
and root weight. In plots that had atrazine treatments of 0 or 2.2 kg'ha'1
in 1989, leaf area, shoot weight, and root weight were not significantly
different (data not presented); however, when atrazine was applied at 1.1
kg ha’1 in 1989, the combination of clomazone plus metribuzin at 1.1 plus

0.4 kg ha‘1 interacted synergistically for leaf area, shoot weight, and

.m n.Hluanes uoou um n.mlu£wHos
uoonm “ ao «Haldane mmoH "oolemucmHn me moHapoc assess ”oue3 mucwHa poueenua: you moaHm> owmue><s
.wcHucmHa Hound mxooslm<§e

 

 

 

 

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ww mm mm MOH m «.0 cHuanuuoE
+ N.N + MOHnoch
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no Na Na NHH H o.o CouscHH
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om Ha Ha em 0 m.o cHnanuuoz
mm mm om om MN H.H encumEOHo
mm mm mm ¢0H NH «.9 eGONmEOHo
................ caucuses: we a------------------- a we: ma
uanoa uanoz mews rmumea me husncH mama uaoauseua
uoom uoofim meoH moHdpoz HmSmH> opHoHnuom
........................ m<3 N-------------------- --m<3 m--
.aoomH CH mumcuou huoon: um coHumcHHEoo CH can ocon pmHHmmm mopHoHnuoS ou newsman mo uncommom .m oHan

38

root weight (Table 9). Combinations of clomazone plus metribuzin at 1.1
plus 0.3 kgha'1 and clomazone plus linuron also interacted synergistically
for leaf area. When atrazine was applied at the highest rate of 3.4 kg
ha'1 in 1989, applications in 1990 of clomazone plus metribuzin at 1.1 plus
0.3 kgha‘1 interacted synergistically and reduced leaf area, shoot weight,
and root weight.

The soybean injury observed in this research may be partially
explained by soil and herbicide sorption characteristics. Metribuzin is
adsorbed by both clay and soil organic matter (12), while clomazone (9)
and linuron (3) availability are primarily influenced by organic matter
content. Injury from combinations of clomazone plus metribuzin and
clomazone plus linuron occurred at Hickory Corners in 1988 and 1989 on
soils of high sand content, and low organic matter and clay content.
Injury was greater in 1989 than in 1988 perhaps because adequate rainfall
allowed for herbicide uptake by germinating soybean seedlings. Soil
organic matter alone did not appear to decrease soybean injury in 1989
because the organic matter content of the soil at East Lansing was 4.4%,
yet soybean injury was observed. Injury was less severe in 1990, despite
adequate rainfall, at both locations because soils had higher clay
contents. Thus soil type and.weather conditions appear to impact soybean
response to clomazone, metribuzin, and linuron
Temperature effects Temperature during soybean germination and emergence
did not influence soybean response to herbicide treatment (data not shown)
and data was combined over temperature regime. Metribuzin applied PPI or
PRE reduced shoot dry weight compared to untreated plants (Table 10). In

both cool and warm temperature regimes, clomazone applied with metribuzin

39

Table 9. Response of soybean 7 WAP to herbicides applied alone and in
combination at atrazine rates of 1.1 kg ha‘1 and 3.4 kg ha'1 at East
Lansing in 1990'.

------------------ Atrazine Rate------------------

 

1.1 kg ha'1 3.4 kg ha'1
Leaf Shoot Root Leaf Shoot Root
Treatment Rate area weight we i ght area weight weight
kg ha'1 --% of untreated”-- --% of untreatedF--
Clomazone 1.1 143 142 111 125 121 112
Metribuzin 0.3 109 111 92 111 123 114
Metribuzin 0.4 138 124 96 74 79 92
Linuron 0.6 97 102 83 73 78 81
Alachlor 2.2 114 102 95 60 61 75
Clomazone + 0.8 +
metribuzin 0.4 90 110 90 95 112 98
Clomazone + 1.1 +
metribuzin 0.4 58' 63' 66' 90 91 96
Clomazone + 1.1 +
metribuzin 0.3 112' 107 84 54' 63' 78'
Clomazone + 1.1 +
linuron 0.6 77' 101 72 80 86 90
Alachlor + 2.2 +
linuron 0.6 94 85 76 113 97 111
Alachlor + 2.2 +
metribuzin 0.4 164 157 112 91 95 90
LSD(0.05) 47 34 NS 35 29 22

 

aWAP-weeks after planting.

asterisk.

bAverage values of untreated plants were: leaf area-1169 c

weight-8.6 g; root weight-2.4 g.

cAverage values of untreated plants were: leaf area-926 c

weight-7.2 g; root weight-1.8 g.

n?;

shoot

shoot

Synergistic interactions are denoted with an

40

Table 10. Response of soybean shoot weight to applications of
herbicides applied alone and in combination averaged over two
temperature regimes‘b.

 

Application Herbicide Shoot
Treatment method? rate weight
kg ha‘1 % of untreated0
Clomazone PPI 1.1 84
Metribuzin PPI 0.4 60
Metribuzin PRE 0.4 72
Linuron PRE 0.6 103
Alachlor PPI , 2.2 91
Clomazone + PPI + 1.1 +
metribuzin PPI 0.4 14'
Clomazone + PPI + 1.1 +
me tr ibuz in PRE o . a 26‘
Clomazone + PPI + 1.1 +
linuron PRE 0.6 89
Clomazone + PPI + 1.1 +
alachlor PPI 2.2 86
Untreated -- -- 100
LSD(0.05) 13

 

.Plants were harvested when the third trifolialate leaf was fully
expanded.

”Temperature regime was not significant in the analysis of variance.
Means are combined over temperature regime. Synergistic interactions
are denoted with an asterisk.

cPPI-preplant incorporated; PRE-preemergence.

dAverage shoot weight of untreated plants was 0.7 g.

41

either PPI or PRE reduced shoot weight further to levels of 14 and 27% of
untreated plants, respectively and these interactions were determined to
be synergistic.

Temperature has been found to alter herbicide uptake in soybean.

Penner (10) found that soybean translocated.more linuron and atrazine from
the root to the shoot at 30 C than at 20 C resulting in decreased plant
height and dry weight. The results in this study did not reflect any
effect from the different temperature regimes.
Herbicide placement Response of soybean to clomazone plus metribuzin was
similar regardless of the placement of the herbicide-treated soil (Table
11). Conversely, for combinations of clomazone plus linuron and atrazine
plus metribuzin, placement of herbicide-treated soil in the same zone as
the seed was necessary for a reduction in leaf area and dry shoot weight
to occur.

These results suggest that the severity of injury from clomazone
plus metribuzin is not dependent upon movement of the these herbicides in
the soil to the area of the germinating soybean seed. Clomazone plus
linuron, however, is similar to a combination of atrazine plus metribuzin
in that movement of these herbicides in the soil to where the germinating
seed is present may significantly increase the level of injury from these
herbicides. As movement of herbicides downward in the soil profile is
primarily dependent on rainfall, heavy rainfall shortly after planting,and
preemergence herbicide application may move the herbicides into the seed

zone, resulting in increased injury to soybean.

42

Table 11. Response of soybean to placement of soil treated with
clomazone plus metribuzin, clomazone plus linuron, or atrazine plus
metribuzin in relation to the seed.

Herbicides Placement Leaf area Shoot weight

------------- % of untreatedP----------

Clomazone +

metribuzin
Above 61 58
With 52 52
Below 66 66
Clomazone +
linuron
Above 70 73
With 48 45
Below 69 69
Atrazine +
metribuzin
Above 69 71
With 37 32
Below 65 65
LSD(0.05) 22 17

 

aAverage values of untreated plants: leaf area-291 cmF; shoot weight-1.4
g.

43

The field studies support observations in producers fields in 1986,
our own field studies, and studies in eastern Arkansas (17). Rainfall
increased injury from linuron supporting the observation that it is
necessary for this herbicide to be in the seed zone. Metribuzin placement
was not critical and.with PPI applications the herbicide was available to
the soybean seed.

The field studies further indicate that a potential for a
synergistic interaction from combinations of clomazone plus metribuzin and
clomazone plus linuron exists, even though it may not appear every year.
Other factors such as soil characteristics, primarily clay content, and
rainfall play a role in determining if the interactions of clomazone plus
metribuzin and clomazone plus linuron will be additive or synergistic.
Temperature at the time of soybean germination and emergence, however,
does not appear to be a factor in the interactions. Atrazine residues may
enhance the interactions of clomazone plus metribuzin and clomazone plus
linuron but this only occurred one location in one year and was not
consistent with increasing rates of atrazine or the levels of atrazine
extracted or detected. In the field studies we were able to detect the
synergistic interactions by visual examination and quantify it by
measuring leaf area, shoot weight, and root weight. Early season stand
count was a poor predictor of the interaction as the herbicides did not
usually prevent soybean emergence, but instead killed plants before the
expansion of the first trifoliolate leaf: 'Visual injury, leaf area, shoot
weight, and root weight did not predict yield loss, probably because of

the ability of soybean to compensate for early season injury (6).

44

While these experiments did not directly examine the interactions of
clomazone plus metribuzin and clomazone plus linuron on any weed species,
the response of soybean to these herbicide combinations suggest that a
similar response could occur in weed species. Weeds such as common
cocklebur (Xanthium strumarium L.) or redroot pigweed (Amaranthus
retroflexus L.), which are marginally affected by clomazone, metribuzin,
and linuron, could exhibit increased injury from combinations of these
herbicides. Research will need to be conducted to determine if these
herbicides are synergistic in weed species. If synergism does exist,
reduced rates of these herbicides could result in acceptable weed control
without injury to soybeans. The behavior of these herbicides in both
soybean, and. weed species will also provide insight as to ‘how the

interaction is occurring.

10.

11.

12.

13.

14.

45

LITERATURE CITED

Caprial, P., A. Haisch, and S. U. Khan. 1985. Distribution and
nature of bound (nonextractable) residues of atrazine in a mineral

soil nine years after the herbicide application. J. Agric. Food
Chem. 33:567-569.

Colby, S. R. 1967. Calculating synergistic and antagonistic
responses of herbicide combinations. Weeds 15:20-22.

Dubrey, H. D. and J. F. Freeman. 1964. Influence of soil
properties and microbial activity on the phytotoxicity of linuron
and diphenamid. Soil Sci. 97:334-340.

Dunigan, E. P., J. P. Frey, L. D. Allen, Jr., and A. McMahon. 1972.
Herbicide effects on the nodulation of Glycine max (L.) Merrill.
Agron. J. 64:806-808.

Fink, R. J. and O. H. Fletchall. 1969. Soybean injury from
triazine residues in soil. Weed Sci. 17:35-36.

Hagood, E. 8., Jr., J. L. Williams, Jr., and T. T. Bauman. 1980.
Influence of herbicide injury on the yield potential of soybeans
(Glycine max). Weed Sci. 28:40-45.

Hamill, A. S. and D. Penner. 1973. Interaction of alachlor and
carbofuran. Weed Sci. 21:330-335.

Ladlie, J. S., W. F. Meggitt, and D. Penner. 1977. Effect of
atrazine on soybean tolerance to metribuzin" Weed Sci. 25:115-121.

Loux, M. M., R. A. Liebl, and F. W. Slife. 1989. Adsorption of
clomazone on soils, sediments, and clays. Weed Sci. 37:440-444.

Penner, D. 1971. Effect of temperature on phytotoxicity and root
uptake of several herbicides. Weed Sci. 19:571-576.

Peter, C. J. and J. B. Weber. 1985. Adsorption, mobility, and
efficacy of metribuzin as influenced by soil properties. Weed Sci.
33:868-873.

Savage, K. E. 1977. Metribuzin persistence in soil. Weed Sci.
25:55-59.

Sharom, M. S. and G. R. Stephenson“ Behavior and fate of metribuzin
in eight Ontario soils. Weed Sci. 24:153-160.

Smith, A” E. 1981. Comparison, of solvent systems for the
extraction of atrazine, benzoylprop, flamprop, and trifluralin from
weathered field soils. J. Agric. Food Chem. 29:111-115.

15.

16.

17.

46

Wax, L. M. 1977. Incorporation depth and rainfall on weed control
in soybeans with metribuzin. Agron J. 69:107-110.

Werling, V. L. and D. D. Buhler. 1988. Influence of application

time on clomazone activity in no-till soybeans, Glycine max. Weed
Sci. 36:629-635.

Westberg, D. E., L. R. Oliver, and.R. E. Frans. 1989. Weed control
with clomazone alone and with other herbicides. Weed Tech. 3:678-
685.

CHAPTER 3

INTERACTION OF CLOMAZONE AND METRIBUZIN IN SOYBEAN, COMMON COCKLEBUR, AND

REDROOT PIGWEED

ABSTRACT

Experiments were conducted in the greenhouse to determine if a
synergistic interaction.occurred.in.soybean, common.cocklebur, and.redroot
pigweed to combinations of clomazone plus metribuzin. The soybean
experiment was conducted on two silt loam soils, one with 2.5% organic
matter (OM) and the other with 4.4% OM. Reductions in soybean shoot dry
weight occurred in the soil with 2.5% OM soil metribuzin at 280 and 560 g
ai ha'1 plus clomazone at 1680 g ai ha”, and from metribuzin at 560 g ha’1
plus clomazone at 420 and 840 g ha”. Soybean shoot dry weight was reduced
by metribuzin at 560 g ha“ plus clomazone at 420, 840, or 1680 g ha'1 in
the 4.4% OM soil. The clomazone plus metribuzin interactions in soybean
were synergistic. Metribuzin applied at 70 g ha'1 plus clomazone at 420
or 840 gha‘1 synergistically decreased common cocklebur dry shoot weight.
Redroot pigweed dry weight was severely reduced from clomazone alone and

an interaction. between clomazone plus metribuzin was not evident.

Nomenclature: clomazone, 2-[(2-chloropheny1)methyl]-4,4-dimethyl-3-
isoxazolidinone); metribuzin, 4-amino-6-(l,l dimethylethyl)-3-
(methylthio)-1,2,4-triazin-5(4H)-one; common cocklebur, Kanthium

strumarium L. # XANST; redroot pigweed, Amaranthus retroflexus L. #

AMARE; soybean, Glycine max (L.) Merr. 'Century'.

47

48

INTRODUCTION

Researchers have observed a synergistic response in soybean to
applications of clomazone plus metribuzin (9, 12, 14). Clomazone is
adsorbed to organic matter (OM) whereas metribuzin is adsorbed to clay and
OM (7, 8, 11), thus an interaction at a given application rate may be
dependent on soil type.

Herbicide interactions, definitions of synergism, and methods of
measuring synergism have been reviewed by Hatzios and Penner (6).
Difficulty has come in developing methods of measuring synergism. Care
must be taken in examining the assumptions implicit in the method chosen.
A test developed by Colby (2) has been used frequently because of its
simplicity. It is based on a multiplicative survival model which assumes
that the two compounds under study have different modes of action.

Clomazone and metribuzin have different sites of action in a plant.
Metribuzin inhibits photosynthesis by blocking electron transport in
photosystem II (1, 3). The site of action of clomazone has not been
conclusively determined, but Duke et a1. (4) suggest that clomazone blocks
diterpene and tetraterpene synthesis. Specifically, clomazone inhibits
the conversion of geranylgeranyl pyrophosphate from isopentenyl
pyrophosphate (10). Symptomology of sensitive plant species, including
bleaching of the leaves, indicate that clomazone is not a photosynthetic
inhibitor. Therefore a multiplicative model for evaluating the
interaction is appropriate.

The purpose of this study was to: (a) determine the range of
application rates at which soybean demonstrated the synergistic

interaction of clomazone and metribuzin on two soils with differing OM

49

levels, and (b) determine if this synergistic interaction of clomazone and
metribuzin could be exploited in providing common cocklebur and redroot
pigweed control, two weeds that are not usually controlled when either of

these herbicides are applied singly.

MATERIALS AND METHODS

The study was conducted with three soils. For the soybean study,
two Capac loam soils (mixed, mesic Aeric Ochraqualf) that varied in
organic matter (OM) content were used. Both soils contained 46 to 48%
sand, 39 to 40% silt, and 13 to 14% clay. Soil pH was 6.5 and OM 2.5% for
one soil while pH was 6.0 and OM 4.4% for the other soil. A Spinks sandy
loam soil (mixed, mesic Psammentic Hapludalf, 71% sand, 19% silt, 10%
clay, pH-6.2, 0.8% OM) was used for the study with common cocklebur and
redroot pigweed. Plastic pots 24-cm tall and ll-cm in diameter with a
total volume of 946 ml were filled with untreated soil to a level 10 cm
below the top of the pot. Soil to be treated was put into smaller 473 m1
plastic pots and herbicides were applied with a stationary, air
pressurized greenhouse pot sprayer in 206 L ha'1 of water at 207 kPa. A
factorial design was utilized with treatments consisting of clomazone at
0, 420, 840, and 1680 gha'1 for all species and metribuzin.at 0, 140, 280,
and 560 g ha‘1 for soybeans,; 0, 70, 140, 280 g ha‘1 for common cocklebur;
and 0, 35, 70, and 140 g ha‘1 for redroot pigweed. Clomazone rates were
selected based on the standard field recommendation of 840 g ha“.
Metribuzin rates were based on the standard field recommendation of 280 g
ha‘1 for soybeans, and on preliminary common cocklebur and redroot pigweed

experiments. Treated soil was mixed for 30 sec with a soil mixer and then

50

used to fill the larger pots within 4 cm of the pot top. Three 'Century'
soybean seeds or three common cocklebur burs were planted 2 cm deep in the
treated soil in each pot and thinned to one plant per pot following
emergence. Redroot pigweed seed was stirred into the soil. Emergence of
redroot pigweed was erratic and plants were not thinned.

Pots were weighed after planting and herbicide application. Soil
moisture levels based on 18% by soil weight were calculated and recorded.
When the soil surface was observed to be dry, soil was irrigated to 18%
moisture. This procedure precluded varying soil moisture levels from
influencing herbicide availability.

Pots were placed in the greenhouse where natural sunlight was
supplemented by sodium halide lights with an intensity of 300 ME m"sec‘2
set for a 15 hour day. Pots were arranged in a completely randomized
design with four replications and the experiment was repeated twice in
time. Soybean plants were harvested at the soil level at the when the
second trifoliolate leaf was fully expanded; common cocklebur at the
fourth leaf stage; and redroot pigweed 4 weeks after planting. The
cotyledons were removed and shoots dried and weighed. Data was subjected
to analysis of variance and Fishers Protected Least Significant
Differences (LSD) calculated at the 5% probability level. For the
herbicide combinations the expected mean was calculated following the
method of Colby (2) and the modified LSD test developed by Hamill and
Penner (5) used to determine if the response to the herbicide combinations

was synergistic.

51

RESULTS AND DISCUSSION

Soybean. Response of soybean to clomazone plus metribuzin was dependent
on soil OM content (Table l). Clomazone alone did not reduce soybean
shoot dry weight on either soil, regardless of application rate.
Metribuzin alone at 560 g ha4 reduced soybean shoot dry weight only when
the soil OM was 2.5%, indicating a use rate too high for this soil and
masking any herbicide interaction. Clomazone applied at 420, 840, and
1680 g ha“ when combined with metribuzin application of 280 and 560 g ha‘1
severely injured soybean grown in the 2.5% OM soil. When the soil OM was
4.4%, severe injury to soybean occurred only when metribuzin was applied
at 560 g ha'1 plus any rate of clomazone. Several rates of metribuzin and
clomazone were calculated to be acting synergistically.

In examining the analysis of variance for both organic matter
levels, the F-value for metribuzin was much greater than the F-value for
clomazone. This indicates that the rate of metribuzin had a greater
influence in reducing shoot weight than the rate of clomazone.

Some of the synergistic interactions that were calculated for
soybean dry shoot weights were for values equal or greater than the dry
shoot weight of untreated plants. This is because the formula developed
by Colby is based on data from plants treated with one of the herbicides
in the combination. In this study soybean dry shoot weight from plants
treated with clomazone alone at 420 and 840 g ha“ and metribuzin at 140
and 280 g hat’1 on both soils were greater than plants treated with no
herbicide. Using these observed values in Colby's formula results in
expected values for the herbicide combinations being,much larger than the

observed values. When the observed value for a herbicide combination is

52

Table 1. Dry shoot weight of soybean treated with clomazone and/or
metribuzin grown in Capac silt loam soil with 2.5% or 4.4% organic
matter contents“.

Clomazone Metribuzin Shoot dry weightb

------ Organic Matter------

 

2.5% 4.4%
----- g ai ha4----- -----g ai ha4----- -----% of untreatedf-----
420 0 121 112
840 O 135 123
1680 0 97 118
0 140 135 113
0 280 112 114
0 560 32 90
420 140 100' 104'
420 280 40' 112
420 560 17 53'
840 140 104' 112'
840 280 55' 101'
840 560 32 50'
1680 140 67' 108'
1680 280 38' 87'
1680 560 15 6'
LSD(0.05) 28 23

 

aSoybean plants were harvested when the second trifolialate leaf was
fully expanded.

bCombinations determined to be synergistic are denoted with an asterisk.
cAverage weight of untreated plants in 2.5% OM soil was 0.9 g. Average
weight of untreated plants in 4.4% OM soil was 0.7 g.

 

(‘1‘

8)

th

 

to
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dr
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a1
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he

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53

greater or similar to the observed value for untreated plants, any
calculated synergism may not be of great consequence.

The data from applications of clomazone alone and metribuzin alone
at low rates would suggest that these herbicide have a stimulatory effect
on soybean shoot weight. The consistency of this response indicates that
the phenomena was indicative of a true stimulatory effect. Sublethal
doses of herbicides such as 2,4-D [(2,4-dichlorophenoxy)acetic acid] have
been shown to stimulate plant development (12).

Common cocklebur. Increasing rates of clomazone or metribuzin applied
alone decreased shoot dry weight (Table 2). Plants were stunted and
extensive whitening (clomazone injury) or chlorosis (metribuzin injury) of
the leaves were observed. Metribuzin alone at 280 kg ha'1 killed common
cocklebur seedlings. A synergistic response occurred when metribuzin at
70 g ha" was combined with clomazone at 420 or 840 g ha".

Redroot pigweed. Clomazone at 420 and 840 g ha" decreased redroot pigweed
dry shoot weight 89 and 100%, respectively (data not presented).
Metribuzin at 140 g ha’1 decreased redroot pigweed shoot dry weight 74% and
all herbicide combinations resulted in complete kill of the seedlings. No
synergism in redroot pigweed response was detected from any of the
herbicide interactions in this study, because of the high degree of
response of redroot pigweed to clomazone.

These experiments indicate that clomazone and metribuzin can
interact synergistically in both soybean and common cocklebur. The
metribuzin application rate should be reduced when combined with clomazone
to reduce the potential for soybean injury. Synergism noted on common

cocklebur would be beneficial for a broader spectrum weed control than

54

Table 2. Dry shoot weight of common cocklebur grown in soil treated

with clomazone and metribuzinP.

 

 

Clomazone Metribuzin Shoot dry weightb
------- g ai ha4----- -------g ai ha4----- ---% of untreated9---
420 O 68
480 0 41
1680 O 30
0 70 86
0 140 20
0 280 0
420 70 21'
420 140 0
420 280
840 70 11'
840 140 O
840 280 0
1680 70 0
1680 140
1680 280 0
LSD(0.05) 22

 

aCommon cocklebur plants were harvested with four fully expanded leaves.
bCombinations determined to be synergistic are denoted with an asterisk.
cAverage dry shoot weight of untreated plants was 0.4 g.

55

from.either herbicide alone. Other weeds such as eastern black nightshade
(Solanum ptycanthum Dun.), that are poorly controlled by either herbicide
alone, may be adequately controlled by the herbicides combined. Synergism
could be exploited for weed control, without causing injury to the soybean

crop.

10.

ll.

12.

13.

56

LITERATURE CITED

Boger, P. and U. Schlue. 1976. Long-term effects of herbicides on
the photosynthetic apparatus 1. Influence of diuron, triazines and
pyridazinones. Weed Res. 16:149-154.

Colby, S. R. Calculating synergistic and antagonistic responses of
herbicide combinations. Weeds 15:20-22.

Draber, W., K. H. Buchel, K. Dickore, A. Trebst, and E. Pistorius.
1969. Structure-activity correlation of 1,2,4-triazinones, a new

group of photosynthetic inhibitors. Prog. Photosyn. Res.
2:1789-1795.

Duke, 8. 0., W. H. Kenyon, and R. N. Paul. 1985. FMC 57020 effects
on chloroplast development on pitted morningglory (Ipomoea lacunosa)
cotyledons. Weed Sci. 33:786-794.

Hamill, A. S. and D. Penner. 1973. Interaction of alachlor and
carbofuran. Weed Sci. 21:330-335.

Hatzios, K. K. and D. Penner. 1985. Interaction of herbicides with
other agrochemicals in higher plants. in Reviews of Weed Science.
Vol. 1 p. 1-63.

Loux, M. M., R. A. Liebl, and F. W. Slife. 1989. Adsorption of
clomazone on soils, sediments, and clays. Weed Sci. 37:440-444.

Peter, C. J. and J. B Weber. 1985. Adsorption, mobility, and

efficacy of metribuzin as influenced by soil properties. Weed Sci.
33:868-873.

Salzman, F. P. and K. A. Renner. 1991. The synergistic
interactions of clomazone plus metribuzin and clomazone plus
linuron. WSSA Abstr. 31:6.

Sandmann, G. and P. Boger. 1987. Interconversion of prenyl
pyroposphates and subsequent reactions inithe presence of FMC 57020.
Z. Naturforsch. 42c:803-807.

Savage, K. E. 1977. Metribuzin persistence in soil. Weed Sci.
25:55-57.

Wax, L. M., L. A. Knuth, and F. W. Slife. 1969. Response of
soybeans to 2,4-D, dicamba, and picloram. Weed Sci. 17:388-393.

Werling, V. L. and D. D. Buhler. 1988. Influence of application
time on clomazone activity in no-till soybeans, Glycine max. Weed
Sci. 36:629-635.

57

14. Westberg, D. E., L. R. Oliver, and R. E. Frans. 1989. Weed control
with clomazone alone and with other herbicides. Weed Tech. 3:678-
685.

CHAPTER 4
ABSORPTION, TRANSLOCATION, AND METABOLISM OF CLOMAZONE, METRIBUZIN, AND
LINURON IN SOYBEAN AND COMMON COCKLEBUR
ABSTRACT

Research was conducted, using soybean and common cocklebur as test
species, to determine if the synergistic interactions of clomazone plus
metribuzin and clomazone plus linuron were due to the effect of one
herbicide on the uptake, partitioning, or metabolism of the other.
Treatments consisted of 1‘C-clomazone alone and combined with metribuzin
or linuron, 14C-metribuzin alone and combined with clomazone, and 1‘C-
linuron alone and combined with clomazone. There were only slight
differences in uptake and partitioning of clomazone applied alone in
soybean and common cocklebur compared to clomazone plus metribuzin or
linuron in soybean and common cocklebur. No differences were noted in
uptake and partitioning in both species when either metribuzin or linuron
were applied alone or combined with clomazone. Levels of parent clomazone
were higher in common cocklebur roots when clomazone was combined with
metribuzin and linuron compared to clomazone alone. The percent of parent
metribuzin was higher in soybean roots, and counnon cocklebur roots and
shoots when clomazone was combined with metribuzin compared to metribuzin
alone. Levels of parent linuron were greater in soybean shoots when
linuron was applied with clomazone compared to linuron alone. These
results indicate that metribuzin and linuron metabolism are altered when
clomazone is also applied, leading to increased phytotoxicity.
Nomenclature: clomazone , 2 - [ (2 -chloropheny1)methyl] -4 , 4-dimethyl - 3 -

isoxazolidinone; linuron, N ' - (3 , 4-dichlorophenyl) -N-methoxy-N-methylurea;

58

59
metribuzin, 4-amino-6-(1,1-dimethy1ethyl)-3-(methylthio)-l,2,4-triazin-
5(4H)-one; common cocklebur, Xanthium strumarium L. # XANST; soybean,

Glycine max (L.) 'Century'.

60

INTRODUCTION

Clomazone plus metribuzin and clomazone plus linuron provide broad
spectrum weed control in soybeans. However, research in the field and
greenhouse has indicated a synergistic interaction in soybean and common
cocklebur to these herbicide combinations (11, 12, 16, 17). The response
of soybean to clomazone plus metribuzin was neither temperature dependent,
occurring under both cool and warm temperature regimes, nor was the
response dependent on placement of the herbicides in the soil. The
response of soybean to combinations of clomazone plus linuron was
dependent on placement of the herbicides in the soil (ll). Symptomology
of soybean and common cocklebur growing in soil treated with metribuzin
plus clomazone or linuron plus clomazone was typical of metribuzin and
linuron injury, respectively.

Linuron is initially demethylated in soybean, and eventually
metabolized to 3,4-dichloroaniline. This reaction is catalyzed by a
mixed-function oxidase enzyme (9) . Despite early reports indicating that
metribuzin is metabolized to a deaminated diketo form and eventually
conjugated to glucose (8, 10, 13), it is now proposed that the major
metabolic pathways for metribuzin in soybean involves either the formation
of a homoglutathione conjugate or degradation into the diketo form that is
incorporated into insoluble residues (4) It was further suggested that
the capacity of soybean to form a N-glucose conjugate is limited (4).

The pathway of clomazone metabolism in higher plants is not well
established. Explanations include conjugation with glucose in tomato
(Lycopersicon esculentum) (18) . Conjugation with glutathione in vitro has

been accomplished leading to the conjecture that this could be a

61

deactivation mechanism in plants (15). Metabolism of these herbicide in
common cocklebur, a weed species only suppressed by these herbicides
individually, has not been determined.

A potential explanation for the synergistic interactions between
clomazone plus linuron and clomazone plus metribuzin in soybean and common
cocklebur may include one herbicide altering the uptake and/or
partitioning of another. Alternatively, plant metabolism may be altered
by competition between herbicides for a particular conjugate or enzyme
that is specific for the deactivation of each.

The purpose of this study was to: (a) determine if the basis for
the synergism in soybean and common cocklebur to clomazone plus metribuzin
and clomazone plus linuron was due to differences in uptake, partitioning,
and/or metabolism; and (b) determine if there were differences in uptake
and partitioning of clomazone, linuron, and metribuzin between soybean and

common cocklebur.

MATERIALS AND METHODS

Soybean and common cocklebur were germinated in silicone sand in the
greenhouse under natural light supplemented with sodium vapor lights with
an intensity of 300 uE m""sec’1 and set for a 15 hr day. Sand was kept
moist with water until emergence of the seedlings, after which the plants
were irrigated with modified Hoaglands solution (1). When the first
trifoliolate leaf of the soybean was fully expanded and common cocklebur
had four fully expanded leaves, each plant was transferred to a jar

containing 50 m1 of modified Hoaglands solution that was continuously

62

aerated. Preliminary experiments indicated that this system allowed
plants to grow and develop normally for 10 days.

The plants were allowed to acclimate for 24 hr in the Hoaglands
solution. The solution was then changed and as a preconditioning
treatment herbicides were added to the solution at concentrations of 6.5
M for clomazone, 1.0 pH for linuron, 1.0 pH for metribuzin, 6.5 plus 1.0
M for clomazone plus linuron, or 6.5 plus 1.0 M for clomazone plus
metribuzin. The plants were allowed to remain in the preconditioning
solutions for 24 hr.

The nutrient solution was again changed and the herbicide treatment
added to the solutions. Treatments consisted of 1‘C-uniformly phenyl ring-
labelled clomazone (specific activity was 1,036,000 Bq mmol",
radiochemical purity was 94%), “C-uniformly ring-labelled linuron
(specific activity was 2,257 Bq mg", radiochemical purity was 95%), or “C-
uniformly ring-labelled metribuzin (specific activity was 769,600 Bq
mmol", radiochemical purity was 98%) alone; “C-clomazone plus metribuzin;
1‘C-metribuzin plus clomazone; “C-clomazone plus linuron; and 1‘C-linuron
plus clomazone. The total amount of activity in each jar was 3.7 MBq.
Final concentrations of 6.5 [AM for clomazone and 1.0 M for metribuzin and
linuron were achieved by the addition of technical grade herbicide.
Plants were left in the treatment solutions for 18 hr after which the
plants were removed and immediately placed on dry ice. Plants were kept
frozen at -20 C until sampled for partitioning or metabolism or used for
autoradiography. There were 15 plants of each species for each treatment

and the experiments were repeated twice.

63

Six plants were divided into shoot meristem, leaves, stem, and roots
and combusted in a biological oxidizer1 to “602. The CO2 was trapped in
a solution of scintillator"’:CO2 absorber3 (2:1 v/v). Total radioactivity
in as sample was determined by liquid scintillation counting} (LSC).

Three plants were exposed for 5 weeks to X-ray films. After the
film was developed, visual comparisons were made between treatments for
evidence of differences in uptake or partitioning.

Metabolites were extracted from six plants of each treatment. Two
plants from each treatment were extracted simultaneously. The extraction
procedures used were described by Weston and Barrett for clomazone (18),
by Kuratle et al. for linuron (7), and by Falb and Smith for metribuzin
(2). These methods are summarized as follows. The extraction solvents
were methanol for clomazone, acetone for linuron, and ethanol:water (4:1
v/v) for metribuzin. Plants were separated into roots and shoots and the
plant portions extracted separately. Plants were homogenized in solvent
for 4 min in a tissue homogenizer’. The homogenate was filtered through
filter paper7 to remove the plant residue from the filtrate.

Radioactivity in the residue was determined by oxidizing a pmeweighed

 

1OX-300. R. J. Harvey Instrument Corp. Patterson, NJ 07642.

2Safety-Solve. Research Products International Corp. Mount Prospect,
IL 60056.

3Carbo-Sorb II. Packard Instrument Co. Meriden, CT 06450.
4Model 1500. Packard Instrument Corp. Downers Grove, IL 60515.
EX-OMAT. Eastman Kodak Co. Rochester, NY 14650.

°VirTis 45. The VirTis Co., Inc. Gardiner, NY 12525.

7Whatman #1. Whatman International Ltd. Maidstone, England.

64

portion of the sample and back calculating. The filtrate was placed in a
flask and evaporated to about 5 ml by rotoevaporation“. Samples were
transferred to a small test tube, and the flask was rinsed three times
with 1 m1 of solvent each time. Metribuzin samples were evaporated to the
water phase. The filtrate was transferred to separatory funnels and
extracted twice with an amount of benzene equivalent to the water in the
funnel each time. The amount of water was recorded and the radioactivity
in a 1 ml aliquot of the water was determined by LSC to quantify the polar
metabolites. The water phase of the extraction was not used further.
After drying samples in a stream of nitrogen, samples were resuspended in
1 ml of solvent. Efficiencies of extraction were 74% for clomazone, 75%
for metribuzin, and 78% for linuron. Solvents used were methanol for
clomazone samples, acetone for linuron samples, and benzene:acetonitrile
(9:1 v/v) for metribuzin samples. Two 100 pl aliquot of each sample was
spotted on a prechanelled, 15 nm silica gel thin layer chromatography
(TLC) plate9 and developed to 15 cm in solvent solution. Developing
solutions were chloroform:methanol (7:1 v/v) for clomazone samples,
chloroformzmethanol:pyridine (100:5:1 v/v/v) for linuron samples, and
benzene:chloroformzp-dioxane (4:3:3 v/v/v) for metribuzin samples. Parent
compounds were also spotted on TLC plates and the plates developed to
identify the parent compound. After developing, plate channels were
scraped in 1 cm increments and the activity measured by LSC.

All data were subjected to analysis of variance. The data for total

activity was analyzed on a Bq mg‘1 basis. The partitioning data was

 

aBfichi R110. Brinkmann Instruments, Inc. Westbury, NY 11590.

9LKSDF. Whatman, Inc. Clifton, NJ 07014.

65

converted to the percent of the total in all plant organs. The metabolite
tiata was converted to the percent of the total in the gel scraped from the
TLC plate. All data converted to percent was analyzed after an arc sin
transformation had been performed. Means were separated by Fishers
‘protected least significant differences test at the 5% level of
probability to compare differences between species and between a single

herbicide and herbicide combinations.

RESULTS AND DISCUSSION

Common cocklebur took up more clomazone than soybean, but retained
a greater percentage of clomazone and metabolites in the roots than did
soybean (Table 1). In the field and greenhouse common cocklebur will show
symptoms of leaf whitening from clomazone while soybean will not. This
data indicates that differential partitioning of clomazone and its
metabolites within the plants did not account for the expression of
clomazone injury in common cocklebur leaves.

In soybean, a greater percentage of clomazone was retained in the
roots when metribuzin was in the nutrient solution compared to clomazone
alone. Accordingly, a greater percentage of clomazone was translocated to
the leaves when clomazone alone was in nutrient solution then when
metribuzin was added. No differences occurred in partitioning of
clomazone alone compared to clomazone plus linuron. Therefore, the
increased injury observed from combinations of clomazone plus linuron and
clomazone plus metribuzin cannot be explained by increased levels of

clomazone in the leaves and meristems.

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cocoons so emocmsHmca mm new ocon couscaH ecu .causnuuuma .mcoumaoao co wcacoduuuuma ecu manna: .H «Hats

67

There was no difference in the percentage of activity in soybean and
common cocklebur tissues from linuron alone compared to linuron plus
clomazone. The percentage of metribuzin in soybean and common cocklebur
tissues was similar from treatments of metribuzin alone compared to
metribuzin plus clomazone. Partitioning of metribuzin and linuron and
their metabolites did not account for the increased injury observed from
combinations of clomazone plus linuron and clomazone plus metribuzin.

There were no detectable visual differences in the autoradiographs
of plants treated with 1‘C-herbicide alone or in combination with another
herbicide in either soybean or common cocklebur. More clomazone appeared
in the roots than in the shoots of common cocklebur and in the shoots,
clomazone was concentrated in the veins and surrounding areas. In
soybean, clomazone was evenly distributed between roots and shoots and was
evenly distributed throughout the leaf. Metribuzin appeared to be more
evenly distributed between root and shoots in both species and in the
shoots was found primarily in the veins and surrounding leaf portions. In
both species, more linuron appeared to be in the roots then in the shoots.
In the shoots linuron was concentrated in the veins in soybean and to a
lesser extent in common cocklebur.

Differences between treatments in radioactivity in the plant
residues were slight (Table 2). More radioactivity was detected in common
cocklebur roots from “C-clomazone alone than from “C-clomazone plus
linuron. No other differences among treatments in plant roots and shoots
were significant. There was more radioactivity in the residue of soybean

shoots from “C-linuron alone compared to “C-linuron plus clomazone.

68

 

 

'Table 2. Unextracted radioactivity in plant residues‘.
Species Organ Treatment % of total “C
Soybean root 14C-clomazone 15.9 a
1"C-clomazone + metribuzin 16.7 a
1‘C-clomazone + linuron 16.0 a
shoot 1‘C-clomazone 7.1 bc
14C-clomazone + metribuzin 7.2 bc
“C-clomazone + linuron 5.3 cd
Common cocklebur root 1‘C-clomazone 8.5 b
1‘C-clomazone + metribuzin 6.7 bcd
“C-clomazone + linuron 5.1 cd
shoot 1‘C-clomazone 5.6 bcd
1"C-clomazone + metribuzin 6.4 bcd
1‘C-clomazone + linuron 4.8 d
Soybean root 1‘C-metribuzin 19.4 bcd
1"C-metribuzin + clomazone 25.0 abc
shoot 1‘C-metribuzin 12.1 d
1‘C-metribuzin + clomazone 18.6 cd
Common cocklebur root 1‘C-metribuz in 27 . 8 ab
“C-metribuzin + clomazone 32.1 a
shoot 1‘C-metribuzin 14.0 d
1‘C-metribuzin + clomazone 14.5 d
Soybean root 1‘C-linuron 4.9 ab
1‘C-linuron + clomazone 4.1 b
shoot “C-linuron 7.3
'“C-linuron + clomazone 3.7 b
Common cocklebur root “C- linuron 4 . 2 b
1‘C-linuron + clomazone 4.6 ab
shoot 1‘C-linuron 4.8 ab
‘“C-linuron + clomazone 7.1 a

¥

 

I'Means followed by the same

Differences at 5%.

are not val id .

letter within the same “C-herbicide grouping
are not significantly different by Fishers Protected Least Significant
Comparisons between herbicide treatment groupings

69

Differences in the level of radioactivity between roots and shoots
did exist (Table 2). Activity of “C-clomazone averaged 16% of the total
recovered in soybean roots, and 6% in soybean shoots. Activity of “C-
clomazone was less than 9% of the total in common cocklebur roots and
shoots. Significantly more radioactivity’was detected.in.common cocklebur
roots from treatments of 1‘C-metribuzin then in shoots. Differences in
soybean from 1“(J-metribuzin treatments between roots and shoots were not
significant. Radioactivity in plant residues accounted for less than 8%
of the total recovered from “C-linuron treatments in organs of both
species.

Clomazone (R, 0.93) and two metabolites (R, 0.13 and 0.33) were
detected.in soybean (Table 3). 'Three metabolites (R,0.l3, 0.33, and.0.47)
were detected in common cocklebur. Vencill and coworkers (14) found two
major metabolites of clomazone in soybean.and.the three Amaranthus species
they examined. 'They also found. that levels of clomazone and. the
metabolites did not change significantly over a period of 12 to 96 hr.

‘A.higher percentage of the parent clomazone extracteduwas present in
soybean shoots than in roots. However, a greater percentage of metabolite
l (R,CL13) was present in the roots than in the shoots. Metabolism of
clomazone occurred in soybean roots 18 hr after addition of “C-clomazone
to the nutrient solutiont Despite the high level (83%) of parent compound
present in soybean shoots, injury symptoms are not usually present in
plants treated with clomazone in the field or greenhouse. In common
cocklebur, similar distribution of metabolites was found in roots and
shoots. More parent clomazone than metabolites were present in root and

shoot of both species.

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71

No differences in levels of metabolites were observed when
metribuzin or linuron were added to clomazone in soybean or common
cocklebur, indicating that the synergistic interactions between the two
combinations is not based on altered clomazone metabolism.

Three metabolites (R, 0.13, 0.43, and 0.67) of metribuzin (R, 0.90)
were detected in soybean and four metabolites (R,(L13, 0.27, 0.43, and
0.67) were detected in common cocklebur (Table 4). Falb and Smith (2)
identified four metabolites in soybean, in order of decreasing R,velue:
6-tert-buty1-3-(methylthio)-as-triazin-5(4H)-one, 6-tert-butyl-as-triazin-
3,5-(2H,4H)-dione, 4-amino-6-tert-butyl-l,2,4-triazin-3,5(2H,4H)-dione,
and an unidentified metabolite, using this extraction and developing
procedure. In soybean, a greater percentage of activity was present as
parent compound in shoots than in the roots. A greater percentage of
metabolite l (R, 0.13) and metabolite 4 (R, 0.67) were found in soybean
roots than in shoots. This indicates that metabolism.of'metribuzin occurs
in soybean roots after uptake. A greater percentage of parent metribuzin
and a lesser percentage of metabolite 1 (R,(L13) were found in soybean
roots treated with 1‘C-metribuzin plus clomazone than in roots treated with
only 1‘C-metribuzin. Differences in the levels of parent compound and
metabolite 1 were not apparent between treatments in soybean shoots. In
preliminary experiments, it took up to 5 days for injury symptoms to
appear, therefore differences in metabolism in the shoot may take longer
than 18 hr to be detectable or it may take longer for parent metribuzin to
accumulate in the shoots.

In common cocklebur, there was a greater percentage of total

activity present as parent metribuzin and a lesser percentage of

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73

metabolite 1 (R, 0.13) in roots and shoots of plants treated with 1‘C-
metribuzin plus clomazone than in plants treated with only “C-metribuzin.
In preliminary experiments, common cocklebur exhibited injury symptoms
from combinations of clomazone plus metribuzin. before soybean. did,
indicating the movement of parent compound to the shoots of cocklebur is
more rapid than in soybean. Total radioactivity in the water-soluble
portion of the extract was greater in soybean roots treated with 1‘C-
metribuzin plus clomazone than from treatments of 1‘C-metribuzin alone
(data not presented). No differences were noted in soybean shoots or in
common cocklebur. This water-soluble portion has been found to include
metribuzin conjugates containing carbohydrates and often amino acids and
lipids, and small amounts of the deaminated, diketo metabolites and
deaminated metabolites (3).

The differences in metribuzin metabolism in soybean root and common
cocklebur root and shoot could explain the synergistic interaction from
clomazone plus metribuzin. The higher percentage of parent metribuzin,
the active form, is consistent with the injury symptoms of metribuzin
observed in treated plants in preliminary experiments. Metabolite l (R,
0.13) is likely a large molecule, possibly the homoglutathione conjugate
of metribuzin, the end product of metribuzin metabolism in soybean. The
decrease in the percentage of this metabolite when clomazone is present
indicates that clomazone is somehow interfering with metribuzin
metabolism. The same explanation may be true for common cocklebur.
Injury appears more quickly in common cocklebur because the parent

compound is more rapidly transported to the shoot.

74

In both soybean and common cocklebur the majority of activity from
the linuron treatments was parent compound.(R,0.87) (Table 5). Four other
metabolites were also detected (R, 0.13, 0.33, 0.53, and 0.67).
Identification of metabolites and R, values for linuron and metabolites in
soybean have been published (6, 7). Metabolites identified, in order of
highest R, value to lowest include linuron, 3,4-dichloraniline, 3-(3,4-
dichlorophenyl)-1-methoxy urea, 3-(3,4-dichloropheny1)-1-ethyl urea, and
3-(3,4-dichlorophenyl) urea. In soybean shoots, a greater percentage of
parent linuron was present in plants treated with mC-linuron plus
clomazone than in plants treated with “C-linuron alone. Higher
percentages of metabolite 1 (R, 0.13) and metabolite 3 (R, 0.53) were
detected in soybean shoots treated with “C-linuron alone than those
treated with 1‘C-linuron plus clomazone. This suggests that in soybean
shoots the clomazone may be interfering with linuron metabolism. In
common cocklebur, a greater percentage of parent linuron was present in
the root than in the shoot, however there was no difference in the
percentages of metabolites between treatments in either plant organ
indicating that a synergistic interaction between the two herbicides would
not be expected it this species.

These experiments demonstrate that the synergism observed from the
combinations of clomazone plus metribuzin and clomazone plus linuron may
be due to differences in metabolism of metribuzin and linuron when
clomazone is also present in the plant, and not due to differences in the
uptake or partitioning of metribuzin or linuron although there were some
differences between species. Evidence suggests that the initial

detoxication of both metribuzin and linuron are catalyzed by mixed-

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76

function oxidases (5, 9). If clomazone detoxication is also catalyzed by
a mixed-function oxidase, there could be competition for the reduced form
of'nicotinamide adenine dinucleotide phosphate (NADPH) and/or activatedO2
that is required for mixed function oxidase activity, or for the active
sites on the mixed-function oxidase. With the reduction in quantity of
NADPH from the action of linuron and metribuzin in the light reaction,
competition for NADPH could become acute. This research suggests that a
mixed-function oxidase is involved in the detoxication of clomazone and
that clomazone is preferentially metabolized by that enzyme over
metribuzin and linuron in soybean and common cocklebur. Future research
will need to elucidate the detoxication pathway(s) of clomazone and the

enzymes involved .

10.

ll.

12.

13.

77

LITERATURE CITED

Blankendaal, M., R. H. Hodgson, D. G. Davis, R. A. Hoerauf, and R.
H. Shimabukuro. 1972. Growing plants without soil for experimental
use. USDA Misc. Pub. 1251. 17 pp.

Falb, L. N. and A. E. Smith, Jr. 1984. Metribuzin metabolism in
soybeans: Characteristics of the intraspecific differential
tolerance. J. Agric. Food Chem. 32:1425-1428.

Falb, L. N. and A. E. Smith. 1987. Metribuzin metabolism in
soybeans: Partial characterization of the polar' metabolites.
Pestic. Biochem. Physiol. 27:165-172.

Frear, D. S., H. R. Swanson, and E. R. Mansager. 1985. Alternate
pathways of metribuzin metabolism in soybean: Formation of N-
glucoside and monoglutathione conjugates. Pestic. Biochem. Physiol.
23:56-65.

Hatzios, K. K. and D. Penner. 1988. Metribuzin, Pages 191-243. in
P. C. Kearney and D. D. Kaufman, eds. Herbicides: Chemistry,
Degradation, and Mode of Action. Marcel Dekker, Inc. New York.

Katz, S. E. 1967. Determination of linuron and its known and/or

suspected metabolites in crop materials. J. Assoc. Off. Anal. Chem.
50:911-917.

Kuratle, H., E. M. Rahn, and C. W. Woodmansee. 1969. Basis for
selectivity of linuron on carrot and common ragweed. Weed Sci.
17:216-219.

Mangeot, B. L., F. E. Slife, and C. E. Rieck. 1979. Differential
metabolism of metribuzin by two soybean (Glycine max) cultivars.
Weed Sci. 3:267-269.

Nashed, R. B. and R. D. Ilnicki. 1970. Absorption, distribution,
and metabolism of linuron in corn, soybean, and crabgrass. Weed
Sci. 18:25-28.

Oswald, T. H., A. E. Smith, and D. V. Phillips. 1978.
Phytotoxicity and detoxification of metribuzin in dark-grown
suspension cultures of soybeans. Pestic. Biochem. Physiol. 8:73-83.

Salzman, F. P. and K. A. Renner. 1989. Interaction of clomazone
and metribuzin in soybean. Proc. NCWSS 44:86.

Salzman, F. P. and K. A. Renner. 1991. The synergistic
interactions of clomazone plus metribuzin and clomazone plus
linuron. Proc. WSSA 31:18.

Smith, A. E. and R. E. Wilkinson. 1974. Differential absorption,
translocation, and, metabolism of metribuzin [4-amino-6-tert-3-

14.

15.

16.

17.

18.

78

(methylthio)-as-triazine-5(4H)one] by soybean cultivars. Physiol.
Plant 32:253-257.

Vencill, W. K., K. K. Hatzios, and H. P. Wilson. 1990. Absorption,
translocation, and metabolism of “C-clomazone in soybean (Glycine
max) and three Amaranthus weed species. J. Plant Growth. Regul.
9:127-132.

Vencill, W. K., K. K. Hatzios, and H. P. Wilson. 1990.
Interactions of the bleaching herbicide clomazone with reduced
glutathione and other thiols. Z. Naturforsch. 45c:489-502.

Werling, V. L. and D. D. Buhler. 1988. Influence of application
time on clomazone activity in no-till soybeans, Glycine max. Weed
Sci. 36:629-635.

Westburg, D. E., L. R. Oliver, and.R. E. Frans. 1989. Weed control
with clomazone alone and with other herbicides. Weed Tech. 3:678-
685.

Weston, L. A. and. M. Barrett. 1989. Tolerance of tomato
(Lycopersicon esculentum) and bell pepper (Capsicum annum) to
clomazone. Weed Sci. 37:285-289.

79

SUMMARY

These studies indicate that environmental factors, including soil
characteristics, influence the synergistic interactions of clomazone plus
metribuzin and clomazone plus linuron. The most severe reductions in
soybean leaf area, shoot weight, root weight, and yield were noted in
field soils of low organic matter and clay contents. These soils may
allow more herbicide to remain available for plant uptake. The
combination of clomazone plus linuron is most injurious to soybean when
the herbicides are in the same soil zone as the seed, compared to
herbicides in the soil above or below the seed. Combinations of clomazone
plus metribuzin are equally injurious despite placement of these herbicide
in the soil. Thus, a heavy rainfall after herbicide application could
move clomazone and linuron in the soil to a zone where increased uptake by
soybean results in increased injury. Ambient air temperature does not
appear to influence these interactions. By examining the various rates of
clomazone and metribuzin that interact synergistically, it appears reduced
rates of clomazone and metribuzin could be used to control common
cocklebur yet not harm soybeans. Studies conducted on the uptake,
translocation, and metabolism of clomazone, metribuzin, and linuron alone
and in combination in soybean and common cocklebur indicate that uptake
and translocation of clomazone, metribuzin, and linuron are similar when
applied alone compared to when clomazone was combined with metribuzin or
linuron, linuron was combined with clomazone, or metribuzin was combined
with clomazone, respectively. Metabolism of metribuzin was altered in
soybean and common cocklebur when clomazone was present while metabolism

of linuron was altered by the presence of clomazone only in soybean.

80

Clomazone detoxification.was not significantly altered by the addition of
metribuzin or linuron. This indicates that clomazone, metribuzin, and
linuron are all detoxified.in the plant similarly. Evidence suggests that
metribuzin and linuron are deactivated in the plant by a mixed-function
oxidase enzyme. Clomazone may also be deactivated in soybean and common

cocklebur by a mixed-function oxidase enzyme.

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