• TABLE OF CONTENTS
HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Introduction
 Literature review
 Materials and methods
 Experimental results
 Discussion
 Summary and conclusions
 Bibliography
 Biographical sketch






Group Title: Factors contributing to the herbicidal resistance of Black titi (Cliftonia monophylla) /
Title: Factors contributing to the herbicidal resistance of Black titi (Cliftonia monophylla)
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 Material Information
Title: Factors contributing to the herbicidal resistance of Black titi (Cliftonia monophylla)
Physical Description: Book
Language: English
Creator: Beers, Walter Leon, 1927-
Publication Date: 1969
Copyright Date: 1969
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Bibliographic ID: UF00102035
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ltuf - ACK3423

Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
    Literature review
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
    Materials and methods
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
    Experimental results
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
    Discussion
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
    Summary and conclusions
        Page 61
        Page 62
    Bibliography
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
    Biographical sketch
        Page 68
        Page 69
        Page 70
Full Text









FACTORS CONTRIBUTING TO THE HERBICIDAL

RESISTANCE OF BLACK TITI (Cliftonia monophylla)














By
WALTER L. BEERS, JR.













A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY














UNIVERSITY OF FLORIDA
1969



















To

my wife

Barbara Merrill Beers

and to the

memory of

W. D. "Bill" Smith

former Woodlands Manager, The

Buckeye Cellulose Corporation

Perry, Florida














ACKNOWLEDGEMENTS

The author acknowledges with sincere thanks the advice,

comments, and assistance of Dr. S.H. West, Dr. T.E. Humphreys,

and Dr. M. Wilcox given during this work. Committee service

of Dr. D. McCloud and Dr. R.G. Stanley is likewise appreci-

ated.

Acknowledgement is also made to Mrs. Grace B. Colson,

Mr. W.L. Dickerson, Mr. Karl Jurbergs, Mr. J.E. Morris,

Mr. Vasco Murray, Mr. G. Olree, and Dr. C. Schenker, all of

The Buckeye Cellulose Corporation, for their help and encour-

agement.


iii



















TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS . .


LIST OF TABLES. . .


LIST OF FIGURES . .


INTRODUCTION . . . . .. . ..


LITERATURE REVIEW . . . . . .


MATERIALS AND METHODS . . . . .


Anatomical study of leaves. . . .
Electron microscopy of leaf surfaces.
Leaf absorption of 2,4-D-1-14C. .
Metabolic degradation of 2,4-D-1-14C.
Preliminary bell jar experiment .
Absorption train experiment . .


EXPERIMENTAL RESULTS . . . . .


Anatomical study of leaves. . . .
Electron microscopy of leaf surfaces.
Leaf absorption of 2,4-D-1-14C. . .
Metabolic degradation of 2,4-D-1-14C.
Preliminary bell jar experiment .
Absorption train experiment .


DISCUSSION . . . . . . .


SUMMARY AND CONCLUSIONS . .. . .


LITERATURE CITED . . . . . .


iii


v
V
vi


. . . 1


* *


. .





- 43
. 45


. . .








r r



* 4 *
. .
. .


. .
a *


* . .














LIST OF TAB3!ES


Table Page

1. Major anatomical difference between (S) and
(R) titi leaves. . . . . . . 37

2. Analysis of 2,4-D-1--14C absorption by (S) and
(R) titi leaves and significance of the
differences. . . . . . . . 44

3. Results of preliminary bell jar experiment in
which evolved '"CO2 was absorbed in potas--
sium hydroxide solution . . . .. 46

4. Uptake and degradation of 2,4-D--1-4C by de-
tached shoots of (S) and (R) titi during
approximately 140 hours in the dark. . 47

5. Data for computing standard deviation from
the curvilinear regression equation Y
-15.7677 + 28.8199X + (-3.4952) X2 . . 50

6. Data for computing standard deviation from
the linear regression equation Y = 14.2043
+ 4.2602X. . . . . . . . . 51

7. Comparison between mean squares for linear
and curvilinear regression---(R) titi decar-
boxylation data. . . . . . ... 52

8. Analysis of variance for curvilinear regress-
ion--2,4-D-1-14C uptake and breakdown data
for (R) titi trials in the absorption
train . . . . . . . . 53














LIST OF FIGURES


Figure Page

1. Foliage and fruit of black titi and white
titi . . . . . . . . . 2

2. White titi selectively controlled amid re-
sistant black titi, following aerial
treatment with 2,4-D ester applied at the
rate of 6 pounds active ingredient in 5
gallons of spray per acre. . . . .. 4

3. Absorption train used in 2,4-D-1-14C metab-
olism experiments with (S) and (R) titi
shoots . . . . . . . . .27

4. Mass correction curve for barium carbonate
precipitates . . . . . . 29

5. Leaf cross-section of (S) titi (Cyrilla race-
miflora) at mid-rib, showing cuticle (C),
epidermis (E), bundle sheath extension
(BSE), internal phloem (IP), xylem (X), ex-
ternal phloem (EP), palisade mesophyll (PM),
spongy mesophyll (SM), and stoma (S) . 32

6. Leaf cross-section of (R) titi (Cliftonia
monophylla) at mid-rib, showing cuticle (C),
epidermis (E), bundle sheath extension
(BSE), xylem (X), phloem (P), palisade
mesophyll (PM), and spongy mesophyll (SM).
Note the absence of sheath extensions on
the secondary vein to the right of the
main vascular bundle . . .. . . 34

7. Leaf cross-section of (S) titi (Cyrilla race-
miflora), showing secondary vein with
bundle sheath extensions (BSE) leading to
upper and lower epidermis, xylem (X), and
phloem (P) . . . . . . . . 35







Figure


8. Electron micrograph showing the smooth adax-
ial surface of an (S) titi leaf . . 39

9. Electron micrograph of the adaxial surface of
an (R) titi leaf, showing a very rough,
ridgy surface. . . . . . . .. 41

10. The dry residue of 10 ul drops of 2,4-D-1-14C
containing 1 per cent Tween 20, applied to
the center of the adaxial surfaces of (S)
and (R) titi leaves . . . . .. 42

11. Cumulative counts per minute evolved as Bal4C03
by (S) and (R) titi during the indicated
dark periods . . ... . . . 48

12. The relationship between 2,4-D (2,4-D-1--14C
uptake and breakdown (decarboxylation) by
(R) titi shoots during approximately 140
hours in the dark . . . . .. .... 54


vii


Page














INTRODUCTt IONi

Black titi cliftonia monophvlla (Lam.) Britt. is a

multiple-stemmed, semi-deciduous shrub that occurs naturally

in the wet lowlands of the Southern Coastal Plain ranging

from West Florida to Texas. The eastward extension of black

titi in Florida appears limited by the Aucilla River in

Jefferson County. A similar plant, white titi (Cyrilla

racemiflora L.) intermingles with black titi over its range,

but extends throughout North Central Florida and beyond,

into Southern Virginia. Both species belong to the family

Cyrillaceae. Where they overlap, floral differences easily

distinguish the two. Black titi bears a panicled raceme,

and white titi, a spike raceme. The persisting fruit

affords another means of identification. Black titi pro-

duces clusters of winged, nut-like drupes, while white titi

develops small globose capsules born on slender spikes

(Fig. 1).

Because of the dense cover it creates, black titi must

be eradicated, or at least severely retarded, prior to re-

forestation of wet lowlands by seeding or planting. Titi

can be top-killed by fire, but vigorous resprouting in-

1























































Fi.. 1. Foliage and fruit of black titi (top) and white
titi (bottom).








variably occurs from adventitious buds at the root collar.

Although reforestation is possible following a fire, re-

surging titi in the pine understory constitutes a contin-

uing fire hazard to developing plantations. Also, the

brush cover would doubtless increase logging costs in the

mature forest.

In swamps, where poor flotation hampers mechanical

equipment ordinarily used for land clearing, chemical con-

trol is the more practical approach to the problem. How-

ever, repeated attempts to control black titi with 2,4-

dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxy-

acetic acid (2,4,5-T) and mixtures of the two were unsuc-

cessful. White titi scattered through black titi thickets

was selectively controlled by 2,4-D, and severely damaged

by 2,4,5-T alone and when combined with 2,4-D (Fig. 2)

black titi was clearly resistant and white titi susceptible

to these phenoxy herbicides.

The research efforts reported herein were directed

towards elucidation of factors contributing to the herbi-

cidal resistance of black titi. Hereafter, resistant

black titi will be designated "(R) titi," and susceptible

white titi, "(S) titi."















































Fig. 2. White titi plant (foreground) selectively con-
trolled amid resistant black titi following aerial
treatment with 2,4-D ester applied at the rate of 6
pounds active ingredient in 5 gallons of spray per acre.














LITERATURE REVI EW

This review will cover some of the problems of wetting

and penetration of leaf surfaces by herbicidal sprays,

followed by a sampling of the literature on metabolism of

2,4-D by plants. Pertinent reviews have been written pre-

viously by Norman, Minarik, and Weintraub (1950); Blackman,

Templeton, and Halliday (1951); Crafts (1953); van Overbeek

(1956); Woodford,. Holly, and McCready (1958); Hilton, Jansen,

and Hull (1963); Franke (1967); and Casida and Lykken (1969).

Spray solutions applied to leaves must first penetrate

the cuticle. Superficially this structure amounts to a skin-

like covering on the leaf that functions to prevent evapor-

ative loss of moisture from underlying tissues. The cuticle

is a complex of polymerized alcohols and long-chain fatty

acids. Cutin makes up the framework of the cuticle. It

consists of polymerized dicarboxylic and hydroxy carboxylic

acids (van Overbeek, 1956) which are cross-linked in a

three-dimensional system featuring intermolecular spaces

(Franke, 1967). Except at the uppermost surface, hemi-

cellulosic and pectic substances are distributed throughout

the cuticle. Wax rods anrd platelets extend upward from the







highly hydrophobic cuticular surface (Leopold, 1964). A

polarity gradient exists from the outer to the inner region

of the cuticle. The low polarity of the wax layer gradually

intergrades to regions of higher polarity internally in the

region of the outer epidermal walls. Working with pear

leaves, Norris and Bukovac (1968) demonstrated the presence

of pectic substances along the cuticle/cell wall interface.

The basal portion of the cuticular layer consists of a

hydrophilic cellulose matrix in which are embedded lipo-

philic platelets. This matrix appears to be in close con-

tact with ectodesmata of the epidermal wall (Jansen, 1964;

Franke, 1967).

The formal and polarity of surfaces waxes car have a very

marked influence on the ease with which a leaf can be wetted.

Such waxes appear to be extruded onto the cuticular surface

at random (Schieferstein and Loomis, 1959) where they harden

into widely varying, characteristic forms (Mueller, Carr,

and Loomis, 1954; Schieferstein and Loomis, 1956). Wax pro-

trusions prevent solid contact between spray droplets and

the cuticular surface (Holly, 1964). The degree of spray

droplet spreading on cuticular surfaces is characterized by

its contact angle (van Overbeek, 1956). Foy and Smith (1965)

presented a method for calculating contact angles from drop

outlines on the leaf. The magnitude of the contact angle







seems dependent upon the following factors: (1) the degree

of polarity of the surface wax, (2) its configuration, or

degree of roughness, and (3) whether an air film occurs

below the drop in the crevices of wax extrusions.

Penetration of the cuticular surface by herbicidal

solutions was reviewed by Currier and Dybing (1959).

Van Overbeek (1956) considered the unbroken cuticle an

improbable point of entry for spray solutions. He proposed

the following alternative routes of entry: (1) through

modified cells which overlie veins, (2) stomates, (3)

cuticular cracks, (4) areas of cuticle stretched thin by

underlying tissue, and (5) a combination of the above.

Recent work indicates that penetration of the cuticular

surface is a limiting step in the passage of growth-regula-

tor compounds through the cuticle, and that this penetration

is a physical process. Norris and Bukovac (1969) studied

the penetration of naphthaleneacetic acid (NAA) through

enzymatically isolated pear leaf cuticle (Pyrus communis L.

cv. Bartlett). The penetration of NAA was found strongly

temperature related, exhibiting a temperature coefficient

(Q]0) of 5.6 between 15 and 25 C. The temperature effects

on permeability were found fully reversible on isolated leaf

discs. This was cited as evidence that cuticular penetration

is a physical process. The authors felt the high Q10 values








observed were probably related to the lipoidal nature of the

cuticular membrane. They did not preclude the possibility

that temperature-induced changes in the cuticle or the

surface waxes may have resulted in higher rates of pene-

tration at higher temperatures. This was not considered

very likely, however, in view of previous work (Norris and

Bukovac, 1968) wherein cuticular waxes were determined to

undergo no visible changes between 5 and 35 C.

The moisture status of leaves may have some role in

spray penetration of astomatous surfaces. When leaves are

exposed to humid conditions the inner hydrophilic region

swells, pulling the hydrophobic wax rodlets apart. This

could create numerous channels for solution movement (Jansen,

1964). Crafts, Currier, and Drever (1958) had previously

shown that leaf absorption of maleic hydrazide maximized in

a humid atmosphere. By contrast, Morton (1966) found

humidity had no effect upon absorption of 2,4,5-T by mes-

quite (Prosopis juliflora var. glandulosa) seedlings. He

attributed this unique result to the xerophilic character

of mesquite and its ability to adapt quickly to humidity

changes. Badiei, Basler, and Santelmann (1966) found that

absorption and translocation of 2,4,5-T decreased with in-

creasing moisture stress in oak seedlings.

Penetration of waxy crevices and stomates is greatly








enhanced by addition of a surface active agent (surfactant)

to the spray solution. The term "surfactant" encompasses

a wide range of chemicals classified as emulsifiers, de-

tergents, and wetting agents (Behrens, 1964). All such

materials exhibit activity at surfaces or interfaces.

Behrens (1964) discussed at length the physical and chemical

properties of surfactants, relating these to effects upon

herbicidal formulations. He visualized a surfactant mole-

cule as one composed of two opposing forces, one compatible

with water and the other with oily substances hydrophilicc

and lipophilic). This two-tailed nature of the molecule

allows it to accumulate at an interface. Although many

examples of surfactant enhancement of herbicides appear in

the literature, in no case has the precise mechanism of

action been elucidated.

Jansen, Gentner, and Shaw (1961) conducted an extensive

study of surfactant activity when they studied the inter-

action between 63 surface active agents and dalapon, 2,4-D,

and amitrol. The surfactants involved were representative

of cationic, anionic, non-ionic, and ampholytic classes.

In general the surfactants increased herbicidal toxicity

with logarithmic increases in concentration. Some brought

about progressive decreases in herbicidal activity, while

others were completely ineffective. Some surfactants tested







independent of the herbicide were phytotoxic in their own

right, while others actually stimulated growth of Zea mays

and Glycine max. The authors found no correlation between

any of the surfactants and the ionogenic class to which they

belonged. This study emphasized the necessity of grooming

a particular surfactant-herbicide mixture to the species

and spray system at hand.

Foy (1962) was able to show a marked surfactant

enhancement of 2,2-dichloropropionic acid (dalapon) uptake

by Zea mays leaves. 14C labelled herbicide was applied with

and without surfactant (0.1% sodium dioctylsulfosuccinate,

Vatsol OT). At intervals of from 15 seconds to 3 hours,

the basal portion of treated leaves was sectioned and

radioactivity determined either by autoradiography or by

extraction and counting in a windowless flow counter.

Treatments with Vatsol OT additive sorbed almost instanta-

neously and moved to the basal portion of the leaf in

approximately five minutes. Those without surfactant showed

delayed absorption and practically no movement from the

treatment locus during the 30-minute evaluation period.

In a later paper, Foy and Smith (1965) studied the

effects of seven different surfactants upon the enhancement

of dalapon on corn. These were: polyoxyethylene sorbitan

monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 80),







x-77 (80% alkylaryl polyoxyethylene glycols, free fatty

acids, and isopropanol), polyoxyethylene polyoxypropylene

polyols (T-1947), 75% sodium dioctylsulfosuccinate (Vatsol

OT), Dynawet, and sodium lauryl sulfate. Materials were

used at concentrations ranging from 0.001 to 5.0%. All

surfactants enhanced herbicidal activity as the rate was

increased. Sodium lauryl sulfate and Dynawet gave the most

pronounced enhancement. In two papers, Jansen (1964, 1965a)

gave several examples of surfactant enhancement of 4,6-

dinitro-6-sec-butylphenol (DNBP), 3-amino-l,2,4-triazole

(amitrole), dalapon, and 2,4-D. In these, the author

attempted to interrelate the physical-chemical properties

of the resulting sprays and the degree of enhancement. He

found, for example, that in one surfactant family, the

alkylbenzenesulfonates (ABS), herbicidal enhancement in-

creased as the benzene ring was moved toward C5 and C6

carbons of the n-dodecyl alkyl group. Also, among the

ethoxylated alkyl amine cationic surfactants certain

variations in the alkyl structure affected herbicidal

toxicity.

It is possible to form a 2,4-D molecule having both

surfactant and herbicidal properties by forming long chain

alkyl-amine salts (Jansen, 1965b).

Crafts and Yamaguchi (1958) studied the uptake and







distribution of labelled 2,4-D by SZbrina and Tradescantia.

They applied 10 pl drops containing 5 pg of carboxyl-

labelled 2,4-D in 50% alcohol with 0.1% Nonic 218 as a

surfactant to either upper or lower leaf surfaces. Direc-

tion and rate of movement were determined by autoradiography.

Material applied to the lower surface moved readily to the

roots; that applied to the upper surface moved predominantly

upward in the plant and outward to the leaf tips. Anatom-

ical study of the two plants involved would probably reveal

the phloem is located on the underside of the vascular

bundles. This would account for downward movement by lower

leaf surface applications. Either phloem is lacking on the

upper side of the vascular bundles, or it is protected by

associated tissue. Autoradiographic analysis seemed to

indicate labelled 2,4-D bypasses mature leaves that are ex-

porting foods. They found that flowers and young fruits are

extremely active sinks, accumulating a high concentration of

tracer.

Radwan, Stocking, and Currier (1960) studied the trans-

location of labelled 2,4-D and other herbicides by means of

histoautoradiography. Carboxyl-labelled 2,4-D (specific

activity: 6.03 mc/mM) was applied to broad bean (Vicia faba)

in 50% ethanol with either X-77 or Vatsol OT as surfactants.

From this study, it was found that leaf-treated plants







always showed activity in the phloem. But as the distance

from the application point increased, phloem activity de-

creased and xylem activity increased. Since herbicidal

action is usually dependent upon phloem transport, these

results suggest a mechanism of immobilization by plants.

Also, it is reasonable to assume that during period of high

transpiration the tendency for lateral transfer between

phloem and xylem might be accentuated.

Jaworski and Butts (1952) treated potted bean plants

(Phaseolus vulgaris, var. Black Valentine) with alpha-

methylene or carboxyl 14C-labelled 2,4-D. At intervals of

1 to 42 days treated plants were sectioned into leaf and

stem material, homogenized, and extracted with 80 per cent

ethanol. Chromatographic analysis showed the 2,4-D was

rapidly converted to two unknown, radioactive compounds. At

the end of two weeks approximately 65 per cent of the applied

2,4-D was converted to "unknown 1" and 9 per cent to "unknown

2." About 20 per cent of the 2,4-D was recovered, unchanged.

Both unknowns were water-soluble, and ether-insoluble. Un-

known 1 was hydrolizable with 2 N HC1, Takadiastase, and

emulsin. The authors felt it was possibly a glucosidic

detoxification product. The small amount of 2,4-D not

accounted for (6%) was presumed lost to decarboxylation or

transported to the roots.








In looking for factors of selectivity, Fang and Butts

(1954) extended the work of Jaworski and Butts (1952) to two

2,4-D resistant plants, corn (Zea nar~y saccharata var.

Golden Bantam) and wheat (Triticum vulgare var. Hard Winter

White). Using similar treatments and analytical methods the

authors found in addition to unknowns 1 and 2, a third,

which they called "unknown 3." As were unknowns 1 and 2,

unknown 3 was water soluble and ether-insoluble. The authors

concluded that the formation of unknown 3 along with slower

rates of absorption and translocation could account for the

selective resistance of corn and wheat to 2,4-D.

Jaworski, Fang, and Freed (1955) reported later that a

2,4-D complex, presumed to be the "unknown 1" of Jaworski

and Butts (1952), was formed equally by normal and etiolated

bean plants. In addition they found that although 2,4-D was

absorbed by etiolated leaves, its transport was dependent

upon exogenous supplies of sucrose, glucose, and various

other sugars. Since all sugars facilitated the transport of

2,4-D, the authors concluded this action is mediated by food

movement in general.

Using 2,4-D-2-14C and 2,4-D--114C, Williams, Slife, and

Hanson (1960) studied absorption, translocation, and metab-

olism in several annual broadleaved weeds. Absorption was

determined by floating leaf and stem section on radioactive







solutions, washing, drying, and counting. Analysis for

radiocarbon was done by collecting CO2 evolved by Van Slyke

combustion and determining the radic-activity with a

vibrating reed electroscope. Translocation was studied by a

combination of wet combustion analysis and autoradiography.

Side-chain catabolism was studied by sealing the treated

plants in a jar and collecting the evolved CO2 in one ml of

20% KOH. The KOH capsules were collected, dried under a

heat lamp and counted in a G-M counter. The amount of 14CO2

evolved compared to amount absorbed was so slight (less than

0.02%) that this mechanism of 2,4-D detoxification was not

considered to occur generally. According to Williams et al.

(1960), Weintraub (1953) had observed similar results.

Bach and Fellig (1961) showed that growth of excised

bean sections treated with 2,4-D was correlated with dis-

appearance of free 2,4-D from the tissue. They found, how-

ever, that approximately 97 per cent of the herbicide re-

mained in the tissue in a form chromatographically distinct

from pure 2,4-D. It was called merely a "detoxification

product." In a following paper, Bach (1961) attempted to

isolate this "detoxification product." Four-week-old bean

(Phaseolus vulgaris) stems were cut into six-inch lengths

and vacuum infiltrated with a 2 per cent sucrose solution

containing 10-4M 2,4-D-1-14C (2-22 mc/mM). Stems were







incubated at room temperature for four days. After lyoph-

ilization, soxlet extraction, ether soluble and ether in-

soluble fractions were obtained. A host of chemical tests

were run on chromatographed components of both fractions.

Only radioactive isolates were analyzed. Bach found that

approximately 50 per cent of the original radioactivity was

distributed among 10 compounds in the ether extract. The

ether insoluble fraction contained six components. Various

breakdown pathways were postulated. These included ring

fissure, hydroxylation, and decarboxylation.

Luckwill and Lloyd-Jones (1960a) studied the metabolism

of 2,4-D and other phenoxy materials by susceptible black

currant (Ribes nigrum L.) and resistant red currant (Ribes

sativum Syme). 2,4-D, labelled with 14C in either the

methylene or carboxyl position was applied to detached

leaves via the petioles. Evolution of CO2 in darkness was

followed over a period of one week. After this, leaves were

extracted by maceration in sodium bicarbonate or dilute

ammonium hydroxide and analyzed by paper chromatography.

During 139 hours in the metabolism chamber, resistant

currant decarboxylated 48 per cent of the carboxyl and 20

per cent of the methylene carbon from the side chain of

2,4-D. During the same period, susceptible currant degraded

only 2 per cent of the applied material. The authors con-








cluded that differential decarboxylation was the basis of

selective 2,4-D toxicity. Ancillary experiments with

2,4,5-T, 4-chlorophenoxyacetic acid (4-CPA), 2-methyl-4-

chlorophenoxyacetic acid (MCPA), and 2-chlorophenoxyacetic

acid (2-CPA) showed all but the latter compound were

decarboxylated by resistant red currant. 2-CPA was thought

to be rapidly bound or converted to another form not

vulnerable to decarboxylation. Even though extensively

decarboxylated, 2,4,5-T was found decidedly toxic to 2,4-D-

resistant red currant. The authors suggested this species

was unable to cope with the probable decarboxylation pro-

duct, 2,4,5-trichlorophenol, but could selectively inacti-

vate 2,4-dichlorophenol.

In a supporting study Luckwill and Lloyd-Jones (1960b)

were able to correlate 2,4-D resistance with decarboxylation

of the molecule by an apple variety (Cox's Orange Pippin)

and its relatives and three strawberry varieties (Royal

Sovereign, Cambridge Favourite, and.Talisman). As with red

currant, Talisman strawberry was susceptible to 2,4,5-T even

though decarboxylation occurred. Only one of sixteen other

test species known to be resistant or susceptible to 2,4-D

carried out any appreciable decarboxylation. Of special

interest was the fact that hawthorne (Crataeus oxycantha)

carried out no side-chain degradation, even though highly







resistant to 2,4-D. As did Williams et al. (1960), the

authors concluded that resistance via the decarboxylation

route does not universally occur and that other groups of

plants must have other resistance mechanisms.

Edgerton and Hoffman (1961) discovered that substitu-

tion of fluorine for chlorine in the 4-position of 2,4-D

(2-chloro, 4-fluorophenoxyacetic acid; 2,4-F) inhibited

decarboxylation of the compound in leaves that otherwise

rapidly metabolized unsubstituted 2,4-D. Detached shoots

of resistant McIntosh apple were treated via the trans-

piration stream with 2,4-D-1-14C and 2,4-F-1-14C then

placed in a darkened metabolism chamber for collection of

evolved 14CO2. In 24 hours only 4 per cent of the 14C was

recovered as 14CO2 from shoots treated with 2,4-F-1-14C.

During the same period, those treated with 2,4-D-1-14C

degraded 33 per cent of the 14C supplied. A parallel study

with 2,4-D-susceptible Staymen apple showed that even in

this species, where 2,4-D is very slowly decarboxylated, a

slight further reduction in breakdown of 2,4-F occurred

(0.14 vs. 0.56%). Basler (1964) showed that 2,4-D, 2,4,5-T

and 2,4-F were not extensively decarboxylated by excised

leaves of blackjack oak (Quercus marilandica Muenchh),

persimmon (Diospyros virqiniana L.), winged elm (Ulmus alata

Michx.), sweet gum (Liquidamvbar styraciflua L.) or green ash








(Fraxinus pennsylvalnica Marsh.).

Wilcox, Moreland, and KlingTnan (1963) discovered that

excised oat, barley, and corn roots produced a ring-

hydroxylated metabolite from phenoxy-n-aliphatic acids,

acetic through hexanoic. The metabolite, identified as

4-hydroxy-phenoxyacetic acid, was produced from compounds

bearing even-numbered side chains. Since there was no

evidence of hydroxylated products of chain lengths longer

than acetic, the authors concluded that beta oxidation of

the side chain may have preceded the hydroxylation. The

greater hydroxylation activity observed in oat, barley, and

corn compared to that in peanut, soybean, and alfalfa roots

suggested ring hydroxylation may be a detoxication mechanism

in the former. Thomas, Loughman, and Powell (1963) isolated

the same metabolite from mesocotyl segments of Avena sativa.

In a later paper, the same authors (Thomas, et al., 1964a)

reported that phenoxyacetic acids with an unsubstituted four

position were hydroxylated there, and the resulting product

was accumulated as a glucoside. Phenoxyacetic acids bearing

a chlorine atom in the four position were not hydroxylated

to the same extent.

Faulkner and Woodcock (1964) isolated a 2,4-D metabolite

from Aspergillus niger, which they identified as 2,4-dichloro-

5-hydroxy-phenoxyacetic acid. A minor metabolite proved to








be 2,5-dichloro-4-hydroxy-phenoxyacetic acid. The formation

of this metabolite was unusual in that it not only involved

a hydroxyl-chlorine replacement, but also a chlorine shift

from the four to five position. Thomas et al. (1964b) dis-

covered that a similar chlorine shift occurred in seedlings

of Phaseolus vulgaris var. Canadian Wonder. A phenolic

glucoside was isolated. After treatment with beta-glucosidase,

the ether-soluble aglycone was found to consist of a mixture

of 2,5-dichloro-4-hydroxy-phenoxyacetic acid and 2,3-dichloro-

4-hydroxy-phenoxyacetic acid.

Crosby (1964) studied the metabolism of 2,4-D by red

kidney bean (Phaseolus vulgaris L.). The major chlorine-

containing ether-soluble plant fraction proved to be free

2,4-D. The ether-insoluble fraction yielded 2,4-D and another

chlorinated compound upon acid hydrolysis. A small amount of

2,4-dichloroanisole was isolated, but no 2,4-dichlorophenol

was found.

An interesting detoxication mechanism for jimsonweed

(Datura stromonium) was discovered by Fites, Slife, and Hanson

(1964). They used carboxyl-labelled 2,4-D-1-14C (specific

activity: 1 mc/mM). A 10 microliter drop of solution con-

taining 22.1 mg of 2,4-D in 2 ml of 50 per cent ethanol was

applied to the first nearly mature leaf of experimental plants.

At different intervals, treated plants were harvested and








divided into stems, roots and leaves. Following ether ex-

traction the changes in relative activity of various plant

portions were evaluated. After 21 days most of the activity

resided in the lower stems and in the roots. The nutrient

solutions used were found to be radioactive, suggesting con-

siderable 2,4-D leakage occurred from the roots. A follow-up

experiment confirmed that approximately 25 per cent of the

original 2,4-D applied had been expelled intact from the

roots. The net result was a transfer of material from the

leaf surface to the medium. As 2,4-D in the apical region

of the plant reached a certain tolerable concentration,

growth resumed.














MATERIALS AND M1ETIHODS

Anatomical Stud oof Leaves

Mature leaves of (S) and (R) titi were collected from

the natural habitats of the two species in Taylor and

Franklin counties, respectively. The base and apex of

sample leaves were snipped free leaving a central segment

approximately 1 cm long. After dehydration in a graded

series of ethanol solutions leaf segments were embedded in

Fisher Tissuemat (melting point, 52-54.5 C). Leaf cross-

sections 12 microns thick were prepared with a Spencer

Model 815 rotary microtome. Resulting sections were

mounted on glass microscope slides and stained with

safranin-fast green according to the procedure of Johansen

(1940). Photographs of the stained sections were made

with a Spencer Model 675 photomicrographic camera.

Electron Microscopy of Leaf Surfaces

Preparation and microscopy of titi leaves were done at

The Buckeye Cellulose Corporation, Cellulose and Specialties

Division laboratory, Memphis, Tennessee, by Mr. K.A. Jurbergs

and his staff.








Mature leaves from potted two-year-old (S) and (R)

plants grown from cuttings were prepared for electron

microscopy by means of a two-step replication technique.

Adaxial leaf surfaces were first impressed into a poly-

styrene film softened by heating,. After the film hardened

the leaves were stripped off and discarded. A chromium

layer approximately 150A thick was deposited on the leaf

impressions by vacuum evaporation of metallic chromium.

Another layer of about the same thickness was deposited

on the chromium by vacuum evaporation of carbon. Desired

areas were cut from the layered replicas and placed on

copper specimen grids with the polystyrene film in contact

with the grid. The polystyrene was then dissolved away in

a solution consisting of 75 per cent ethylene dichloride

and 25 per cent xylene. Final specimens consisted of a

chromium replica supported by carbon. Micrographs were

made with a Phillips Model EM-75B electron microscope.

14
Leaf Absorption of 2,4-D-- 14C

Mature (S) and (R) titi leaves were harvested from

two-year-old greenhouse-grown plants and inserted into

one-inch cubes of florist's foam plastic hydrated with

0.1 M glucose solution. Four petri dishes were prepared,

each holding four leaves each of (S) and (R) titi. Just

prior to insertion, a fresh face was cut on the petioles







with a sharp scalpel to ensure good continuity between con-

ducting tissues and the hydrating solution. After a five-

minute equilibration period 10 yil drops of 0.1 M radio-

labelled 2,4-D (2,4-D---I-14C; S.A. 0.014 pc/)jl) solution,

as the sodium salt, were applied to the center of the

adaxial leaf surfaces. The uncovered dishes were illumi-

nated with approximately 200 f.c. of incandescent light.

After 2 hours, the dry herbicide residue was washed from the

leaves into stainless steel planchets with 5 per cent ammo-

nium hydroxide solution. Pianchets were dried under a heat

lamp and counted in a Nuclear-Chicago Model 8775 scaler.

The scaler was attached to a Nuclear-Chicago Model 3053

sample -changer, which housed a thin-window Geiger tube.

2,4-D-1-14C uptake was estimated from the difference

between radio-activity applied and that recovered from leaf

washings. Count data were converted to micrograms of

2,4-D-1-14C taken up. The experiment was analyzed as a

paired "t" test as outlined by Snedecor and Cochran (1956).

Metabolic Degradation of 2,4-D-l-14C

Preliminary Bell Jar Experiment

A preliminary experiment similar to that of Williams

et al. (1960) was devised to check for metabolic degradation

of 2,4-D-1-14C by (S) and (R) titi via the decarboxylation

route.







An (R) titi shoot 10.cm in length was stripped of all

but five terminal leaves and placed in a 125 ml Erlenmeyer

flask containing distilled water. The shoot was steadied

in a vertical position with a cotton plug placed in the

neck of the flask. The leaves to be treated were held in

a horizontal plane with cellophane tape. A 10 ul drop of

0.1 M 2,4-D-1-14C (S.A. = 0.014 uc/10 ul) solution con-

taining 1 per cent v/v polyoxyethylene sorbitan monolaurate

(Tween 20) was applied to the center of the adaxial sur-

faces of four leaves, with the fifth being left untreated.

The treated plant and flask were sealed with a two-liter

bell jar along with a planchet containing 1 ml of 20 per

cent potassium hydroxide solution. The assembly was placed

in a controlled temperature room (72 + 30F) and exposed to

the following light and dark periods: 9.5 hours' light, 8

hours' dark, 16 hours' light, and 8 hours' dark. After

this period, 41.5 hours in all, the planchet containing

potassium hydroxide was removed from the bell jar, dried

under a heat lamp, and counted in the scaler. Treated

leaves were washed individually into planchets with 5 per

cent ammonium hydroxide for determination of residual radio-

activity.

Another replicate of the same experiment was carried

out with a comparable shoot collected from an (S) plant.







The light and dark periods; were essentially the same as

those for (R) titi.

Absorption Train Ex2p riLme nt

An absorption train (Fig. 3) similar to that of Morgan

and Hall (1962) was assembled for measuring the incremental

evolution of 14C02 from (S) and (R) titi shoots treated with

a 0.01 M solution of 2,4-D--1-4C (S.A. = 1 mc/mM).

Detached (S) and (R) titi shoots 8 to 10 cm long,

bearing seven terminal leaves were used in this experiment.

Herbicidal treatment was made by placing the shoots in a

2 ml volumetric flask containing a weighed amount of

2,4-D-l-14C solution, and allowing them to absorb the

labelled material via the transpiration stream. The neck

of the flask was tightly plugged with cotton to prevent

loss to evaporation. The assembly was placed 10 inches

from a slowly moving six-inch fan, and illuminated with

approximately 200 f.c. of incandescent light during the

uptake period. Periods were varied from 10 minutes to 3

hours to give a range of herbicide dosages in the experi-

mental material. Uptake was estimated from the loss in

weight of the flask and solution. After treatment, the

shoot was transferred to a 5 ml volumetric flask containing

distilled water. The neck of the flask was plugged with

cotton as before. The assembly was placed in the metab-





















































Fig. 3. Absorption train used in 2,4-D-1-14C metabolism
experiments with (S) and (R) titi shoots.








olism chamber, which then was covered with a light-tight

box for the duration of the test. At the start of the

metabolism period, the chamber w:s purged of atmospheric

CO2 by pulling in a supply of air through scrubbers con-

taining 1 N solutions of sodium hydroxide. The chamber was

then sealed off with screw clamps. Each treated shoot

remained in the chamber for approximately 140 hours. Per-

iodically during this time the chamber air was absorbed into

200 ml of 0.1 N sodium hydroxide. The absorber solution was

transferred to a 500 ml Erlenmeyer flask. To this was added

about 15 ml of 2 N barium chloride to recover any evolved

14CO2 as precipitated Ba14CO3. The precipitate was collect-

ed by vacuum filtration onto a sintered stainless steel disc

covered with a thin layer of Celite filter aid. After

drying under the heat lamp, the precipitate and disc were

placed in a copper planchet and counted in the scaler.

Counts were corrected for precipitate mass by means of the

curve shown in Fig. 4.

Seven metabolism experiments were run with (R) and

three with (S) titi. A slight modification was made in the

fourth metabolism experiment, designated R-4. Just prior

to treatment with 2,4-D-1-14C solution, the shoot was

allowed to take up, via the transpiration stream, 2 ug of

fluorine from a solution 100 p.p.m. in sodium fluoride.
























100

90
so
4 80

u 70
-w4I



g so
i4



0
S30

20

10


0 10 20 30 40 50 60 70 80

Sample Density- -g/cm2














Fig. 4. Mass correction curve for barium carbonate
precipitates.













EXPERIMENTAL RESULTS

Anatomical Study of Leaves

Microscopic examination of leaf cross-sections of (S)

and (R) titi (Figs. 5 and 6, respectively) revealed many

anatomical differences.

(S) titi has a thin cuticle (2.4 microns) overlying an

epidermis consisting of large, thin-walled cells loosely

arranged above the main vascular bundle. The main and

secondary vascular bundles are collateral, having both in-

ternal and external phloems. A well'-defined bundle sheath

extension pyramids upward from the main vascular bundle to

the loosely arranged region of the epidermis. Bundle sheath

extensions lead from secondary veins to both upper and lower

leaf surfaces (Fig. 7). The mesophyll is bifacial. Two

layers of palisade parenchyma are evident, with a third

intergrading into spongy parenchyma below. Stomates are

lacking on the upper leaf surface but occur frequently on

the lower.

(R) titi has a thick cuticle (13 microns) overlying an

epidermis consisting of small, thick-walled cells compactly

arranged above the main vascular bundle. Main and secondary

30























Fig. 5. Leaf cross-section of (S) titi (Cyrilla racemiflora) at mid-rib, x 330, showing
cuticle (C), epidermis (E), bundle sheath extension (BSE), internal phloem (IP), xylem
(X), external phloem (EP), palisade mesophyll (PM), spongy mesophyll (SM), and stoma (S).






















Fig. 6. Leaf cross-section of (R) titi (Cliftonia monophylla) at mid-rib, x 330, showing
cuticle (C), epidermis (E), bundle sheath extension (BSE), xylem (X), phloem (P), pali-
sade mesophyll (PM), and spongy mesophyll (SM). Note the absence of sheath extensions
on the secondary vein to the right of the main vascular bundle.

































S100


Fig. 7. Leaf cross section of (S) titi (Cyrilla race-
miflora), x 330, showing secondary vein with bundle
sheath extensions (BSE) leading to upper and lower
epidermis, xylem (X), and phloem (P).







vascular bundles are bicollateral, with internal phloem

lacking. A poorly defined sheath extension leads upward

from the main vascular bundle to the epidermis. Secondary

veins are deeply embedded in the leaf, without sheath

extensions. As with (S) titi, the mesophyll of (R) titi

is bifacial. Stomates are absent on the upper leaf surface

and only sparsely present on the lower.

The major anatomical differences are summarized in

Table 1.

Electron Microscopy of Leaf Surfaces

Examination of electron micrographs of (S) and (R) titi

leaf surfaces show marked differences in cuticular texture.

(S) titi (Fig. 8) has a relatively smooth, homogeneous

surface through which the dim outlines of epidermal cell

walls can be seen. (R) titi (Fig. 9), by contrast, exhibits

a rough, ridgy surface, probably waxy and very hydrophobic.

This would suggest (R) titi leaf surfaces are much more

difficult to wet by herbicidal sprays, as considerable re-

duction in spray droplet surface tension would be required

to cause them to relax into the waxy crevices and other

irregularities. On the other hand, (S) titi surfaces could

be wetted with comparative ease. This difference in wett-

ability is illustrated in Fig. 10, which shows the extent

of spread of 10 ul drops (0.01 M 2,4-D-1-14C, 1 per cent v/v








Table 1. Major anatomical differences between (S) and (R) titi leaves.


Feature (S) Titi (R) Titi


cuticle 2.4 microns thick 13 microns thick

epidermis large, thin-walled cells small, thick-walled cells
loosely arranged above compactly arranged above
main vascular bundle main vascular bundle

vascular bundles collateral bicollateral

bundle sheath present on all veins weakly defined on main vas-
extensions cular bundle but lacking on
others

stomates plentiful on lower epidermis sparsely distributed on lower
epidermis

























Fig. 8. Electron micrograph showing the smooth adaxial surface of an (S) titi leaf, x
20,000.












Vh








^ ( 4 0.*ev


IrFb

















































Fig. 10. The dry residue of 10 ul drops of 2,4-D-1-14C
containing 1 per cent Tween 20, applied to the center
of the adaxial surfaces of (S) and (R) titi leaves.








in Tween 20) applied to the center of the leaves of the two

species. On the (S) titi leaf, the droplet spread quickly

in both directions along the mid-vein and migrated toward

the lateral margins before drying. On the (R) titi surface,

the droplet dried essentially in place, with little or no

spreading from the point of application.

Leaf Absorption of 2,4-D-1-14C

Results and analysis of the 2,4-D-1- 4C absorption

trials with excised leaves of (S) and (R) titi are given

in Table 2.

(S) titi leaves absorbed on the average, 18.61 + 7.32

ug of the 221 ug of 2,4-D-1-- C applied in 10 ul drops.

This amounted to an absorption of 8.4 per cent. Under the

same experimental conditions, (R) titi absorbed 14.54 +

2.32 ug, for an uptake of 6.6 per cent. A comparison of

the two averages shows that (S) titi took up 1.28 times

more 2,4-D-1-14C than (R) leaves. This difference was

significant at the 5 per cent level of t.

14
Metabolic Degradation of 2,4-D-- 14

Preliminary Bell Jar Experiment

As mentioned earlier the bell jar experiment was

instituted to check for decarboxylation of 2,4-D-1-14C by

the titi species as a prelude to a more extensive absorption

train experiment. Results of the bell jar trials are








Table 2. Analysis of 2,4-D-1-14C absorption by (S) and (R)
titi leaves and significance test of the differences.



Leaf 2,4-D-1-14C Absorption Difference
Pair No. (S) Titi (R) Titi D = X1 X2

ug ug ug

1 10.5210 4.9098 5.6112
2 50.5008 4.9098 45.5910
3 17.5350 11.9238 5.6112
4 25.6011 17.5350 8.0661
5 10.5210 34.3686 -23.8476
6 19.2885 21.3927 2.1042
7 11.5731 15.0801 3.5070
8 46.6431 4.9098 41.7333
9 83.1159 20.6913 62.4246
10 17.1843 12.6252 4.5591
11 88.0257 9.8196 78.2061
12 26.3025 19.9899 6.3126
13 24.1983 10.8717 13.3266
Total 431.0103 189.0273 241.9830
Mean 18.6141 14.5405 18.6140


Calculation of "t" and Test of Siqnificance


2
S2 = D2 n
n 1


= 14784.9320 -


2
(241.9830)2
S 13


= 856.7201


2 = S2 = 856.7201 = 65.9015 S- = 8.1182
d d d
--- 13
n


t = d uD = 18.6140 0 = 18.6140 0 = 2.2928*
S- 8.1182 8.1182
d


d.f. = n 1 = 12; t.05 = 2.160







summarized in Table 3, below. Both (S) and (R) titi carried

out some decarboxylation (2.4 and 5.4%, respectively) and

(S) titi absorbed more 2,4-D--1-14C than (R) titi (5.43 vs

3.10%). Because of the lack of replication, this experiment

did not lend itself to statistical analysis.

Absorption Train Experiment

2,4-D-1-14C breakdown data from the absorption train

trials are given in Table 4. Breakdown was expressed as a

per cent of 2,4-D-1-14C taken up via the transpiration

stream. Depending on dosage, (R) titi degraded from 5 to

13.9 per cent of the 2,4-D-1-14C during dark periods of

approximately 140 hours duration. Two of the three (S) titi

trials resulted in no degradation. A third, S-2, showed a

breakdown of 0.8 per cent. A marked difference in

2,4-D-1-14C metabolism exists between (S) and (R) titi

(Table 4). This difference is shown more strikingly in

Fig. 11, where cumulative counts per minute evolved as

Bal4CO3 are plotted over time for the trials listed in Table

4. Trial R-4 was omitted from the plot because of the pre-

treatment it received with sodium fluoride solution.

In order to evaluate the relationship between

2,4-D-1-14C uptake and breakdown, the data in Table 4 were

analyzed by both linear and curvilinear regression analysis.

The linear regression, Y = 14.2043 + 4.2602X, was rejected















Table 3. Results of preliminary bell jar experiment in
which evolved 14CO2 was absorbed in potassium hydroxide
solution.


Plant


(S) Titi

(R) Titi


Total
Activity
. Applied
c/10a

23451

27160-


Activity Absorbed
by Leaves
c/10 %/b

1256 5.43

821 3.10


Apparent 2,4-D
Breakdown
c/10 /c

32 2.40

45 5.40


amounts per 10 minutes.

bExpressed as a per cent of total activity applied.

CExpressed as a per cent of activity absorbed.


---















Table 4. Uptake and degradation of 2,4-D-1-14C by detached
shoots of (S) and (R) titi during approximately 140 hours
in the dark.




Trial and 2,4-D---14C 2,4-D-1-14C
Plant Uptake Breakdown


R-1

R-2

R-3

R-4a

R-5

R-6

R-7

S-1

S-2

S-3


ug

356.2

237.2

103.9

254.4

602.0

158.2

124.0

697.7

305.0

65.4


ug

46.6

29.6

14.4

23.6

30.3

17.5

14.2

0

2.4

0


%

13.1

12.5

13.9

9.3

5.0

11.1

11.4

0

0.8

0


apretreated with 2 ug flourine as sodium fluoride.
















17
R-1

16


IS
R-5

14


13


12


If


II
O
X R-2
X 9

0


R-6

U

6R-3






4




2




S-2


S-1


0 10 20 30 40 ISO 0 70 O 90 100 110 120 130 1l0
Hours in Dark






Fi. 11. Cumulative counts per minute evolved as Ba 4C03

by (S) and (R) titi during the indicated dark periods.








by means of the calculations given in Tables 5, 6, and 7.

A significant F value was determined (Table 7) when the

mean square deviations from the linear and curvilinear

regressions were compared. The second degree curve,

2
Y 15.7677 + 28.8199X + (-3.4952) X2, was considered to

fit the data best, due to this significant reduction in the

sums of squares.

The analysis of variance for curvilinear regression

(Table 8) showed that a significant amount of the vari-

ability was accounted for by the second degree curve.

Further, the coefficient of determination (R2) of 0.93

(Table 8) indicated that approximately 96 per cent of the

variability in breakdown was accounted for by differences

in 2,4-D-1-14C uptake by the plants.

Herbicide breakdown (Fig. 12) in the (R) titi shoot

maximized at a calculated level of 400 pg total, or 57 pg

per leaf (all shoots bore seven leaves). This result

suggested that toxic levels were reached at a 2,4-D con-

centration of 57 pg per leaf. By extrapolation of the

curve it can be estimated that little or no breakdown would

occur beyond a concentration of about 800 ug per shoot, or

114 pg per leaf.

Confidence intervals were calculated for the degrada-

tion curve (Fig. 12) to determine whether 2,4-D-1-14C









Table 5. Data for computing standard deviation from the curvilinear regression
equation Y = -15.7677 + 28.8199X + (-3.4952) X2.


2,4-D-1-14C 2,4-D-1-14C
Plant and Uptake Breakdown
Trial X Y Y dyx dyx2
yx yx


ug x 102 ug ug ug ug

R-l 3.562 46.6 42.5424 +4.0576 16.4641

R-2 2.372 29.6 32.9281 -3.3281 11.0762

R-3 1.039 14.4 10.4031 +3.9969 15.9752

R-5 6.020 30.3 31.0606 .-0.7606 0.5785

R-6 1.582 17.5 21.0779 -3.5779 12.8013

R-7 1.240 14.2 14.5947 -0.3947 0.1557


57.0510


2
S = Ed 2 = 57.0510 = 19.0170
yx yx
n-3 3

Syx = + 4.3608


Sum









Table 6. Data for computing standard deviation from the linear regression
equation Y = 14.2043 + 4.2602X.


2,4-D-1-14C 2,4-D-1-14C
Plant and Uptake Breakdown
Trial X Y Y dyx d 2



ug x 10 ug ug ug ug

R-1 3.562 46.6 29.3791 17.2209 296.5593

R-2 2.372 29.6 24.3094 5.2906 27.9904

R-3 1.039 14.4 18.6306 -4.2306 17.8979

R-5 6.020 30.3 39.8507 -9.5507 91.2158

R-6 1.582 17.5 20.9439 -3.4439 11.8604

R-7 1.240 14.2 19.4869 -5.2869 27.9513


473.4751


2
Syx = dyx2 = 473.4751 = 118.3687
n-2 4

S = + 10.8797


Sum









Table 7. Comparison between mean squares for linear and curvilinear
regression--(R) titi decarboxylation data.


Analysis of Variance

Source of Variation d.f. SS MS


Deviations from Linear Regression 4 473.4751

Deviations from Curvilinear Regression 3 57.0510 19.0170


Curvilinearity of Regression


416.4241


416.4241


F = 416.4241 = 21.8974*
19.0170

F .05; n = 4,3 = 9.12















Table 8. Analysis of variance for curvilinear regression -
2,4-D-1-14C uptake and breakdown data for (R) titi trials
in the absorption train.




Analysis of Variance
Source of
Variation d.f. SS MS F


Total 5 799.9333 159.9867

Curvilinear
Regression 2 742.8917 371.4458 19.535*

Error 3 57.0416 19.0139


R2 = 742.8917
799.9333

= 0.93


F = 371.4458 = 19.535*
19.0139

F 05; n = 2,3 = 9.55
.05






















SBLACK TITI (Cliftonia onoPhvlla)

S= -15.7677 + 28.8199X + (-3.4952)X2
R2 0.93





a .
so -







10 -



0 1 3 4 7
2,4-D Uptake x 10-2 -





Fiq. 12. The relationship between 2,4-D (2,4-D-1-14C)
uptake and breakdown (decarboxylation) by (R) titi
shoots during approximately 140 hours in the dark.







breakdown by the R-4 shoot, pretreated with 2 ug of fluoride

ion, departed significantly from predicted values based on

the amount of herbicide taken up. The standard deviation

from curvilinear regression (Syx), calculated in Table 5,

was determined to be + 4.3608 ug. Shoot R-4 took up

254.4 ug and degraded 23.6 ug of the applied material

(Table 4). Based on the regression in Fig. 12, the pre-

dicted breakdown for an uptake of 254.4 ug was found to be

34.9 ug. The difference between actual and predicted

values was 1.1.3 ug, or 2.60 times greater than one standard

deviation (11.3290 e 4.3608 = 2.60). Since the value of

t.05 for five degrees of freedom is 2.57, we can say the

quantity of 2,4-D-1-14C degraded by shoot R-4 departed

significantly from the other six shoots evaluated in the

absorption train. Presumably this departure, amounting to

a 32 per cent reduction in breakdown, was fluorine-related,

as experimental conditions were otherwise held constant.














DISCUSSION

Unquestionably, leaf anatomy plays no small role in

determining the degree of herbicidal susceptibility of any

given plant. Before a spray deposit bearing systemic

materials can produce the desired effect, it must first

penetrate into the leaf. Foliar absorption must occur

directly through the leaf cuticle and through stomates if

present. However, once into stomatal chambers, the spray

solution is still confronted by a cuticular lining which

must be crossed (Norris and Bukovac, 1968). While being

absorbed, the herbicide must be kept in a fluid state. The

quantity of active material absorbed is a function of its

concentration in the spray droplet, leaf area covered, and

the rate of evaporation of the solution.

After crossing the cuticle, the herbicide passes through

and between the cells surrounding the main and secondary

veins gaining access to them through bundle sheath extensions

when present (Esau, 1953; van Overbeek, 1956). Upon entering

the veins, solution movement is bi-directional, part of it

moving upward in the xylem via the transpiration stream, and

part toward the root in the phloem, in the food transport

56







system. Thereafter, the herbicide must reach a sensitive

site and accumulate to toxic concentration in some active

form.

From the standpoint of herbicidal susceptibility, (S)

titi (Fig. 5) has an ideal leaf anatomy. A spray droplet

landing above the main vein is confronted by a thin, smooth

cuticle overlying loosely arranged epidermal cells. Droplet

spreading (Fig. 10) and solid contact are favored by the

lack of wax protrusions on the leaf surface (Fig. 8). Due

to the large voids between epidermal cells, the herbicide

probably reaches the upper bundle sheath extension via an

apoplastic route. Once in the bundle sheath extension, the

herbicide solution is funnelled directly to the internal

phloem. Sheath extensions on secondary veins (Fig. 7) like-

wise channel spray materials to the phloem, although some are

doubtless intercepted by xylem tissue overlying the external

phloem (internal phloem was found lacking in secondary veins

of (S) titi).

In contrast to (S) titi, (R) titi (Fig. 6) has many of

the anatomical attributes of a resistant species: thick,

ridgy cuticle, compact epidermis, scanty phloem, and deeply

embedded veins lacking sheath extensions to the epidermis.

Since internal phloem is lacking in (R) titi, the upper

bundle sheath extension leads directly from the epidermis








to the xylem of the main vascular bundle. Herbicide solution

entering the leaf in the vicinity of mid-rib is probably

carried to the xylem elements where it is sidetracked into

the transpiration stream. Solutions entering the leaf at

points other than near the mid-rib probably move through the

mesophyll with difficulty, as bundle sheaths are lacking on

secondary veins in this species.

Results given in the present study showed that (S) titi

absorbed 28 per cent more 2,4-D-1-14C (sodium salt) than (R)

titi under the same experimental conditions. This difference

is surprisingly small considering the disparity in cuticular

thickness observed (2.4 vs. 13.0 microns). However, as in-

dicated earlier (Norris and Bukovac, 1969), the limiting step

in leaf absorption is apparently the initial penetration of

the cuticular surface, not the thickness of the cuticle pae

se.

Since the uptake trials involved mid-rib placement of

droplets, the results could have been slightly biased in fa-

vor of (R) titi. Close scrutiny of Fig. 6 will show light

streaks bisecting the cuticle along the concave surface of

the leaf above the main vein. The streaks, which terminate

at the anticlinal walls of epidermal cells, possibly function

as tension zones to accommodate changes in leaf size due to

variable turgidity. One might expect that solutions would







preferentially enter such tension zones in the cuticle.

Despite this possible bias, the difference in uptake was

significant, and it was correlated with susceptibility.

This result was at variance with those of Fang (1958),

Luckwill and Lloyd-Jones (1960a, 1960b), Foy (1962), and

others, who observed no such correlation between susceptible

and resistant species.

The amount of decarboxylation determined for (R) titi

was low compared to that of resistant red currant, straw-

berry, and apple, reported by Luckwill and Lloyd-Jones

(1960a, 1960b), and Edgerton and Hoffman (1961). It is pro-

posed here that (R) titi is herbicidally resistant by virtue

of the combined effects of poor leaf absorption and the

ability to carry out some decarboxylation of 2,4-D. As pre-

viously stated, (R) titi was able to withstand aerial

applications of 2,4-D ester applied at the rate of 6 pounds

active ingredient in 5 gallons of spray per acre. The

application equipment used produced spray droplets averaging

about 400 microns in diameter. With the above spray volume,

and allowing 20 per cent spray loss to the atmosphere, this

would yield about 75 drops per square inch. The concentra-

tion used would supply 48 x 10-2 mg 2,4-D per square inch.

(R) titi leaves individually cover an average area of 0.875

square inches. Allowing for inclination and overlap, the







effective leaf area is probably reduced to approximately 0.5

square inches. Thus it is estimated that each (R) titi would

intercept (0.5)(48 x 10-2) 24 x 10.2 mg or 240 ig 2,4-D

via the commercial application. To reach the toxic threshold

of 57 pg per leaf, the commercially applied material would

need to penetrate the (R) titi leaf at a level in excess of

23 per cent. This is a reasonable level of penetration which

should be attainable through surfactant enhancement.

Introduction of fluoride ion into the (R) titi leaf just

prior to treatment with 2,4-D-l-14C had a sparing effect on

decarboxylation. This result,though similar, is not fully

equatable to that of Edgerton and Hoffman (1961) since they

utilized a fluorine substitution in the 2,4-D molecule rather

than the free ion for inhibition of the systems) involved.

Addition of fluoride ion to spray mixtures may provide a use-

ful means of potentiating 2,4-D in (R) titi. Also, it would

be interesting to check for the selective toxicity of 2,4,5-T

as observed by Luckwill and Lloyd-Jones (1960a, 1960b) in

other decarboxylating species.














SUMMARY AND CONCLUSIONS

The failure to control black titi (Cliftonia mono-

phvlla) with aerial applications of 2,4-D led to studies

of the factors of resistance, using closely related

2,4-D-susceptible (S) white titi (Cyrilla racemiflora) for

comparative analysis.

Anatomical study of leaves showed resistant (R) titi

to have a thick cuticle, compactly arranged epidermal cells,

no internal phloem in the main vascular bundle, and no

bundle sheath extensions. (S) titi had a thin cuticle,

loosely arranged epidermal cells, internal phloem just under

the epidermis, and well-defined bundle sheath extensions on

all veins. Ultra-structure studies of adaxial leaf sur-

faces revealed those of (R) titi to be rough and ridgy, and

those of (S) titi, relatively smooth and homogeneous.

14
Ten il drops of 2,4-D-1- C solution containing Tween

20 surfactant were applied to excised leaves of (S) and (R)

titi. (R) titi absorbed 6.6 per cent and (S) titi, 8.4 per

cent of the applied material. The difference was signifi-

cant at the 5 per cent point of t.

Detached shoots of (S) and (R) titi were fed 2,4-D-1-14C

61








solution via the transpiration st-ream then sealed in a dark-

ened metabolism chamber for measur:iement of evolved 14CO2.

Depending on dosage, (R) titi shoots degraded from 5 to 13.9

per cent and (S) titi, 0 to 0.8 per cent of the applied her-

bicide during approximately 140 hours. Curvilinear regress-

ion analysis of (R) titi degradation data suggested 2,4-D

breakdown maximized at a leaf concentration of 57 pg, and

decreased thereafter.

Pretreatment of an (R) titi shoot with 2 y.g of fluorine

as sodium fluoride resulted in a significant reduction in

decarboxylation of 2,4-D-1-14C.

From the foregoing studies it can be concluded that:

1. Major factors contributing to the herbicidal re-

sistance of black titi include its thick, ridgy

cuticle, peculiar arrangement of internal leaf

tissues, and comparatively low and high rates of

2,4-D penetration and decarboxylation, respectively.

2. Enhancement of herbicide penetration is the pro-

bable key to control of black titi, as economical

levels of 2,4-D were apparently leaf-toxic when

applied via the transpiration stream.

3. As a practical measure, fluoride ion supplied sep-

arately to black titi may lead to a significant

reduction in the toxic concentration indicated for

control of this species.















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BIOGRAPHICAL SKETCH

Walter Leon Beers, Jr. was born June 28, 1927, in

Greensburg, Westmoreland County, Pennsylvania, the son of

Margaret E. and Walter L. Beers, Sr. He attended the

Greensburg public schools and was graduated from the

Greensburg High School in June, 1945. Immediately there-

after he enlisted in the United States Marine Corps for a

period of four years.

In July, 1949, he married Barbara Jean Merrill. They

have three children, Walter Bradley, Barry Leon, and

Rebecca Sue.

In September, 1949, he entered the Pennsylvania State

University and in February, 1953, he was conferred the

degree of Bachelor of Science with a major in Forestry.

He then entered the Graduate School of the same institution

for one term, majoring in Forest Management.

In June, 1953, he accepted employment with The Buckeye

Cellulose Corporation, Perry, Florida, and until now has

worked as a Research Forester in the Woodlands Department.

He entered the University of Florida Graduate School

in February, 1962, majoring first in Botany and later in








Agronomy. The degree of Doctor of Philosophy was conferred

on him in December, 1969.

He is a member of the Society of American Foresters,

Kiwanis International, and Xi Sigma Pi, Gamma Sigma Delta,

Phi Sigma, and Alpha Zeta honorary organizations.










This dissertation was prepared under the direction of

the chairman of the candidate's supervisory committee and

has been approved by all members of that committee. It was

submitted to the Dean of the College of Agriculture and to

the Graduate Council, and was approved as partial fulfill-

ment of the requirements for the degree of Doctor of

Philosophy.



December, 1969




CfDean, College of Agriculture




Dean, Graduate School



Supervisory Committee:




Chairman




s/I/f2/,




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