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This is to certify that the

dissertation entitled

REFINING POSTEMERGENCE WEED CONTROL IN CORN:
HERBICIDE SELECTIVITY AND TIMING

presented by

James Boyd Carey

has been accepted towards fulfillment
of the requirements for

Ph.D. . Crop and Soil Sciences
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MSU I. An Afﬁrmative Action/Equal Opportunity Institution
Wanna-m

REFINING POSTEMERGENCE WEED CONTROL IN CORN:
HERBICIDE SELECTIVITY AND TIMING

By

James Boyd Carey

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1994

ABSTRACT

REFINING POSTEMERGENCE WEED CONTROL IN CORN:
HERBICIDE SELECTIVITY AND TIMING

By

James Boyd Carey

Nicosulfuron and primisulfuron are sulfonylurea herbicides that display
differential selectivity despite similar chemical structures and a common site of
action in susceptible plants. Corn is tolerant and johnsongrass is sensitive to
both herbicides. Barnyardgrass and giant foxtail are sensitive to nicosulfuron and
tolerant to primisulfuron. Eastern black nightshade is tolerant to nicosulfuron
and sensitive to primisulfuron.

14C-radiolabelled herbicides were used to determine if differences in whole
plant responses to nicosulfuron and primisulfuron were due to differential
herbicide absorption, translocation, or metabolism. Nicosulfuron and
primisulfuron selectivity in corn, johnsongrass, barnyardgrass, and giant foxtail
was primarily due to differential herbicide metabolism rate. Tolerant species
metabolized the herbicide more rapidly and extensively than sensitive species.
Differential herbicide absorption, translocation, or metabolism did not account for
differential sensitivity of eastern black nightshade to nicosulfuron and
primisulfuron.

Further experiments were conducted to determine if the difference in

eastern black nightshade sensitivity to nicosulfuron and primisulfuron was due

to a difference at the herbicidal site of action: the acetolactate synthase (ALS)
enzyme. Greater sensitivity of eastern black nightshade to primisulfuron was due
to greater ALS inhibition by primisulfuron. Eastern black nightshade and
johnsongrass ALS sensitivity to nicosulfuron were similar, despite differences in
whole plant response. Eastern black nightshade tolerance to nicosulfuron was
due to a combination of greater ALS level and less herbicide translocation.
Field studies were conducted in 1992 and 1993 to determine if weed
interference prior to herbicide application reduces corn yield with a total
postemergence herbicide program. Nicosulfuron plus bromoxynil plus nonionic
surfactant applied to 5-, 10-, or 15-cm weeds provided nearly complete weed
control. Weed interference did not reduce corn height or corn grain yield when
postemergence applications were made to small weeds (_<_ 10 cm). Weed
interference reduced corn height and grain yield in 1992 when applications were
made to 15-cm weeds even though weed control was nearly complete. Weed
control was incomplete and corn height and grain yield reduced both years when

applications were delayed until weeds were 20 cm tall.

ACKNOWLEDGEMENTS

Sincere appreciation is extended to Dr. James J. Kells for granting the
author the opportunity to pursue a graduate degree at Michigan State University,
and for dedicating so much time and effort to my graduate and professional
careers.

I would also like to extend a special thank you to Dr. Donald Penner for
providing guidance, advice, and support throughout my research. Appreciation
is extended to Dr. Bruce Branharn and Dr. Matthew Zabik for serving on the
author’s committee and for their guidance in my research. I would like to thank
Dr. Michael S. DeFelice, Dr. Gordon K. Roskamp, and Dr. Wally A. Neys for
their direction in my education and career.

Special thank you’s are extended to the many people who participated in
my research and from whom I have learned so much: Joe Bruce, Andy Chomas,
Christy Sprague, Karen Geiger, Rick Schmenk, Steve Hart, Eric Spandl, Jeff
Stachler, and Troy Bauer. Of all the beneﬁts I have received at Michigan State
University, the friendships I have established are the most valuable.

I would like to thank my parents Jim and Dianna, and my brother Jason.
Their support and inspiration over the years has made it possible for me to
pursue a graduate education. I would also like to thank Grace B. Knight for her

support and encouragement.

iv

TABLE OF CONTENTS

Chapter 1. Review of Literature.

Introduction .................................... 1
Sulfonylurea Herbicides ........................... 3
Mode of Herbicide Action ..................... 3
Basis For Selectivity ......................... 4
Postemergence Weed Control in Corn ................ lO
Nicosulfuron and Primisulfuron ................ 10
Weed Interference ......................... 10
Herbicide Application Timing ................. 12
Literature Cited ................................ 14

Chapter 2. Physiological Basis For Nicosulfuron and Primisulfuron
Selectivity in Five Plant Species.

Abstract ..................................... 24
Introduction ................................... 27
Materials and Methods ........................... 29
Plant material ............................. 29
Species sensitivity ......................... 3O
Absorption, translocation, and metabolism ........ 31
Acetolactate synthase activity ................. 36
Results and Discussion ........................... 39
Species sensitivity ......................... 39
Foliar absorption .......................... 4O
Translocation ............................. 41
Metabolism .............................. 42
ALS enzyme properties ...................... 44
Acknowledgements ............................. 48
Literature Cited ................................ 49

Chapter 3. Timing of Total Postemergence Herbicide Applications for
Weed Control and Corn Yield.

Abstract ..................................... 65
Introduction ................................... 67
Materials and Methods ........................... 69
Results and Discussion ........................... 73
Crop injury .............................. 73
Weed density ............................. 74
Weed control ............................. 75
Weed interference with corn .................. 76
Literature Cited ................................ 79

LIST OF TABLES

Chapter 2. Physiological Basis For Nicosulfuron and Primisulfuron
Selectivity in Five Plant Species.

Table 1. Plant sensitivity to nicosulfuron and

primisulfuron ......................... 54
Table 2. 1“C recovery .......................... 55
Table 3. Nicosulfuron and primisulfuron absorption in

ﬁve plant species ...................... 56
Table 4. Nicosulfuron and primisulfuron translocation

in ﬁve plant species ..................... 57
Table 5. Nicosulfuron and primisulfuron metabolism

in ﬁve plant species ..................... 58
Table 6. ALS activity, protein levels, and I80 values

for eastern black nightshade and johnsongrass . . 59
Table 7. Factors which contribute to plant tolerance to

nicosulfuron or primisulfuron .............. 60

Chapter 2. Timing of Total Postemergence Herbicide Applications for
Weed Control and Corn Yield.

Table l. Postemergence herbicide application

information ........................... 82
Table 2. Weed densities at herbicide application ....... 83
Table 3. Corn injury from bromoxynil plus nicosulfuron . 84
Table 4. Inﬂuence of herbicide application timing on

corn height and corn grain yield ............ 85
Table 5 Inﬂuence of weed canopy height on weed control
with bromoxynil plus nicosulfuron .......... 86

vii

LIST OF FIGURES

Chapter 2. Physiological Basis For Nicosulfuron and Primisulfuron
Selectivity in Five Plant Species.

Figure 1. Inhibition of eastern black nightshade

ALS activity .......................... 61
Figure 2. Inhibition of johnsongrass ALS activity ....... 62
Figure 3. Inhibition of ALS activity by nicosulfuron ..... 63
Figure 4. Inhibition of ALS activity by primisulfuron . . . . 64

Chapter 2. Timing of Total Postemergence Herbicide Applications for
Weed Control and Corn Yield.

Figure l. Weed biomass accumulation at postemergence
application timings in corn ................ 87

viii

REVIEW OF LITERATURE

INTRODUCTION

According to United Nations statistics, the global food supply must
increase by 75% to feed the projected world population in the year AD 2000.
To feed such numbers, agriculture must become even more efﬁcient and
productive than in the past. The judicious use of agrochemicals, including
pesticides, is essential to insure optimum yields of high quality crops. To
produce and use pesticides safely and effectively, it is crucial to understand how
they interact with target species (31).

The importance of foliar applied (postemergence) herbicides has increased
in the last decade. Numerous issues and production practices have driven this
trend. Postemergence herbicides are commonly used in place of mechanical
cultivation to control weeds in no-tillage (59,89,106). Atrazine [6-chloro-N-
ethyl-N’-(1-methylethyl)—1,3,5-t1iazine-2,4-diamine] has historically been an
economical, effective means of controlling weeds in corn (Zea mays L.).
However, the continuing spread and development of triazine resistant weed
species (88) and environmental concerns over the presence of atrazine in surface
and groundwater have prompted corn producers and weed scientists to seek

alternatives. Total postemergence programs are also attractive to promoters of

2

integrated weed management systems since they allow corn growers to determine
the extent of the weed problem before making a herbicide application (45,104).

Nicosulfuron {2-[[[[(4,6-dimethoxy-2-pyrimidinyl)—amino]-carbonyl]-
amino]sulfonyl]-N, N-dimethyl-3-pyridinecarboxamide} and primisulfuron {2-
[[[[[4,6-bis(diﬂuoromethoxy)-2-py1imidinyl]amino]carbonyl]-amino]sulfonyl]-
benzoic acid} are sulfonylurea herbicides introduced to the corn market in 1990.
Their introduction represented a major breakthrough in corn weed control.
Nicosulfuron and primisulfuron provide selective control of emerged perennial
and annual grasses and some broadleaf weeds at usage rates of 35 and 40 g a.i.
ha“, respectively (1,2,45). Such low usage rates help solve handling, application
and container-disposal issues, while reducing the amount of chemical applied to
the ﬁeld by a factor of 100-1000 over conventional herbicides (15). The
sulfonylurea class of herbicides are also attractive from an environmental
standpoint due to their low mammalian toxicity (3,4,45).

The combination of nicosulfuron or primisulfuron with postemergence
broadleaf herbicides may provide single-application total postemergence weed
control in corn (25,38,72). This type of weed control program was previously
not available, and provides another option to the corn grower. However, the
effects of early season weed competition with corn raise signiﬁcant questions
about this type of weed control program, since weeds are allowed to germinate

and grow with the crop until the herbicides are applied.

3

The speciﬁcity and weed spectrum of the sulfonylureas is unpredictable
(75). Although nicosulfuron and primisulfuron have similar chemical structures,
usage rates, and a common site of action in susceptible plants, these herbicides
display differential selectivity (3,4). An understanding of the basis for this
differential selectivity might provide insight to improve herbicidal activity on
tolerant weed species and to optimize their potential uses in weed control

systems.

SULFONYLUREA HERBICIDES

Mode of herbicide action. Sulfonylurea herbicides are absorbed and
translocated to their site of action where they inhibit the acetolactate synthase
(ALS) enzyme in susceptible plants (8,27,28,55,64,80,82,83,87,102).
Acetolactate synthase, also known as acetohydroxyacid synthase (AHAS), is a
key enzyme in the branched-chain amino acid biosynthetic pathway of higher
plants. ALS catalyzes the condensation of two pyruvate molecules to form CO2

and (ac-acetolactate, which leads to valine and leucine synthesis, and the

condensation of one molecule of pyruvate with oc-ketobutyrate to form CO2 and

oc-aceto-oc-hydroxybutyrate, which leads to isoleucine formation (8). The ALS

enzyme resides in the chloroplasts of plant cells (69).

4
Little is known about how inhibition of ALS results in plant death.

Depletion of the branched-chain amino acid pool will contribute to inhibition of
protein synthesis, but this may not be the primary pathway for inhibition of plant
growth (30,84). The control of plant cell division has also been implicated as a
possible contributor to plant death by sulfonylureas (8,10). Other research has

implicated the buildup of the intermediate a-ketobutyrate as an important
component of sulfonylurea herbicide action (65,87). oc-Ketobutyrate has been

shown to be toxic to bacteria cells, but little is known about the effects of high

levels of this intermediate in higher plants (65,84,87).

Basis for selectivity. To exert toxicity, all postemergence herbicides must: 1)
be absorbed into the foliage, and 2) move to the site of action. At the site of
action the herbicide must be present in an adequate concentration and in the
proper toxic form for sufficient duration of time (46,56). This sequence can fail
in numerous places, thereby preventing adequate control of the weed (56).
Any factor which interrupts this sequence may contribute substantially to
differential selectivity of postemergence herbicides between species, between
species within the same genus, and different herbicides within the same species.
Differential herbicide absorption, translocation, or metabolism are common
contributing factors to differential selectivity (13,57,92,107). Properties of the
ALS enzyme are also important factors which might contribute to the selectivity

of herbicides whose mode of action is inhibition of this enzyme.

5

The speciﬁcity and weed spectrum of the sulfonylureas is unpredictable
(75). Crop-selective sulfonylurea herbicides have been commercialized for use
in wheat (T riticum aestivum L.), barley (Hordeum vulgare L.), rice (Oryza sativa
L.), corn, soybeans [Glycine max (L.) Merr.], and oilseed rape (Brassica napus
L.), with additional crop-selective compounds in cotton (Gossypium hirsutum

L.), potatoes (Solanum tuberosum L.), and sugarbeet (Beta vulgaris L.) (15).

Metabolism. The primary basis of selectivity for these herbicides is differential
rate of herbicide metabolism. Tolerant species are able to rapidly detoxify
sulfonylureas to herbicidally inactive products, while metabolism is much slower
and less extensive in susceptible species (14,49,58,75,77,80,105,110).
Metabolites of sulfonylurea herbicides have been shown to be inactive against
plant ALS (14,16,58).

Metabolism is accomplished by a wide variety of processes. The relative
importance of these processes, and the sites at which they occur, can differ
signiﬁcantly for the same compound in different species, or for close analogs in
a given species (8). Pathways by which sulfonylurea herbicides are inactivated
in plants include aryl and aliphatic hydroxylation followed by glucose
conjugation, sulfonylurea bridge hydrolysis and sulfonamide bond cleavage,

oxidative O-demethylation and direct conjugation with (homo)glutathione (15).

6

Absorption and translocation. Absorption and translocation of sulfonylurea
herbicides is generally quite slow (5,6,17,23,42,66). Some research has
correlated differences in absorption and translocation with plant sensitivity, but
these processes were normally operating in conjunction with differences in
metabolism (5,40,85,110). In some instances, translocation of sulfonylureas is
greater in tolerant than in sensitive species (105). Generally, it is unlikely that
species selectivity in this herbicide class results, primarily, from differential

uptake and translocation (15,105).

ALS sensitivity. ALS enzyme sensitivity in sensitive plant species is similar to
that in tolerant species, further emphasizing metabolism as the basis of selectivity
(40,58,75,82,83,105). However, ALS sensitivity has been shown to be the basis
for herbicide resistance in genetically engineered plants as well as weeds that
have developed resistance through mutagenesis (7,27,28,47,52,53,54,67,81,91).
This resistance results from production of an altered form of ALS that is
enzymatically functional but much less sensitive than the normal enzyme to
inhibition by sulfonylurea herbicides (27).

Although the vast majority of research has emphasized herbicide
metabolism and de-emphasized ALS enzyme sensitivity as the basis for
sulfonylurea selectivity, recent research has shown the level of tolerance to

sulfonylureas may also be related to the amount or speciﬁc activity of ALS

7

present in the tissues (44). Elevated levels of a target enzyme may provide the
basis for tolerance to certain herbicide molecules (39,44,73,96). Researchers
found greater ALS content in roots than in shoots of certain corn inbreds. They
concluded that the naturally occurring differences in ALS levels in the roots of
the investigated inbred lines contribute largely to the differential in vivo response
observed to chlorsulfuron {2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-

yl)amino]carbonyl]benzenesulfonamide} (44). Differential speciﬁc activity of
ALS was correlated with in vivo tolerance to chlorsulfuron as well. In vivo corn

tolerance to chlorsulfuron was greater with greater ALS speciﬁc activity (44).

Nicosulfuron and primisulfuron. Most research investigating absorption,
translocation, metabolism, or ALS enzyme inhibition by nicosulfuron or
primisulfuron has focused on corn, johnsongrass, or bamyardgrass [Echinochloa
crus-galli (L.) Beauv.] (23,43,48,77).

Absorption of 14C-nicosulfuron applied to a tolerant species (corn) or
susceptible species (johnsongrass) was similar (about 40%). About 20-30% of
the absorbed 1“C-nicosulfuron translocated beyond the treated leaf of rhizome
johnsongrass at 21 days. Nicosulfuron was almost completely metabolized
within 20 h in corn, while there was no perceptible metabolism in the treated

leaves of johnsongrass even after 24 h (77).

8
Another study reported limited absorption of both primisulfuron and

nicosulfuron by rhizome johnsongrass, but more nicosulfuron was absorbed than
primisulfuron. Translocation out of the treated leaf was similar and less than
20% for both herbicides. Prevention of johnsongrass regrowth in the greenhouse
by nicosulfuron but not by primisulfuron was attributed to differential absorption
(23).

ALS from bamyardgrass and com, and numerous weeds is similar in
sensitivity to primisulfuron. Barnyardgrass is reported to be tolerant to
primisulfuron because it can rapidly metabolize the herbicide into metabolites
which do not inhibit ALS (75). Metabolism of primisulfuron in corn occurs by
hydroxylation of the phenyl and pyrimidine rings followed by sugar conjugation.
This process is catalyzed by a cytochrome P450-dependent monooxygenase
system located in the microsomal membranes (43).

Other research has investigated corn hybrids with great differences in
sensitivity to sulfonylurea herbicides. One study found ALS from normal and
susceptible corn plants was equally inhibited by both nicosulfuron and
primisulfuron. Herbicide uptake and initial translocation of nicosulfuron in both
were also the same, but metabolism differed and was identiﬁed as the process
responsible differential sensitivity (37). Variation in varietal response to

nicosulfuron and primisulfuron was also due to metabolism (22).

9

Another study investigating corn inbreds and hybrids found greater than
40 000-fold differences in sensitivity to primisulfuron, despite similar ALS
sensitivity (48). These results further support metabolism as the primary basis
for differences in response. However, results from the same research found
nicosulfuron was much less active than primisulfuron or thifensulﬁrron {3-[[[[(4-
methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-
thiophenecarboxylic acid} at the enzyme level in certain corn hybrids. These
researchers concluded that the lower enzyme activity by nicosulfuron is probably
the major factor in its greater corn tolerance. They also stated that the ALS
enzyme in weeds is also less sensitive to nicosulfuron, but did not present the
data (48).

Nicosulfuron and primisulfuron exhibit differential selectivity between
species, and between varieties within species (1,2,22,37,48,75). Herbicide
metabolism has been identiﬁed as the primary basis for these responses in many
species (22,37,48,75,77), however, other factors have been implicated as possible
contributors (23,48). A greater understanding of the interactions of nicosulfuron
and primisulfuron with economically important weed species may lead to

maximization of their potential uses for weed management in corn.

10
POSTEMERGENCE WEED CONTROL IN CORN

Nicosulfuron and primisulfuron. Nicosulfuron and primisulfuron were
introduced to the corn herbicide market in 1990. Prior to their introduction,
effective and reliable postemergence weed control in corn was limited to the use
of selective herbicides which are effective against only broadleaf weeds, or
directed sprays which require special application equipment to avoid crop
interception of the herbicide during application (12).

Nicosulfuron and primisulfuron were the ﬁrst selective postemergence
herbicides for control of several grass weed species in corn (1,2). These
herbicides also provided control of historically troublesome weeds in corn such
as johnsongrass [Sorghum halepense (L.) Pers.] and quackgrass [Elytrigia repens
(L.) Nevski] which were controlled by few preemergence herbicides and no
selective postemergence herbicides (9,23,24,45,74,77). Nicosulfuron or
primisulfuron in combination with postemergence broadleaf herbicides may

provide single-application total postemergence weed control in corn (25,38,72).

Weed interference. The ability of weeds to interfere with the crop and reduce
corn yields is well documented (51,61,62,63,71,76,98,99,100). Interference
consists of 1) direct competition by weeds for light, water, and nutrients or 2)

allelopathy which is the inhibition of plant growth through production of

11
biological toxins by the weeds (90). The degree to which weeds may interfere

with a crop depends upon the weed species (19,20,2l,29,35,79,86,94,97,103),

weed density (l1,19,20,21,26,29,32,34,35,36,62,71,93,98,103,1ll), weed grth
and rate of development (29,50,60,70,95), environmental conditions
(33,34,41,50,68,93,111), edaphic conditions (18,20,41,76,99,100,101), control of
insects and diseases (18), and duration of interference (51,61,63,78,100). Perhaps
the most signiﬁcant effect of weed interference is crop yield reduction.

The term "critical period" has been used to describe the duration of weed
control (51) or weed interference (109) which may occur without signiﬁcant yield
reduction as a result of weed interference. There are two recognized components
of the critical period. The ﬁrst component is the length of time the crop must
remain weed-free to prevent crop yield loss. The second component is the length
of time weeds can remain in the crop before they interfere with crop growth and
ultimately reduce yield (104,108,109,112).

With a total postemergence weed control program in corn, we are
concerned with the period of time the weeds interfere with the crop until the
postemergence herbicide is applied. The longer application is delayed, the
greater the duration of weed interference and the more likely the "critical period"
will be exceeded and yields reduced.

Competitive effects of giant foxtail (Setaria faberi Herrm.) with corn are

important early in the growing season (61). Increasing duration of giant foxtail

12

interference reduced yields gradually (61). Signiﬁcant yield reductions occurred
when giant foxtail was allowed to interfere with corn until weeds were 15-23 cm
in height and corn was 41-61 cm in height with 9-10 leaves (61).

A study in Ontario, Canada found the critical period to occur from the 4-
leaf to 14-leaf stage in corn. The critical period varied with environment, weed

species, and weed density (51).

Herbicide application timing. Traditional studies investigating duration of
weed interference in corn have employed hand pulling or hoeing as the method
of weed removal to end the period of weed interference. This method ceases
weed competition with the crop abruptly and completely. This is not an accurate
approximation of herbicide action. A postemergence herbicide, even when
completely effective, does not terminate weed competition in such a fashion.
Sulfonylurea herbicides in particular kill plants relatively slowly.
Complete plant death in susceptible species occurs in 7-21 days (3) and 10-30
days (4) with nicosulfuron and primisulfuron respectively, depending upon
growing conditions, weed species, and growth stage of the weeds. Weed
interference could still occur with the crop after herbicide application. It would
also seem possible that an effective herbicide treatment could completely control
weeds after the critical period of weed interference has expired. Weed

interference would have irreversibly reduced corn yield in such a situation.

13

The grower has some level of control over the duration of weed
interference with the crop through the choice of weed control methods and time
of control. If a grower adopts a total postemergence weed control program in
com, the time of herbicide application is important not only to insure successful
weed control, but to control the duration of weed interference and prevent crop
yield reduction. Knowledge of the maximum duration of interference allowable
before yield reduction occurs would be valuable information needed to effectively

implement total postemergence herbicide programs in corn.

10.

14
LITERATURE CITED

Anonymous. 1993. Accent® herbicide product label. E.I. duPont de
Nemours and Co., Agricultural Products, Wilmington, DE 19898.

Anonymous. 1993. Beacon® herbicide product label. CIBA-GEIGY Corp.,
Agricultural Division, Greensboro, NC 27419.

Anonymous. 1990. Accent® Herbicide. Technical Bull. E.I. duPont de
Nemours and Co., Agricultural Products, Wilmington, DE 19898. 12 p.

Anonymous. 1989. Beacon® Herbicide. Technical Release. Agricultural
Division, CIBA-GEIGY Corp., Greensboro, NC 27419. 8 p.

Baird, J.H., J.W. Wilcut, GR. Wehje, R. Dickens, and S. Sharpe. 1989.
Absorption, translocation, and metabolism of sulfometuron in
centipedegrass (Eremochloa ophiuroides) and bahiagrass (Paspalum
notatum). Weed Sci. 37:42-46.

Beckett, TH. and E.W. Stoller. 1991. Effects of methylammoniurn and
urea ammonium nitrate uptake of thifensulfuron in velvetleaf (Abutilon
theophrasti). Weed Sci. 39:333-338.

Bedbrook, J., R.S. Chaleff, S.C. Falco, B.J. Mazur, and N. Yadav. 1988.
Nucleic Acid Fragment Encoding Herbicide Resistant Plant Acetolactate
Synthase. Eur. Patent Appl. 0257993.

Beyer, E.M. Jr., M.J. Duffy, J.V. Hay, and DD. Schleuter. 1988.
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Herbicides: Chemistry, Degradation and Mode of Action. 3rd ed., Marcel-
Dekker, New York.

Bhowmik, P.C., B.M. O’Toole, and J. Andaloro. 1992. Effects of
nicosulfuron on quackgrass (Elytrigia repens) control in corn (Zea mays).
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Blair, AM. and TD. Martin. 1988. A review of the activity, fate and mode
of action of sulfonylurea herbicides. Pest. Sci. 22:195-219.

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15

Bloomberg, J.R., B.L. Kirkpatrick, and L.M. Wax. 1982. Competition of
common cocklebur (Xanthium pensylvanicum) with soybean (Glycine
max). Weed Sci. 30:507-513.

Bode, LE. 1987. Spray application technology. p. 85-109 in C.G.
McWhorter and MR. Gebhardt, eds. Methods of Applying Herbicides.
Weed Science Society of America, Charnpaign, IL.

Boldt, RF. and AR. Putnam. 1981. Selectivity mechanisms for foliar
applications of diclofop-methyl. II. metabolism. Weed Sci. 29:237-241.

Brown, HM. and SM. Neighbors. 1987. Soybean metabolism of
chlorimuron ethyl: physiological basis for soybean selectivity. Pestic.
Biochem. Physiol. 29:112-120.

Brown, HM. 1990. Mode of action, crop selectivity, and soil relations of
the sulfonylurea herbicides. Pestic. Sci. 29:263-281.

Brown, H.M., V.A. Wittenbach, D.R. Forney, and SD. Strachan. 1990.
Basis for soybean tolerance to thifensulfuron methyl. Pestic. Biochem.
Physiol. 37:303-313.

Bruce, J.A., D. Penner, and II. Kells. 1993. Absorption and activity of
nicosulfuron and primisulfuron in quackgrass (Elytrigia repens) as affected
by adjuvants. Weed Sci. 41:218-224.

Buchanan, GA. 1981. Management of weeds in cotton. p. 215-242 in D.
Pirnental, ed. Handbook of Pest Management in Agriculture. Vol. III. CRC
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Physiological Basis For Nicosulfuron and Primisulfuron
Selectivity in Five Plant Species1

J. BOYD CAREY, DONALD PENNER, and JAMES J. KELLS2

Abstract. Greenhouse and laboratory studies were conducted to determine the
physiological basis for selectivity of nicosulfuron and primisulﬁrron in ﬁve plant
species. Differential sensitivity of the species was quantiﬁed by determining
GR,0 values (herbicide rate required to reduce plant growth by 50%) for each
species/herbicide combination. GR50 data indicated the following levels of
sensitivity; corn - tolerant to both herbicides, seedling johnsongrass - sensitive
to both herbicides, barnyardgrass - sensitive to nicosulfuron and tolerant to
primisulfuron, giant foxtail - sensitive to nicosulfuron and tolerant to
primisulfuron, and eastern black nightshade - tolerant to nicosulfuron and
sensitive to primisulfuron. Studies utilizing 14C-radiolabelled herbicides were
conducted to determine if differential herbicide absorption, translocation, or
metabolism contributed to whole plant responses. Nicosulfuron and

primisulfuron selectivity in corn, johnsongrass, barnyardgrass, or giant foxtail

 

 

1Received for publication . and in revised form

2Grad. Res. Asst., Prof, and Prof, Dep. Crop and Soil Sci., Michigan State
Univ., East Lansing, MI 48824.

24

25

was primarily due to differential herbicide metabolism rate. Tolerant species
metabolized the herbicide more rapidly and extensively than sensitive species.
Differential herbicide absorption, translocation, or metabolism did not explain
differential sensitivity of eastern black nightshade to the herbicides. Further
studies indicated that differential eastern black nightshade response to
nicosulfuron and primisulfuron was due to differential ALS enzyme sensitivity.
The ALS sensitivity of johnsongrass and eastern black nightshade was similar in
the presence of nicosulfuron. A combination of ALS level and herbicide
translocation are factors which contribute to differential selectivity of
nicosulfuron for eastern black nightshade and johnsongrass. Nomenclature:
Nicosulfuron, 2-[[[[(4,6-dimethoxy-2-pyrirnidinyl)amino]carbonyl]amino]-
sulfonyl]-N, N-dimethyl-3-pyridinecarboxamide; primisulfuron, 2-[[[[[4,6-
bis(diﬂuoromethoxy)-2-pyrimidinyl]amino]carbonyl]amino]sulfonyl]benzoic acid;
barnyardgrass, Echinochloa crus-galli (L.) Beauv. #3 ECHCG; eastern black
nightshade, Solanum ptycanthum Dun. # SOLPT; giant foxtail, Setaria faberi
Herrm. # SETFA; johnsongrass, Sorghum halepense (L.) Pers. # SORHA; corn,

Zea mays L. ’Pioneer 3751’.

 

3Letters following this symbol are a WSSA-approved computer code from
Composite List of Weeds, Revised 1989. Available from WSSA, 309 West
Clark Street, Champaign, IL 61820.

26

Additional index words. Absorption, acetolactate synthase, corn, GRSO,
metabolism, sulfonylurea, tolerance, translocation, ECHCG, SETFA, SOLPT,

SORHA.

27
INTRODUCTION

Nicosulfuron and primisulfuron are sulfonylurea herbicides introduced to
the US. corn market in 1990. They were the ﬁrst selective postemergence
herbicides which would effectively control problem perennial and annual grasses
as well as some broadleaf weeds in corn (3,4,17). As members of the
sulfonylurea family of herbicides, nicosulfuron and primisulfuron have similar
chemical structures, usage rates, and the same site of action (1,2,3,4,17).
Sulfonylurea herbicides inhibit the acetolactate synthase (ALS)4 enzyme in
susceptible plants (5,12,l3,23,26,34,36,37,38,42). Despite all the similarities,
there is a difference in the selectivity of nicosulfuron and primisulfuron for
species they will or will not control (3,4).

The speciﬁcity and weed spectrum of this family of herbicides is
unpredictable (3 2). Sulfonylurea herbicides are used in wheat (Triticum aestivum
L.), barley (Hordeum vulgare L.), rice (Oryza sativa L.), corn, soybeans [Glycine
max (L.) Merr.], and oilseed rape (Brassica napus L.), with additional crop-
selective compounds in cotton (Gossypium hirsutum L.), potatoes (Solanum

tuberosum L.), and sugarbeet (Beta vulgaris L.) (8).

 

4Abbreviations: ALS, acetolactate synthase; PPF D, photosynthetic photon
ﬂux density; NIS, nonionic surfactant; GRSO, herbicide rate required to reduce
plant growth by 50%; LSS, liquid scintillation spectrometry; TLC, thin-layer
chromatography; FAD, ﬂavin adenine dinucleotide; 180, herbicide concentration
required to inhibit enzyme activity by 80%; HAT, hours after treatment.

28

To exert toxicity, all postemergence herbicides must be absorbed into the
foliage and move to the site of action where they must be present in an adequate
concentration and proper toxic form for a sufficient duration of time (18,24).
Any factor which does not allow this sequence to occur may account for
differential selectivity of herbicides between species, or differential sensitivity of
a species between herbicides. Properties of the ALS enzyme are also important
factors which can affect the selectivity of herbicides whose mode of action is
inhibition of this enzyme.

The primary basis for selectivity of sulfonylurea herbicides is differential
rate of herbicide metabolism. Tolerant species are able to rapidly detoxify
sulfonylureas to herbicidally inactive products, while metabolism is much slower
and less extensive in susceptible species (7,20,25,32,33,34,43,45). Metabolites
of sulfonylurea herbicides have been shown to be inactive against plant ALS
(7,9,25,32). Nicosulfuron was rapidly metabolized within 20 h in corn (tolerant)
while there was no perceptible metabolism in johnsongrass (sensitive) even after
24 h (33). Barnyardgrass is tolerant of primisulfuron because it can rapidly
metabolize the herbicide into products which do not inhibit ALS (32).

ALS from tolerant and sensitive plants is normally similar in sensitivity
to nicosulfuron and primisulfuron (14,19,32), further emphasizing metabolism as
the primary basis for differences in response. However, nicosulfuron is much

less active than primisulfuron at the enzyme level in certain corn hybrids and

29
unspeciﬁed weed species. This differential enzyme sensitivity is probably the

major factor in the greater tolerance of these corn hybrids to nicosulfuron (19).
The level of tolerance to sulfonylureas may also be related to the amount or
speciﬁc activity of ALS present in tissues. Naturally occurring differences in
ALS levels from the roots of certain corn inbreds contribute largely to the
differential in vivo responses to chlorsulfuron (l6).

Nicosulfuron and primisulfuron display differential selectivity between
species, between varieties within species, and between herbicides within species
(1,2,10,14,19,32). Herbicide metabolism has been identiﬁed as the primary basis
for these responses (10,14,19,32,33), however, other factors have been implicated
as possible contributors (11,19).

The objective of this research was to determine the physiological basis for
nicosulfuron and primisulfuron selectivity in corn, johnsongrass, barnyardgrass,

giant foxtail, and eastern black nightshade.

MATERIALS AND METHODS

Plant material. Barnyardgrass and giant foxtail seed were obtained from a

commercial seed supplier’. Johnsongrass seed was collected in the state of

 

5F & J Seed Service, PO. Box 82, Woodstock, IL 60098-0082.

30

Mississippi and eastern black nightshade was collected on the Michigan State
University Crop and Soil Science Research Farm at East Lansing. The corn
variety investigated was Pioneer 37516. The same seed material was used for
all studies.

Plants used for all experiments were grown in the greenhouse using 945-
ml plastic pots with a commercial potting mix7. Environmental conditions were
maintained at 24 i 2 C in a 16-h photoperiod of natural and supplemental metal
halide lighting with an average midday photosynthetic photon ﬂux density
(PPFD)4 of 700 uE m‘2 5". Plants were watered and fertilized as needed to
insure maximum growth. Plants were thinned after emergence to uniform

numbers and growth stages.

Species sensitivity. Individual greenhouse experiments were conducted to
compare the sensitivity of each plant species to nicosulfuron and primisulfuron.
Plants were grown to the stages presented in Table 1. Growth stages were
chosen as representative of those normally treated with a ﬁeld application of

either herbicide. Commercial formulations of nicosulfuron or primisulfuron were

 

6Pioneer Hi-Bred International, Inc., Des Moines, IA.

7Baccto Professional Planting Mix. Michigan Peat Co., PO. Box 980129,
Houston, TX 77098

31

applied at a range of rates with nonionic surfactant8 (NIS)4 at a rate of 0.25%
(v/v). All herbicides were applied with a laboratory sprayer ﬁtted with a 8001E
ﬂat fan nozzle9 delivering 234 L ha’l at 170 kPa. Root uptake of herbicide was
prevented by covering the soil surface with vermiculite before herbicide
application. The vermiculite was removed after treatment.

Herbicide injury was rated visually at 10 to 12 d after treatment with 0
representing no visible injury and 100 representing plant death. Above ground
biomass was harvested and dry weight determined immediately after visual
ratings. Experiments were conducted and analyzed separately for each species.
Nonlinear regression analysis was conducted [Y = B(l) x em”) " X) + B(3)] and
GR50“ values (herbicide rates required to reduce plant growth by 50%) calculated
based on both visual ratings and percent dry weight reduction. GR50 values
(Table 1.) are means from two experiments each with four replications, with the
exception of eastern black nightshade dry weight reduction which is from one
experiment. Paired t-tests at a = 0.05 were used to compare GRso values for
nicosulfuron and primisulfuron within a species. No test was conducted for corn

since no GR50 was identiﬁed for either herbicide.

 

8X-77‘19-Nonionic-type spreader and activator. Principle functioning agents:
Alkylaryl polyoxyethylene, free fatty acids, glycols, isopropanol. Constituents
effective as spray adjuvant-90%. Constituents ineffective as spray adjuvant-10%.
Valent U.S.A. Corp., 1333 N. California Blvd., PO. Box 8025, Walnut Creek,
CA 94596-8025.

9Spraying Systems Co., North Avenue, Wheaton, IL 60188.

32

Absorption, translocation, and metabolism. A single experiment utilizing l4C-
radiolabelled herbicides was designed to compare absorption, translocation, and
metabolism of nicosulfuron and primisulfuron among all ﬁve plant species.
Plants were initially grown in the greenhouse under the conditions
described previously. Plants were transferred to a uniform environment in
growth chambers approximately 7 days before treatment. Environmental
conditions in the growth chambers were maintained at 26/22 C day/night
temperatures and 45/55 % day/night relative humidities. Lighting was provided
by ﬂuorescent and incandescent lamps at 500 uE m"2 s'1 PPFD with a 16-h
photoperiod. Plants were grown to the same stages as used in GR50 studies.
The youngest fully developed leaf of each species was chosen for
treatment with l“C-herbicide. This was the third true leaf of corn, johnsongrass,
and barnyardgrass, and the fourth and ﬁfth true leaf of giant foxtail and eastern
black nightshade, respectively. To achieve sufficient uptake of radioactivity for
metabolism research, a minimum of 3.7 x 103 Bq would need to be applied to
the treated leaf. Preliminary spray retention studies were conducted to determine
the amount of spray solution and herbicide dosage that the leaf chosen for 14C
treatment intercepts during a herbicide application. A version of the technique
reported by Boldt and Putnam (6) was followed. Consideration of this
information in conjunction with the speciﬁc activity of the radiolabelled

herbicides allowed us to determine the concentration and amount of l“C-spotting

33

solution to apply as the treatment. None of the species received a 14C treatment
which exceeded the normal herbicide dosage for the treated leaf by more than
1.5 X.

The leaf targeted for l4C-herbicide application was covered with
cellophane and the remainder of the plant treated with unlabelled nicosulfuron
at 35 g ai ha'l or primisulfuron at 40 g ai ha'1 and N18 at 0.25% v/v. Herbicides
were applied with the same equipment and conditions as used for spray retention
and GRso studies. The cellophane was removed and l‘iC-herbicides applied
immediately thereafter to the youngest fully developed leaf.

The radiolabelled spotting solution contained pyrirnidine-Z-“C-
nicosulfuron (2.3 x 106 Bq mg'l speciﬁc activity, 98.8% purity) or phenyl-”C-
primisulfuron (1.9 x 106 Bq mg'l speciﬁc activity, 97.2% purity), with
appropriate amounts of formulation blank, NIS, and water diluent to approximate
a normal spray solution. Each treatment consisted of six droplets of solution at
2 uL each for a total of 3.7 x 103 Bq of radioactivity applied to the adaxial
surface of the treated leaf. Pursuant to results of the spray retention studies,
johnsongrass, barnyardgrass, and giant foxtail treatments were split between two
plants by applying three droplets to each plant. Multiple plant parts were
combined for analysis.

Plants were returned to growth chambers immediately after treatment.

Treated leaves were excised from the plant at 12, 72, or 168 h after treatment.

34

The leaf was rinsed in methanolzdistilled water (2:1, v/v) to remove unabsorbed
herbicide. The rinse solution was radioassayed by liquid scintillation
spectrometry (LSS)‘. All plant parts were immediately frozen on dry ice and
stored at -30 C until further analysis.

All plant parts excluding the treated leaf were oxidized in a biological
sample oxidizer10 using a mixture of carbon dioxide absorbent and scintillation
ﬂuid (1:2 ratio) to trap evolved COZ. Samples were radioassayed by LSS.

Treated leaves were the only plant sections found to contain sufficient
amounts of ”C-herbicide for detection of metabolites, therefore metabolism was
determined in the treated leaf only. Treated leaves were ground in a tissue
homogenizerll with 20 ml of cold acetonezwater (80:20, v/v). The homogenate
was then vacuum ﬁltered12 and the residue rinsed with additional solvent. The
rinsate volume was recorded and two l-ml aliquots were radioassayed with LSS
to determine total extractable 1“C. The residue along with the ﬁlter paper was
air dried and oxidized to determine unextractable radioactivity.

The ﬁltrate was evaporated to a volume of 1 to 5 ml with a rotary
evaporator at 42 and 35 C for nicosulfuron and primisulfuron extracts,

respectively. The solution was transferred to a test tube and brought back to

 

10R.J. Harvey Instruments Corp, 123 Patterson St., Hillsdale, NJ 07642
“Sorvall Omni-mixer. Sorvall, Inc., Newton, CT.

l2Whatman #1. Whatrnan International Ltd., Maidstone, England.

35

volume with acetonitrile. The sample was then concentrated under a stream of
nitrogen and ﬁltered again through a 0.2 pm millipore ﬁlter”.

Twenty to ﬁfty uL of the concentrated extract containing 15 to 50 Bq of

radioactivity was spotted onto 20- by 20-cm silica gel thin layer chromatography
(TLC)4 plates for metabolite separation. Plates with nicosulfuron extracts14
were developed to a l3-cm solvent front in dichloromethanezmethanolzammonium
hydroxide (165:30:5, v/v/v). Plates with primisulfuron extracts15 were ﬁrst
developed to a 13-cm solvent front in chloroform:methanol:formic acidzwater

(70:25:422, v/v/v/v). Plates were air dried and then rotated 90° counterclockwise

and developed a second time to a 13-cm solvent front in chloroform:methanol:
ammonium hydroxide:water (80:30:422, v/v/v/v). l“C-nicosulfuron and
primisulﬁrron standards were also spotted and chromatographed in the same
manner as their respective extracts to determine the Rf values for the parent
herbicides. Radioactive positions, proportions, and their corresponding Rfvalues
were determined by scanning TLC plates with a radiochromatogram scanner16
Herbicide absorption was calculated as the total 1“C recovered in the plant

divided by the 1“C applied. l“C translocation out of the treated leaf was

 

”Gelman Sciences Inc., Ann Arbor, MI 48106.

l“Whatman® Linear-K Preadsorbent, Whatrnan International Ltd., Maidstone,
England.

”Fisherbrand Redi/Plate®, Fisher Scientiﬁc, 711 Forbes Avenue, Pittsburgh,
PA, 15219-4785.

”Ambis Systems, Inc., 3939 Ruffm Road, San Diego, CA 92123.

36

calculated as the 14C recovered in plant parts excluding the treated leaf divided
by the total 14C recovered in the plant. Herbicide metabolism in the treated leaf
was calculated by dividing the extractable l“C remaining as intact herbicide by
the total 14C in the treated leaf.

A randomized complete block design with a two factor (herbicide by
species) factorial arrangement of treatments and four replications was used. The
experiment was conducted twice and the combined results presented. Data
within each harvest time were subjected to analysis of variance and means

separated using Fisher’s Protected Least Signiﬁcant Difference Test at a = 0.05.

1“C recovery averaged over all species and harvest times was 89% for both

nicosulfuron and primisulfuron (Table 2).

Acetolactate synthase activity. ALS activity levels were determined in the
leaves of eastern black nightshade and johnsongrass grown to heights of 5 and
15 cm, respectively. Plant were grown in the greenhouse as previously
described.

ALS was extracted and enzyme activity levels measured in the presence
of nicosulfuron or primisulfuron with a modiﬁcation of the methods outlined by
Ray (37), Shaner (40), and Hart et al. (21,22). All extraction, centrifugation, and
column separation procedures were conducted on ice or at 4 C. Two 10 g

samples were taken from each species. Samples were a composite of newly

37

formed plant leaves excised from the apex of several plants. Each 10 g sample
was homogenized in 40 ml of cold homogenization buffer [0.1 M KQHPO4, 1.0
mM sodium pyruvate, 0.23 mM MgC12, pH 7.5, 0.5 mM thiamine pyrophosphate,
10 M ﬂavin adenine dinucleotide (FAD)“, 10% by vol glycerol] with 2.5 g of
polyvinylpolypyrrolidone in the homogenization vessel. The homogenate was
ﬁltered through eight layers of cheesecloth and then centrifuged at 27 000 g for
20 min. The supernatant fraction was brought to 50% saturation with cold
(NH,,)2SO4 and allowed to stand 1 h on ice. The mixture was then centrifuged
at 18 000 g for 15 min. The supernatant was discarded and the precipitated
pellet dissolved in resuspension buffer (0.1 M KZHPO4, 20 nM sodium pyruvate,
0.23 mM MgC12, pH 7.5). This solution was passed through a Sephadex“ G-
25M PD-10l7 column equilibrated with the same buffer. The desalted enzyme
preparation was immediately used for enzyme assays.

ALS enzyme assays were carried out in a ﬁnal volume of 1.5 ml
containing the enzyme preparation, reaction buffer (25 mM KQHPO4, 25 mM
sodium pyruvate, 0.29 mM MgC12, pH 7.0, 0.625 mM thiamine pyrophosphate,

1.25 M FAD), and technical grade nicosulfuron or primisulfuron at 0, 5, 50,

500, or 5 000 nM concentrations. Reaction tubes were incubated for l h at
35 C when the reaction was stopped with the addition of 50 uL of 6 N H2S04.

The reaction tubes were then heated for 15 min at 60 C to facilitate

 

”Pharmacia, 800 Centennial Avenue, Piscataway, NJ 08855-1327.

38

decarboxylation of acetolactate to acetoin. Then 0.5 ml of 0.5% wt by vol

creatine in 2.5 N NaOH and 0.5 ml of 5% wt by vol a-naphthol freshly prepared

in 2.5 N NaOH were added consecutively to each tube. The solutions were
heated for an additional 15 min at 60 C. Acetoin content was then determined
by spectrophotometric analysis as described by Westerﬁeld (44). Protein
concentration was determined by the method of Lowry (29). ALS activity is
expressed as nM acetoin h'l mg’1 protein.

The experiment was repeated with four replications of each herbicide
concentration per experiment. ALS enzyme activity is presented as a percent of
control assays using a log10 transformation of herbicide concentrations. Paired

t-tests were conducted at or = 0.01 to determine if means at each herbicide

concentration were signiﬁcantly different. Tests were conducted to determine if
ALS activity in response to the log of the herbicide concentration was linear or
quadratic. A quadratic model was ﬁt to eastern black nightshade ALS activity
in response to nicosulfuron concentration with a coefficient of determination of
0.88. A linear model was ﬁt to johnsongrass ALS activity in response to
nicosulfuron concentration with a coefficient of determination of 0.94. Quadratic
models were ﬁt to eastern black nightshade and johnsongrass ALS activity in
response to primisulfuron concentration with coefﬁcients of determination of 0.92
and 0.97, respectively. 1304 values (herbicide concentration required to inhibit

enzyme activity by 80%) were calculated from these equations. Comparisons

39

between eastern black nightshade and johnsongrass were made in terms of ALS

activity and protein level with paired t-tests at a = 0.01.

RESULTS AND DISCUSSION

Species sensitivity. Plant species were chosen based on wide variation in
species sensitivity to nicosulfuron and primisulfuron applications in ﬁeld trials.
Greenhouse experiments were conducted to quantify and validate the
observations made in ﬁeld trials.

Evaluation of GR50 values calculated from visual ratings or from percent
dry weight reductions led to similar conclusions. GR50 values could not be
obtained with several species/herbicide combinations with rates as high as 480
g ha", while GR,o values for some combinations were less than 5 g ha". The
ﬁeld application rates for nicosulfuron and primisulfuron are 35 and 40 g ha",
respectively (1,2).

Corn was tolerant to both herbicides (Table 1.). No comparisons between
herbicides was made since GR,0 values could not be determined for either
herbicide. Johnsongrass was sensitive to both herbicides with GR50 values well

below the ﬁeld application rates for either herbicide. GR50 values calculated on

40

a percent dry weight reduction basis indicated no difference in sensitivity to the
herbicides.

Barnyardgrass and giant foxtail were both more tolerant to primisulfuron.
The difference in tolerance to the two herbicides was greater with barnyardgrass
than with giant foxtail. Giant foxtail was not as tolerant to primisulfuron as
barnyardgrass or corn. Eastern black nightshade was tolerant to nicosulfuron and

sensitive to primisulfuron.

Foliar absorption. Foliar absorption of l4C-nicosulfuron or l4C-primisulfuron
by johnsongrass was less than or equal to absorption of either herbicide by com
at 72 or 168 HAT”, even though johnsongrass was sensitive and corn was
tolerant to both herbicides (Tables 1 and 3).

Barnyardgrass and giant foxtail both absorbed more l4C-primisulfuron than
l4C-nicosulfuron at each harvest time even though both species are tolerant to
primisulfuron and sensitive to nicosulfuron. l“C-nicosulfuron absorption was
equal to or greater than l4C-primisulfuron in eastern black nightshade at each
harvest time even though the species is tolerant to nicosulfuron and sensitive to
primisulfuron.

Eastern black nightshade is extremely tolerant to nicosulfuron yet absorbed
more l“C-nicosulfuron than an extremely sensitive species (johnsongrass) at each

harvest time. Barnyardgrass and giant foxtail are tolerant to primisulfuron yet

41

each absorbed more l‘iC-primisulfuron than an extremely sensitive species
(johnsongrass) at each harvest time.

Foliar herbicide absorption by itself cannot account for differential
selectivity of nicosulfuron or primisulfuron in any of the species investigated, or

for differences in sensitivity to the herbicides by any of the species.

Translocation. Nicosulfuron and primisulfuron movement within the plant was
approximated by measuring 1“C translocation out of the treated leaf. Greater l“C
translocation did not consistently correlate with greater sensitivity of any species
to nicosulfuron or primisulfuron (Table 4). Corn treated with primisulfuron
translocated more 1“C thanicom treated with nicosulfuron at 12 or 72 HAT. l4C
translocation in nicosulfuron or primisulfuron treated corn was equivalent at 168
HAT. Johnsongrass treated with primisulﬁrron translocated more 1"C than
johnsongrass treated with nicosulfuron at 12 HAT. At 72 or 168 HAT,
johnsongrass treated with nicosulfuron translocated more 1"C than johnsongrass
treated with primisulfuron. Neither corn nor johnsongrass displayed differential
sensitivity between nicosulfuron and primisulfuron even though there were
differences in translocation (Tables 1 and 4). Differences in 1"C translocation
between barnyardgrass or giant foxtail plants treated with nicosulfuron and
primisulfuron do not correlate with sensitivity or are not great enough to account

for whole plant differences in sensitivity.

42

14C translocation in nicosulfuron treated plants was greater in sensitive
species (johnsongrass, barnyardgrass, and giant foxtail) than in tolerant species
(corn and eastern black nightshade) (Table 4). The greatest l“C translocation (>
20%) occurred in johnsongrass plants treated with nicosulfuron at 72 and 168
HAT. The least l“C translocation (5 3%) occurred in eastern black nightshade
treated with nicosulfuron. This difference in translocation could contribute to the
difference in johnsongrass and eastern black nightshade sensitivity to
nicosulfuron.
Plants tolerant to primisulfuron (corn, barnyardgrass, and giant foxtail)
translocated similar or greater amounts of 1“C than plants sensitive to
primisulfuron (johnsongrass and eastern black nightshade). Translocation does

not appear to contribute to selectivity of primisulfuron in the species investigated.

Metabolism. Metabolism of both herbicides occurred in each species by 12
HAT (Table 5). Three distinct metabolites of l“C-nicosulfuron were separated
from the parent herbicide. Their Rf values in order of appearance over time were
0.88, 0, and 0.20. l“C-nicosulfuron had an Rf value of 0.40. The relative
abundance and rate of formation of the metabolites was speciﬁc to each species,
however, the order of appearance of the metabolites was the same for each

species (data not shown).

43

Numerous l“C-primisulfuron metabolites were formed by the species
investigated. l“C-primisulfuron had Rf values of 0.87 and 0.74 for the ﬁrst and
second developments, respectively. The presence, relative abundance, and order
of primisulfuron metabolite formation was species speciﬁc. The most common
primisulfuron metabolites among species had ﬁrst/second development Rf values
of 077/058, 090/059, and 059/017. As many as 10 distinct primisulfuron
metabolites were formed in giant foxtail alone (data not shown). The same
sulfonylurea herbicide may be metabolized by different pathways in different
plants (25,32,43). No attempt was made to identify the metabolites of either
herbicide in this research.

Corn metabolized both herbicides more rapidly and extensively than any
other species by 12 HAT (Table 5). There were no differences in the amount of
nicosulfuron or primisulfuron remaining in the treated corn leaf, nor was there
greater than 20% parent herbicide remaining at any harvest time. Johnsongrass
did not metabolize either herbicide as rapidly or extensively as corn. Greater
than 50% of both herbicides remained as parent at 12 HAT, compared to less
than 20% for corn. The percentage of parent nicosulfuron or primisulfuron in
johnsongrass decreased over time but there was no difference between the
percentage of parent herbicides remaining at any harvest.

Both barnyardgrass and giant foxtail metabolized primisulfuron more

rapidly than nicosulfuron. More nicosulfuron remained in both species at 12 and

44

72 HAT. Both species continued to metabolize each herbicide over time with the
difference between the percentage of parent herbicides remaining diminishing in
barnyardgrass by 168 HAT. At 72 HAT the percentage of primisulfuron
remaining in barnyardgrass and corn was equivalent.

The rate of herbicide metabolism is a major factor which determines
whether corn, johnsongrass, barnyardgrass, or giant foxtail is tolerant or sensitive
to nicosulfuron or primisulfuron. Species tolerant to a herbicide were able to
metabolize it more rapidly and extensively than sensitive species. Previous
researchers have identiﬁed herbicide metabolism as the primary basis for
differential selectivity of nicosulfuron and primisulfuron (10,14,19,32,33).

Eastern black nightshade did not metabolize either herbicide as quickly or
extensively as any other species. The percentage of parent nicosulfuron in the
treated leaf of eastern black nightshade was greater than or equal to that in
species sensitive to nicosulfuron (johnsongrass, barnyardgrass, and giant foxtail)
at each harvest. The percentage of parent nicosulfuron remaining in eastern
black nightshade was greater than or equal to the percentage of parent
primisulfuron at each harvest, even though this species is tolerant to nicosulfuron
and sensitive to primisulﬁrron. Differential foliar herbicide absorption,
translocation, or metabolism cannot account for the differential tolerance of
eastern black nightshade to nicosulfuron and primisulfuron. Differential foliar

absorption or metabolism cannot account for differential selectivity of

45

nicosulfuron in the species investigated. Differential translocation of
nicosulfuron may contribute, but by itself cannot totally account for the

selectivity of nicosulfuron in the species investigated.

ALS enzyme properties. ALS activity was studied in eastern black nightshade
to determine if differential enzyme sensitivity could account for diﬁerential
whole plant response to nicosulfuron and primisulfuron. ALS activity in
johnsongrass was used for comparison since this species is sensitive to both
herbicides and the ALS enzyme would be sensitive to inhibition by both
herbicides as well.

ALS from both eastern black nightshade and johnsongrass was more
sensitive to inhibition by primisulfuron than nicosulfuron at 50 and 500 nM
concentrations (Figures 1 and 2). Calculated I80 values demonstrate the
differential sensitivity in both species as well (Table 6). Other researchers have
found nicosulfuron to be much less active than primisulfuron at the enzyme level
in certain corn hybrids and unspeciﬁed weed species (19). They concluded that
the lower enzyme activity is probably the major factor in the greater tolerance
of the corn hybrids to nicosulﬁrron. In the bacteria Salmonella typhimurium and
Escherichia coli there are several isozymes of ALS each encoded by a separate
gene (5). Two of these isozymes have been shown to be sensitive to the

sulfonylurea sulfometuron methyl {methyl 2-[[[[(4,6-dimethyl-2-

46

pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoate}, while a third isozyrne is
not (27,28). Weeds that have developed resistance through mutagenesis have
an altered form of ALS that is enzymatically functional but much less sensitive
than the normal enzyme to inhibition by sulfonylurea herbicides (30,35,39).
Consideration of the variability of ALS forms and sensitivity in bacteria and
plants makes it reasonable to assume that variations in ALS sensitivity among
species is likely and could contribute to differential sensitivity to herbicides.

In this research, the lack of differences in herbicide absorption,
translocation, or metabolism emphasize the difference in enzyme sensitivity as
the basis for differential tolerance of eastern black nightshade to nicosulfuron and
primisulfuron.

Comparisons of eastern black nightshade and johnsongrass ALS sensitivity
in the presence of nicosulfuron or primisulfuron revealed very similar enzyme
inhibition patterns (Figures 3 and 4). Eastern black nightshade and johnsongrass
ALS sensitivity to nicosulfuron was roughly equivalent at the enzyme level even
though whole plant response is drastically different (Table 1).

The speciﬁc activity of the ALS from johnsongrass was nearly three times
that of eastern black nightshade ALS (Table 6). However, eastern black
nightshade contained nearly three times as much protein per mg of tissue fresh
weight as johnsongrass. ALS activity on a unit of tissue fresh weight basis was

equivalent in the two species. This indicates that differences in speciﬁc activity

47

and protein level in eastern black nightshade and johnsongrass offset each other
and the ratio of protein content is a valid estimation of the ratio of ALS content.
Consequently, the tissue of eastern black nightshade had nearly three times as
much ALS per unit of tissue as johnsongrass. In addition, less nicosulfuron
translocation occurred in eastern black nightshade (3%) compared to
johnsongrass (> 20%) at 72 or 168 HAT (Table 4). Other research determined
that in johnsongrass plants treated with l"C-nicosulfuron, a large portion of
translocated l“C accumulated in the growing point of the shoot (33). Metabolites
of sulfonylurea herbicides have been shown to be inactive against plant ALS
(7,9,25), suggesting that accumulation of 14C in the growing points represents
translocated nicosulfuron. The combination of less ALS and greater translocation
resulted in greater concentration of nicosulfuron per unit of ALS at the growing
point in johnsongrass compared to eastern black nightshade. This would account
for differential selectivity of nicosulfuron in these two species.

Elevated levels of a target enzyme may provide the basis for tolerance to
certain herbicide molecules (15,31,41). A recent study concluded that naturally
occurring differences in ALS levels from roots of certain corn inbreds contributed
largely to differential in vivo response to the sulfonylurea herbicide chlorsulfuron
(l6).

Herbicide metabolism rate was the major factor which contributed to the

tolerance of corn, barnyardgrass, and giant foxtail to nicosulfuron or

48

primisulfuron (Table 7). Johnsongrass did not display tolerance to either
herbicide (Table 1). Differential ALS enzyme sensitivity was the major
contributing factor which explained the differential tolerance of eastern black
nightshade to nicosulfuron and primisulfuron on the whole plant level (Table 7).
The combination of ALS level and herbicide translocation rate are factors which
contribute to differential selectivity of nicosulfuron in eastern black nightshade
and johnsongrass.

The results of this research indicate that tolerance to sulfonylurea
herbicides is not always a function of herbicide metabolism rate. Other factors
can contribute to or be responsible for selectivity. A complex interaction of
several factors may determine the degree of sensitivity of a particular plant

species to any one sulfonylurea herbicide.

ACKNOWLEDGMENTS

The authors wish to thank Christy L. Sprague and Karen A. Geiger for
their technical assistance in this research. Appreciation is also extended to
CIBA, E.I. duPont, and the Michigan State University Pesticide Research Center

for their support.

10.

49
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Hay, J.V. 1990. Chemistry of sulfonylurea herbicides. Pestic. Sci. 29:247-
261.

Hess, ED. and RH. Falk. 1990. Herbicide deposition on leaf surfaces.
Weed Sci. 382280-288.

Hutchison, J.M., R. Shapiro, and PB. Sweetser. 1984. Metabolism of
chlorsulfuron by tolerant broadleaves. Pestic. Biochem. Physiol. 22:243-
247.

LaRossa, RA. and SC. Falco. 1984. Amino acid biosynthetic enzymes as
targets of herbicide action. Trends Biotechnol. 22158-161.

LaRossa, RA. and J .V. Schloss. 1984. The sulfonylurea herbicide
sulfometuron methyl is an extremely potent and selective inhibitor

of acetolactate synthase in Salmonella typhimurium. J. Biol. Chem.
259:8753-8757.

LaRossa, RA. and DR. Smulski. 1984. ilvB-encoded acetolactate
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Lowry, O.H, N.S. Rosebrough, A.L. Farr, and RS. Randall. 1951. Protein
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Mallory-Smith, C.A., D.C. Thill, and M.J. Dial. 1990. Identiﬁcation of
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Technol. 42163-168.

Nafziger, B.D., J.M. Widholrn, H.C. Steinrt'icken, and J.L. Killrner. 1984.
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Plant Physiol. 76:571-574.

Neighbors, S. and LS. Privalle. 1990. Metabolism of primisulfuron by
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33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

52

Obrigawitch, T.T., W.H. Kenyon, and H. Kuratle. 1990. Effect of
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Petersen, RI. and BA. Swisher. 1985. Absorption, translocation, and
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Technol. 42169-172.

Ray, TE. 1986. Sulfonylurea herbicides as inhibitors of amino acid
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Ray, TE. 1984. Site of action of chlorsulfuron: inhibition of valine and
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sulfonylurea resistance in the broadleaf weed, Kochia scoparia. Plant
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Biol. Chem. 161:495-502.

45.

53

Wilcut, J.W., G.R. Wehtje, M.G. Patterson, T.A. Cole, and TV. Hicks.
1989. Absorption, translocation, and metabolism of foliar-applied
chlorimuron in soybeans (Glycine max), peanuts (Arachis hypogaea), and
selected weeds. Weed Sci. 37:175-180.

54

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Table 2. 1“C recovery“.

 

 

 

 

 

 

 

Recoveryb
Species Nicosulfuron Primisulfuron
%
12 hours after treatment
Corn 93 d 99 a
Johnsongrass 92 d 96 ab
Barnyardgrass 88 e 93 cd
Giant Foxtail 94 bed 96 abc
Eastern Black Nightshade 92 d 96 abc

72 hours after treatment

 

 

 

 

Corn 90 a 90 a
Johnsongrass 89 a 90 a
Barnyardgrass 88 ab 83 (1
Giant Foxtail 89 a 85 cd
Eastern Black Nightshade 86 be 90 a
168 hours after treatment

Corn 85 be 79 f
Johnsongrass 90 a 85 bcd
Barnyardgrass 86 bc 81 cf
Giant Foxtail 88 ab 82 def
Eastern Black Nightshade 83 cde 87 ab

 

llMeans may be compared within or across columns within harvest times.
Means followed by the same letter are not signiﬁcantly different according to
Fisher’s Protected LSD Test (or = 0.05).

bRecovery expressed as % of total 14C recovered from all processes divided
by ”C applied.

56

Table 3. Nicosulfuron and primisulfuron absorption in ﬁve plant species“.

 

Absorption
Species Nicosulfuron Primisulfuron

 

 

 

% of applied l"C —
12 hours after treatment

 

 

Corn 8 g 3 h
Johnsongrass 10 f 11 cf
Barnyardgrass 12 ef 22 a
Giant Foxtail 16 c 19 b
Eastern Black Nightshade 13 de 14 cd

 

72 hours after treatment

 

 

 

Corn 12 e 12 e
Johnsongrass 12 e 14 e
Barnyardgrass 15 de 28 b
Giant Foxtail 18 d 23 c
Eastern Black Nightshade 33 a 32 ab
168 hours after treatment
Corn 18 e 23 cd
Johnsongrass 15 e 16 e
Barnyardgrass 15 e 26 c
Giant Foxtail 17 e 22 (1
Eastern Black Nightshade 38 a 33 b

 

“Means may be compared within or across columns within harvest times.
Means followed by the same letter are not signiﬁcantly different according to
Fisher’s Protected LSD Test (a = 0.05).

57

Table 4. Nicosulfuron and primisulfuron translocation in ﬁve plant species“.

 

Translocation”

 

Species Nicosulfuron Primisulfuron

 

 

% of absorbed l“C —
12 hours after treatment

 

 

Corn 4 ef 13 abc
Johnsongrass 12 c 15 a
Barnyardgrass 9 d 12 be
Giant Foxtail 12 bc 14 ab
Eastern Black Nightshade 2 f 5 e

 

 

72 hours after treatment

 

 

Corn 7 d 15 bc
Johnsongrass 23 a 18 be
Barnyardgrass 16 bc 16 bc
Giant Foxtail 18 b 14 c

Eastern Black Nightshade 3 e 4 de

168 hours after treatment

Com , 6 cf 8 e

Johnsongrass 21 a 16 be
Barnyardgrass 19 ab 13 cd
Giant Foxtail 19 ab 16 be
Eastern Black Nightshade 3 f 10 de

 

“Means may be compared within or across columns within harvest times.
Means followed by the same letter are not signiﬁcantly different according to
Fisher’s Protected LSD Test (or = 0.05).

lyTranslocation of 1“C out of the treated leaf.

58

Table 5. Nicosulfuron and primisulfuron metabolism in ﬁve plant species“.

 

 

 

 

 

Metabolismb

Species Nicosulfuron Primisulfuron
% parent
12 hours after treatment

Corn 18 e 16 e
Johnsongrass 51 c 55 be
Barnyardgrass 58 be 33 (1
Giant F oxtail 69 a 54 bc
Eastern Black Nightshade 69 a 64 ab

72 hours after treatment

 

 

 

 

Corn 13 g 19 fg
Johnsongrass 39 d 48 cd
Barnyardgrass 60 b 24 cf
Giant Foxtail 59 b 29 e
Eastern Black Nightshade 70 a 54 be
168 hours after treatment

Corn 12 e 20 de
Johnsongrass 38 ab 31 bed
Barnyardgrass 32 bc 22 cde
Giant Foxtail 46 a 31 bed
Eastern Black Nightshade 48 a 50 a

 

“Means may be compared within or across columns within harvest times.
Means followed by the same letter are not signiﬁcantly different according to
Fisher’s Protected LSD Test (a = 0.05).

bMetabolism expressed as % of 14C within the treated leaf remaining as
intact parent.

59

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61

 

10

80

- + nicosulfuron

60 _ -O— primisulfuron

ALS enzyme activity (% of control)

 

 

 

 

4o — *
a 31:
20 —
0 1 I
0 5 50 500 5000

Herbicide Concentration (nM)

Figure 1. Inhibition of eastern black nightshade ALS activity. Means with a *

are different at the corresponding herbicide concentration according to paired t-
test at a = 0.01

62

 

 

 

 

 

111l
3 so __ \ + nicosulfuron
:
3 _ + primisulfuron
‘8
5‘; 60 — *
b
g _
i .. ..
E s
0
a 20 ~—
0 l 1
0 5 50 500 5000

Herbicide Concentration (nM)

Figure 2. Inhibition of johnsongrass ALS activity. Means with a * are
different at the corresponding herbicide concentration according to paired t-test
at or = 0.01

63

 

 

 

 

 

10
g + eastern black nightshade
l- _.
‘5 so + johnsongrass
a .
9
5 so —
b
I: _
‘3
3 4o —-
E
E
3 20 _
<1
0 L l l
0 5 50 500 5000

Herbicide Concentration (nM)

Figure 3. Inhibition of ALS activity by nicosulfuron. Means are not different
at each herbicide concentration according to paired t-test at or = 0.01

64

 

     

 

 

 

 

10

‘e‘

E so + eastern black nightshade

8 _ + johnsongrass

‘5

5 60 —

3'

g ..

g 40 —

0

a 20 —
0 ﬂ
0 5 50 500 5000

Herbicide Concentration (nM)

Figure 4. Inhibition of ALS activity by primisulfuron. Means with a * are
different at the corresponding herbicide concentration according to paired t-test
at or = 0.01

Timing of Total Postemergence Herbicide Applications

for Weed Control and Corn Yield1

J. BOYD CAREY and JAMES J. KELLS2

Abstract. Postemergence grass and broadleaf herbicides are available for
effective, single-application total postemergence weed control in corn. Field
experiments were conducted in 1992 and 1993 to determine the effects of weed
interference prior to herbicide application on corn yield. Nicosulfuron plus
bromoxynil was applied at 5, 10, 15, or 20-cm weed canopy heights in plots with
or without weed interference. Both experiments were conducted on sites with
extremely heavy natural weed infestations. Crop injury was more severe when
herbicides were applied to smaller corn. Injury was temporary and did not
reduce corn yield. Herbicide applications made to 5-, 10-, or 15-cm weeds
provided nearly complete weed control. Weed interference did not reduce corn

height or grain yield when postemergence applications were made to small weeds

 

‘Received for publication . and in revised form

2Grad. Res. Asst. and Prof, respectively, Dep. Crop and Soil Sci., Michigan
State Univ., East Lansing, MI 48824.

65

66

(g 10 cm). Weed interference reduced corn height and grain yield in 1992 when
applications were made to 15-cm weeds even though weed control was nearly
complete. Weed control was incomplete and corn height and grain yield were
reduced when applications were delayed until weeds were 20 cm tall.
Nomenclature: nicosulfuron, 2-[[[[(4,6-dimethoxy-2-pyrimidinyl)-
amino]carbonyl]amino]sulfonyl]-N,N-dimethyl-3-pyridinecarboxamide;
bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile; corn, Zea mays L. ’Pioneer

3753’.

Additional index words: Bromoxynil, nicosulfuron, interference, weed

biomass, ABUTH, AMARE, AMBEL, CHEAL, SETFA.

67
INTRODUCTION

Nicosulﬁrron and primisulfuron are sulfonylurea herbicides which provide
selective postemergence control of many grass and some broadleaf weed species
in corn (1,2). Prior to their introduction, effective and reliable postemergence
weed control in corn was limited to the use of selective herbicides which
controlled only broadleaf weeds, or directed sprays which require special
application equipment to avoid crop interception of the herbicide during
application (6).

Tank-mixing nicosulfuron with postemergence broadleaf herbicides can
broaden the spectrum of weed control and provide single-application total
postemergence weed control in corn (10,13). This type of program provides an
effective alternative to the traditional practice of using soil applied herbicides as
a preventative approach to weed control. Total postemergence programs allow
the corn grower to assess the extent of the weed problem before making a
herbicide application (14,22).

However, since weeds are allowed to germinate and grow with the crop
until the herbicide application is made, signiﬁcant questions are raised about the
effect of early season weed interference on the corn. The competitive effects of

weeds on corn are important early in the growmg season (16,24). Signiﬁcant

68

yield reductions occurred when giant foxtail (Setaria faberi Herrm. #3 SETFA)
was allowed to interfere with corn until weeds were 15-23 cm in height and corn
was 41-61 cm in height with 9-10 leaves (16).

Researchers have attempted to identify what is called a "critical period"
for com (15). In terms of total postemergence programs, the critical period is the
period of time weeds may interfere with a crop before signiﬁcant yield reduction
will occur (23). Studies show that the critical period for weed competition in
corn is difﬁth to deﬁne, and varies with enviromnent, weed species, and weed
density (15,24).

Traditional studies investigating duration of weed interference in corn have
employed hand pulling or hoeing as the method of removal to end the period of
weed interference (15,16). This method ceases weed competition with the crop
abruptly and completely. Most postemergence herbicides, even when completely
effective, do not terminate weed competition in such a fashion. Sulfonylurea
herbicides in particular kill plants relatively slowly. Complete plant death in
susceptible species occurs in 7 to 21 days with nicosulfuron, depending upon
growing conditions, weed species, and growth stage of the weeds (4). Weed
interference could still occur with the crop after herbicide application. It would

also seem possible that an effective herbicide treatment could completely control

 

3Letters following this symbol are a WSSA-approved computer code from
Composite List of Weeds, Revised 1989. Available from WSSA, 309 West
Clark Street, Champaign, IL 61820.

69

weeds after the critical period had expired, having already allowed irreversible
interference and subsequent yield reduction.

The corn grower has some level of control over the duration of weed
interference with the crop by choosing the time and method of weed control. If
a grower adopts a total postemergence weed control program in com, the time
of herbicide application is important not only to insure successful weed control,
but to control the duration of weed interference and prevent crop yield reduction.
Knowledge of the duration of interference allowable before yield reduction
occurs would be valuable to effectively implement total postemergence weed
control programs in corn.

The objective of this research was to determine if weed interference prior
to herbicide application reduces corn yield with a total postemergence herbicide

program.

MATERIALS AND METHODS

Experiments were conducted in 1992 and 1993 at two separate locations
on the Michigan State University Crop and Soil Science Research Farm at East
Lansing, M1 to evaluate total postemergence herbicide application timing in

conventional tillage corn. Both studies were conducted on a Capac (fme-loamy,

70

mixed, mesic, Aeric Ochraquolfs) soil with a pH of 7.2 and 3.1% organic matter
in 1992, and a pH of 6.6 and 3.3% organic matter in 1993.

Tillage at the sites consisted of moldboard plowing corn stalks the fall
prior to disking and ﬁeld cultivation the spring of 1992, and chisel plowing
soybean stubble followed by disking and cultivation the spring of 1993. Pioneer
3753 corn was planted at 60 540 seeds ha‘l on May 14, 1992 and May 17, 1993.
Prior to spring ﬁeld cultivation 305 kg ha’1 of 46-0-0 was applied broadcast and
365 kg ha'1 of 6-24-24 was applied as a banded treatment 5 cm below and 5 cm
beside the corn seed at planting. Teﬂuthrin [(2,3,5,6-tetraﬂuoro-4-

methylphenyl)methyl-( l or,3 or)-(Z-(i)-3 -(2-chloro-3 ,3 ,3-triﬂuoro-1-propenyl)-2,2-

dimethylcyclopropanecarboxylate] insecticide was applied at 1.22 kg ai ha’l at
planting in 1992.

Plots consisted of four rows spaced 76 cm apart with lengths of 10.7 and
9.1 m in 1992 and 1993, respectively. A randomized complete block design was
used with a factorial arrangement of treatments replicated six times. The ﬁrst
factor consisted of plots with (weedy) or without (weed-free) weed interference.
Weed-free plots received a preemergence application of atrazine [6-chloro-N-
ethyl-N ’-(1-methylethyl)-1,3,5-triazine-2,4-diamine] at 1.12 kg ai ha‘l plus
metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-
methylethyl)acetamide] at 2.24 kg ai ha'1 and handweeding as necessary to

prevent any weed interference with the crop throughout the growing season.

71

Weedy plots received a postemergence application of bromoxynil at 0.42 kg ai
ha’l plus nicosulfuron at 0.035 kg ai ha" plus nonionic surfactant4 at 0.25%
(v/v) applied at application timings of 5, 10, 15, or 20-cm measured weed canopy
heights. Two additional treatments not included in the factorial arrangement
were a handweeded treatment with no herbicides, and a preemergence application
of atrazine plus metolachlor applied at the same rates used in the weed-free plots
but without handweeding. All herbicides were applied with a tractor-mounted
compressed-air sprayer calibrated to deliver 206 L ha’l at 207 kPa using 8003
ﬂat-fan nozzles’. Postemergence herbicide application information is presented
in Table l.

Weed density by species was determined at each application timing by
counting all weeds within a 929 cm’2 quadrant at four random locations within
each plot to be treated. Weed biomass was harvested at each application timing
in each plot to be treated, and at 30 days after the last treatment (DALT)6 in all
treated plots. At each harvest, all weeds within a single 0.25 by 2.3 m quadrant

were cut at ground level, oven dried at 49 °C for 7 days, and weighed. Harvests

 

4X-77’ -Nonionic-type spreader and activator. Principle functioning agents:
Alkylaryl polyoxyethylene, free fatty acids, glycols, isopropanol. Constituents
effective as spray adjuvant-90%. Constituents ineffective as spray adjuvant-10%.
Valent U.S.A. Corp, 1333 N. California Blvd, PO. Box 8025, Walnut
Creek,CA 94596-8025.

5Spraying Systems Co., North Avenue, Wheaton, IL 60188.

6Abbreviations: DALT, days after the last treatment; DAT, days after
treatment.

72

at application and at 30 DALT were taken at fixed locations 1 and 1.4 m from
the back of the plot, respectively.

Weed control by species and corn injury were evaluated visually in weedy
plots with 0 representing no visible injury and 100 representing complete plant
death. Corn injury was evaluated 4 days after treatment (DAT)6 and 14 DALT.
Weed control was evaluated at 14 DALT.

Corn height was determined 30 DALT by measuring ﬁve randomly chosen
plants within each plot. Corn grain yield was determined by harvesting the
center two rows of each plot with a plot combine. Seed weight was corrected
to 15.5% moisture.

Weed densities at each application are presented in Table 2. Nonlinear
regression analysis was performed on weed biomass data taken at application and
the best ﬁt models are presented in Figure 1. All other data were subjected to
analysis of variance and means separated using Fisher’s Protected Least

Signiﬁcant Difference Test at a = 0.05. Weed biomass data obtained 30 DALT

were transformed to the square root and visual weed control data transformed to
the arcsin before analysis of variance and mean separation. Original means are

presented. Results are presented by year due to year by factor interactions.

73
RESULTS AND DISCUSSION

Crop injury. Corn injury at 4 DAT was primarily leaf burn typical of
bromoxynil injury (3). The injury was much more severe when applied to
smaller corn at the 5- and 10—cm application timings (Table 3). Corn grew
rapidly and injury symptoms were much less evident by 14 DALT. Applications
at 5 or 10-cm weed canopies were made to 20-cm or smaller corn with 3 or
fewer fully collared leaves (Table 1). Bromoxynil is more injurious to smaller
corn and tank mixing bromoxynil with nicosulfuron requires addition of a
nonionic surfactant which may also result in increased initial crop leaf burn (3).

Effects of herbicide injury can be assessed independent of weed
interference. The factorial arrangement of treatments allows corn height and
grain yield comparisons between application timings within weed-free plots
(Table 4).

Herbicide injury reduced corn height at 30 DALT in weed-free plots when
applications were made to 5 or lO-cm weeds in 1992, and to 5,10, or 15-cm
weeds in 1993 (Table 4). However, corn grain yield was not reduced by
herbicide injury from any application in 1993, and in 1992 herbicide injury
reduced yields in the 5 and 10-cm plots when compared to the untreated weed-
free plots. A severe frost injured corn 3 days after corn emergence and 9 days

prior to the 5-cm application in 1992. The combination of environmental stress

74

and herbicide injury to small corn may explain why yield reduction occurred in
5 and 10-cm treatments in 1992 but not 1993.

The effects of the atrazine plus metolachlor portion of the weed-free plots
on corn were assessed by analyzing all treatments as a randomized complete
block. The atrazine plus metolachlor plus handweeding treatment provided corn
heights and corn yields equivalent to the handweeded treatment (data not shown).
From these results, we concluded that the methods used to maintain plots free

ﬁ'om weed interference did not have any adverse effects on corn growth or yield.

Weed density. Weed densities were extremely high both years of the study
(Table 2). Giant foxtail was the most predominant weed species present both
years. More broadleaf weeds were present in 1993, in particular common
lambsquarters (Chenopodium album L. # CHEAL) and redroot pigweed
(Amaranthus retroflexus L. # AMARE). Relatively small populations of
common ragweed (Ambrosia artemisiifolia L. # AMBEL) and velvetleaf
(Abutilon theophrasti Medik. # ABUTH) were present in both years.

Weed biomass increased exponentially over the time period between
planting and the last application timing both years (Figure 1). Between the ﬁrst
and last application timings, l9 and 14 days elapsed in 1992 and 1993,
respectively (Table 1). Weed biomass increased by factors of 26 and 17,

respectively over these time periods (Figure 1).

75

Weed control. Weeds were controlled at 14 DALT by postemergence
applications of bromoxynil plus nicosulfuron plus nonionic surfactant made at 5,
10, or 15-cm canopy heights (Table 5). Giant foxtail was not controlled
completely either year when weed canopies reached 20 cm before application.
Giant foxtail control was less in the 5-cm treatment than in the 10 or 15-cm
treatment in 1992. Weeds present in the 5-cm treatment at 30 DALT germinated
after the initial population was controlled by the herbicide treatment. Neither
bromoxynil nor nicosulfuron have residual soil activity (1,3). Greater weed
biomass in the 5-cm plots at 30 DALT in 1992 was primarily a result of giant
foxtail germination after the postemergence application since this was the most
abundant species and the other weeds were controlled. The 20-cm treatment
reduced weed biomass less than any of the other treatments in 1992, due
primarily to incomplete giant foxtail control at the time of application.

Giant foxtail, common lambsquarters, and redroot pigweed control was
inadequate with the 20-cm treatment in 1993 and contributed to greater weed
biomass than in any of the other treatments (Table 5). The 5 and lO-cm
applications provided the greatest control of giant foxtail and redroot pigweed
and the greatest reduction of weed biomass.

Common ragweed and velvetleaf control was generally adequate both
years (Table 5). Their populations were relatively low and appeared to

contribute relatively little to total weed biomass when compared to giant foxtail,

76

common lambsquarters, or redroot pigweed (Table 2). Weed biomass was
reduced by all postemergence treatments when compared to the untreated plots

(Table 5).

Weed interference with corn. Effects of weed interference can be assessed
independent of crop injury. The factorial arrangement of treatments allows corn
height and corn yield comparisons between weedy and weed-free plots in which
each received the postemergence herbicide application at the same timing (Table
4).

Corn height reduction at 30 DALT was closely correlated with yield
reduction from weed interference (Table 4). Other research has demonstrated
corn height reduction from increasing weed density (17). Weed interference
reduced corn height at 30 DALT and corn grain yield at harvest both years in
untreated plots. Weed interference reduced corn height and corn grain yield in
the 20-cm treatment both years. Weed interference reduced corn height and grain
yield in the 15-cm treatment only in 1992. Late season germination of weeds
after effective initial control with the 5- and 10-cm applications in 1992 did not
reduce corn yields. These results are similar to previous ﬁndings in which
signiﬁcant yield reduction occurred when giant foxtail was allowed to interfere
with corn until weeds were 15 to 23 cm in height, and late season competition

effects were not as great as early season (16).

77

Corn height and grain yield reduction due to weed interference in the 20-
cm treatrnents can be explained by the inability to control weeds at that advanced
growth stage, thereby allowing weed interference to continue throughout the
season (Tables 4 and 5). However, the reduction in corn height and corn yield
caused by weed interference in the 15-cm treatment in 1992 occurred even
though weed control from the postemergence treatment was complete (Table 5).

The critical period is deﬁned as the duration of weed interference with a
crop that is allowable before yield reduction will occur ( 15,24). Previous
research has shown that the critical period for weed interference in corn is
difﬁcult to deﬁne (15,24), and that it varies with environment, weed species, and
weed density (15). In our study, no attempt was made to deﬁne a critical period.
However, our results suggest that the critical period was exceeded when the
postemergence application was made to 15-cm weeds in 1992.

This study was not intended to deﬁne the mechanisms by which yield
reductions occur. However, differences in relative proportions of weed species
at the 1992 and 1993 sites may help explain why yield reduction with the 15-cm
treatment occurred in 1992 but not 1993. Weed densities were extremely high
both years, but the 1993 site had a greater proportion of broadleaf weeds than the
1992 site which was infested almost entirely by giant foxtail (Table 2). Weed
species differ in their ability to interfere with crops (7,8,9,11,12,18,19,20,21).

Signiﬁcant allelopathic effects from giant foxtail on corn have also been

78

demonstrated (5). The likely contribution of environmental and other
uncontrolled factors is also recognized.

This study suggests that weed interference will not reduce corn yields
under normal environmental conditions if weeds are controlled in a timely
manner with postemergence herbicides. Even with intense weed pressure, no
yield reductions from weed interference occurred when bromoxynil plus
nicosulfuron plus nonionic surfactant was applied to weeds at growth stages
recommended by the respective herbicide labels ( 1,3). Delaying postemergence
herbicide applications increases the possibility of inadequate weed control and
yield reduction. With intense weed pressure, later applications (>10-cm weeds)

- may result in yield reductions even though weed control is complete.

10.

ll.

79
LITERATURE CITED

Anonymous. 1993. Accent® herbicide product label. E.I. duPont de
Nemours and Co., Agricultural Products, Wilmington, DE 19898.

Anonymous. 1993. Beacon® herbicide product label. CIBA-GEIGY Corp.,
Agricultural Division, Greensboro, NC 27419.

Anonymous. 1993. Bromoxynil® Product Label. Rhone-Poulenc Ag Co.,
Research Triangle Park, NC 27709.

Anonymous. 1990. Accent® Herbicide. Technical Bull. E.I. duPont de
Nemours and Co., Agricultural Products, Wilmington, DE 19898. 12 p.

Bell, D.T., and DE. Koeppe. 1972. Noncompetitive effects of giant foxtail
on growth of corn. Agron. J. 64:321-325.

Bode, LE. 1987. Spray application technology. p. 85-109 in C.G.
McWhorter and MR. Gebhardt, eds. Methods of Applying Herbicides.
Weed Science Society of America, Champaign, IL.

Buchanan, GA. and ER. Burns. 1971. Weed competition in cotton 1.
sicklepod and tall morningglory. Weed Sci. 19:576-579.

Buchanan, GA. and ER. Burns. 1971. Weed competition in cotton 11.
cocklebur and redroot pigweed. Weed Sci. 19:580-582.

Buchanan, G.A., R.H. Crowley, J.E. Street, and J.A. McGuire. 1980.
Competition of sicklepod (Cassia obtusifolia) and redroot pigweed
(Amaranthus retroﬂexus) with cotton (Gossypium hirsutum). Weed Sci.
28:258-262.

Carey, J .B., P.B. Knoerr, and I]. Kells. 1990. Herbicide interactions with
nicosulfuron and primisulfuron in corn. Abstr. Weed Sci. Soc. Am. 32:12.

Chandler, J.M. 1977. Competition of spurred anoda, velvetleaf, prickly
sida, and Venice mallow in cotton. Weed Sci.25:151-158.

12.

l3.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

80

Crowley, RH. and GA. Buchanan. 1978. Competition of four
morningglory (Ipomoea spp.) species with cotton (Gossypium hirsutum).
Weed Sci. 26:484-488.

Dobbels, AF. and G. Kapusta. 1992. Postemergence weed control in corn
with nicosulfuron combinations. Proc. North Cent. Weed Sci. Soc. 47:47.

Foy, CL. and H.L. Witt. 1990. Johnsongrass control with DPX-V9360 and
CGA-136872 in corn (Zea mays) in Virginia. Weed Technol. 42615-619.

Hall, M.R., C.J. Swanton, and G.W. Anderson. 1992. The critical period
of weed control in grain corn (Zea mays). Weed Sci. 40:441-447.

Knake, EL. and F .W. Slife. 1969. Effect of time of giant foxtail removal
from corn and soybeans. Weed Sci.17:281-283.

Moolani, M.K., E.L. Knake, and F.W. Slife. 1964. Competition of smooth
pigweed with corn and soybeans. Weeds 12:126-128.

Patterson, D.T. and ER Flint. 1983. Comparative relations, photosynthesis,
and grth of soybean (Glycine max) and seven associated weeds. Weed
Sci. 31:318-323.

Shurtleff, J .L. and H.D. Coble. 1985. The interaction of soybean (Glycine
max) and ﬁve weed species in the greenhouse. Weed Sci. 33:669-672.

Snipes, C.E., G.A. Buchanan, J.E. Street, and J.A. McGuire. 1982.
Competition of common cocklebur (Xanthium pensylvanicum) with cotton
(Gossypium hirsutum). Weed Sci. 30:553-556.

Street, J.E., G.A. Buchanan, R.H. Crowley, and J.A. McGuire. 1981.
Inﬂuence of cotton (Gossypium hirsutum) densities on competitiveness of

pigweed (Amaranthus retroﬂexus) and sicklepod (Cassia obtusifolia).
Weed Sci. 29:253-256.

Swanton, OJ. and SF. Weise. 1991. Integrated weed management: the
rationale and approach. Weed Technol. 52657-663.

Weaver, SE. and CS. Tan. 1983. Critical period of weed interference in
transplanted tomatoes (Lycopersicon esculentum): growth analysis. Weed
Sci. 31:476-481.

24.

81

Zimdahl, R.L. 1988. The concept and application of the critical weed-free
period. p. 145-155 in MA. Altieri and M. Liebman, eds. Weed

Management in Agroecosystems: Ecological Approaches. CRC Press, Boca
Raton, FL.

82

Table l. Postemergence herbicide application information.

 

Corn Growth Stage‘

 

 

 

 

 

Date Height Cﬁtilairgg
Treatment” 1992 1993 1992 1993 1992 1993
cm —- cm -— —- no. -—
5 6-3 6-10 8 10 2 2
10 6-9 6-16 15 20 3 3
15 6-16 6-21 30 38 5 4
20 6-22 6-24 46 46 6 4

 

“Corn planted 5-14-92, and 5-17-93.

bWeed canopy height at postemergence application of bromoxynil at 0.42
kg ha'1 plus nicosulfuron at 0.035 kg ha‘1 plus nonionic surfactant at 0.25% (v/v).

 

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Table 3. Corn injury from bromoxynil plus nicosulfuron“.

 

 

 

 

 

 

 

Corn Injury
4 DATb 14 DALTb

Treatment° 1992 1993 1992 1993

cm %

5 33 b 38 b 3 a 9 a

10 35 a 43 a 2 ab 10 a

15 7 c 19 c 2 abc 6 b

20 8 c 7 d 1 be 5 b
Untreated 0 d 0 e 0 c 0 c

 

“Means within a year followed by the same letter are not signiﬁcantly
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compared across years.

bDAT = days after treatment, DALT = days after the last treatment.

°Weed canopy height at postemergence application of bromoxynil at 0.42
kg ha’1 plus nicosulfuron at 0.035 kg ha‘1 plus nonionic surfactant at 0.25% (v/v).

85

 

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0 1993

Y = 30.32 * e""” ‘ x’ - 33.72: R“: 0.96
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Y = 3.87 "' cw” ' x) - 5.17: R2 = 0.95

Dry Weight (g m")
H
8
I

 

 

1
5 10 15 20

Weed Canopy Height at Application (cm)

:3

Figure 1. Weed biomass accumulation at postemergence application timings in
com.

V LIBRARIES

MICHIGAN STATE UNI .
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