PHYSIOLOGICAL EFFECTS OF DIFLUBENZURON'

[— 1(4 - CHLORPH ENYL) ~3 - (2,6 - DIFLUOROBENZOYL) UREA]
AND FENTIN HYDROXIDE (TRIPHENYLTIN HYDROXIDE)
0N SOYBEAN {GLYCINE MAX.(L.) MERRJ
AND RICE (ORYZA SATTIVA L)

Thesis for the Degree of M. S.

MICHIGAN STATE UNIVERSITY

KRITON KLEANTHIS HATZIOS
1977 ' '

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ABSTRACT

PHYSIOLOGICAL EFFECTS OF
DIFLUBENZURON [1-(4-CHLOROPHENYL)-3-(2,6-DIFLUOROBENZOYL)UREA]
AND FENTIN HYDROXIDE (TRIPHENYLTIN HYDROXIDE)
ON SOYBEAN [GLYCINE MAX (L.) MERR.] AND RICE ORYZA SATIVA L.)

 

 

by

Kriton Kleanthis Hatzios

The effect of the insecticide diflubenzuron [l-4—chlor0pheny1)-3-

(2,6-dif1uorobenzoyl)urea] on photosynthesis, respiration, and leaf

ultrastructure of soybean [Glycine max (L.) Merr., cv. Swift] and of

 

the fungicide fentin hydroxide (triphenyltin hydroxide) on photo-
synthesis, respiration, and leaf ultrastructure of soybean and rice

(Oryza sativa L., cv. Starbonnet) was examined. In addition, the

 

effect of these two pesticides on seed quality was examined for five
soybean cultivars, namely "Coker 102", "Cobb", "Tracy", "Ransom" and
"Bragg".

Soybean plants at the second trifoliate leaf stage were treated
with 0, 0.067, or 0.269 kg active ingredient/ha of diflubenzuron and
0, 0.56, or 2.24 kg active ingredient of fentin hydroxide/ha. Rice
plants, 25 cm tall, were treated with 0, 0.56, or 2.24 kg active
ingredient/ha of fentin hydroxide.

Photosynthesis and respiration were measured with an infrared

C02 analyzer in an open flow system, prior to pesticide application

Kriton Kleanthis Hatzios

and at 4, 24, 48, and 96 hours after treatment. Leaf tissue samples
from both plant species were examined for ultrasturcture changes
by transmission electron microscopy 9 days after treatment.

Seeds from five soybean cuitivars, obtained from plots treated
with 0, 0.035, 0.069 and 0.14 kg/ha of diflubenzuron for "Coker 102",
0 and double application of 0.56 kg/ha of fentin hydroxide for "Cobb",
"Tracy", and "Ransom" and 0, 0.266, 0.532 and 1.064 kg/ha of fentin
hydroxide, single and in combination with 0.067 kg/ha of diflubenzuron
for "Bragg", were analyzed for their chemical composition, expressed
in terms of lipid, protein and carbohydrate content, by using Nuclear
Magnetic Resonance (NMR), Kjeldahl, and anthrone procedures. Seed
viability and seedling vigor were determined by applying a regular
germination test, an accelerated aging test, and a cold test.

Neither diflubenzuron nor fentin hydroxide appeared to have any
detrimental effect on photosynthesis, respiration, and leaf ultra-
structure of soybean and rice with the exception that diflubenzuron
at the high rate (0.269 kg/ha) stimulated respiration in a transitory
manner.

In one study, fentin hydroxide at 0.56 kg/ha caused rice plants
to become greener and taller than the control and the 2.24 kg/ha
treated plants. This could not be repeated in successive greenhouse
studies.

Both diflubenzuron and fentin hydroxide showed no negativeeffects
on the production of healthy seedlings. Fentin hydroxide, in some
instances, increased the percent of healthy seedlings following the

accelerated aging stress.

Kriton Kleanthis Hatzios

No effect of either pesticide on soybean seed lipid, protein and
carbohydrate content was evident with the exception that fentin
hydroxide treated "Ransom" and one set of treated "Bragg" soybeans

showed greater and lower carbohydrate content respectively.

PHYSIOLOGICAL EFFECTS OF
DIFLUBENZURON [-l(4-CHLORPHENYL)—3-(2,6-DIFLUOROBENZOYL)UREA]
AND FENTIN HYDROXIDE (TRIPHENYLTIN HYDROXIDE)

ON SOYBEAN [GLYCINE MAX (L.) MERR.] AND RICE (ORYZA SATIVA L.)

 

 

By

Kriton Kleanthis Hatzios

A THESIS

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

'MASTER OF SCIENCE

Department of Crop and Soil Sciences

1977

ACKNOWLEDGEMENTS

I offer special thanks to my academic advisor Dr. Donald Penner,
whose skilled guidance and trusted friendship have contributed a lot
to my education and this research. Sincere appreciation is expressed
to Dr. G. S. Ayers and L. 0. Copeland for their assistance and service
in the guidance committee. Dr. G. Hooper and Mrs. J. Mack are also
appreciated for their assistance in the electron microscope studies.
I am grateful to Dr. Bouyoukos for offering me his scholarship, by
means of which I was able to finish my studies. I also wish to
mention Dr. G. A. Mourkides, without whose advise and assistance
I might never have begun this master's program.

Thanks are also expressed to Thompson-Hayward Chemical Company

for granting this study.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . .
LIST OF FIGURES. . . . . . . .
INTRODUCTION . . . . . . . . .
CHAPTER 1. LITERATURE REVIEW.

List of References. . .

CHAPTER 2.

MERR.]
STRUCTURE. . . . .

Abstract . . . . . . . .
Introduction . . . . . .
Materials and Methods. .
Results and Discussion .
References . . . . . . .

CHAPTER 3.

THE EFFECT OF FENTIN HYDROXIDE

HYDROXIDE) 0N SOYBEAN [GLYCINE
RICE (ORYZA SATIVA L.) PHOTOSYNTHESIS,

 

THE EFFECT OF DIFLUBENZURON l-(4-CHLOROPHENYL)-3-
(2,6-DIFLUOROBENZOYL)UREA ON SOYBEAN [GLYCINE MAX (L.

 

PHOTOSYNTHESIS, RESPIRATION AND LEAF ULTRA-

(TRIPHENYLTIN
MAX (L.) MERR.]AND

 

AND LEAF ULTRASTRUCUTRE.

Abstract. 0 O O O O O O O O 0

Introduction. . . . . .

Materials and Methods .

Results and Discussion.

References. . . . . . .

iii

RESPIRATION

Page

vi

13
13
l3
l4
17

22

24
24
24
25
28

38

Page

CHAPTER 4. THE EFFECT OF DIFLUBENZURON l-(4-CHLOROPHENYL)-3-
(2, 6- -DIFLUOROBENZOYL)UREA AND FENTIN HYDROXIDE
(TRIPHENYLTIN HYDROXIDE)ON SOYBEAN [GLYCINE MAX (L. )

MERR. ] SEED QUALITY . . . . . . . . . . . 40
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . 40
Introduction. . . . . . . . . . . . . . . . . . . . . . . 41
Materials and Methods . . . . . . . . . . . . . . . . . . 41
Results and Disscusion. . . . .'. . . . . . . . . . . . . 43
References. . . . . . . . . . . . . . . . . . . . . . . . 53

CHAPTER 5. SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . 55

iv

 

 

1mg}! £355... .32. Iauikgfi!

LIST OF TABLES
Page
CHAPTER 2

l. The effect of diflubenzuron on total photosynthesis of
soybean plants in the second trifoliate leaf stage . . . . l9

2. The effect of diflubenzuron on respiration of soybean
plants in the second trifoliate leaf stage. . . . . . . . 19

CHAPTER 3

1. The effect of fentin hydroxide on total photosynthesis of
soybean plants in the second trifoliate leaf stage . . . . 30

2. The effect of fentin hydroxide on respiration of soybean
plants in the second trifoliate leaf stage. . . . . . . . 30

3. The effect of fentin hydroxide on total photosynthesis
of rice plants . . . . . . . . . . . . . . . . . . . . . . 31

4. The effect,of fentin hydroxide on respiration of rice
plants 0 O O O O O O O O O O O O O O O O O O O O O O O O O 31

CHAPTER 4

1. Soybean seed, cv. "Coker 102", viability and vigor follow-
ing treatment with diflubenzuron. . . . . . . . . . . . . .45

2. Soybean seed viability and vigor following treatment with
fentin hydroxide . . . . . . . . . . . . . . . . . . . . . 46

3. Soybean seed, cv. "Bragg", viability and vigor following
treatment with fentin hydroxide. . . . . . . . . . . . . . 47

4. Soybean seed, cv. "Bragg", viability and vigor following
treatment with fentin hydroxide and diflubenzuron. . . . . 48

5. Soybean seed, cv. "Coker 102", composition following treat-
ment with diflubenzuron. . . . . . . . . . . . . . . . . . 49

6. Soybean seed compositon following treatment with fentin
hydroxide I O O O O O I I O O O O O O O O O O O O O O O O O 50

7. Soybean seed, cv. "Bragg", compositon following treatment
with fentin hydroxide. . . . . . . . . . . . . . . . . . . 51

8. Soybean seed, cv. "Bragg", composition following treatment
with fentin hydroxide and diflubenzuron. . . . . . . . ... 52

V

LIST OF FIGURES

CHAPTER 2

1. Chemical structures of the herbicides dichlobenil and

monuron and of the insecticide diflubenzuron . .

2. a. Electronmicrograph of the upper trifoliate leaf of
soybean plant treated 9 days earlier with water
(control). . . . . . . . . . . . . . . . . .

b. Electronmicrograph of the upper trifoliate leaf of
soybean plant treated 9 days earlier with 0.269 kg/ha
of diflubenzuron . . . . . . . . . . . . . .. .

c. Electronmicrograph of the lower trifoliate leaf of
soybean plant treated 9 days earlier with 0.269 kg/ha
of diflubenzuron. . . .

CHAPTER 3

1. Chemical structures of the fungicides fentin hydroxide

and triarimol. . . . . . . . . . . . . . . . . . .

2. a. Electronmicrograph of rice leaf tissue from plant
treated 9 days earlier with water (control). .

b. Electronmicrograph of rice leaf tissue from plant
treated 9 days earlier with 0.56 kg/ha of fentin
hydroxide (1X rate). . . . . . . . . . . . . .

c. Electronmicrograph of rice leaf tissue from plant
treated 9 days earlier with 2.24 kg/ha of fentin
hydroxide (4X rate). . . . . . . . . . . .

3. a. Electronmicrograph of soybean leaf tissue from
lower trifoliate leaf treated 9 days earlier with
water (control). . . . . . . . . . . . . . . .

b. Electronmicrograph of soybean leaf tissue from the

upper trifoliate leaf of plant treated 9 days earlier

with 0.56 kg/ha of fentin hydroxide (1X rate).

vi

0

Page

15

21

21

21

26

33

33

33

35

35

Electronmicrograph of soybean leaf
trifoliate leaf of plant treated 9
2.24 kg/ha of fentin hydroxide (4X

Electronmicrograph of soybean leaf

trifoliate leaf of plant treated 9
2.24 kg/ha of fentin hydroxide (4X

vii

tissue from upper
days earlier with
rate)

tissue from lower
days earlier with
rate)

Page

32

32

INTRODUCTION

Pesticides are toxic materials intended to be physiologically
active only on the target organism. Success for many lies on being
relatively toxic to pests and non-toxic to treated crops (28)1.

Thus, herbicides designed to kill weeds, which are higher plants,
may not affect non-vascular plants such as algae or fungi, because of
differences in shape, form, and physiology existing between these two
plant divisions. Similarly, fungicides may not affect higher plants
and insecticides may not affect non-vascular or vascular plants.

However, selectivity is not always complete. There is documented
evidence that certain herbicides affect fungi and insects, and certain
fungicides and insecticides may have effects on higher plants. These
interactions have been recently reviewed by Putnam and Penner (32).

Numerous pesticides affect respiration in animal and plant tissues
(11) and many herbicides act on photosynthesis (8, 11). The effect on
these physiological systems may or may not be involved in the pest—
icidal action of the compound. This effect also, may be observed only
on the susceptible organism or in other instances, on both, the
susceptible organism and the tolerant crop species. If observed in
the tolerant species, the effect is generally transitory and its
duration may be related to the rate of pesticide detoxication by the

tolerant species.

 

1 References at the end of "Literature Review".

2

Numerous attempts have been made to relate the effects of
pesticides on physiological systems to altered cellular structure (4).
These relationships have not always been straight-forward as occa—
sionally the ultrastructure may be affected without any measurable
change in photosynthetic or respiratory rate.

Pesticides which cause serious deleterious effects on plant
growth may affect the chemical composition of seeds in a detrimental
manner (28). Failure of pesticides to control pests, resulting in
growth stress on the crop may also affect seed quality. Seed quality
can be divided into two basic parameters, the chemical composition
of the seed and the quality of the seed in terms of germination and
production of a vigorous, healthy seedling.

Production of vigorous seedlings may be controlled by the chemical
composition of the seed as well as by hormones, inhibitors, and pest-
icide residue levels. Seeds that have been overdosed with toxic
fungicides such as the organic mercurial compounds, commonly produce
abnormal seedlings (17). Plants treated or accidentally subjected to
certain pesticides such as 2,4-D (2,4-dichlorophenoxyacetic acid) and
phenolic compounds may produce abnormal or nonviable seedlings (23).
Pesticides which affect seed moisture content or microorganisms may
influence the health of future seedlings.

The insecticide diflubenzuron [l-(4-chlorophenyl)-3-(2,6—
difluorobenzoyl)urea] and the fungicide fentin hydroxide (triphenyltin
hydroxide), have shown promise in several tests to control insects and
fungi diseases on many crops (5, 6).

Prior to registration of these two pesticides for widespread use,
considerable testing is necessary to insure their effectiveness on the

insects and fungi and their nontoxicity to nontarget organisms.

3
This study was primarily designed to determine the physiological
effects of diflubenzuron and fentin hydroxide on soybean [Glycine

max (L.) Merr.] and rice, (Oryza sativa L.) and to evaluate seed quality

 

from treated plants.

CHAPTER I

LITERATURE REVIEW

1. Substituted Ureas as Insecticides

 

The herbicidal properties of various derivatives of urea are well-
known and many of these compounds are employed in agriculture (8, 24).

In terms of insecticidal activity substituted ureas were thought
to be insignificant until 1972, when van Daalen g£_§l, (41) introduced
the first substituted urea insecticide, namely DU 19111 [l-(2,6-
dichlorobenzoyl)—3-(3,4-dichlorophenyl)urea]. One year later, the
synthesis of other disubstituted ureas and their evaluation as insect—
icides were reported (44, 45). Of these compounds diflubenzuron
[1-(4-chlorophenyl)-3-(2,6-difluorobenzoy1)urea] and its dichloro-
analogue (PH 6038) were found to be the most active.

With respect to their mode of action, disubstituted urea insect-
icides interfere with cuticle formation in insect larvae during the
process of ecdysis (27) and chitinization. However, it is not entirely
clear whether this is due to inhibition of chitin synthesis (30, 31)
or to increased chitinase activity (16). Since these compounds are
effective in early stages of insect development, their possible applica-
tion to stored grain has been suggested (34).

Studies on diflubenzuron degradation showed photodegradation to
be an important factor in the eventual environmental degradation of
this compound (25, 35). The degradative pathways usually involved

cleavage between the carbonyl and amide groups of the urea bridge (25).

5

Mammalian toxicity of the disubstitued ureas appears to be low
(5, 41). Feeding diflubenzuron to cows did not have any adverse effect
during the feeding period (26) and this insecticide has been considered
as a food additive for cows to control flies larvae in manure. Diflu-

benzuron did not bioconcentrate in the mosquito fish Cambustia affinis,

 

through the food chain as did DDT (25).

Diflubenzuron is chemically related to the herbicides monuron
[3-(p-chloropheny1)-l,l-dimethylurea] and dichlobenil (2,6-dichlo-
benzonitrile) (see Figure 1). The structural similarity raises the
possibility of phytotoxicity of diflubenzuron or of its potential
breakdown products.

However, the aforementioned compound DU 19111 which is chemically
related to the herbicides dichlobenil and diuron [3-(3,4-dichlorophenyl)
-1,1—dimethy1urea], appeared to be non-phytotoxic since the leaves of
the French bean, tomato, sugar beet, and oats and as well a large
variety of weeds remained unaffected when sprayed with a 1% acetone
solution (41). The same authors also found that the germination of
seeds was not inhibited after a preemergence application of DU 19111.

II. Triphenyltin Compounds as Fungicides

 

In general, organometalic compounds, or metal compounds in which
a metal is linked with at least one carbon of an organic group, are
more active as pesticides than the metal alone. Their application in
agriculture has been reviewed by Kaars Sijpestein £5 £1. (19).

The first tests on the biocidal activity of organotin compounds
were done in 1886, but their insecticidal properties were only dis-
covered in 1929 (24). Later, van der Kerk and Luijten in 1954 (42),
investigated the greater fungicidal properties of trisubstituted tin

compounds, i.e. R3Snx, compared to the other three basic groups,

6
RSnX3, RZSnXZ, R4Sn. In terms of structure-activity relationships,
it was found that the nature of the organic group, R, which may be
simple aliphatic or aromatic radicals, appeared to be more important
in contrast to the nature of the fourth group, X, which may be a
halide, hydroxide, 0R, SH, SR, or acyl radicals and did not apparently
influence activity (14, 17, 19, 22, 42).

Within the effective compound group, R3SnX, the trialkyltin
compounds proved to be more effective than triphenyltin compounds in_
vitrg, but the results were the opposite under field conditions.
Triphenyltin compounds appeared to be more effective in_yixg_despite
their moderate fungitoxicity.igugi££g (14,29). Triphenyltin compounds
were also found to be less phytotoxic, though not sufficiently
innocuous to many crops, as compared to trialkyltin compounds which
appeared to be too phytotoxic at concentrations required to control
fungi (14, 29).

As a consequence, particular attention was given to triphenyltin
compounds for possible application in agriculture as commercial pest—
icides. The ultimate decomposition of organotin compounds under the
action of light and air to harmless inorganic tin, coupled with its
effectiveness on crops (20), promoted efforts for commercial applica-
tion of these compounds.

The potential use of triphenyltin compounds in insect control has
been reviewed by Ascher and Nissim (7). The action of triphenyltin
compounds as insect reproductory inhibitors (20) and as antifeeding
agents for some chewing insects (24) has been reported. The possible
application of organotins as herbicides has also been mentioned (11,

24), but much more importance has been given to the use of triphenyltin

compounds as agricultural fungicides. Two of them fentin acetate
(triphenyltin acetate) and fentin hydroxide (triphenyltin hydroxide)

are used in Europe to control Phytophthora infestans on potato

 

 

(Solanum tuberosum L.), Cercospora beticola on sugar beet (Beta

 

vulgaris L.), and Septoria apii on celery (Apium graveolens L.)

 

 

(7, 14, 19, 29, 39, 40). Phytotoxicity of these compounds has limited

their widespread use. Tomato (Solanum lycopersicum L.), fruits, vine,

 

greenhouse plants and ornamental plants have been reported as being
sensitive (7, 14, 21).

Efforts to reduce phytotoxicity of trisubstituted tin compounds
revealed that the nature of X, exerted a considerable influence.
Pieters (29) reported that triphenyltin acetate caused more leaf
damage than did the triphenyltin hydroxide on potatoes. Proper form-
ulation of organotin compounds can also reduce the degree of phyoto—
toxicity (22, 29). Kubo (21) reported that introduction of diphenyl
phosphine moiety into trialkyl— or triphenyl-tin derivatives reduced
phytotoxicity considerably.

Trisubstituted tin compounds are considered to exert their mode
of action by inhibiting oxidative phosphorylation in mitochondria of

animal tissues (1, 2, 3, 38). Kaars Sijpestein gt_§l, (18) assumed

that inhibition of oxidative phosphorylation is also responsible for

the antifungal effect. Although such in_zi£rg_activity may be
sufficient to explain the in_yigg effects, no experiments have been
done with pests (11).

The amazing increase in yield after treatments with fentin
acetate, observed in sugar beet and celery (13, 36) has given rise
to the hypothesis of a possible growth promoting effect of fentin

acetate on these plants (7).

8

0n the other hand, absorption and translocation studies of labeled
113Sn-triphenyltin acetate by kidney beans (Phaseolus vulgaris L.)
celery, and sugar beet indicated that this compound was not absorbed
by plants, leaves or roots (9, 10, 15). Nevertheless, effective
concentrations for growth regulation may be very low. Baumann (9, 10)
ascribes the total increase in yield observed in sugar beet and celery
treated with fentin acetate to the excellent fungicidal activity.

Since triphenyltin acetate is converted to triphenyltin hydroxide
(19, 40) and there is well-documented evidence for the growth-regulating
action of the fungicide triarimol [a—(2,4-dichloropheny1)-a-pheny1-5-
pyrimidinemeyhanol] (12, 33, 37) which structurally resembles fentin
hydroxide (see Figure l) the possibility that fentin hydroxide has

growth-regulating effects on plants merits examination.

10.

11.

12.

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Aldridge, W. N. and J. E. Cremer. 1955. The biochemistry of
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Aldridge, W. N. and B. W. Street. 1964. Oxidative phosphorylation.
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55: 514-516.

 

*Unread published material. Information from Baumann is depending
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l3.

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10

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Ishaaya, I. and J. E. Casida. 1974. Dietary TH 6040 alters
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11

Mulder. R. and M. G. Gijswijt. 1973. The laboratory evaluation
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Pieters, A. J. 1962. Triphenyltin hydroxide, a fungicide for the
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fungus diseases. Proc. Brit. Insecticide Fungicide Conf.
Brighton, England. 2: 461—470.

 

Post. L. C. and W. R. Vincent. 1973. A new insecticide inhibits
chitin synthesis. Naturwissenschaften. 9: 431-432.

Post, L. C., B. J. DeJong, and W. R. Vincent. 1974. 1-(2,6-
disubstituted benzoyl)-3—pheny1urea insecticides: Inhibitors of
chitin synthesis. Pestic. Biochem. Physiol. 4: 473-483.

Putnam, A. R. and D. Penner. 1974. Pesticide interactions in
higher plants. Res. Reviews. 50: 73-110.

Ragsdale, N. N. and H. D. Sisler. 1972. Inhibition of ergos-
terol synthesis in Ustilago maydis by the fungicide triarimol.
Biochem. Biophys. Res. Commun. 46: 2048-2053.

 

Rowlands, D. G. 1975. The metabolism of contact insecticides in
stored grains. III. 1970-1974. Res. Reviews. 58: 113-155.

Ruzo, L. 0., M. J. Zabik, and R. D. Schuetz. 1974. Photochemistry
of bioactive compounds. l-(4-chlorophenyl)-3-(2,6-dihalobenzoyl)
ureas. J. Agr. Food Chem. 22: 1106-1108.

Schupp, F.* 1957. Bod. Obst. u. Gargenbaur U. 189-190.

Shive, J. B., Jr. and H. D. Sisler. 1976. Effects of ancymidol
(a growth regulator) and triarimol (a fungicide) on the growth
sterols and gibberellins of Phaseolus vulgaris (L.). Plant
Physiol. 57: 640-644.

 

Stockdale, M., A. P. Dawson, and M. J. Selwyn. 1970. Effects of
trialkyltin and triphenyltin compounds on mitochondrial respira-
tion. Eur. J. Biochem. 15: 342-351.

Stoner, H. B. 1966. Toxicity of triphenyltin. Brit. J. lndustr.
Med. 23: 222-229.

Thomas, B. and H. L. Tann. 1971. Pesticide residues on food-
stuffs in Great Britain. XV. Triphenyltin residues on potatoes.
Pestic. Scil 2: 45-47.

 

*Unread published material. Information from Schupp is depending
on Hartel (14) and Ascher gt 31. (7).

12

41. van Daalen, J. J., J. Meltzer, R. Mulder, and K. Wellinga.

1972.
A selective insectide with a novel mode of action. Naturwissen-
schaften. 7: 312-313.
42. van der Kerk, G. J. M., J. G. A. Juijten. 1954. Investigations

on organotin compounds. III. The biocidal properites of
organotin compounds. J. Appl. Chem. 4: 314-319.

43. van der Kerk, G. J. M., J. G. A. Luijten, J. C. van Egmond, and

J. G. Noltes. 1962. Fortschritte auf dem organozinngebiet.
Chimia. 16: 36-42.

44. Wellinga, K., R. Mulder, and J. J. van Daalen. 1973. Synthesis

and laboratory evaluation of l-(2,6-disubstitued benzoyl)-3-
phenylureas, a new class of insecticides. I. 1-(2,6-Dichloro-
benzoy1)-3-phenylureas. J. Agr. Food Chem. 21: 348-354.
45. Wellinga, K., R. Mulder, and J. J. van Daalen. 1973. Synthesis
and laboratory evaluation of l-(2,6-disubstituted benzoyl)-3-

phenylureas, a new class of insecticides. II.

Influence of the
acyl moiety on insecticidal activity. J. Agr. Food Chem.
21: 993-998.

CHAPTER 2

The Effect of Diflubenzuron [l—(4—chlorophenyl)-3-(2,6-

difluorobenzoyl)urea] on Soybean [Glycine max (L.) Merr.]

 

Photosynthesis, Respiration and Leaf Ultrastructure

ABSTRACT
The effect of the insecticide diflubenzuron [1-(4-chlorophenyl)-
3-(2,6-difluorobenzoyl)urea] on photosynthesis, respiration and leaf

ultrastructure of soybean [Glycine max (L.) Merr., cv. Swift] was ex-

 

amined on plants treated at the second trifoliate leaf stage with 0,
0.067 and 0.269 kg active ingredient/ha of diflubenzuron. Photosynthesis
and respiration were measured with an infrared C02 analyzer in an open
flow system prior to diflubenzuron application and at 4, 24, 48, and

96 hours after treatment with diflubenzuron. Diflubenzuron had no effect
on soybean photosynthesis at any rate examined. Respiration was stimu-
lated by the high rate (0.269 kg/ha) in a transitory manner.

Tissue samples removed from both old and new leaves, 9 days after
diflubenzuron application, were used for the ultrastructure study with
the transmission electron microscope. The lower trifoliate leaves con-
tained more starch grains than the upper being formed after treatment,
but no aberrations or degradation of leaf ultrastructure due to diflu-

benzuron treatment were evident.

INTRODUCTION
l-(2,6-Disubstituted benzoyl)-3-phenylureas are a new class of
selective insecticides with activity of the growth regulator type (1,

2, 3). They have been reported successful in inhibiting biosynthesis

13

14

and deposition of chitin (homopolymer of N-acetyl-D-glucosamine), which
makes up the cuticle-like shell of mature insects (4, 5, 6, 7). Diflu-
benzuron [l-(4-chlor0pheny1)-3-(2,6-dif1uorobenzoyl)urea] has shown
promise in several tests on lepidopterous insects of soybean [Glycine
max (L.) Merr.] (8).

Inhibition of the Hill reaction of photosynthesis is generally
acknowledged to indicate the primary site of action of the substituted
urea herbicides (9, 10). Application of this knowledge to basic re-
search has contributed markedly to the elucidation of our current in-
formation of photosynthetic pathways. Diflubenzuron is chemically
related to the herbicides monuron [3-(p-chlorophenyl)-l,l-dimethylurea]
and dichlobenil (2,6-dichlorobenzonitrile) (Figure l). The latter com-
pound has been used for the preparation of diflubenzuron (3). The afore-
mentioned similarity raises the possibility of phytotoxicity of diflu-
benzuron or of its potential breakdown products.

Ultrastructural examination correlated with biochemical or physio-
logical investigations may provide insight into possible mechanisms
of pesticidal action that the latter alone could not provide (11).

The objectives of this study were to determine whether difluben-
zuron affected soybean photosynthesis, respiration, and leaf ultra-

structure.

MATERIALS AND METHODS

Plant material. Soybeans [Glycine max (L.) Merr., cv. Swift] were seeded

 

5 seeds per 947 m1 pot in greenhouse soil. After emergence the plants

were thinned to 1 plant per pot. When the plants had developed to the

15

 

iii
c Eli
0|
2,6 —iiiciilorolionzonitiilc 3-(p—cliloropiiony|)-l,l-dlmethylurca
dichlobenil monur on

2 —=
°=T

I: —=

‘2

O i

l -(4-chloiophcnyi)-3-(2,6-iiiilucrohenzoyliutca

diflubenzuron

Figure 1. Chemical structures of tile homicides dichlobenil and monuron and
till insecticide diflulicnzum

16

stage where the second trifoliate leaf was about 1/3 expanded, the plants
were treated with diflubenzuron.

Photosynthesis and respiration measurement. Photosynthesis and respira-
tion were measured with an infrared CO2 analyzer in an open flow system,
500cc/min. Measurements were made of the whole plants prior to diflu-
benzuron application. Two plants were then sprayed with water (controls),
two with the proposed use rate or 1X rate of 0.067 kg active ingredient/
ha of diflubenzuron, and two plants with the 4X rate of 0.269 kg a.i./ha
of diflubenzuron. Photosynthesis and respiration were measured 4, 24,
48, and 96 hours after the diflubenzuron application. The data were
calculated as the percent of the original photosynthetic and respiratory
level. Data presented are the means of two experiments.

Transmission electron microscopy study. Tissue samples were removed

from areas adjacent to the midvein near the apex of the center leaflet

of the first trifoliate leaf and from a similar area from the center
leaflet of the third trifoliate leaf 9 days after the application of
diflubenzuron. The third trifoliate leaf had developed since the appli-
cation of diflubenzuron.

These tissues were incubated for 2 hr at 25°C in the following
fixation solution: 5%(v/v) glutaraldehyde, Sorensen's phosphate buffer,
pH 7.2 (12). The material was then washed in the same buffer and fixed
for one hr in 1%(v/v) osmium tetroxide. After washing, the tissues
were stained in 0.5%(w/v) aqueous uranyl acetate for 2 hr. Then, the
material was dehydrated in ethyl alcohol and embedded in eponaraldite

(13). Thin sections were stained with lead citrate and examined in

17

a PHILIPS 300 Transmission Electron Microscope at 60 KV.

RESULTS AND DISCUSSION

Photosynthesis in young soybean plants was not significantly altered
at the 5% level by postemergence application of diflubenzuron (Table
1). After the application of the 4X rate (0.269 kg/ha) of diflubenzuron,
the normal increase in the photosynthetic rate with time was not evident;
however, there were no significant differences at the 5% level. The
data indicates that this substituded urea insecticide is not a photo-
synthetic inhibitor, and if degraded rapidly by plants its metabolites
also appear to be without effect on soybean photosynthesis.

Diflubenzuron degradation studies in model systems showed the
degradative pathways to be almost entirely through cleavage between
the carbonyl and amide group of the urea bridge (14, 15). Some of the
identified metabolites in those studies appeared to be the same to those
identified in metabolism studies of the herbicides monuron (l6) and
the fluoro-analogues of dichlobenil (17) in plants. None of these meta-
bolites has been reported to act as photosynthetic inhibitor. Thus
far the metabolic fate of diflubenzuron in plants has not been elucidated.

Following the application of the 4X rate of diflubenzuron, there
was a significant increase in the respiration rate (Table 2). Two days
or 48 hr after treatment the rate had dropped to the level observed
for the water control, indicating that the effect was transitory. Thus,
only the high rate stimulated respiration and then only in a transitory

manner 0

Examination of the electronmicrographs of the leaf sections of

18

trifoliate leaves, both old and new, from soybean plants 9 days after
treatment with 0, 0.067 and 0.269 kg/ha of diflubenzuron revealed no
obvious abnormalities due to the application of this compound (Figure 2
a, b, c). The lower trifoliate leaves contained more starch than the
upper (Figure 2 b and c), but no aberrations or degradation of ultra-
structure due to diflubenzuron application were evident in either.

The upper trifoliate leaves had deve10ped after the treatment with di-
flubenzuron. Since these were normal, diflubenzuron does not appear

to have any subtil morphogenic effects on soybean leaf cellular structure.

19

Table 1. The effect of diflubenzuron on total photosynthesis of soybean

plants in the second trifoliate leaf stage.

Hours after treatment

 

Treatment 4 24 48 96
(CO2 uptake as % of original level)
Water 93 128 .134 149
0.067 kg/ha 121 123 166 124
0.269 kg/ha 116 107 105 108

 

Table 2. The effect of diflubenzuron on respiration of soybean plants

in the second

trifoliate leaf stage.

Hours after treatment

 

Treatment 43 24 48 96
(02 uptake as % of original level)
Water 117‘ 159 165 180
0.067 kg/ha 156’ 146 175 170
0.269 kg/ha 182* 182 154 156

 

a An asterisk in the first column indicates significant difference

from the initial level at the 5% level of significance.

20

Figure 2.

a. Electronmicrograph of the upper trifoliate leaf of
soybean plant treated 9 days earlier with water (control).

Magnification: 52,000 X, Chloroplast and mitochondria.

b. Electronmicrograph of the upper trifoliate leaf of
soybean plant treated 9 days earlier with 0.269 kg/ha
of diflubenzuron.

Magnification: 21,000 X, chloroplasts showing stacks

of grana and starch grains.

c. Electronmicrograph of the lower trifoliate leaf of
soybean plant treated 9 days earlier with 0.269 kg/ha
of diflubenzuron.

Magnification: 25,000 X, starch-filled chloroplast.

 

21

 

3.

22

REFERENCES

J. J. van Daalen, J. Meltzer, R. Mulder, and K. Wellinga, A new
insecticide with a novel mode of action, Naturwissenschaften
7,312 (1972).

K. Wellinga, R. Mulder, and J. J. van Daalen, Synthesis and
laboratory evaluation of l-(2,6-disubstituted benzoy1)-3-phenyl-
ureas, a new class of insecticides I: 1-(2,6-dichlorobenzoy1)-
3-pheny1ureas, J. Agr. Food Chem. 21,348 (1973).

K. Wellinga, R. Mulder, and J. J. van Daalen, Synthesis and labora-
tory evaluation of 1-(2,6-disubstituted benzoyl)-3-phenylureas,

a new class of insecticides II: Influence of the acyl moiety on
insecticidal activity, J. Agr. Food Chem. 21, 993 (1973).

I. Ishaaya and J. E. Cassida, Dietary TH 6050 alters composition
and enzyme activity of housefly larval cuticle, Pestic. Biochem.
Physiol. 4, 484 (1974).

R. Mulder and M. J. Gijswijt, The laboratory evaluation of two
promising new insecticides which interfere with cuticle deposition,
Pestic. Biochem. Physiol. 4, 737 (1973).

L. C. Post and W. R. Vincent, A new insecticide inhibits chitin
synthesis, Naturwissenschaften 9, 431 (1973).

L. C. Post, B. J. DeJong, and W. R. Vincent, 1-(2,6-disubstituted
benzoyl)-3-phenylurea insecticides: Inhibitors of chitin synthesis,

Pestic. Biochem. Physiol. 4, 473 (1974).

10.

ll.

12.

13.

14.

15.

16.

17.

23

Anonymous. TH 6040, insect growth regulator. Mimeogr. Tech. Inf.
brochure issued by Thompson-Hayward Chemical Company, Kansas.
p. 25. (1974).

F. M. Ashton and A. S. Crafts, "Mode of Action of Herbicides,"
p. 367, John Wiley and Sons, New York, 1973.

J. R. Corbett, "Biochemical Mode of Action of Pesticides," p. 60,
Academic Press, New York, 1974.

J. L. Anderson and W. W. Thomson, The effects of herbicides on the
ultrastructure of plant cells, Residue Reviews, 47, 167 (1973).

G. Gomori, Preparation of buffers for use in enzyme studies, in
"Methods in Enzymology" (S. P. Colowick and N. 0. Kaplan, Eds),
Vol. 1, p. 143, Academic Press, New York, 1955.

C. J. Dawes, "Biological Techniques in Electron Microscopy," p. 72,
Barnes and Noble, New York, 1971.

R" In Metcalf, iPo-Yung Lu, and S. Bowlus, Degradation and environ-
mental fate of l-(2,6-dif1uorobenzoyl)-3-(4-chlorophenyl)urea,

J. Agr. Food Chem. 23, 359 (1975).

L. 0. Ruzo, M. J. Zabik, and R. D. Schuetz, Photochemistry of
bioactive compounds. l-(4-chlor0phenyl)-3-(2,6-dihalobenzoyl)
ureas, J. Agr. Food Chem. 22, 1106 (1974).

B. S. Frear, H. R. Swanson, and F. S. Tanaka, Herbicide metabolism
in plants, in "Recent Advances in Phytochemistry" (V. C. Runeckles
and T. C. Tso, Eds) Vol. 5, p. 225, Academic Press, New York, 1972.

A. VerloOp and W. B. Nimmo, Absorption, translocation and metabolism

of dichlobenil in bean seedlings, Weed Res. 9, 357 (1969).

CHAPTER 3

The Effect of Fentin Hydroxide (Triphenyltin Hydroxide) on

Soybean [Glycine max (L.) Merr.] and Rice (Oryza sativa L.)

 

 

Photosynthesis, Respiration and Leaf Ultrastructure

ABSTRACT

Soybean [Glycine max (L.) Merr., cv. Swift] plants at the second

 

trifoliate leaf stage and rice (Oryza sativa L. cv. Starbonnet) plants

 

25 cm tall were treated with 0, 0.56 and 2.24 kg/ha of fentin hydroxide
(triphenyltin hydroxide) to determine the effect of this fungicide on
photosynthesis, respiration, and leaf ultrastructure. Photosynthesis
and respiration were measured with an infrared C02 analyzer in an open
flow system prior to fentin hydroxide application and at 4, 24, 48, and
96 hours after treatment with fentin hydroxide. No significant detri-
mental effects on photosynthesis or respiration were evident in either
soybean or rice through 96 hours after treatment.

Tissue samples from soybean and rice plants, 9 days after fentin
hydroxide application, examined for ultrastructure changes with the
transmission electron microscope showed no effect due to the fungicide

treatment.

INTRODUCTION
The fungicide fentin hydroxide (triphenyltin hydroxide) has been

used in Europe to control the fungi Phytophthora infestans on potato

 

(Solanum tuberosum L.), Cercospora beticola on sugar beet (Beta vulgaris

 

 

L.) and Septoria apii on celery (Apium graveolens L.) (1, 2, 3, 4, 5).

 

 

Application of fentin hydroxide for the control of fungus diseases on

24

25

soybean [Glycine max (L.) Merr.] and rice (Oryza sativa L.) has also

 

 

been suggested (5).

Interference with respiration due to marked inhibition of oxidative
phosphorylation in rat liver mitochondria caused by triphenyltin com-
pounds is well documented (6). The same mode of action has been assumed
for the antifungal effect (7), but this is not certain since no experi-
ments have been done with pests (8).

Phytotoxicity of triphenyltin compounds limits their application
in agriculture as fungicides (l, 2, 3). 0n the other hand, the possible
growth promoting action of triphenyltin fungicides in certain plants is
of interest. Increases in yields of sugar beet and celery after treat-
ment with fentin acetate (triphenyltin acetate) have been reported (1).
Since absorption and translocation studies using 113Sn-triphenyltin
acetate indicated no absorption either by leaves or roots in both sugar
beet and celery (9), some contribution is evident. Since triphenyltin
acetate is easily hydrolyzed to triphenyltin hydroxide (3) and the growth
regulating activity of the fungicide triarimol [a-(2,4-dichlorophenyl)-
a-phenyl-S-pyrimidinemethanol] has been established (10, ll, 12), which
structurally resembles fentin hydroxide (Figure l), the growth promoting
effect of triphenyltin fungicides bears reexamination.

The purpose of this study was to determine whether triphenyltin
hydroxide affected photosynthesis, respiration, or leaf ultrastructure
in soybean and rice plants.

MATERIALS AND METHODS

Plant material. Soybeans [Glycine max (L.) Merr., cv. Swift] and rice

 

 

26

0|" Oil
811
CI
l
iiipiieiiyltin hydroxide a (2,4-dicliloroplienyli—a-plieiiyl-S-pytimidinemetiiancl
Fentin hydroxide Triarimol

’Figure '|. Chemical :tnictures oi the fungicides fentin hydroxide and triaiiiiiel

27

(Oryza sativa L., cv. Starbonnet) were seeded 5 and 40 seeds per 947 ml

 

pot, respectively, in greenhouse soil. After emergence the plants were
thinned to 1 plant per pot for soybeans and 20 plants per pot for rice.
When the soybean plants had developed to the stage where the second tri-
foliate leaf was about 1/3 expanded and the rice was about 25 cm tall,
the plants were used for this study.
Photosynthesis and respiration measurement. Photosynthesis and reSpira-
tion rates were measured with an infrared C02 analyzer in an open flow
system, 500 cc/min. Measurements were made of the whole plants prior
to fentin hydroxide treatment. Two plants were then treated with water
(controls), two with the proposed use rate or 1X rate of 0.56 kg active
ingredient/ha of fentin hydroxide, and two plants with the 4X rate of
2.24 kg a.i.Iha of fentin hydroxide. Photosynthesis and respiration were
measured at 4,24, 48, and 96 hours after the application of fentin hy-
droxide. The data were calculated as the percent of the original and
respiratory level. Data presented are the means of two experiments.
Transmission electron microscopy study. Nine days after fentin hydroxide
application, soybean tissue samples were removed from area adjacent to
the midvein near the apex of the center leaflet of the first trifoliate
leaf and from a similar area from the center leaflet of the third trifoli-
ate leaf. The latter had developed since the application of fentin hydroxide.
Tissue samples from rice leaves were obtained from an area 6 cm away from
the leaf tip 9 days after the treatment with this fungicide.

These tissues were incubated for 2 hr at 25°C in the following fix-
ation solution: 5%(v/v) glutaraldehyde, Sorensen's phosphate buffer,

pH 7.2 (13). The material was then washed in the same buffer and fixed

28

for one hr in 1%(v/v) osmium tetroxide. After being washed, the tissues
were stained in 0.5%(w/v) aqueous uranyl acetate for 2 hr. Then the
material was dehydrated in ethyl alcohol and embedded in eponaraldite
(14). Thin sections were stained with lead citrate and examined in a

PHILIPS 300 Transmission Electron Microscope at 60 KV.

RESULTS AND DISCUSSION

The treatment with fentin hydroxide had no visual effects on soy-
bean. However, rice plants receiving the 0.56 kg/ha or 1X rate appeared
noticeably taller and greener than the control or the plants treated with
the 4X rate. There were no visual differences between the control rice
plants and the 4X fentin hydroxide treated plants. This effect on plant
color appears similar to that observed by Shive and Sisler for triarimol
on beans (12). Attempts to duplicate the result in greenhouse studies
were unsuccessful. Whether this failure was due to inactivation of the
compound by air or by light, it is not known.

Fentin hydroxide did not inhibit soybean photosynthesis (Table 1).
However, a significant increase of the photosynthetic rate was evident
4 hr after application for the plants treated with the 4X rate of 2.24
kg/ha of fentin hydroxide as compared to the control plants treated
with water. The low value for the water control 4 hr after treatment is
indicative of partial stomatal closure at that measurement. Soybean
respiration, rice photosynthesis, and rice respiration were not sig-
nificantly affected by fentin hydroxide up to 96 hr after treatment
(Tables 2, 3, and 4). Thus, the aforementioned possible effect of fentin

hydroxide on rice growth and coloration did not appear to be due to a

29

rapid stimulation of photosynthesis or inhibition of respiration. _Fentin
hydroxide did not appear to have any deleterious effect on soybean or
rice photosynthesis and respiration.

Electronmicrographs of control and fentin hydroxide treated rice
plants are shown in Figure 2 (a, b, c). Examination of numerous sections
revealed no differences between control plants and plants receiving 0.56
kg/ha of fentin hydroxide. A certain amount of grana disarray was evident
in a section from rice plants treated with the 4X rate of 2.24 kg/ha (Fig-
ure 2c). This disarray is characteristic of a number of stresses and
occurs during the fixation process according to Dr. G. Hooper (15). Whether
the stress causing this particular effect was drought, insect, disease,
temperature, or fentin hydroxide treatment cannot be assigned with any
degree of certainty.

Electronmicrographs of control and fentin hydroxide treated soybean
plants are shown in Figures 3 (a, b) and 4 (a, b). Fentin hydroxide
treatments did not appear to have any detrimental effect on cellular
ultrastructure of soybean tissues. In Figure 3b a certain amount of grana
disarray is evident. Again, these are stresses induced and occur during
fixation. The contributing stresses cannot be identified at this point.
It is doubtful that fentin hydroxide is responsible since the 4X appli-
cation rate failed to produce this effect as seen in Figure 4a, and it
was observed in only one plant receiving the 0.56 kg/ha rate of fentin

hydroxide.

30

Table l. The effect of fentin hydroxide on total photosynthesis of soybean

plants in the second trifoliate leaf stage.

Hours after treatment3

Treatment 4 24 48 96

 

(C02 uptake as % of original level)

Water 74 a 92 abc 96 abc 88 abc
0.56 kg/ha 99 abc 103 abc 101 abc 79 ab
2.24 kg/ha 110 c 117 c 113 c 107 bc

 

3Means with similar letters are not significantly different at the

5% level by Duncan's multiple range test.

Table 2. The effect of fentin hydroxide on respiration of soybean plants

in the second trifoliate leaf stage.

Hours after treatment

Treatment 4 24 48 96

 

(02 uptake as % of original level)
Water 120 117 125 119
0.56 kg/ha 153 110 110 105

2.24 kg/ha 147 101 120 118

 

31

Table 3. The effect of fentin hydroxide on total photosynthesis of rice plants.

Hours after treatment

Treatment 4 24 48 96

 

(CO2 uptake as % of original level)

Water 89 91 87 92
0.56 kg/ha 93 89 99 107
2.24 kg/ha 84 99 98 100

 

Table 4. The effect of fentin hydroxide on respiration of rice plants.

Hours after treatment

Treatment 4 24 48 96

 

(02 uptake as % of original level)
Water 114 99 95 104
0.56 kg/ha 107 93 88 100

2.24 kg/ha 99 114 93 122

 

32

Figure 2

a. Electromicrograph of rice leaf tissue from plant treated
9 days earlier with water (control).
Magnification: 32.000 X, dark lipid bodies, grana,

starch grains, plasmadesmata.

b. Electronmicrograph of rice leaf tissue from plant treated
9 days earlier with 0.56 kg/ha of fentin hydroxide (1X rate).
Magnification: 21.000 X, chloroplasts, starch grains,

plasmalemma, grana.

c. Electronmicrograph of rice leaf tissue from plant treated
9 days earlier with 2.24 kg/ha of fentin hydroxide (4X rate).
Magnification: 10,000 X, lipid bodies, starch grains,

grana.

33

 

34

Figure 3.
a. Electronmicrograph of soybean leaf tissue from trifoliate
lower leaf treated 9 days earlier with water (control).
Magnification: 52.000 X, ferritin in chloroplast,

lipid body, grana.

b. Electronmicrograph of soybean leaf tissue from the upper
trifoliate leaf of plant treated 9 days earlier with
0.56 kg/ha of fentin hydroxide (1X rate).

Magnification: 12,500 X, chloroplast.

 

 

 

Figure 4.

a.

36

Electronmicrograph of soybean leaf tissue from the upper
trifoliate leaf of plant treated 9 days earlier with
2.24 kg/ha of fentin hydroxide (4X rate).

Magnification: 12,500 X, grana.

Electronmicrograph of soybean leaf tissue from the lower
trifoliate leaf of plant treated 9 days earlier with
2.24 kg/ha of fentin hydroxide (4X rate).

Magnification: 21,000 X, cell wall, mitrochondria,

chloroplast, grana, starch grains.

 

 

38

REFERENCES

L. R. S. Ascher and S. Nissim, Organotin compounds and their poten-
tial use in insect control, World Rev. Pest Control 3, 189 (1964).

K. Hartel, Triphenyltin compounds, Agr. Vet. Chem. 3, 19 (1962).

A. Kaars Sijpestein, J. G. A. Luijten, and G. J. M. van der Kerk,
Organometalic Fungicides, in "Fungicides" (D. C. Torgeson Ed.),
Vol. 2,80. 331, Academic Press, New York, 1969.

A. J. Pieters, Triphenyltin hydroxide, a fungicide for the control
of Phytophthora infestans on potatoes and some other fungus diseases,
Proc. Brit. Insecticide Fungicide Conf. Brighton, England, 2, 461
(1962).

Anonymous. Du-Ter. Mimeogr. Tech. Inf. brochure issued by Philips-
Duphar B. V., The Netherlands. (ca 1973), pp 7.

M. Stockdale, A. P. Dawson, and M. J. Selwyn, Effects of trialkyltin
and triphenyltin compounds on mitochondrial respiration, Eur. J.
Biochem. 15, 342 (1970).

A. Kaars Sijpestein, F. Rijkens, J. G. A. Luijten, and L. C. Willemsens,
0n the antifungal and antibacterial activity of some trisubstituted
organogermanium, organotin and organolead compounds, Antonie van
Leeuwenhoek, J. Microbiol. Serol. 28, 346 (1962).

J. R. Corbett, "Biochemical Mode of Action of Pesticides," p. 48,
Academic Press, New York, 1974.

J. Herok and H. Gotte, Radiometrische unterrsuchungen uber das
verhalten von treiphenylzinnacetat in pflanze und tier, Int. J.

Appl. Radiat. 14, 461 (1963).

39

R. P. Covey, Jr., Orchard evaluation of two new fungicides for the
control of apple powdery mildew, Plant Disease Rept. 55, 514 (1971).
N. N. Ragsdale and H. D. Sisler, Inhibition of ergosterol synthesis

in Ustilago maydis by the fungicide triarimol, Biochem. Biophys.

 

Res. Commun. 46, 2048 (1972).
J. B. Shive, Jr. and 1L.D. Sisler, Effects of ancymidol (a growth
retardant) and triarimol (a fungicide) on the growth, sterols and

gibberellins of Phaseolus vulgaris (L.), Plant Physiol. 57, 640 (1976).

 

G. Gomori, Preparation of buffers for use in enzyme studies, in
Methods in Enzymology," (S. P. Colowick and N. 0. Kaplan, Eds),
Vol. 1, p. 143, Academic Press, New York 1955.

C. J. Dawes, "Biological Techniques in Electron Microscopy," p. 72,
Barnes and Noble Inc., New York, 1971.

G. Hooper. 1976. Pesticide Research Center, Michigan State Uni-

versity, East Lansing. Personal communication.

CHAPTER 4
The Effect of Diflubenzuron [l-(4-chlorophenyl)-3-
(2,6-difluorobenzoy1)urea] and Fentin Hydroxide (triphenyltin

hydroxide) on Soybean [Glycine max (L.) Merr.] Seed Quality

 

ABSTRACT

Seeds of five soybean [Glycine max (L.) Merr.] cultivars, namely

 

'Coker 102', 'Cobb', 'Tracy', 'Ransom' and 'Bragg', obtained from plots
treated with 0, 0.035, 0.069, and 0.14 kg/ha of diflubenzuron [l-(4-
chlorophenyl)-3-(2,6-difluorobenzoy1)urea] for 'Coker 102', 0 and two
applications of 0.56 kg/ha of fentin hydroxide (triphenyltin hydroxide)
for 'Cobb', 'Tracy', and 'Ransom' and 0, 0.266, 0.532, and 1.064 kg/ha

of fentin hydroxide, single and in combination with 0.067 kg/ha of di-
flubenzuron for 'Bragg' were tested for their quality. Chemical com-
position expressed in terms of lipid, protein, and carbohydrate content
was determined by Nuclear Magnetic Resonance (NMR), Kjeldahl and Anthrone
procedures. No effect of either pesticide on soybean seed lipid, protein,
and carbohydrate content was evident with the exception that fentin
hydroxide treated'Ransom and one set of treated 'Bragg' soybeans

showed greater and lower carbohydrate content respectively.

Seed viability and seedling vigor were determined by applying a
regular germination test, an accelerated aging test, and a cold test.
Neither fentin hydroxide nor diflubenzuron had any effect on the pro-
duction of healthy seedlings. In several seed tests fentin hydroxide
increased the percent of healthy seedlings following the accelerated

aging stress.

40

41

INTRODUCTION

Seed quality can be divided into two basic parameters: the chemi-
cal composition and the quality of the seed in terms Of germination
and production of a vigorous, healthy seedling. Chemical composition
of seeds can be affected in a detrimental manner by pesticides which
cause serious deleterious effects on plant growth (1). Production of
vigorous seedlings may be controlled by the chemical composition of
the seed as well as by hormones, inhibitors, and pesticide residue
levels (2). Pesticides which affect seed moisture content or micro-
organisms may also influence the health of future seedlings.

Diflubenzuron [l-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea]
is a substituted urea with growth regulator insecticidal activity and
has shown promise in lepidopterous insect control for soybean [Glycine
max_(L). Merr.] (3).
A Fentin hydroxide (triphenyltin hydroxide) is a fungicide which
has been extensively used in Europe to control fungus diseases on many
crops (4, 5, 6, 7, 8). It has also been suggested for control of diseases

on soybean and rice (Oryza sativa L.) (8).

 

The objectives of this study were to evaluate the influence of
diflubenzuron and fentin hydroxide on soybean seed quality, examining

both chemical composition and production of vigorous seedlings.

V

MATERIALS AND METHODS
Seed material. Seeds of five different soybean cultivars, namely 'Coker'

102', 'Cobb', 'Tracy', 'Ransom', and 'Bragg', were obtained from plots

42

located in Florence, South Carolina. Cultivar 'Coker 102' had been
treated during the growing season with 0, 0.035, 0.069, and 0.14 kg/ha
of diflubenzuron, cultivars 'Cobb', 'Tracy', and 'Ransom' had been treated
with 0 and double application of 0.56 kg/ha of fentin hydroxide, and
cultivar 'Bragg' had been treated with O, 0.266, 0.532, and 1.064 kg/ha
of fentin hydroxide, single and in combination with 0.067 kg/ha of di-
flubenzuron.

Testing the seed viability and seedling vigor. The effects of these
pesticide treatments on seed viability and seedling vigor were evalu-
ated as follows. A regular germination test was run on four lots of
100 seeds each from each sample. The seeds were placed on moist paper
towels at 25°C for 7 days. Only healthy germinated seeds were recorded.
Viability and vigor were also tested by stressing the seeds.

In the accelerated aging Egg; (9) four lots of 100 seeds each from
each sample were subjected to 42°C for 3 days at 100% relative humidity.
They were then transferred to the conditions for the regular germination
test and the number of healthy seedlings 7 days later reported as percent

germination. A second manner of stressing the seed, the cold test (9),

 

was accomplished by seeding in moist unsterilized soil, 50 seeds per

16 oz cup, and placing them in a refrigerator at 10°C for 5 days.

The cultures were then transferred to a control environment with a 16-
hr day at 30°C and 8-hr night at 20°C for 6 days. Percent germination,
emergence, and seedling height were recorded.

Testingythe chemical composition of seeds. The effect of the pesticide

treatments on seed composition was evaluated in this study by determining

43

the lipid, protein, and the carbohydrate content of the seeds. The

lipid content was determined by nuclear magnetic resonance (NMR) pro-

cedures at the University of Illinois. The protein content was determined

by Kjeldahl procedure and the carbohydrate content was determined by

the anthrone method adapted from.Yemm.and Willis (10). The data for

lipid and protein content are the means of two determinations. The

data for the carbohydrate content are the means of four determinations.
The moisture content of the samples was 4.4:0.3. The reason for

the low level of moisture was the storage of the seeds during the winter

time.

RESULTS AND DISCUSSION

The germination percentages for the pesticide-treated and the un-
treated seeds were low for the 'Coker 102', 'Tracy', and 'Ransom' vari-
eties (Tables 1 and 20. The varieties 'Cobb' and 'Bragg' showed higher
germination levels (Tables 3 and 4). Since only normal healthy seed-
lings were recorded, the actual number of seeds which germinated could
have been considerably higher. Neither fentin hydroxide nor diflubenzuron
had any marked negative effect on the viability parameters measured.
Absence of any negative effect on germination of seeds of French beans,
tomato, sugar beet, and oats and as well of a large variety of weeds
after preemergence application of DUl9lll [l-(2,6-dichlorobenzoyl)-
3-(3,4-dichlorophenyl)urea], another substituted urea insecticide, has
also been reported (11). The data in the Tables 2 and 3 indicate that
fentin hydroxide in some instances markedly increased the percent of

healthy seedlings following the accelerated aging stress. Reduction

44

of microorganisms associated with the seed by the earlier fungicidal
action of fentin hydroxide during the growing season may account for
the observed results.

' Cultivar differences were observed in the lipid and protein con-
tent of the soybean seedlings, but no effect of either pesticide tested
on soybean seed lipid and protein content was evident (Tables 5 to 8).

Fentin hydroxide and diflubenzuron had no effect on carbohydrate
content with the exception that fentin hydroxide treated 'Ransom' and
one set of treated 'Bragg' soybeans showed greater and lower carbohy-

drate content, respectively (Tables 5 to 8).

45

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53

‘ REFERENCES

National Academy of Sciences. 1968. Principles of plant and
animal pest control. Vol. 6. Effects of Pesticides on Fruit
and Vegetable Physiology. p. 90.

Mayer, A. M. and A. Poljakoff-Mayber. 1975. Dormancy, ger-
mination, inhibition and stimulation. .ln "The Germination of
Seeds," 2nd ed. Pergamon Press, Oxford. p. 192.

Anonymous. 1974. TH 6040, insect growth regulator. Mimeogr.
Tech. Inf. brochure issued by Thompson-Hayward Chemical Co.,
Kansas. p. 25.

Ascher, L. R. S. and S. Nissim. 1964. Organotin compounds and
their potential use in insect control. World Rev. Pest Control,

Hartel, K. 1962. Triphenyltin compounds. Agr. Vet. Chem., 3:19-24.

Kaars Sijpestein, A., J. G. A. Luijten, and G. J. M. van der Kerk.
1969. Organometalic fungicides. IE_D. C. Torgeson (ed.) "Fungicides."
Academic Press, N.Y., Vol. 2, pp 331-366. A

Pieters, A. J. 1962. Triphenyltin hydroxide, a fungicide for the
control of Phytophthora infestans on potatoes and some other fungus
diseases. Proc. Brit. Insecticide Fungicide Conf., Brighton, England
2:461—470.

Anonymous. ca 1973. Du-Ter. Mimeogr. Tech. Inf. brochure issued
by Philips-Duphar B. V., The Netherlands, p. 7.

Copeland, L. O. 1976. "Principles of Seed Science and Technology."

Burgess Publishing Co, p. 369, chaptef 7.

 

 

159a 1......1 7. 3i. ,K‘K Ivy-‘14. .3353

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54

10. Yemm, E. W. and A. J. Willis. 1954. The estimation of carbohy-
drates by anthrone. Biochem. J. 57:508-514.

11. van Daalen, J. J., J. Meltzer, R. Mulder, and K. Wellinga. 1972.
A selective insecticide with a novel mode of action. Naturwissen-

schaften. 7:312-313.

CHAPTER 5

SUMMARY AND CONCLUSIONS

Photosynthesis, respiration, and leaf ultrastructure of soybean
and rice plants appeared to be unaffected by the insecticide diflu-
benzuron and the fungicide fentin hydroxide. The only exception was
the transitory stimulation of soybean respiration caused by diflu-
benzuron at the rate of 0.269 kg/ha. The positive effect of fentin
hydroxide at the rate of 0.56 kg/ha on rice height and coloration,
observed in one study, it is of interest but more work is needed before
definite conclusions can be made.

Neither diflubenzuron nor fentin hydroxide showed any negative
effect on the production of healthy seedlings. Moreover, in some seed
tests fentin hydroxide increased the percent of healthy seedlings,
following the accelerated aging stress.

Lipid, protein, and carbohydrate content of soybean seeds from
five different cultivars were unaffected by both pesticides with the
exception that fentin hydroxide treated "Ransom" and one set of treated
"Bragg" soybeans showed greater and lower carbohydrate content respec-
tively.

In conclusion, it can be said that both diflubenzuron and fentin
hydroxide appeared to have no detrimental effects on' the physiological

functions of soybean and rice examined in this study.

55

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