STUDIES GK THE FEERMCEDAL ENHANCEMENT
OF 3¢AMINQ:‘E, 2, LTREAZOLE
BY AMMQNEUM THIOCYANATE WITH
AGROPYRQN REFENS

 

Them For Hm Dom-cc 0‘ pk. D.
MECHIGAN STATE ‘JNWERSITY
William F. Donnalley

1964

THESIS

This is to certify that the

thesis entitled

"Studies on the Herbicidal Enhancement of
3-Amino- l, 2 , 4-Triazole by Ammonium
Thiocyanate with Agropyron Repens"

 

presented by

William F. Donnalley

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in HortiCUIture

. N 7
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. // (Z ,11,

Major"~ firofessor

Date June 9, 1964

 

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LIBRARY

MlCllt-I’Wl State
UMAVCI'Slty

 

ABSTRACT
STUDIES ON THE HERBICIDAL ENHANCEMENT

OF-3AMINO—l,2,4-TRIAZOLE BY
AMMONIUM THIOCYANATE WITH AGROPRYON REPENS

by William F. Donnalley

Foliar applications of amitrole-T, a 1:1 mixture of
3-amino-1,2,4-triazole (amitrole) and ammonium thiocyanate
(NH4SCN), have proven to be more effective in eradicating
quackgrass than amitrole alone. However, the manner in which
NH4SCN enhances the phytotoxicity of amitrole has not been
determined. In view of the benefits which might be derived
from a better understanding of herbicidal enhancement,
laboratory and field studies were conducted to determine the
manner in which NH4SCN augments the herbicidal activity of
amitrole.

The viability and sprouting of quackgrass buds
following foliage and rhizome applications of amitrole was

not altered by the addition of NH SCN.

4
Root and foliage applications of amitrole and NH4SCN,
to plants in solution cultures, indicated that the enhancement
response was influenced by the application site of the com-
pounds. Both the amount of chlorosis and reduction in dry

weight of the rhizomes and roots was greater following foliage

application of the mixture than from root application.

William F. Donnalley

Foliage and root applications of NH SCN alone had no effect

4

on growth.

Split applications relative to time of amitrole and

NH4SCN, in greenhouse and field investigations, indicated

that amitrole activity was influenced by the time of NH4SCN

application. Foliar applications of NH SCN before or in

4

combination with amitrole resulted in greater quackgrass

control than amitrole alone. However, NH SCN applied after

4

amitrole failed to increase the activity of the herbicide.

Foliage applications in the field indicated that the
degree of amitrole enhancement from different rates of NH4SCN
was dependent on the amount of NH4SCN applied. Irrespective
of the rate of amitrole employed (1 to 8 lb/A), increasing

rates of NH4SCN from 1 to 4 lb/A progressively increased the

phytotoxicity. However, 8 lb/A of NH SCN with all rates of

4

amitrole was no better than amitrole alone. The optimum

rate of NH4SCN for all amitrole levels was between 2 and

4 lb/A.
The herbicidal effectiveness of amitrole (2 lb/A) in
controlling quackgrass, either applied alone or in combin-

ation with NH SCN (2 lb/A), was not influenced when the pH

4
of the spray solutions was varied from 4.6 to 9.6.

The absorption of both C14

labeled amitrole (21.6%)
and NH4SCN (17.1%), 96 hours following foliar application,

was not altered when the compounds were combined. Ammonium

William F. Donnalley

thiocyanate applied before or after amitrole had no influence

on the amount of amitrole absorbed.

Translocation of Cl4 labeled amitrole, 96 hours

following foliar application, was markedly increased by the

addition of NH4SCN. When NH4SCN at 1,250 and 5,000 ppm was

combined with amitrole at 5,000 ppm, 27.9 percent of the
absorbed amitrole was translocated compared to only 15.4 per-
cent following application of amitrole alone. However,

NH SCN at 20,000 ppm did not alter the amount of amitrole

4

translocated. Amitrole translocation was also increased when

NH SCN at 5,000 ppm was applied 24 hours prior to amitrole

4
(25.3%), but did not influence translocation when applied 24

hours after amitrole (14.1%).

14

Translocation of C labeled NH SCN at 5,000 ppm

4
(7.1%) was not altered by the addition of amitrole at 5,000
ppm.

These studies suggest that NH4SCN enhances the ami-

trole activity on quackgrass by increasing the amount of

herbicide translocated within the plant.

STUDIES ON THE HERBICIDAL ENHANCEMENT
OF 3-AMINO—l,2,4-TRIAZOLE
BY AMMONIUM THIOCYANATE WITH

AGROPYRON REPENS

 

By .g‘

J

‘.
..\
“ .'

William F. Donnalley

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1964

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation
to Dr. S. K. Ries for his advice, guidance and assistance
during the preparation of this thesis. Appreciation is also
expressed to Dr. S. H. Wittwer for the use of his laboratory
facilities and guidance in writing this manuscript, and to
Dr. R. S. Bandurski, Dr. H. K. Bell, Dr. A. L. Kenworthy and
Dr. W. F. Meggitt for serving on the guidance committee.

Special appreciation is due to my wife, Helen, for
her encouragement and typing of the manuscript.

The financial support of the Amchem Products Company

is also gratefully acknowledged.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . .‘. . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . 3
Growth Characteristics of Quackgrass 3
Bud Dormancy of Quackgrass Rhizomes 7
Comparative Effectiveness of Amitrole
plus NH4SCN 9
Amitrole Absorption, Translocation and
Mode of Action 12
Thiocyanate Effects of Plants 21

MATERIMJS AND METHODS O O O O O Q O O O O O O O O O O 24

Response of Quackgrass Rhizomes to

Amitrole and NH4SCN 24
Localization of the Morphological Site of
Enhancement in Quackgrass 28

Response of Quackgrass to Split and Simul—

taneous Foliar Applications of Amitrole

and NH4SCN 30
Effect of Rate and pH of Foliar Applications

of Amitrole and NH4SCN,on the Enhancement

Response in the Field 32
Foliar Absorption and Translocation of

Amitrole and NH4SCN 34
Statistical Analysis 39

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 40

Response of Quackgrass Rhizomes to Amitrole

and NH4SCN , 40
Localization of the Morphological Site of
Enhancement in Quackgrass 45

Response of Quackgrass to Split and

Simultaneous Foliar Applications of

Amitrole and NH4SCN ‘ 48
Effect of Rate and pH of Foliar Applications

of Amitrole and NH4SCN on the Enhancement

Response in the Field 53
Foliar Absorption and Translocation of
Amitrole and NH4SCN in Quackgrass 57

iii

Page

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 75

LITERATURE CITED . . . . . . . . . . . . . . . . . . . 78

iv

LI ST OF TABLE S

Table Page

1. Effect of amitrole and NH4SCN, alone and
in combination, on the percent sprouting
and bud viability of quackgrass rhizomes . . 41

2. Effect of foliar applications of amitrole
(2 1b/A) and NH4SCN’(2 lb/A) applied
alone, in combination and as split appli—
cations on the control 0f quackgrass in
the field . . . . . . . . . . . . . . . . . 43

3. Percent viability of quackgrass rhizomes
in the field following foliar applications
of amitrole (2 lb/A) and NH4SCN (2 lb/A)
applied alone, in combination and as
split applications . . . . . . . . . . . . . 44

4. Effect of root applications of amitrole and
NH4SCN, alone and in combination, on the
growth of quackgrass . . . . . . . . . . . . 46

5. Effect of root and/or foliage applications
of amitrole and NH4SCN, applied alone
and in combination, on the growth of '
quackgrass plants . . . . . . . . . . . . . 47

6. Influence of pH on the herbicidal effective—
ness of foliar applications of amitrole and
amitrole—NH4SCN mixtures on quackgrass . . . 56

7. Foliar absorption,expressed as percent of
total applied, of C14 labeled amitrole
(5,000 ppm) and NH4SCN (5,000 ppm) alone
and in combination in quackgrass . . . . . . 58

8. Influence of varying concentrations of
NH4SCN on the foliar absorption, ex—
pressed as percent of total applied, of
amitrole* in quackgrass . . . . . . . . . . 64

Table

9. Influence of foliar applications of NH4SCN
(5,000 ppm), applied 24 hours before or
after amitrole (5,000 ppm), expressed as
percent of total applied On the absorption
of amitrole* . . . . . . . . . . . . . . .

vi

Page

68

Figure

LIST OF FIGURES

Influence of foliar applications of
NH4SCN, applied before or after
amitrole, on the regrowth of quack-
grass in the greenhouse . . . . . . . .

Effect of foliar applications of
NH4SCN, applied before or after
amitrole, on the regrowth of quack-
grass in the field . . . . . . . . . . .

Regrowth of quackgrass, three weeks
after plowing, as affected by various
rates of foliar applied mixtures of
amitrole and NH4SCN . . . . . . . . . .

Translocation of C14 labeled amitrole and
NH4SCN, alone and in combination, in
quackgrass following foliar applications

Quackgrass plants and autoradiograms
showing the distribution of foliar ab-
sorbed amitrole* 96 hours following
application . . . . . . . . . . . . . .

Quackgrass plants and autoradiograms showa
ing the distribution of foliar absorbed
NH4SCN* 96 hours following application .

Translocation of foliar applied amitrole*
in quackgrass as influenced by varying
concentrations of NH4SCN . . . . . . . .

Quackgrass plants and autoradiograms showa
ing the distribution of foliar absorbed
amitrole* and NH4SCN 96 hours folloWing
application . . . . . . . . . . . . . .

Translocation of foliar applied amitrole*

in quackgrass as influenced by the time
of NH4SCN application . . . . . . . . .

vii

Page

50

51

S4

59

61

63

65

67

69

‘f‘
-u
4

Figure Page

10. Quackgrass plants and autoradiograms
showing the distribution of foliar
absorbed amitrole* and NH SCN 96
hours following applicatién . . . . . . . . 70

viii

f.

LL

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INTRODUCTION

One of the most challenging phases of herbicide re—
search is the study of the mechanism by which certain
adjuvants enhance the activity of herbicides. The observed
increase in the herbicidal activity of 3-amino-l,2,4-triazole
(amitrole) by ammonium thiocyanate (NH4SCN) in certain weed
species is one of the outstanding examples of herbicidal
enhancement by a non-phytotoxic compound. Numerous field
studies have demonstrated that the amitrole—NH4SCN mixture
is from two to four times more effective than amitrole alone
for the control of quackgrass.

Quackgrass is probably the most serious perennial
weed in the north central and northeastern states. It not
only competes with crops for light, nutrients and moisture
but also exudes a toxic substance from its rhizomes which
may be harmful to subsequent crops. Quackgrass readily in-
fests agricultural crop lands, particularly where long ro-
tation programs are utilized. Since mechanical methods of
control have proved to be both costly and ineffective, con—
siderable effort has been directed towards chemical control.
However, few herbicides have proven practical and effective
.fOr control of quackgrass.

Due to the importance of quackgrass control in horti-

CUltural areas and benefits which might be derived from a

1

better understanding of herbicidal enhancement, studies were

conducted to determine the manner in which NH4SCN increases

the herbicidal activity of amitrole in quackgrass.

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REVIEW OF LITERATURE

Growth Characteristics of Quackgrass

Quackgrass (Aqropyron repens (L.) Beauv.) has become
a serious weed in crop lands in the northern latitudes of
the United States since its introduction from Europe in the
18th century (56). Quackgrass has been found in every state
north of Florida and Arizona, but rarely causes trouble as a
~weed south of the latitude of Washington, D. C. and St. Louis,
Mo. It occurs northward to the limits of cultivation, al-
though quackgrass in not common above 2,000 feet, or in arid
or semi-arid regions. The areas of most serious infestation
include the north central and north eastern states where
moderate summer temperatures and adequate rainfall during the
growing season are favorable for quackgrass growth (102).

Quackgrass is a troublesome weed because it spreads
rapidly, is difficult to eradicate and tends to take complete
possession of any field where it establishes itself. This
is especially true in crops where long rotations are used,
such as orchards or pastures. In these crops, the infestation
of quackgrass often spreads from a few scattered patches to
all parts of the field. During the time when land is in
grain, hay, or pasture, control or eradication of quackgrass

is impractical or impossible.

'v

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Although quackgrass is far from a prolific seed pro-
ducer, its spread to uninfested areas may occur through this
medium, Kephart (56) reported that an average head contains
about 25 viable seeds. According to Dunham §£.§l. (27) the
seeds retain their ability to germinate for at least four
years in storage or in soil. When buried in the soil, most
of the seeds in the top three inches either germinate or die
within two years. However, those buried at a greater depth
may persist for five or more years and germinate when brought
to the surface. QuackgraSs plants head and bloom in the
latter part of June or early July and seeds ripen in July (8).

Quackgrass populations increase mainly from rhizome
distribution in the field. Rhizomes multiply rapidly and
very soon exceed the combined length of all above—ground
parts (56). Numerous buds are produced on these rhizomes,
each of which is capable of producing a new plant. If the
rhizomes are broken or cut, growth continues because of the
stored up food supply. Most forms of tillage will break up
and spread pieces of rhizomes to new areas (96). The
seriousness of this method of disemination is brought out by
the fact that most forms of control and eradication are aimed
at killing the rhizomes.

In an undisturbed area, Evans and Ely (29) reported
that the majority of rhizomes are found in the upper three
to four inches of soil while in plowed fields the rhizomes
are distributed throughout the furrow slice. New rhizomes

begin to form in May and June and there may be another period

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of initiation in August and September, depending on the
environmental conditions (26, 29, 106). Welcott (106) re-
ported that new rhizomes up to one-half inch in length have
been observed during field preparation about the middle of
May. Other workers (26,29) report that new rhizomes were
just beginning to form in early June.

The rhizomes of quackgrass originate almost entirely
from buds at the nodes of other rhizomes, the chief area
being the first two or three nodes below the crown near the
surface of the soil on older rhizomes (29). These workers
also report that the newly forming horizontal rhizomes may
produce branch rhizomes from buds present, and this may
continue until the original rhizome finally turns upward and
develops into a shoot. Most rhizomes develop during the
summer. The periods of greatest rhizome and shoot growth may
overlap, but do not coincide. Palmer (77) has observed that
aerial-shoot formation is preceded by a change in the response
of the rhizome to gravity, the tip becoming negatively geo—
tropic and curving into a vertical position. Rhizomes could
be induced to change prematurely into aerial—shoot only by
placing the rhizomes so that they were pointed upwards.

After one season, the fresh wieght of rhizomes by
November has been estimated to be approximately eight tons
per acre in a well infested field (7). Kephart (56) has
reported finding over two and one—half tons of rhizomes
(fresh weight) per acre and Fail (30) found that one acre

may contain 80 miles of rhizomes. The result is an area

interwoven with numerous rhizomes and buds, each of which,

is capable of producing an above-ground shoot or another
rhizome branch. Many of the buds, however, never grow during
the entire life of the rhizome (7). It has been reported

. that the individual rhizomes rarely live more than 15 months
(56). However, formation of rhizomes is a rapid and con-
tinuous process in the spring and fall and thus quackgrass
can continue vegetatively almost indefinitely.

Most of the work that has been done on the composition
of quackgrass rhizomes deals primarily with carbohydrate
reserves. This is because workers have been looking for
periods in the growth cycle when the reserves in the rhizomes
reach low levels, thus indicating ideal times for starting
eradication procedures. Although the carbohydrate reserves
in roots of some perennial weeds are at a minimum in late
spring, measurements of the stored material in quackgrass
rhizomes reveal that with this species there is no time of
the year when reserves are particularly low (9, 26, 83).

Fertilizers, particularly nitrogen, affect rhizome
growth. Dexter (25) has shwon that although quackgrass
growth was approximately equal on both heavily nitrogen
fertilized and unfertilized areas, the subsequent growth of
rhizomes was much different. Rhizomes growing under high
nitrogen levels were more vigorous, sprouted more readily.
were more sensitive to clipping treatments and drought con-
dition, and when weakened by excessive sprouting or exposure

to drought were more susceptible to decomposition. Ries (89)

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also found that quackgrass was more readily controlled with
herbicides if a nitrogen fertilizer was applied prior to
herbicide application.

Extracts of degraded rhizomes have been reported to
have a deleterious effect on other weeds (38), small grains
(8, 57, 103, 42, 60), grasses (75), and legumes (58, 62).
It is not certain, however, that it is the toxins in these
extracts which inhibit the growth of subsequent crops (58,

103).

Bud Dormancy onuackgrass Rhizomes

Observations of quackgrass rhizomes during any time
of the year will reveal a large number of dormant buds.
Under normal undisturbed conditions the majority of these will
not grow during the entire life of the rhizome. Any dis—
turbance of the plant, such as plowing, cultivating or disk-
ing, which destroys the tops and causes the rhizomes to be
broken up or cut, results in breaking this dormant condition
(27, 56). This, however, does not occur at all times of the
year. Dexter (26) observed a definite seasonal variation in
the sprouting ability of buds. Even when the rhizomes were
disturbed by digging, removing top growth, or providing
favorable growing conditions, a period of low sprouting
occurred in May, June, and early July. Other studies have
also revealed a Sharp decrease in the ability of the rhizome
buds to produce shoots and new growth during May and early

June (110). Wolcott (106) reported that rhizomes which were

cultivated and partially exposed during mid-June formed no
shoots in the following three weeks in spite of three inches
of rain during that period.

Johnson and Buchholtz (54), have proposed that there
are two types of bud dormancy in quackgrass. The first type.
correlation inhibition, occurs during most of the year in
undisturbed rhizomes and is similar to apical dominance.
where the buds are kept dormant due to factors arising out—
side of the buds. The second type occurs during early summer
and is due to conditions within the buds themselves, since
the buds remain dormant even though disturbed, broken and
provided with favorable growing conditions. Hay (41) also
found a correlation inhibition present in the buds of new
rhizomes collected from fertile land during the summer. Some
buds developed on all rhizomes when the tips were-removed.
Lower buds tended to be inhibited by buds sprouting near the
apical ends. In no case, however, did buds fail to develop
as was reported by Johnson and Bucholtz (54).

Although few studies have been reported on the
factors that influence bud dormancy in quackgrass rhizomes,
certain related studies have provided some findings which
should be considered. Johnson and Dexter (52), observed
that rhizomes which had been growing under high nitrogen
conditions sprouted more freely than rhizomes under low
nitrogen levels when detached and grown.in.yit£g under favor—
able conditions. Johnson and Buchholtz (53) have also re-

ported that bud dormancy could be overcome by applying

nitrogen fertilizers in the fall or spring prior to collect-
ing rhizome sections, or by transferring the sections to
agar containing various nitrogenous compounds.

Meyer and Buchholtz (71) found that under the range
of conditions normally occurring in the field, C02 and 02
levels had no appreciable effect on bud sprouting. Optimum
soil temperature for bud activity and shoot growth of quack-
grass occurred between 20 and 27°.
Comparativeggffectiveness of Amitrole
and Amitrole plus NH4SCN

,Amitrole is a water-soluble heterocyclic compound
composed of a five membered ring having three nitrogen and
two carbon atoms (4). Its growth regulating properties were
discovered by Allen in 1952 (5). Later, Hall.g§.al. demon~
strated that amitrole was active as a defoliant and inhibitor
in tests with cotton. Subsequent testing by many investi-
gators soon established its potential as a non—selective
herbicide. At high concentrations, amitrole causes rapid
contact killing; the foliage turns brown and the plant may
die. At lower levels the leaves become chlorotic. Regrowth
is characterized by a high degree of chlorosis and the plant
may slowly decline and eventually die. Amitrole is commonly
used as a foliar spray to control Canada thistle (35, 37),
quackgrass (64, 76, 79) and poison-ivy (ll, 84). It has al—
so been used to some extent to control nutgrass (76), horse

nettle (l3) and common milkweed (64). Since amitrole is

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effective as a foliar spray, it is often combined with pre-
emergence type herbicides such as simazine and diuron to
control established annual and perennial weeds (22).

In 1958, Melander gt.al. (70) demonstrated that the
herbicidal activity of amitrole was greatly increased by the
addition of NH4SCN even though the latter compound was non-
phytotoxic at the concentration employed in the mixture.
Since then, extensive field tests have shown that the combin-
ation of amitrole plus NH4SCN was far more effective in the
control and eradication of certain weed species such as
quackgrass and Bermuda grass than amitrole alone. Conse-
quently, most weed control recommendations now include the

amitrole—NH SCN mixture in preference to amitrole alone for

4
the control of these weeds.

The most outstanding example of NH4SCN augmenting
the herbicidal activity of amitrole can be found in tests
with quackgrass. Several studies (49, 70, 88) have indicated

that the addition of NH SCN to amitrole spray solutions more

4
than doubled the effectiveness of amitrole. Based on the
amount of regrowth following foliar applications, Raleigh

(88) reported that the amitrole—NH SCN mixture was twice as

4
effective on quackgrass as amitrole alone. Also, the best
control was obtained when the ratio of amitrole to NH4SCN
was 1:3, 2:3, and 4:3 lb/A. This indicates that the amount
of NH4SCN is more important in the enhancement response than

the amitrole rate. Greenhouse investigations have also shown

the importance of the rate of NH4SCN in the enhancement

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response. Holly and Chancellor (49) have reported that the

optimum NH SCN rate was between 1 and 4 lb/A, but that

4
dosages as low as 0.25 lb/A were effective in increasing ami-
trole activity on quackgrass. Other field studies (3, 70),
have demonstrated that l lb/A of amitrole plus 3 lb/A of
NH4SCN was equally as effective as 8 lb/A of amitrole alone.
The thiocyanate ion appears to be the important moiety in
the enhancement response since the ammonium salt was no more
effective than either the lithium, potassium or sodium salts
(70). In all of these studies, quackgrass did not respond to
NH4SCN when applied alone at rates from 0.25 to 16 1b/A.

The enhancement phenomenon has also been observed on
Bermuda grass and Russian knapweed, but the magnitude of the
response was considerably lower than that obtained on quack-
grass (3). In general, foliar applications of the amitrole-

NH SCN at 2 lb/A were equally as effective in the control of

4
these species as amitrole alone at 4 1b/A. In other perennial
weed species, such as poison—ivy, Canada thistle, milkweed

and horse nettle there is no appriciable increase in amitrole
activity when NH4SCN is included (3).' Also, the enhancement
response is not apparent on annual weeds (69, 82). Mixtures

of NH SCN with several other types of herbicides, such as

4
phenoxy acids, triazines, substituted ureas and carbamates

failed to increase their activity (99).

12

Amitrole Absorption, Translocagion and
Mode of Agtion

‘Apsorption.--Amitrole is absorbed readily through
both foliage and roots. Bondarenko and Willard (12) found

14 labeled amitrole1 to

that it took at least one hour for C
be absorbed and translocated from the leaf surface to the
stem in Canada thistle. Similar results were reported by
Anderson (6) for southern nutgrass. Herrett and Link (45)
observed that the uptake of amitrole by both Canada thistle
and field bindweed was relatively constant through the period
required for the droplet to dry (45 to 90 minutes) and was

of the same order for both plants (18.8% for thistle, 14.4%
for bindweed). However, upon drying the rate of absorption
per hour for bindweed (2.7%) was considerably less than that
observed for thistle (7.6%). The penetration of the chemical
was primarily through the cuticle. They proposed that
differences in the uptake of amitrole between thistle and
bindweed accounted in part for the difference in sensitivity
between the two species.

Several other factors also affect the absorption of
amitrole. In Zebrina pendula and Tradescantia fluminensis,
which have stomates only on the lower leaf surface, amitrole
was absorbed to a greater extent by the lower leaf surface,
(20). Absorption studies with amitrole* indicated that the

repression of ionization on the basic side enhanced movement

 

fi'

lHereafter, radioactive compound will be designated
by an asterisk.

13

in the leaves (19). The presence or absence of light either
preceding or during the time period tested had no influence
on the absorption of amitrole in Canada thistle or field
bindweed (45). On the other hand, Carter and Naylor (15)
found that absorption of amitrole* by beans (Phaseolus
vulgaris), honeysuckle (Lonicera japonica), and sugar maple
(ager.§accharinum) was greater in the light than in the dark.

Freed and Montgomery (32) have shown that the use of
surfactants increased the absorption of amitrole in beans.
They used both alkylaryl sulfonate and alkylaryl polyoxy-
ethlene types of surfactants. Although absorption probably
was increased because these surfactants reduced the surface
tension, they felt that the relationship of molecular inter-
action between surfactant and the herbicide was perhaps
equal or more important than that of lowering the surface
tension.

The substitution of a different group on the triazole
ring seemed to influence absorption. Replacement of the
amino group by a hydroxyl group reduced absorption in French
dwarf beans (66).

Amitrole*has'been found to be absorbed readily by
broad bean i2121§.£§2§) roots (87) and barley roots (107).
When amitrole* was applied to the active phloem of tree
trunks of manzanita (Arctostaphvlos manzanita), toyon
(Photinia arbutifolia), and buckeye (Aesculeus california),

it was strongly absorbed by the xylem and slightly by the

14

active phloem of all three species, regardless of whether it

was transported by phloem or xylem (109).

Translocation.¥-Amitrole is readily translocated
throughout plants soon after application. The time required
for complete distribution varies from one species_to another,
the amount of chemical applied, and various physiological
and environmental factors.

In honeysuckle plants that were 12 to 14 inches tall,
Carter (14) found that amitrole* could be detected through-
out within 12 hours. Amitrole* applied directly to the pri-
mary leaves of pinto bean seedlings was detected.moving down
the stem within 24 hours (86). Amitrole* was translocated
throughout soybean and Canada thistle plants after 30 hours
(93). The translocation of amitrole* in southern nutgrass
seems to be slower, since 25 hours after application radio-
activity was not present in all areas of the plants (6).
However, after 125 hours labeling was distributed throughout
the entire plant.

The application of amitrole to leaves does not always
result in the translocation of the chemical. Factors such as
the amount applied, cuticle thickness or presence of stomates
all influence the degree of absorption and subsequent trans—
location. Yamaguchi and Crafts (109) have studied the pene—
tration and movement of amitrole* through the upper and lower
leaf surfaces of Zebrina pendula. They found that in order

to Obtain the same degree of translocation, five times the

15

amount had to be applied to the upper surface as compared
with applications to the lower surface of the leaf.

There is good evidence from experiments where ami-
trole* was employed, that very little amitrole*.pgr'§g was
translocated. Most of the radioactivity detected was from
some transformation product or products (14, 15, 43, 86).
Racusen (86) found that after five days, approximately one~
half of the amitrole* radioactivity applied to pinto bean
leaves had migrated elsewhere, and of the total radioactivity,
only seven percent was in the form of amitrole. Carter and
Naylor (15) found no unaltered amitrole* in terminal areas
of bean plants following application to one primary leaf.

Amitrole or its transformation products have been
found to accumulate in largest amounts in the meristematic
tissues such as the root tips, branch roots and shoot apices
(6, 12, 66, 72, 86, 109). Anderson (6) reported that in
short term experiments with southern nutgrass only the root
tips showed a heavy accumulation of amitrole*. In two or
more days all the accumulation sites were heavily labeled.
In Canada thistle, maximum radioactivity was also centered
in the terminal apex of the stem and in the younger leaves
after 48 hours (12). Miller and Hall (72) found acropetal
movement of amitrole* within 24 hours after application to
basal leaves in cotton with increasing amounts of the
chemical present in the young growth up to seven days. When
amitrole* was applied to a primary leaf of pinto bean seed-

lings, the terminal buds accumulated radioactivity at the

16

highest rate (86). Amitrole has been reported to be scarce
or lacking in dormant buds, storage parenchyma and mature
tissues in nutgrass (6).

Kath certain herbicides, such as 2,4-D, it had been
shown that translocation of these compounds from leaves to
other areas will occur only when there is a movement of carbOw
hydrates from the leaves. Most of the information available
also indicates a similar dependence for amitrole movement.
Studies on nutgrass (6) and Canada thistle (12) have shown
that the translocation of amitrole* to all areas of the plant
was incomplete unless a carbohydrate source was available.
Yamaguchi and Crafts (109), using variegated plants of
Tradescantia.£;uminens;§ to study this problem, found that
amitrole* translocation was dependent on the amount of green
tissue present. Penot (80) also reported that amitrole* did
not move out of the branches of Tradescantia viridis kept in
total darkness unless sucrose was injected simultaneously
with amitrole*.

The outstanding discrepancy on this dependence of
carbohydrates for movement is found in the work of Leonard
(61). He observed that both 2,4-D* and amitrole* were trans—
located when the plants were growing: but after growth had
ceased, 2,4-D* failed to translocate appreciably while move-
ment of amitrole* was essentially the same as it was during
the period of active growth. Similar results were also ob—
tained for 2,4-D* and amitrole* in Zebrina growing in

solutions of both high and low nutrient content.

l7

Amitrole can be translocated in either the xylem or
phloem. The vascular system used initially generally depends
on where the herbicide was applied. Amitrole applied to the
leaves was absorbed and moved out of the leaves via the
phloem (17, 21, 22, 43, 66, 87, 109). In one species,
Zebrina (21), the herbicide remained in the phloem during its
translocation; however, in barley (20) and broad bean (87)
there was some lateral transfer of amitrole to the xylem as
the distance from the point of application on the leaf
increased.

Amitrole is also readily absorbed by the roots and
translocated in the transpiration stream (21). Radwan.gt.§l.
(87) placed broad bean roots in amitrole* solutions for six
hours, and 20 hours later made sections through the root and
hypocotyl region. Their histoautoradiograms showed most of
the labeling was in the xylem with only a trace in phloem
tissue.

In addition to transport of amitrole in the vascular
system, movement also has been observed through the apoplast
in bean plants (61) and in storage parenchyma of potato
tubers (109). Amitrole* applied at the base of the blade in
bean plants moved upward and toward the margins of the leaf
without entering the veins.

Although amitrole does not accumulate to any extent
in dormant tissue, it has been observed to be translocated
through rhizomes (6, 40, 93). Rogers (93) noted that ami—

trole* applied to the leaf of one Canada thistle plant

18

traveled some six inches through the rhizome from a treated
to an nontreated Canada thistle plant and entered the shoot.
Similar results have been observed using nutgrass (6). In
this case, chains of three plants were used, a mother plant
and two daughter plants. Amitrole* was shown to be trans—
located from the treated mother plant to the nontreated
daughter plants, provided they were dependent on the mother
plant for assimilates.

Amitrole* distribution in plants was increased when

NH SCN was foliar applied with amitrole* (31, 22, 101).

4

Forde (31) found that NH SCN increased the translocation of

4
amitrole* into quackgrass rhizomes four days following foliar
application of the compounds. Both he and Crafts (22) pro-

posed that the NH SCN lessened the rapid damage to the cells

4
at the absorption site and thereby permitted the foliage to
absorb and translocate amitrole* over a longer period of
time. Van der Zweep (101), using bush bean, found that the
movement of amitrole* from the application site at the base
of a primary leaf was increased by the addition of NH4SCN.

It was also observed that NH4SCN alleviated the necrotic
contact affect of amitrole as well as increasing the amounts
of chlorosis produced in nontreated areas. On the basis of
this evidence, it was proposed that NH4SCN is increasing the
translocation of amitrole* directly or indirectly by increaSF
ing its absorption. Although both of these studies indicate

. * . .
that a greater amount of am1trole was d1str1buted throughout

the plant, the limitations of the autoradiographic technique

19

to distinguish between absorption and translocation makes it

difficult to attribute this increase to either process.

Mode of Action.--Many investigators have attempted
to elucidate the mode of action of amitrole since the dis-
covery that it would inhibit chlorophyll synthesis (91, 92).
After ten years of experiments with plants, microorganisms
and animals, it is still not fully understood how this com—
pound works in these organisms. For many studies it has been
postulated that amitrole interferes with the following physio-
logical and biochemical mechanisms: pigment synthesis (92,
92), ethanol metabolism (63, 73), porphyrin metabolism (91,
92, 98), purine and pyrimidine synthesis and degradation (2,
104), nucleic acid metabolism (107), riboflavin metabolism
(97, 98), glycine and serine metabolism (14, 15, 16), respir-
ation (44, 65, 68) and catalase activity (78, 85). However,
only two mechanisms, plastid development and histidine
metabolism, have been sufficiently studied to warrant dis—
cussion here.

The most obvious effect resulting from amitrole
applications to plants is the appearance of chlorosis in the
leaves developing after spraying. All the foliage prior to
spraying remained green provided a sub-lethal rate of ami-
trole was applied. Rogers (91, 92, 94), presented cytological
data which showed chlorosis in corn, Johnsongrass and rape
plants treated with a sub-lethal dose of amitrole was from a

lack of chloroplasts rather than an effect on chlorophyll

20

per se. Plastids were absent from sections of chlorotic
tissue. nyr m §£.éi- (85) also observed microscopically
that leaf tissue from barley and potato had fewer plastids
than normal tissue.

The majority of evidence from microscopic studies
favors the hypothesis that amitrole interferes with plastid
develOpment. This hypothesis has also been supported by
recent electron microscopy studies (95). Electron micro-
graphs showed that not only were the number of plastids re-
duced in the chlorotic tissue, but also that the internal
structures were missing from the remaining plastids.

Recently, it has been demonstrated that amitrole
interferes with histidine metabolism in yeast (46, 104),
bacteria (104), and in root hairs of Agrostis 312; (50).
Hilton (46) observed that when a supply of L—histidine was
added to yeast cultures, the amino acid produced different
degrees of protection against amitrole inhibition. This pro-
tective effect of L-histidine was observed in four species
of higher plants (corn, oats, wheat and tomatoes). Histidine
also nullifies the effect of amitrole on the growth of
Escherichia coli and Torula cremoris (104). However, with
these yeast adenine had to be added in addition to histidine
to completely overcome the toxicity of amitrole. Jackson
(50) reported that the inhibition of root hair elongation in
redtop by amitrole was partially overcome by the simultaneous
addition of L—histidine. It was observed that root hair inhi-

bition was greatest 90 to 120 minutes following the addition

21

of amitrole. This period of time coincided with the same
period where the greatest degree of reversal was obtained
when the two compounds were added simultaneously.

Jones and Ainsworth (55) reported that 1,2,4-triazole-
3-alanine was a specific histidine antagonist in tests with
certain strains of_§.'ggli. The addition of histidine would
reverse this inhibition. This alanine—triazole complex was
the same compound that Massini (67) isolated from French
beans following amitrole application. When the complex was
applied to the leaves it readily translocated and produced

chlorosis in young tissue.

Thiocyanate Effects on Plants

Thiocyanate salts, particularly NH4SCN, have mainly
been used in agriculture to control weeds and break dormancy
in seeds and vegetative buds. All-thiocyanate salts are
hygroscopic and readily soluble in water.

When used as a herbicide, NH4SCN has been applied as
a foliar spray or in the crystalline form at rates ranging
from 40 to 1600 lb/A, depending on the period of soil ste-
rility desired. Harvey (39) has shown that rates of 1600
lb/A rendered the soil sterile for four months while appli-
cations of 320 lb/A resulted in sterility for only four weeks.
Similar tests (34, 81) have also shown that NH4SCN, applied
as a foliar spray at 40 lb/A, produced rapid contact injury
to the foliage, but failed to prevent regrowth of perennial

weed species.

22

Although the nature of the rapid killing of the cells
is unknown, it is considered to be a protoplasmic poison in-
hibiting certain enzymes such as catalase (59), and coagulaté
ing proteins (39). Ahlgren.gt.al. (1) reported that rates of
750 lb/A were required to control quackgrass. .Even though
the field was first plowed to expose the rhizomes, some of
the rhizomes escaped contact with the chemical and conse—
quently survived. NH4SCN is particularly effective on rage
wort (10), poison—ivy (l) and gooseberry (74). In general,
both annual and perennial grasses are more resistant to this
compound than broadleafs (90). Tests on the various thio—
cyanate salts have shown that the ammonium, potassium and
sodium salts were of equivalent toxicity with the calcium
salt being less toxic (18). Although these investigations
illustrate the extreme phototoxicity of thiocyanates at rates
from 40 to 1600 lb/A, they have not been found toxic at the
2 to 4 lb/A rate employed in the amitrole—NH4SCN mixture.

Since NH4SCN has been effective on certain shallow
rooted perennials, it has led some investigators to speculate
that it is translocated basipetally in plants (90, 34).

This has been confirmed in recent studies by the presence of

NH SCN* in the roots of quackgrass four days following foliar

4
application of the compound (31).

Mixtures containing NH4SCN and one or more chemicals
have been more effective as herbicides than either one alone.

Combinations of NH4SCN at 20 lb/A and 2,4—D at l lb/A caused

greater injury to the foliage of plants than either one

23

alone (47). Similar results were also obtained when NH4SCN
was added to a solution of sodium arsenite (90). It was pro—
posed that NH4SCN was aiding the penetration and subsequent
translocation of the arsenical.

The thiocyanate salts have also proven effective in
increasing the germination rate of certain seeds and breaking
dormancy in newly harvested potatoes (25, 100). Townsend
(100) reported that potato seed pieces, momentarily immersed
in a one percent NH4SCN solution, readily sprouted when ex-
posed to favorable growing conditions. Similar results were
_obtained for both the potassium and sodium salts by Denny
(23). Gemeinhardt (33) observed that thiocyanates were active
in increasing the germination and subsequent growth of beets,
oats and carrots. He also found that the endogenous thio-o
cyanate level varied considerably from one plant species to
another.

To date, however, no one has investigated the effect
of thiocyanates on bud sprouting in quackgrass rhizomes or

its influence on the growth of this species.

MATERIALS AND METHODS

Response ofvguacquass Rhizomes to

Amitrole and NH4SCN

Viability of Single Bud Sections.--The influence of

rhizome applications of amitrole and NH SCN, alone and in

4
combination, on bud viability was studied in the laboratory.

Quackgrass rhizomes were dug from the field on July
24, 1962. The rhizomes were shaken free of soil in the field
and washed in the laboratory. They were then wrapped in
moist cheesecloth to prevent dessication.

Only newly—formed, fully developed rhizomes with
visually uninjured buds were selected. Adventitious roots
and scale leaves at the rhizome nodes were removed to reduce
contamination and to facilitate the observation of new growth
from the bud or rhizome section. The rhizomes were cut in
20 mm sections, each containing one bud. The sections were
then rinsed in distilled water and randomized prior to plac-
ing them in Petri dishes.

Ten sections were placed in a single Petri dish con-

taining two Whatman's No. 2 filter paper saturated with 6 ml

of treatment solution. Aqueous stock solutions of amitrole1

 

lTechnical grade amitrole and NH4SCN, 90% active,
obtained from Amchem Products, Inc., Ambler, Pa.

24

25

and NH4SCN were formulated on a weight-volume basis and the
final concentrations were determined by aliquot. The concen—
tration of the solutions applied singly were as follows:

Amitrole; 10, 100 and 1,000 ppm and NH SCN; 3, 33 and 333

4
ppm. When the two compounds were combined, a 3:1 ratio of
amitrole-NH4SCN was employed. Since there was little evapor—
ation from the closed Petri dishes, no additional solution
was added during the course of the investigation. The study
was conducted at room temperature since a previous study indi-
cated that 20-250C is near optimum for bud growth (71).
Treatments were run in triplicate and each replicate
consisted of a single Petri dish containing ten single bud

sections. Bud viability, based on the appearance of shoot

growth, was recorded two weeks after treatment.

Influence on Rhizome Dormancy.——Lateral bud dormancy
following rhizome application of amitrole and NH4SCN, alone
and in combination, was studied next. Newly-formed rhizomes
were collected in the field in August, 1962 and handled in
the manner previously described.

Rhizome sections, containing a single apical and
eight lateral buds, were severed from the distal portion of
the rhizomes collected in the field. The rhizomes were
rinsed in distilled water following removal of the adventi-
tious roots and scale leaves. Five of these rhizomes were
placed in parallel and between moistened paper towels. The

towels were then tightly rolled and placed vertically in

26

plastic cups with the proximal ends of the rhizomes downward.
Seventy milliliters of treating solution was added to the
cup and the volume maintained by the addition of distilled
water. Preparation of solutions and the concentrations em—
ployed were the same as those in the previous study.

Three weeks following treatment, the toweling was
removed and observations were made on the sprouting of
lateral buds. Unsprouted buds were considered to be in a
dormant condition. Bud viability was also evaluated after
cutting the rhizomes into individual sections to induce
sprouting. Bud sections were placed in Petri dishes on
moistened filter paper and their viability observed after two
weeks.

All treatments were run in triplicate and each repli-
cate consisted of five rhizome sections for studying'sprout-

ing and 25 single bud sections for determining viability.

Rhizome Response Following Foliar Applications.--The
effect of foliar applications of amitrole and NH4SCN on bud.
dormancy and Viability was studied in the field during the
spring of 1962.

The plots were located at the Michigan_State Univer-
sity Horticultural Farm on a Hillsdale sandy loam soil. The
area contained a heavy infestation of quackgrass which had
not been disturbed for several years.

The plots were arranged in a split plot design. The

main plots consisted of nitrogen treatments and were not

27

replicated. Sub—plots were composed of chemical treatments
and replicated three times. Each replicate consisted of 50
square feet (2 x 25 ft.). In the block which received nitro—
gen, 50 lb/A of nitrogen in the form of NH4NO3 was broadcast
one week prior to applying the chemicals. Regardless of the
treatment, amitrole and NH4SCN were applied at a rate of 2
lb/A in 40 gallons of water with a small plot sprayer using
quart bottles as containers and carbon dioxide cylinders as

a source of pressure. Chemicals were applied when the quack-
grass was approximately six inches in height.

Chemical treatments consisted of amitrole and NH4SCN
applied either before or after each other, with time inter—
vals of 1, 4 and 8 days between applications. Whenever ami—
trole was applied as a part of a split application, NH4SCN
was also applied alone and in combination with amitrole. The
latter treatments were necessary in order to properly evalu-
ate the effectiveness of split applications applied under
different environmental conditions occurring in the field.

Quackgrass control ratings, based on the degree of
chlorosis, were made periodically. Two weeks following
chemical applications, observations were made on lateral bud
dormancy. This was accomplished by lifting out several
sections of sod, washing the rhizomes, and counting the num-
ber of lateral buds that had sprouted. The viability of the
rhizomes was determined in Petri dishes as previously

described.

28

Localization of the Morphological Site
of Enhancement in Quackgrass

Root Applications.--In the fall of 1962, a greenhouse
study was initiated to determine whether the observed herbi—
cidal enhancement of amitrole by NH4SCN following foliar
applications could be reproduced when these chemicals were
applied to quackgrass roots.

Single bud sections were sprouted in a No. 8 grade
washed quartz sand. When the plants were approximately three
inches high, they were washed free of adhering sand and trans-
ferred into 2-gallon glazed crocks containing a one-half
strength Hoaglandls solution (48). The plants were supported
by styrofoam holders in the masonite covers and the solutions
aerated by passing air through porous air stones.

The treatment crooks were arranged in a randomized
complete block design on greenhouse benches. Treatments
were run in triplicate and each replicate consisted of three
plants. A flourescent light source, having a minimum inten-
sity of 100 foot-candles, was used to supplement the natural
day length to 16 hours. Air temperatures ranged from 15 to
270C during the course of the study.

Chemicals were applied when the plants were 10 to 12
inches high and had an extensive rhizome system. Aqueous
stock solutions of amitrole, NH4SCN and amitrole plus NH4SCN
were prepared on a weight—volume basis using technical grade
material. Final treatment concentrations were obtained by

pipetting an appropriate quantity of stock solution into the

29

nutrient solutions. The concentrations were as follows:

amitrole; l, 10 and 100 ppm and NH SCN; 0.3, 3.3 and 33.3

4
ppm. When combined, the ratio of amitrole to NH4SCN was 3:1.
Three weeks following application,chlorosis ratings

were taken and the foliage collected for dry weight

determinations.

Root and Foliage Applications.n-Since the previous
study included only separate and simultaneous applications
of amitrole and NH4SCN to the roots, it was of interest to
know if the enhancement phenomenon could be achieved by
simultaneously applying one compound to the foliage and the
other to the roots. Also, recent information indicated that
equimolar amounts of amitrole and NH4SCN produced a greater
degree of enhancement than the 3:1 ratio used in the previous
study (99).

This investigation was initiated during the spring
of 1963. The methods used for growing the plants, the
preparation and application of treatment solutions to the
roots and the experimental design were the same as previously
described. However, environmental conditions varied; air
temperatures ranged from 19 to 32°C and no supplemental
light was added to the natural lS-hour photoperiod.

Foliar treatments were applied using a sprayer which
consisted of a suspended, stationery nozzle positioned over
a variable speed conveyor bed. All foliar concentrations
were delivered at the rate of 85 gallons of water per acre

which was sufficient to wet the foliage to run-off.

30

Amitrole and NH4SCN were applied alone or in combin-
ation to the roots or foliage. In addition, simultaneous
applications of one compound were made to the foliage and to
the roots of the same plant. Both chemicals were foliar
applied at 4,500 ppm with root applications at 10 ppm.

Chlorosis ratings were recorded 12 days following
applications. Five days later, the foliage and rhizomes plus
roots were collected for dry weight determinations.

Response ofygpackgrass to Split and

Simultaneous Foliar Applications of
Amitrole and NH4SCN

#—

Greenhouse Studies.—-Since a previous field study
indicated that the enhancement phenomenon could be achieved
in the field, the following investigation was designated to
establish whether this enhancement could be reproduced in
the greenhouse and also evaluate factors which might influence
its occurrence.

Rhizomes, which had been collected the previous fall
and stored at 38oF, were placed in No. 8 quartz washed sand
to sprout. Twelve days later, single plants were transplanted
in 200 4—inch pots containing a 2—1-1 potting mixture of sand,
peat and soil. The plants were watered with a 0.5 percent
solution of 10—52—17 water soluble fertilizer. Once estab—
lished, the plants received only weekly applications of a
0.1 percent solution of NH4NO3. In order to stimulate tiller

develOpment and greater foliage growth, the plants were

clipped twice at the soil level. At the initiation of this

t.

«C

an

T.

5‘.

Q»

n~.

A. H.

s1

‘1

..
‘s

31

study, they had attained a height of six to seven inches and
were growing vigorously. Greenhouse temperatures ranged from
16 to 290C during the course of the study.

The pots were arranged in a split plot design with
three replicates: the order of randomization was washing
treatment, chemical treatment, time interval between split
applications and rate of chemical. Each pot, which contained
a single plant, was designated as a replicate.

The greenhouse sprayer, previously described, was
used to apply chemical treatments of amitrole and NH4SCN at
rates of l and 3 lb/A. The soil'was fitted with a cardboard
cover prior to spraying in order to prevent any chemical from
striking the soil surface and entering the plants via the
roots. Treatments consisted of amitrole, NH4SCN, amitrole

plus NH SCN and the two compounds applied at intervals of l,

4
4 and 8 days between applications. In the split application
treatments, where the influence of washing between applica-
tions was evaluated, the plants were inverted and the foliage
rinsed under tap water. When the plants were washed, they
were permitted to dry for approximately four hours before ap-
plying the second compound.

Quackgrass control ratings were taken 16 days follow-
ing application. The plants and soil media were lifted from
their original 4—inch pots and placed in 6-inch pots in an
inverted position so that the soil-rhizome mass was facing
upward. The latter process was used to simulate field plowb

ing. Twenty days after inverting, observations were made on

the amount of regrowth.

32

Field Investigations.--In the spring of 1963, a study
was made to further evaluate the enhancement response ob—
served from split applications applied in the field during
the spring of 1962, and in the previous greenhouse
investigation.

The experimental plots were located at the Michigan
State Entomological Farm on a Miami loam soil. The area con-
tained a heavy infestation of quackgrass which had not been
disturbed for several years. The experimental design, treat-
ments and applications of the chemicals were identical to
those used in the 1962 study. However, the size of the plots
were increased to 200 square feet (8 x 25 ft.) to facilitate
tillage operations. Treatments were applied when the quack-
grass foliage was approximately eight inches in height.

The plots were plowed and cross-disked two weeks
following treatment, in order to keep the interval between
application dates and time of plowing constant. Three weeks
after plowing and disking, the amount of regrowth was

recorded.

J

Effect of Rate and pH of Foliar Appli—
cations of Amitrole and NH4SCN on the
Enhancement Response in the Field

Rate of NH4SCN.-—A field study was conducted to ascer-

 

tain whether the rate of NH4SCN or the ratio of NH4SCN to

amitrole was the important criterion for the observed enhance-

ment phenomenon.

33

The plots were arranged in a randomized complete
block design on a site which contained a heavy infestation
of quackgrass. Treatments were replicated three times and
each plot consisted of 200 square feet (8 x 25 ft.). The
quackgrass foliage was seven to nine inches in height when
chemical treatments were applied.

Treatments consisted of foliar applications of

amitrole and NH SCN at l, 2, 4, and 8 lb/A. Both compounds

4
were applied alone and in all possible combinations.
Two weeks later, the plots were plowed and regrowth

observations made after three weeks.

ypH of Foliar Applications.--During the course of the
investigations on the enhancement phenomenon, large differ-
ences were observed between the pH of the commercial formu—
lations of amitrole (9.6) and amitrole plus NH4SCN (4.6).
Since the pH of foliar applied materials could possibly alter
their effectiveness, the effect of pH on the herbicidal
activity of amitrole and amitrole plus NH4SCN was studied.

The test was conducted during the spring of 1963, on
a site adjacent to the preceding study. The experimental de-
sign, plot size, methods of application and tillage Oper-
ations were identical to those in the rate of application
study.

Technical grade amitrole and NH4SCN were applied to
the foliage alone and in combination at three different pH

levels; 4.6, 7.1 and 9.6. The pH of the treatment solutions

ELLA“

cu

34

was adjusted using a 0.1 M solution of NaOH, HCL or H2S04.
Commercial formulations of both amitrole and amitrole plus
NH4SCN were also included in the study. All treatments were
applied at a rate of 2 1b/A. Three weeks after plowing,
quackgrass regrowth was recorded.

Foliar Absorption and Translocation of
Amitrole and NH4SCN in Quackgrass

 

Studies were started in the spring of 1963 to deter—
mine the foliar absorption and translocation of amitrole and
NH4SCN as influenced by: (a) separate and simultaneous ap-

plication of both compounds, (b) various concentrations of

NH SCN on amitrole activity and (c) NH SCN applied before or

4 4

after amitrole application.

Rhizomes were collected from the field, cut into.
single bud sections and sprouted in a No. 8 quartz sand.
They were planted approximately one inch deep and watered
daily with a one—half strength Hoagland's solution. The
plants were retained in the sand for two weeks then trans-
ferred to solution cultures. At this time, the plants were
approximately three inches in height. Twelve gallon solution
culture tanks (9 x 18 x 25 in.), containing a full-strength
Hoagland's solution, were aerated by forcing air through
several porous air stones. The plants were supported by
placing them between fastened styrofoam strips (1 x l x 24
in.) suspended directly over the tanks.

When the plants had attained the five leaf stage,

they were taken to the laboratory and transferred to covered

35

500 ml flasks containing a full-strength Hoagland's solution.
The nutrient solutions were aerated as previously described
and the plants supported in the neck of the flask with a
cotton plug. The entire study was conducted under a day—
night temperature which ranged from 23 to 27°C. A l6-hour
photoperiod was employed using a flourescent light source
with an intensity of 1,000 ft-c at the leaf surface.

The design consisted of a randomized complete block
with single plant replicates. Each treatment was replicated

seven times within a particular time interval.

Absorption.--The specific activity of the Cl4-labeled

amitrole and NH SCN samples was 0.95 and 1.10 millicuries per

4
millimole, respectively. The Cl4-amitrole was labeled in the

5-position of the triazole ring. Labeled treating solutions
consisted of a 5,000 ppm solution of either amitrole or

NH SCN containing 50 microcuries of Cl4 per milliliter. Un—

4

labeled reagent grade amitrole and NH SCN were used to adjust

4
the concentrations of the treating solutions. All treating

solutions were buffered at a pH of 6.2 with a 0.1 M solution
of KH2P04

One day after the plants were transferred into solu—

- NaOH (24).

tion culture flasks, the most recently matured leaf was
treated with one drOp (ca. 0.02 ml) of treating solution by
means of a tuberculin syringe fitted with a No. 27 gauge
stainless steel needle. The drOp was applied to the adaxial

surface on the main vein, midway from the apex to the base.

36

Since considerable more absorption might occur if the leaf
was scratched, special care was exercised so as not to in-
duce leaf injury with the tuberculin syringe needle.

In order to study the influence of one compound on
the absorption of the other, the following two treating solu—
tions were employed: amitrole* plus NH4SCN and NH4SCN* plus
amitrole. The concentration of both compounds was 5,000 ppm.

The influence of NH SCN concentration on the absorp-

4
tion of amitrole* was also studied. Identical procedures
were employed as previously described, except the concen-
trations of NH4SCN were 1,250, 5,000 and 20,000 ppm with
amitrole* held constant at 5,000 ppm.

The final phase of the study dealt with split appli-
cations of NH4SCN and amitrole*. Where split applications
were employed, NH4SCN was sprayed on the upper surface of a
single leaf one day prior to or after the application of
amitrole*. NH4SCN was applied at a concentration of 5,000
ppm with an atomizer to the point of run—off.

The plants were harvested at designated time inter-
vals and the treated surface of the leaf washed. During
washing, the treated area of the leaf was directed toward the
inside of a 1 3/4 ounce paper cup and the treatment site
washed with 20 ml of 50 percent ethanol. This quantity was
supplied drOpwise with a 20 ml pipette. A preliminary study
indicated that 20 ml of 50 percent ethanol was a sufficient
l4

quantity to remove one drop of either C labeled amitrole

or NH4SCN. The volume of the wash solution was then brought

37

to 25 ml. A one milliliter aliquot of the wash solution was
evaporated on a planchet and radioassayed with an end-window
G-M tube and standard scaler unit. The amounts of labeled
amitrole and NH4SCN not recovered in the leaf washings were
considered as absorbed amounts. The absorbed amounts were
expressed as percentages of the total amount applied.

Translocatign.--Plants retained from the absorption
studies were used to study the translocation of labeled
NH4SCN and amitrole.

The amount of Cl4 translocated following foliar ap-
plications of either NH4SCN* or amitrole* was determined using
the technique of Massini (65). The plants were first sec—
tioned into three parts: the treated leaf blade, the remain-
ing foliage and the roots. These parts were cut into one
centimeter sections and first extracted with 40 ml of 70 per-
cent ethanol containing 5 percent formic acid at 83°C. They
were then further extracted with 40 ml of water at room
temperature. The volume of both extracts was adjusted to 50
ml and combined. A one milliliter aliquot of the combined
extracts was evaporated on a planchet and assayed for
radioactivity.

The amount of C14 that moved out of the treated leaf

was considered translocated. This amount was expressed as a

percentage of the total amount absorbed.

Autoradioqraphy.--Autoradiograms were prepared to

follow the pattern of movement of amitrole* and NH4SCN*

38

within the leaf and translocation out of the leaf following
foliar absorption. These studies were carried out concur—
rently with the quantitative studies. The methods employed
were identical to the previous study, except for the specific
activity of the solution used. In this study, a 5,000 ppm
treating solution containing 25 microcuries per milliliter
was employed and treatments were not replicated.

The techniques used for the preparation of plants
prior to autoradiography were similar to those described by
Yamaguchi and Crafts (108). At the end of the time intervals,
the plants were removed from the culture flasks, the roots
rinsed under distilled water, and blotted dry between paper
towels. They were then spread between blotters and dried
under steel plates at 75°C for three hours. The plant
material, being brittle after drying, was placed in a high
humidity chamber to facilitate handling during the mounting
process. The plants were fastened on white blotter paper
with thin strips of Scotch tape. The mounted plants were
then covered with a sheet of cellophane and flattened in a
plant press over—night.

Autoradiography was accomplished by placing the
plants and X-ray films in a lightproof plant press in the
following sequence: First, a masonite board, a piece of X-
ray film, the plant mount, then a double thickness of blotter
paper and another masonite board. After several such layers

were assembled, they were exposed to the Xkray film for 26

39

days. The film was then developed according to standard

Xéray procedure.

Statistical Analysis

Data were statistically evaluated by the Analysis of
Variance. Where a significant F value occurred but main ef-
fects could not be partitioned into single degrees of freedom,
Duncan's Multiple Range Test was used to compare mean
differences.

With certain studies, it was possible to segregate
effects into single degrees of freedom. In these studies,
class and trend comparisons as well as specific interactions

»were partitioned.

RESULTS AND DI SCUS SION

ReSponse offguacquass Rhizomes to

Amitrole and NH4SCN

It has been clearly demonstrated by several investi—

gators (49, 70, 88) that NH SCN increases the herbicidal

4
activity of amitrole when the two compounds are applied in
combination to the foliage of quackgrass. Since this en-
hancement phenomenon is particularly evident on regrowth of
quackgrass following foliar applications of the compounds,

it was hypothesized that NH SCN might be stimulating growth

4
of dormant lateral buds and thereby increase their suscepti—
bility to amitrole.

To study the influence of amitrole and NH4SCN, alone
and in combination, the compounds were applied to rhizome
sections with eight lateral buds (Table 1). All treatments,
regardless of the concentration, failed to alter the sprout—
ing percentage of lateral buds.

The viability of single bud sections was reduced
following application of all solutions containing amitrole
(Table 1). In general, bud viability was inversely related
to the amitrole concentration. In no case, did the addition

of NH4SCN to amitrole solutions decrease bud viability in

comparison to amitrole alone at the same concentration.

40

41

Ammonium thiocyanate alone, regardless of the concentration,

did not effect bud viability.

 

 

 

 

Table 1. Effect of amitrole and NH SCN, alone and in combin—
ation on the percent sprouting and bud viability
of quackgrass rhizones.

Lateral bud Bud viability2
Conc. sprouting 1 (%)

Chemicals (PPm) _ (%) Test 1, Test 2

None --- 17.3 76.3 a 84.0

Amitrole 10 19.3 28.7 b 40.7

Amitrole 100 21.3 8.3 c 4.7

Amitrole 1,000 14.7 0.3 c 3.3

NH4SCN 3 20.0 81.0 a 81.3

NH4SCN 33 ' 14.3 70.7 a 79.3 a

NH4SCN 333 18.7 75.7 a 84.3 a

Amitrole + NH4SCN 10 + 3 18.3 32.7 b 36.7 b

Amitrole + NH4SCN 100 + 33 22.3 21.3 b 8.7 c

Amitrole + NH4SCN 1,000 + 333 16.7 1.0 c 6.7 c

 

f

1F value for treatments not significant at 5% level.

2Percentages with different letters significantly
different at 1% level.

These data indicate that the enhancement phenomenon,
was not achieved following rhizome applications. In no case
was amitrole plus NH4SCN more effective than amitrole alone
in breaking bud dormancy or decreasing bud viability. Al—

though NH SCN had been previously shown to be effective in

4

42

stimulating growth in dormant vegetative buds of other plant
species (25, 108), it had no effect on the sprouting of dor—
mant quackgrass buds.

In a field study, foliar applications of amitrole and
NH4SCN were applied alone and in combination as split appli-

cations. Treatments containing amitrole and NH SCN, either

4
applied in combination or as split applications, resulted in
greater control than amitrole alone (Table 2). Split appli-
cations of amitrole and NH4SCN, irrespective of time interval
between applications, were equally as effective as the combin—
ation treatment. NH4SCN applied alone, had no visual effect
on quackgrass. Differences in the degree of control for
chemical treatments were observed at various application
dates. These differences are attributed to varying environ—
mental conditions occurring in the field or the physiologi-
cal status of the quackgrass at the time of application.

Rhizomes were dug from the test plots and visually
evaluated for lateral bud sprouting. Lateral bud sprouting
ranged from 10 to 22 percent, with no difference between
treatments.

The rhizomes were cut into single bud sections to
induce sprouting and determine bud viability. Bud viability
was decreased following foliar application of all treatments
containing amitrole. The addition of NH4SCN, applied before

or in combination with amitrole, did not increase the ef-

fectiveness of amitrole in reducing bud viability. Foliar

43

applications of NH SCN alone did not alter bud viability

4
(Table 3).

Table 2. Effect of foliar applications of amitrole (2 lb/A)
and NH4SCN (2 lb/A) applied alone, in combination
and as split applications on the control of quack—
grass in the field.1

 

 

 

 

 

Date of application Chem.
Chemical 4/25 4/28 5/2 ave.
None 1.0 1.0 1.0 1.0 a
Amitrole + NH4SCN 7.3 8.2 8.0 7.8 c
Amitrole 4.8 5.3 5.7 5.3 b
NH4SCN 1.0 1.0 1.0 1.0 a
NH4SCN before amitrole2 6.7 ~-- ~-- 6.7 c
NH4SCN before amitrole2 --— 6.7 -—- 6.7 c
NH4SCN before amitrole2 ——- —-— 7.2 7.2 c
Time ave.3 5;o a 5.3 ab 5.5 b

lControl ratings, l.0——no control, 9.0—-complete

control. *

2Split application, NH4SCN applied on April 24 and.
amitrole on above dates.

3Ratings with different letters significantly dif—
ferent at 5% level.

Results from the field study, regarding the effec—
tiveness of amitrole plus NH4SCN and amitrole alone, are in
agreement with those of several investigators (49, 70, 88).

Prior to this study, however, it had not been demonstrated

44

that NH4SCN applied at various time intervals prior to ami-
trole application was equally as effective as the amitrole-

NH4SCN mixture. Whether this enhancement affect can be

achieved by applying NH SCN after amitrole application will

4
be.reported in a later study.

Table 3. Percent viability of quackgrass rhizomes in the
field following foliar applications of amitrole
(2 lb/A) and NH4SCN (2 lb/A) applied alone, in
combination and as split applications.

 

 

Date of application . Chem.

 

 

Chemicals 4/25 4/28 5/2 ave.
None 69 59 77 68 a
Amitrole + NH4SCN 33 59 45 46 b
Amitrole 57 60 43 53 b
NH4SCN 73 67 58 66 a
NH4SCN before amitrolel 45 —-- -—- 45 b
NH4SCN before amitrolel —-- 54 --- 54 b
NH4SCN before amitrolel -—— -—- 48 48 b
Time ave.2 55 a 60 a 54 a

 

lSplit application, NH4SCN applied on April 24 and
amitrole on above dates.

2Percentages with different letters significantly
different at 5% level.

45

The results obtained from laboratory and field in—
vestigations on amitrole and/or NH4SCN applications indicate
that the enhancement phenomenon is only manifested by the
foliage following combination or split application treat—
ments of the two compounds. The addition of NH4SCN to
amitrole treatment solutions, either applied to the foliage
or rhizomes, failed to increase the effectiveness of amitrole
in regard to bud sprouting and viability. In view of these
results, the previously stated hypothesis was rejected and
further studies utilizing this approach discontinued.

Localization of the Morphological Site
of Enhancement

Although the increase in amitrole activity by NH4SCN
is evident following foliage applications, it was not known
if a similar response could be induced via the roots. Conse-
quently, it was believed that studies directed toward identi—
fying the morphological site of amitrole enhancement by
NH4SCN would be valuable in guiding future investigations.

Chlorosis was apparent on all plants that received
root applications of solutions containing amitrole (Tables 4
and 5). In general, increasing the amitrole concentration
resulted in an increasing degree of chlorosis and a reduction
in the dry weight of the foliage (Table 4). The dry weight
of the roots plus rhizomes was not reduced following root
application of amitrole (Table 5). In no case, did the ad-

dition of NH4SCN increase the activity of amitrole in regard

46

to the degree of chlorosis, dry weight of foliage or roots
plus rhizomes. Root applications of NH4SCN alone, regard-
less of the concentration, had no visible affect on plant

growth.

Table 4. Effect of root applications of amitrole and NH
alone and in combination, on the growth of
quackgrass.

4SCN,

 

_V— 1'?

 

 

Treatments Chlorosis Dry wt. of
ratings foliage
Chemical Conc. (ppm) 1'2 (gm)
None --- 1.0 a 5.7 a
NH4SCN 0.3 1.0 a 5.6 a
Amitrole 1.0 3.7 b 5.1 a
Amitrole + NH4SCN 1.0 + 0.3 4.3 b 5.6 a
NH4SCN 3.3 1.0 a 6.0 a
Amitrole 10.0 6.7 b 3.8 b
Amitrole + NH4SCN 10.0 + 3.3 6.3 b 3.7 b
NH4SCN 33.3 1.0 a 5.1 a
Amitrole 100.0 9.0 c 1.5 c
Amitrole + NH4SCN 100.0 + 33.3 9.0 c 1.7 c

 

lChlorosis rating: l.O-—no chlorosis, 9.0-~complete
chlorosis.

2Means with different letters significantly different
at 1% level.

47

Table 5. Effect of root and/or foliage applications of
amitrole and NH4SCN, applied alone and in combin—
ation, on the growth of quackgrass plants.

 

 

Dry wt. of
Chlorosis roots +
Chemical and Application ratings rhizomes
Conc. (ppm) site(s) 1'2 (gm)
None --- 1.0 a 24.3 a
Amitrole
(10) root 6.0 b 21.3 a

NH4SCN

(10) root 1.0 a 23.7 a
Amitrole + NH4SCN

(10) (10) root 5.7 b 21.9 a
Amitrole

(4,500) foliage 6.0 b 22.7 a
NH4SCN
(4,500) foliage 1.0 a 24.7 a
Amitrole + NH4SCN
(4,500) (4,500) foliage 9.0 c 15.3 b
Amitrole, NH4SCN
(4,500) (10) foliage, root 6.0 b 20.9 a
NH4SCN, Amitrole
(4,500) (10) foliage, root 5.7 b 21.8 a

 

T fi'

lChlorosis ratings: l.0——no chlorosis, 9.0-—comp1ete

chlorosis.

2Means with different letters significantly different

at 1% level.

3Same as 2, but at 5% level.

48

All plants that received foliar applications of
amitrole, either alone or in combination with NH4SCN, were
chlorotic (Table 5). However, the addition of NH4SCN to
amitrole solutions reduced the dry weight of the roots plus
rhizomes and markedly increased the degree of chlorosis in
comparison to amitrole alone. Plant response from simul-
taneous applications of amitrole to the foliage and NH4SCN to
the roots, or the reverse, was similar to that of foliar and
root applications of amitrole alone.

This evidence indicates that the enhancement response
occurred only following foliar applications of amitrole plus
NH4SCN. Although this study offers no direct evidence for
the mechanism of enhancement, the differential response be-
tween foliage and root applications could be due to the con-
trasting anatomical and physiological features of these
organs. The foliage possesses barriers to absorption and
translocation which are not present in the roots (105). If

NH SCN decreased one of the barriers to amitrole, greater

4

herbicidal activity could result.

Response of Quackgrass to Split and
Simultaneous Foliar Applications_gf

Amitrole and NH4SCN

The previous greenhouse study established that
NH4SCN only augments the herbicidal activity of amitrole in
quackgrass when the two compounds are applied to the foliage.

This enhancement also occurred in the field when NH4SCN was

49

applied before amitrole. In both of these studies, however,
the enhancement was based on the response of the treated
foliage following application of the compounds. Therefore,
it was of interest to know if the enhancement phenomenon
would occur on quackgrass regrowth when NH4SCN was applied
either before or after amitrole.

In a greenhouse study, the amount of regrowth follow»

ing foliar applications of NH SCN, either before or after

4
amitrole, varied largely in a linear fashion with the time
between applications (Figure 1). Regrowth control was ex—
pressed as the percentage of the amitrole-NH4SCN mixture.

Control was greater when NH SCN was applied one day before

4
amitrole than when applied one day after. vAt longer time
intervals between applications, four and eight days, regrowth
following application of NH4SCN before amitrole was similar
to NH4SCN applied after amitrole.

The rate of amitrole applied and the effect of wash-
ing the foliage prior to applying the second compound had no
influence on the degree of quackgrass control.

Regrowth following foliar applications of NH4SCN in
the field, either before or after amitrole at various time
intervals, is shown graphically in Figure 2. When a one day
interval was used between applications, NH4SCN applied be-
fore amitrole resulted in a greater degree of control than

NH SCN applied after amitrole. The differences in regrowth

4

control between these treatments became progressively less,

50

 

O
O

I

NH4SCN before amitrole

 

— -- NH4SCN after amitrole

NI
0
T

*1
O
I

 

 

 

 

x of Amitrole - M44 SCN Regrowth Rating

Time Interval Between Application (days)

Figure 1. Influence of foliar applications of NH4SCN,
applied before or after amitrole, on the
regrowth of quackgrass in the greenhouse.
Interaction for the linear effect of time
x treatments significant at 1% level.

~~~ ”
~~~~~”’
I L
60
l 4 8

51

 

IOO ,

90’

x of Amitrole -NH4 SCN Roam win Rating

 

 

 

NH‘SCN before amitrole
--- NH4SCN after amitrole .)

 

 

1 I 1

50 l 4 8
Time Interval Between Application (days)

Figure 2. Effect of foliar applications of NH4SCN, applied

before or after amitrole, on the regrowth of
quackgrass in the field.

Interaction for the linear effect of time x
treatments significant at 5% level.

52

in a linear fashion, as the time interval between applications
increased from one to four and eight days.

The above data indicated that NH SCN applied one day

4
before amitrole was as effective in controlling quackgrass
regrowth as the amitrole+NH4SCN mixture. However, when
NH4SCN was applied after amitrole, regardless of the time
interval between applications, it was similar to applying
amitrole alone. These results are in general agreement with

those of Holly and Chancellor (49). They found that NH SCN

4
applied two days prior to amitrole greatly increased the
herbicidal activity of the latter compound on quackgrass.

However, amitrole activity was also increased slightly when

NH SCN was applied two days after amitrole. The lack of

4
complete agreement between these studies might be attributed
to a different response between quackgrass clones or the

fact that they used a surfactant (Tween 20) in the spray
solutions. These studies further suggest that NH4SCN may be
favorably predisposing a physiological or anatomical process
in the foliage which is intimately associated with the herbi—
cidal activity of amitrole. Failure of NH4SCN to augment
activity when applied more than 4 days before amitrole,

might be due to its movement out of the foliage or degradation

prior to applying the herbicide.

53

Effect of Rate and pH of Foliar Appli-
cations of Amitrole and NH4SCN on the

Enhancement Response in the Field

 

Few studies have been devoted to factors that might
influence the occurrence or magnitude of the enhancement re-
sponse, namely: the amount of amitrole and NH4SCN and the
pH of the spray solution. A clearer understanding of the
effect of these factors could be valuable in elucidating the
mechanism of enhancement as well as explaining discrepancies
obtained by different investigators.

The amount of regrowth following foliar applications
of various levels of amitrole and NH4SCN varied with the
amount of NH4SCN employed in the mixture (Figure 3). In
general, the degree of control obtained for all amitrole
levels was progressively greater when the rate of NH4SCN was
increased from 0 to 2 lb/A and remained relatively unchanged
between 2 and 4 lb/A. Increasing the rate of NH4SCN from 4
to 8 lb/A decreased control from all amitrole levels. In no

case, was addition of NH SCN at 8 lb/A better than applying

4
amitrole alone.

The optimum rate of NH4SCN, regardless of the amitrole
level, appears to be between 2 and 4 lb/A. Melander §£.§i-

(70) and Raleigh (88) have also demonstrated the importance

of the rate of NH4SCN rather than the ratio of amitrole to

NH4SCN on the activity of amitrole. Consequently, it appears

that NH4SCN acts independently of amitrole in the enhancement

response. Although there is no direct evidence as to the

54

 

  

Regrowth Ratings

 

I 1 I
~-._ —-- amitrole, 8 Ib/A
I --\\ -— amitrola,4 lb/A —
\ ———— amitrole, 2 lb/A
\ —— amitrole, l lb/A

 

\\
\\

\ —i

 

 

\
\\\‘\
\ _
1 l I
3 O 2 4 8
Rate of Ammonium Thiocyanate (lb/A)
Figure 3.

Regrowth of quackgrass, three weeks after plowing,
as affected by various rates of foliar applied
mixtures of amitrole and NH4SCN.

Interaction for the linear rate of amitrole x
quadratic rate of NH4SCN significant at 1% level.

55

manner of this rate affect, it might be speculated that
NH4SCN is stimulating a physiological process associated
with amitrole activity at low rates.

The influence of spray solution on the performance

of amitrole, alone and in combination with NH SCN, was

4
studied (Table 6). Neither the spray solution pH nor the
reagent used to adjust the pH had any affect on the degree
of control obtained from foliar applications of amitrole and
amitrole plus NH4SCN. Commercial grades of amitrole and
amitrole plus NH4SCN were no different than those formulated
from technical material.

Although it was originally proposed that large
differences in the pH of commercial formulations of amitrole

(9.6) and amitrole plus NH SCN (4.6) might be contributing

4
to the enhancement effect, the results do not support this
hypothesis. Other investigators (28, 49) have reported con-
flicting evidence in regard to the influence of pH on the
activity of amitrole—NH4SCN mixtures in quackgrass. At a pH
of 4.0 or lower, Elliott (28) found that the effectiveness
of the mixture was increased, while Holly and Chancellor (49)
observed a substantial decrease in activity. These differ—
ences could probably be explained on the basis of clonal re-
sponse (49) or environmental conditions. Nevertheless,

under commercial conditions, it is doubtful that pH plays an

important role in the enhancement response since the pH of

the spray solution would be above 4.0.

56

Table 6. Influence of pH on the herbicidal effectiveness of
foliar applications of amitrole and amitrole—
NH4SCN mixtures on quackgrass.

 

v

 

 

Regrowthl 2
Treatment ratings '
Reagent

Chemical pH used

None --- --- 1.0 a
Amitrole3 9.6 —-- 4.0 b
Amitrole 9.6 ' NaOH 4.7 b
Amitrole 7.1 NaOH 4.0 b
Amitrole 4.6 HCL 4.3 b
Amitrole 4.6 H2504 4.7 b
Amitrole + NH4SCN 9.6 NaOH '8.3 c
Amitrole + NH4SCN 7.1 . NaOH 8.3 c
Amitrole + NH4SCN 4.6 HCL 8.7 c
Amitrole + NH4SCN 4.6 H2S04 9.0 c
Amitrole + NH4SCN3 ‘ 4 6 --- 8.3 c

 

lRegrowth ratings 21 days after plowing: l.0——re-
growth, 10.0—-no regrowth.

2Ratings with different letters significantly dif-
ferent at 1% level.

3Commercial formulations, pH not adjusted.

57

Foliar Absorption and Translocation of
Amitrole and NH4SCN in Quackgrass

 

Results obtained from greenhouse and field studies

on the enhancement of amitrole activity by NH SCN have indi—

4
cated that foliar absorption and/or translocation might have
been involved in the occurrence of this response. Therefore,
the absorption and translocation of Cl4 labeled amitrole and
NH4SCN following foliar application1 was studied. Since the
main objective was to correlate the observed enhancement re-
sponse with possible increases in absorption or translocation,
the treatments employed in the following studies were similar
to those used in previous greenhouse and field investigations.
The absorption of amitrole* and NH4SCN*, alone and
in combination with nonlabeled material, was determined at
various times following application (Table 7). Ninety-six
hours after application, absorption of amitrole* was greater
than NH4SCN* when these compounds were applied alone. The
addition of NH4SCN to amitrole* or amitrole to NH4SCN* did
not alter the absorption of either labeled compound in com-
parison to applying them alone. In regard to the amount
absorbed at various times following application, the rate of
absorption for all treatments was greatest during the first

four hours and decreased between 4 and 96 hours. Similar

absorption patterns for amitrole alone have been reported by

 

lFor brevity and clarity, it is assumed that the C14—

products translocated are the same as those applied, even
though this has not been verified.

58

Herrett and Link (45) for Canada thistle and bindweed and by
Anderson (6) on southern nutgrass. Wittwer egqal. (105) have
described the initial rapid rate of uptake of substances as
non—metabolic and the slower following rate as active

absorption.

Table 7. Foliar absorption, expressed as percent of total
applied, of C14 labeled amitrole (5,000 ppm) and
NH4SCN (5,000 ppm) alone and in combination in
quackgrass.

 

Percent of labeled compound

 

Chemical absorbed after 96 hrs.1
Amitrole* . 21.6 a
Amitro1e* + NH4SCN 22.4 a
NH4SCN* 17.1 b
NH4SCN* + Amitrole 18.0 b

 

1Percentages with different letters significantly
different at 1% level.

In contrast to foliar absorption, translocation
studies indicated marked differences among treatments in the
amount of Cl4 transported out of the treated leaf (Figure 4).
The addition of NH4SCN to amitrole* solutions greatly in—
creased the amount of amitrole* translocated after 96 hours
in comparison to amitrole* alone. Although the translocation
was similar for both treatments 24 hours following appli—

cation, differences in amitrole* transport became

59

 

 

 

 

 

300 I l I l T
1'
v ,M””
3 25.0 — / _
8 ---- amitrolo“ + NH4SCN I”
§ —--- omitrolo * ,’
.. NH sc~*+ammolo ,I’
c , - ‘
3 200 __ NH4SCN. ’1’ ‘
E ,I’
< ,’
é l5.0 "' /” --/"' _.
Q- I ""
O ’/ -""
u ‘
2 no.0 — / v” —
8 ll ”/
O
7E I’;/ "'""'"
2 5.0 '- ’I/ ’ —
l— l’ I’ /
"a’

3' /”

o (I l l I

4 8 24 48 96
Hours Following Application

Figure 4. Translocation of Cl4 labeled amitrole and NH4SCN,

alone and in combination, in quackgrass following

foliar applications.
Interaction for treatments x linear time signifi-

cant at 1% level.

6O

progressively greater with time. After 96 hours, trans—

location from the amitrole* plus NH SCN combined was double

4
that of amitrole* alone. Higher values of translocation
following foliar application of amitrole* have been reported
by Racusen (86) in pinto beans, Massini (66) in tomatoes and
Carter and Naylor (15) in broad beans. Anatomical differ-
ences among these plant species and quackgrass, such as
cuticle thickness or wax deposits, might have influenced ab-
sorption and subsequent translocation. No previous quanti-
tative studies on the translocation of amitrole* in combin—
ation with NH4SCN have been reported.

Autoradiography was next employed to confirm the
previous translocation study and determine possible differw
ences in the distribution pattern resulting from treatments.
Autoradiograms (Figure 5), made 96 hours after application,
indicated that amitrole* translocation was greater following
amitrole* + NH4SCN application than from amitrole* alone.
The addition of NH4SCN to amitrole* treatment solutions sub—
stantially increased both acropetal transport to the leaf
tip and basipetal movement into the roots. Although the
quantity of amitrole* translocated was increased by the

addition of NH SCN, the distribution pattern was similar to

4
amitrole* application alone. With both treatments, amitrole*
was readily translocated and accumulated in meristematic
tissue, such as the basal sheath of expanding leaves, root

tips and young shoots. Radioactivity was scarce or lacking

in the lower, more mature leaves.

 

 

Figure 5.

 

61

 

 

 

Quackgrass plants (left) and autoradiograms (right)
showing the distribution of foliar absorbed
amitrole* (0.5 pc) 96 hours following application.
Arrowheads indicate application sites.

(top) amitrole* (5,000 ppm) alone,

(bottom) amitrole* + NH4SCN (5,000 + 5,000 ppm).

62

These results are in agreement with other autoradio-
graphic studies (31, 101). Van der Zweep (101), working

with bush beans, reported that the addition of NH SCN not

4
only increased the translocation of amitrole* but also de—
layed contact injury from amitrole at the site of appli-
cation. In the present study, no consistent difference in
necrosis was observed at the application site when NH4SCN
was combined with amitrole*. Forde (31), using quackgrass,
attributed the increased translocation of amitrole* by
NH4SCN to a lessening of the toxic action at the site of
entry. This, he states, would permit greater penetration
and subsequent translocation of amitrole*. Regardless of
the manner in which NH4SCN is influencing amitrole movement,
it may be concluded that the addition of NH4SCN greatly in—
creased the translocation of amitrole*.

The amount of NH4SCN* translocated, 96 hours follows
ing foliar applications of NH4SCN* alone and in combination
with amitrole, was similar for both treatments (Figures 4
and 6). Compared to the extensive movement from amitrole*
applications, translocation following NH4SCN* applications
was restricted. As indicated by the autoradiograms (Figure
6), the bulk of radioactivity was confined to the treated
leaf with only slight basipetal translocation into the roots.
This experiment confirms the results of Forde (31), who
found that NH4SCN* transport was mainly confined to the

treated leaf with only traces of radioactivity present in

the roots.

63

 

 

 

 

 

Figure 6. Quackgrass plants (left) and autoradiograms
(right) showing distribution of foliar

absorbed NH4SCN (0.5 pc) 96 hours following
application.

Arrowheads indicate application sites.
(top) NH4SCN* (5,000 ppm), alone and
(bottom) NH4SCN* + amitrole (5,000 + 5,000 ppm).

64

The influence of various concentrations of NH4SCN on

the foliar absorption of amitrole* was next studied (Table 8).

Table 8. Influence of varying concentrations of NH4SCN on
the foliar absorption, expressed as percent of
total applied, of amitrole* in quackgrass.

 

 

O *
Percent amitrole

 

Concentration absorbed a ter
Chemical . . . .(ppm) 96 hrs. .
Amitrole* + NH4SCN 5,000 + 1,250 23.8
Amitrole* + NH4SCN 5,000 + 5,000 22.4
Amitrole* + NH4SCN 5,000 + 20,000 ‘ 20.7

W _7 W

1F value for differences among treatments not signi-
ficant at 5% level.

Amitrole* absorption, 96 hours following application, was
not influenced when the concentrations of NH4SCN in the

amitrole* + NH SCN mixture was increased from 1,250 to 5,000

4
and 20,000 ppm. Absorption values at various times following
application were similar to those previously reported for
amitrole* alone.

The amount of amitrole* translocated 96 hours followa
ing foliar application was a function of the concentration
of NH4SCN in the amitrole* + NH4SCN mixture (Figures 7 and
8). Translocation was similar with amitrole* + NH4SCN

mixtures when the concentration of NH4SCN was 1,250 and

5,000 ppm (Figure 7). However, increasing the NH4SCN

65

 

on
9
o

 

 

 

 

 

1 I I r [I
R ---- OMl'r0|Q.(5, OOOppthH‘ SCN(5 OOOppm) ///
Q ——omltrolo‘(5. OOOppml+ +NH :SCNU .250ppm) //
§ 25.0- omllrolo‘(5, 000ppm) muscmzo ,OOOppm) // -
3
K
g 200- .
a
g '5-0' 1
‘6
§ .00. -
3
Q
Q
a

o 4 a 24 4a 96

Hours Following Application

Figure 7. Translocation of foliar applied amitrole* (O. 5 pc)
in quackgrass as influenced by varying concen-
trations of NH SCN.

Interaction for NH SCN concentrations x linear
time significant at 1% level.

66

concentration to 20,000 ppm in the mixture decreased the
amount of amitrole* translocated. Although translocation
from all treatments was similar from four to 24 hours
following application, differences became increasingly
greater at 48 and 96 hours. Autoradiograms (Figure 8), made
96 hours following application of various concentrations of
NH4SCN in combination with amitrole*, were not in complete
agreement with the results obtained from the quantitative
study. The autoradiograms confirmed the fact that amitrole*
translocation was increased by the addition of NH4SCN at
1,250 and 5,000 ppm, but did not reveal any appreciable sup-
pression in translocation at 20,000 ppm other than the
absence of amitrole* in mature leaves. Since treatments in
the autoradiographic study were not replicated and the pro-
cedure itself was qualitative, the results from the quanti—
tative investigation were considered more reliable. Never—
theless, these studies indicated that the concentration of
NH4SCN in the amitrole* + NH SCN mixture was decisive in

4

* translocation.

augmenting amitrole
In studies where amitrole* and NH4SCN were applied
separately to the same leaf, amitrole* absorption after 96
hours was not influenced by the time of NH4SCN application
(Table 9). In all cases, the rate of amitrole* absorption
was greatest during the first four hours and then decreased

between four and 96 hours following application.

67

l \\

 

 

 

‘______n----;-l-l-I---—'-—'

. ’_ --—

7.- -I.

3’

/
/

 

 

14> o
. l
J
/
‘ J
\5
l
l

 

 

Figure 8. Quackgrass plants (left) and autoradiograms (right)

showing distribution of foliar absorbed amitrole
(0.5 pc) plus NH4SCN 96 hours following application.
Arrowheads indicate application sites.

(top) amitrole* + NH4SCN (5,000 + 1,250 ppm);
(bottom) amitrole* + NH4SCN (5,000 + 20,000 ppm).

68

Table 9. Influence of foliar applications of NH4SCN
(5,000 ppm), applied 24 hours before or after
amitrole (5,000 ppm), expressed as percent 0
total applied, on the absorption of amitrole.

 

w , j Y w v W e W
Y .

Percent amitrole*

 

Chemical absorbed after 96 hrs.1
Amitrole* 21.6
Amitrole* + NH4SCN 22.4
NH4SCN before amitrole* 23.0
NH4SCN after amitrole 20.1

 

Y~ m V —v 7*

1F value for difference between treatment not signifi—
cant at 5% level.

Depending on the time of application, NH4SCN altered"
the amount of amitrole* translocated out of the treated leaf
(Figures 9 and 10). When NH4SCN was applied in combination
with amitrole* or one day before amitrole* the amount of
1amitrole* translocated was greater than when amitrole* was
applied alone or one day prior to the application of NH4SCN
(Figure 9). These differences were not apparent at 4, 8, or
24 hours, but were evident at 48 and 96 hours after appli— -
cation. NH4SCN applied in combination with amitrole* or one
day before amitrole* approximately doubled the amount of 014
translocated in comparison to amitrole* alone. This trans-
location pattern was also revealed in autoradioqrams of

plants that received NH4SCN, either 24 hours before or after

amitrole* (Figure 10). Plants that received NH4SCN prior to

69

 

 

 

 

 

 

 

300 l l l 1 l
'0 II/
3 I
§ 25-0 ‘ ----- amitrole“ + NH. SCN ,/ / ‘
a ’ af’
.1 —-—NH.. SCN ldoy before amitrole“ ,’ /
o:- NHqSCN Idoy after amitrolo" ’z’
3 20.0 — —-— amitrole“ ,’ / -—4

I /
E ,’
4 ’I /
2 I/ a”
1 '5'0 * / ,--——-: —
O
U
C
3 I0.0 _ _
0
2
0
C
D
$1 50- _
32
o l l l l
4 8 24 48 96

Hours Following Application

Figure 9. Translocation of foliar applied amitrole* (0.5 pc)
in quackgrass as influenced by the time of NH4SCN
application.

Interaction for chemicals x linear time signifi-
cant at 1% level.

 

 

7O

 

 

 

 

Figure 10.

 

 

 

Quackgrass plants (left) and autoradiograms
(right) showing distribution of foliar absorbed
amitrole* (0.5 pc) and NH4SCN 96 hours follow-
ing application. Chemicals applied at

5,000 ppm.

Arrowheads indicate application sites.

(top) NH4SCN 24 hours before amitrole*,

(bottom) NH4SCN 24 hours after amitrole*.

71

amitrole* accumulated a greater amount of amitrole* in the
young expanding leaves and roots than those that received
NH4SCN after amitrole.* A comparison of autoradiograms
(Figures 5 and 10) indicates that translocation in plants
that received NH4SCN prior to amitrole* was similar to those
that received the amitrole* + NH4SCN mixture, while trans-
location from application of NH4SCN after amitrole* was com-
parable to amitrole* alone.

These results suggest that NH4SCN, depending on the
concentration and time of application, greatly increased
amitrole translocation but not absorption. When compared
with field studies, they offer convincing evidence that the
herbicidal enhancement of amitrole by NH4SCN is associated
with increased translocation of the herbicide. Where similar
applications of amitrole and NH4SCN were employed, the in-
creased translocation of amitrole obtained in laboratory in-
vestigations closely paralleled the increase in herbicidal
activity exhibited in the field. For example, more amitrole
was translocated when NH4SCN at 1,250 and 5,000 ppm was ap-
plied 24 hours prior to or in combination with amitrole.
Comparable applications in the field resulted in greater
quackgrass control. Furthermore, the magnitude of the in-
creased translocation in the laboratory coincided with the
increase in the degree of control observed in the field. In
both instances, the addition of NH4SCN approximately doubled

the response.

72

Although both laboratory and field studies offer evi-
dence that an increase in translocation is involved in the
enhancement phenomenon, they do not explain the manner in
which this translocation response occurs. Consequently, it
is only possible to speculate on this enhancement mechanism.
Crafts (22), using autoradiography, originally proposed that
NH4SCN lessens the rapid damage to the cells at the absorp—
tion site and thereby permitted the foliage to absorb and
translocate the amitrole over a longer period of time. Since
the technique employed does not distinguish between absorp-
tion and translocation and present investigations have indi-
cated that absorption is unaltered, it appears doubtful

NH SCN is influencing amitrole uptake per sq. Van der Zweep

4
(101) also observed that amitrole contact injury was delayed
by NH4SCN, but attributed it to increased translocation
rather than absorption. This latter proposal appears to be
in agreement with the studies reported herein. Increased
amitrole translocation by NH4SCN would also account for the
observed lessening of contact injury, because the amitrole
concentration at the absorption site would be maintained at

a low level through rapid removal.

In View of a recent study (51) and the evidence from
the present investigations, the following hypothesis for in-
creased amitrole translocation by NH4SCN is proposed. Jansen
(51) demonstrated that an amitrole binding substance was

present in leaves. -A1though the chemical nature of this sub-

stance was not identified, it was postulated that it required

73

amitrole saturation before the herbicide was available for
translocation. Since the triazole ring is unreactive and re-
sists cleavage in plants (67, 86, 104), the amino group
might be a common attachment site for the triazole moiety
and the binding substance. This complex, being immobile,
would thus prevent amitrole removal from the application

site. However, the addition of NH SCN, particularly the

4
thiocyanate ion (70), might alter the substance to a form

not capable of binding amitrole. It is also possible that
the thiocyanate ion, having a greater affinity for the sub—
stance than amitrole, would be bound in preference to the
herbicide. Accordingly, a greater amount of amitrole would
remain unbound and readily available for transport. The
specificity of the substance to bind amitrole might also ac—
count for the failure of NH4SCN to enhance the activity of
other herbicides (99).

This hypothesis would be suitable for explaining the
results of the present study. For example, NH4SCN, applied
before or in combination with amitrole, might lessen amitrole
binding and permit a greater amount of amitrole to be trans-
located. However, NH4SCN applied after amitrole would be in-
effective in altering amitrole translocation since the herbi—
cide would have been previously bound. The amount of NH4SCN
required to prevent amitrole binding would depend on the

quantity of binding substance present, rather than the amount

of amitrole. Provided the substance was only present in

74

leaves, this hypothesis might also account for the occurrence
of the enhancement response following application of the com—

pounds to the foliage, but not to the roots.

S UMMARY

The herbicidal enhancement of amitrole activity by
NH4SCN was studied in the laboratory, greenhouse and field.

Regardless of the site of application, the addition
of NH4SCN to amitrole was no more effective than amitrole
alone in reducing bud viability and sprouting. NH4SCN ap-
plied alone to both foliage and roots had no effect on
rhizome growth.

Solution culture studies indicated that the enhance-
ment response was influenced by the application site of
amitrole and NH4SCN. Although chlorosis was apparent followe
ing both foliar and root applications of amitrole, the amount
of chlorosis was only increased by NH4SCN when it was applied
with amitrole to the foliage. Simultaneous, but separate ap—
plications of one compound to the roots and the other to the
foliage were no more effective than amitrole applications
alone to either site.

Split applications of amitrole and NH4SCN to the
foliage of quackgrass, in both greenhouse and field studies,
indicated that the time of NH4SCN application influenced the
activity of amitrole. Application of NH4SCN one day prior to
or in combination with amitrole was more effective in con—
trolling quackgrass than amitrole alone. However, NH SCN

4

75

76

applied after amitrole failed to increase the effectiveness
of the herbicide.

Field studies revealed that the enhancement response
following foliar applications of the amitrole-NH4SCN mixture

was affected by the amount of NH SCN applied. Increasing

4

the amount of NH4SCN from 1 to 2 and 4 lb/A resulted in a
greater degree of control for all amitrole levels. However,

when the rate of NH SCN was further increased to 8 1b/A,

4
control decreased sharply and was similar to that for ami—
trole alone. Regardless of the rate of amitrole applied,
the Optimum rate of NH4SCN was between 2 and 4 lb/A.

The herbicidal activity of foliar applications of
amitrole (2 1b/A), alone and in combination with NH4SCN
(2 lb/A), was not affected by the pH of the spray solution.
Although the amitrole-NH4SCN mixture resulted in a greater
degree of quackgrass control than amitrole alone, its effec-
tiveness was not altered when the pH of the solution was
varied from 4.6 to 7.1 and 9.6.

Amitrole* (labeled with C14) absorption by quack—
grass leaves was not influenced when NH4SCN was applied be—
fore, after or at various concentrations with amitrole. In
all cases, the initial rapid rate of amitrole* absorption was
followed by a slower but steady rate of uptake.

Amitrole* translocation, 96 hours following foliar

application, was greatly enhanced by the addition of NH4SCN.

When the compounds were combined, the amount of amitrole*

77

translocated was approximately doubled by NH4SCN at 1,250
and 5,000 ppm but not at 20,000 ppm. A similar increase in
translocation was also obtained when NH4SCN (5,000 ppm) was
applied 24 hours before amitrole* (5,000 ppm) application.

However, translocation from NH SCN applied 24 hours after

4
amitrole* was similar to that obtained from applications of
amitrole* alone.

Foliar absorption and translocation of NH4SCN*, 96
hours following application, was not altered by the addition
of amitrole. In comparison to amitrole*, NH4SCN* was ab—
sorbed and translocated to a much lesser extent with only
negligible amounts present in the roots.

The results, from both field and laboratory investi-
gations, offer evidence that the greater herbicidal activity

of the amitrole—NH SCN mixture on quackgrass is due to the

4

NH SCN increasing the translocation of amitrole.

4

10.

ll.

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