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thesis entitled

MODE OF ACTION, POSTINFECTION CONTROL CHARACTERISTICS
AND SYSTEMIC PROPERTIES OF SELECTED TRIAZOLE
AND IMIDAZOLE FUNGICIDES FOR USE
AGAINST VENTURIA INQEUQALIS

presented by

 

Randall David Kelley-

has been accepted towards fulfillment
of the requirements for

Master of Sciencgdegreein Botany & Plant Pathology

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MODE OF ACTION, POSTINFECTION CONTROL CHARACTERISTICS
AND SYSTEMIC PROPERTIES OF SELECTED TRIAZOLE
AND IMIDAZOLE FUNGICIDES FOR USE
AGAINST VENTURIA INAEQUALIS

 

By

Randall David Kelley

A THESIS

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1980

c: //é W

ABSTRACT

MODE OF ACTION, POSTINFECTION CONTROL CHARACTERISTICS
AND SYSTEMIC PROPERTIES OF SELECTED TRIAZOLE
AND IMIDAZOLE FUNGICIDES FOR USE
AGAINST VENTURIA INAEQUALIS

 

By
Randall David Kelley

Three fungicides (CGA-6425l, bitertanol, and phenapronil),
for use against apple scab, were evaluated for postinfection control
and effect on lesion development. In greenhouse studies, fungicides
gave complete control when applied up to 3 days after inoculation,
although chlorotic lesions were noted. In field studies, CGA-6425l
and bitertanol gave control if applied within 3 days after an infec-
tion period or if two sprays were applied 1 wk apart, starting either
2 days before sporulation was predicted or 2 days after lesions were
observed. Scanning electron microscope examination of lesions showed
fungicides prevented conidia from maturing. A positive correlation
existed between delay time and percent mature conidia. Attempts to
germinate conidia from sprayed lesions were unsuccessful. SEM exami-
nation of chlorotic lesions revealed subcuticular hyphae and deformed
surface growth. Isolations from washed chlorotic lesions were suc-
cessful. x-ray autoradiographs and scintillation counts showed both
upward and downward movement, with bitertanol least systemic. Equi-

librium of transcuticular movement required from l2 hr to 3 days.

TABLE OF CONTENTS

LIST OF TABLES .
LIST OF FIGURES

GENERAL INTRODUCTION AND LITERATURE REVIEW
LITERATURE CITED
PART I
POSTINFECTION CONTROL OF APPLE SCAB WITH
CGA-64251, BITERTANOL, AND PHENAPRONIL
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LITERATURE CITED
PART II
VOLATILITY AND SYSTEMIC PROPERTIES IN APPLE
OF CGA-64251, BITERTANOL, AND
PHENAPRONIL FUNGICIDES
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION

LITERATURE CITED

ii

Page

iv

13
35
38

41
43
45
57
59

APPENDICES

A.

SCANNING ELECTRON MICROGRAPHS OF LESIONS SPRAYED
WITH CGA- 64251 OR BITERTANOL . . .

SCANNING ELECTRON MICROGRAPHS OF CHLOROTIC LESIONS
SPRAYED WITH CGA- 64251 OR BITERTANOL. .

TRANSMISSION ELECTRON MICROGRAPHS OF PALISADE CELLS

OF APPLE

Page
60

61

76

89

Table

LIST OF TABLES

Page

After-infection control of scab on potted apple trees
in the greenhouse by fungicides applied at various
times after inoculation . . . . . . . . . . 16

Control of scab on McIntosh apple with protective

and postinfection eradicant treatments of CGA- 6425l

l0% W applied in dilute sprays at 18. 7 pg a. i. /ml

under orchard conditions in 1979.. . . . 22

Control of scab on McIntosh apple with protective

and postinfection eradicant treatments of CGA-6425l

l0% W l8.7 ug a.i./ml and bitertanol 50% W 299.6 pg

a.i./ml applied in dilute sprays under orchard

conditions in l980 . . . . . . . . . . . . 26

iv

Figure

6A.

GB.

LIST OF FIGURES

PART I

Germinated conidium of Venturia inaequalis on filter
paper saturated with l2.5 pg of CGA-64251

Apple leaf, with chlorophyll removed, stained with
basic fuchsia to detect subcuticular hyphae (A) and
occasional surface growth (8) of Venturia inaequalis
in chlorotic lesions . . .

Schedule of sprays applied in l979 in relation to
predicted infection periods . . . .

Schedule of sprays applied in 1980 in relation to
predicted infection periods . . . .

Scanning electron micrographs of typical lesions and
conidia of Venturia inaequalis from apple trees
sprayed with bitertanol 50% W 299. 6 pg a. i. O/ml and
CGA-6425l l0% W 18. 7 pg a. 1. /ml . . .

 

Cross-sections of apple leaves taken from unsprayed
trees

Cross-sections of apple leaves taken from trees
sprayed with CGA-64251 on a 7-day schedule
PART II

Radioactive fungicide remaining on cover glasses at
various times after application . . . .

Distribution of the portion of radioactive fungicide
which had moved out of treated leaves, 7 days after
application . . . . . . . . .

Transcuticular movement of radioactive fungicide into
apple leaves . . . .

Page

l4

I8

20

24

27

31

33

46

48

49

Figure

4A.

4B.

4C.

Al.

A2.

A3.

A4.

A5.

A6.

A7.

X-ray film autoradiograph showing the pattern of
movement and distribution of radioactive fungicides
in apple shoots 7 days after application of phena-
pronil to a single leaf (darkest leaf)

X-ray film autoradiograph showing the pattern of
movement and distribution of radioactive fungicides

in apple shoots 7 days after a plication. of CGA- 64251

to a single leaf (darkest leaf . .

X-ray film autoradiograph showing the pattern of
movement and distribution of radioactive fungicides
in apple shoots 7 days after application of
bitertanol to a single leaf (darkest leaf)

APPENDIX A

Lesion (top, X390) of Venturia inaequalis from
untreated trees, showing dense sporulation and normal
conidia (bottom, X4800)

 

Lesion (top, X390) of Venturia inaequalis from pre-
symptom treatment of CGA-64251, taken 2 days after
second spray was applied . . . . . . .

 

Lesion (top, X390) of Venturia inaequalis from pre-
symptom treatment of bitertanol, taken 2 days after
second spray was applied

 

Lesion (top, X440) of Venturia inaequalis from post-
symptom treatment of CGA-6425l, taken 2 days after
first spray was applied . . . . . . .

Lesion (top, X390) of Venturia inaequalis from post-
symptom treatment of bitertanol, taken 2 days after
first spray was applied . . . . . . .

 

Lesion (top, X390) of Venturia inaequalis from post-
symptom treatment of CGA-6425l, taken 2 days after
second spray was applied . . . . . . .

 

Lesion (top, X390) of Venturia inaequalis from post-
symptom treatment of bitertanol, taken 2 days after
second spray was applied . . . . . . .

 

vi

Page

SI

53

55

62

64

66

68

70

72

74

Figure

81.

82.

B3.

B4.

85.

86.

CI.

C2.

APPENDIX B

Chlorotic lesion (top, X390) and deformed surface
growth (bottom, X3040) of Venturia inaequalis from
l4-day treatment of CGA-64251, taken on l0 June

 

Chlorotic lesion (top, X270) of Venturia inaequalis
from 14-day treatment of bitertanOT, taken l0 June

 

Chlorotic lesion (top, X390) of Venturia inaequalis
from after-infection treatment of CGA-6425l, taken
l0 June . . . . . . . . . . . . . . .

 

Chlorotic lesion (top, X270) of Venturia inaequalis
from after—infection treatment of bitertanol, taken
10 June . . . . .

 

Chlorotic lesion (top, X390) and deformed surface
growth (bottom, Xl890) of Venturia inaequalis, from

 

presymptom treatment of bitertanol, taken on 10 June .

Chlorotic lesion (top, X400) of Venturia inaequalis
from postsymptom treatment of bitertanol, taken
l0 June . . . . . . . . . .

 

APPENDIX C

Transmission electron micrographs of longitudinal .
section of apple palisade cells taken from leaves of
7-day schedule of CGA-6425l

Transmission electron micrographs of longitudinal

section of apple palisade cells taken from leaves
of unsprayed trees . . . . . . .

vii

Page

77

79

81

83

85

87

9O

9O

92

GENERAL INTRODUCTION AND LITERATURE REVIEW

Control of apple scab, caused by Venturia inaequalis (Cke.)
Wint., has, in recent years, relied heavily on two fungicides;
benomyl and dodine. Numerous reports of resistance to benomyl,

involving many organisms (10), including 1, inaequalis, indicate

 

that its future as an effective fungicide is limited, and many
orchards already have levels of resistance which make its use
impossible. Dodine resistant strains of the apple scab fungus
exist throughout the northeastern United States (ll, l4, 18). The
loss of these chemicals is particularly distressing because benomyl,
and to a lesser extent dodine, could be applied effectively after
infection had occured, thus facilitating their use in pest manage-
ment schemes.

During the past l2 yr, several fungicides which inhibit
ergosterol biosynthesis and membrane function have been tested for
apple scab control. The pyrimidine fungicides triarimol, fenarimol
and nuarimol and the piperazine fungicide triforine have been
studied most extensively, and were found to control apple scab when
applied after the onset of infection (6, 8, l3, l6, l7). Recently,
certain triazole and imidazole fungicides, possessing protective
and curative activities against many Ascomyceteous fungi, and which
inhibit ergosterol biosynthesis in a manner similar to the pyrimi—
dine fungicides, have become available for testing (l, 2, 20).

l

Two triazole fungicides: CGA-64251 (l-((2-(2,4-Dichloro-
phenyl)-4-ethy1-1,3-dioxolan-2-y1)methy1)-1fl:l,2,4-triazole) and
bitertanol, and the imidazole fungicide phenapronil were chosen for
study, and an integrated set of experiments was initiated to assess
the extent of their postinfection control abilities, and to examine
their effects on apple scab lesion and conidial development and
morphology. In addition, experiments were undertaken to determine
characteristics of movement and localization within the plant; as
the ability of a fungicide to enter the plant and the extent and
speed of its movement after entry determine, to large extent, the
optimal application conditions and the level and type of control
to be expected. Solel and Edgington and Edgington et a1. (5, 19)
have studied transcuticular movement of fungicides on apple leaves,
and Interieri (9) and Fuchs and Ost (6) have reported the use of
x-ray film autoradiographs for visualization of distribution and
uptake.

Nusbaum (12), Preese (15), Corlett et al. (3), and Hammill
(7) have produced excellent studies of germination, infection, and
lesion development of the normal untreated fungus, and a more
recent report by Corlett et a1. (4) dealt with the morphological

effects of benomyl on y, inaequalis. Their description of prepara-

 

tion of specimens and the photographs supplied with the article
suggest that the collapse of surface structures which they noted
is more likely an artifact of preparation than a direct effect of
the fungicide. However, 75-90% of annellides in treated lesions

had ceased development at the primary conidium stage with rounded

immature spores, similar to structures noted in the present study.
A comparable morphological study on fungicide treated Penicillium
of citrus was reported by VanGestel (22).

Szkolnik (21) recently discussed the methods and value of
various approaches to fundamental fungicide research. His analysis
of the various possible relationships between spray timing and the
stages of disease development was used as the logical basis for
portions of the present study.

The data from this integrated set of experiments make it
possible to determine the way in which these new fungicides might

be most effectively incorporated into pest management programs.

LITERATURE CITED

Buchel, K. H., W. Kramer, W. Meiser, W. Brandes, P. E.
Frahberger, and H. Kaspers. 1979. Chemistry and
properties of phenyl-triazole-methanes, a highly
active class of new azole fungicides. Abstract 475
in Abstracts of Papers, IXth Int. Congr. of Plant
Protection, Washington, D.C.

Carley, H. E. 1979. A new multiple use fungicide: a-butyl-
a-phenyl-1H-imidazole-l-propanenitrile. Phytopathology
69:1023.

Corlett, Michael, James Chang, and E. G. Kokko. 1976. The
ultrastructure of the Spilocaea state of Venturia
inaequalis in vivo. Can. J. Microbiol. 22:1144-1152.

 

Corlett, Michael, and R. G. Ross. 1979. Morphology of
Spilocaea pgmi on untreated and benomyl-treated
McIntosh apple leaves. Can. J. of P1. Path. 1:79-84.

 

Edgington, L. V., P. P. 0. deWildt, K. Jacques, and J. Psutka.
1976. The study of transcuticular movement of fungi-
cides, in Proceedings: Apple and pear scab workshop,
pp. 32-35, A. L. Jones and J. D. Gilpatrick, eds.,

N.Y. Agric. Exp. Stn., Special Report 28, 38 pp.

Fuchs, A., and W. Ost. 1976. Translocation, distribution and
metabolism of triforine in plants. Archives of Environ-
mental Contamination and Toxicology 4:30-43.

Hammill, T. M. 1973. Fine structure of annellophores IV.
Spilocaea pomi. Trans. Br. Mycol. Soc. 60:65-68.

Hoch, H. C., and M. Szkolnik. l979. Viability of Venturia
inaequalis in chlorotic flecks resulting from fungicide
application to infected Malus leaves. Phytopathology
69:456-462.

 

Interieri, Cesare, and Kay Ryugo. 1972. Enhanced penetration
and translocation of a fungicide following foliar treat-
ment of seedlings with growth retardants. Plant Dis.
Rep. 56:590-592.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Jones, A. L., and G. R. Ehret. 1976. Tolerance of fungicides
in Venturia and Monilinia of tree fruits, in Symposium:
Resistance of Plant Pathogens to Chemicals. The American
Phytopathological Society, Inc., St. Paul. MN. 52 pp.

Kappas, A., and S. G. Georgopoulos. 1970. Genetic analysis
of dodine resistance in Nectria haematococca (syn.
Hypomyces solani). Genetics 66:617-622.

 

Nusbaum, Charles J., and G. W. Keitt. 1938. The cytological
study of host-parisite relations of Venturia inaequalis
on apple leaves. J. Agri. Res. 56:596-618.

Pearson, R. C., M. Szkolnik, and F. W. Meyer. 1978. Suppres-
sion of cedar apple rust pycnia on apple leaves following
postinfection applications of fenarimol and triforine.
Phytopathology 68:1805-1809.

Polach, F. J. 1963. Genetic control of dodine tolerance in
Venturia inaequalis. Phytopathology 63:1189-1190.

 

Preese, T. F. 1963. Micro-exploration and mapping of apple
scab infections. Trans. Br. Mycol. Soc. 46:523-529.

Prusky, 0., Benjamin Jacoby, and A. Dinoor. 1979. Is the
accumulation of a systemic fungicide at the infection
site related to its eradicative action? Pestic. Biochem.
Physiol. 12-75-78.

Ragsdale, Nancy N. 1975. Specific effects of triarimol on
sterol biosynthesis in Ustilago maydis. Biochemica et
Biophysica Acta 380:81-96.

 

Ross, R. G., and R. J. Newbery. 1977. Tolerance of Venturia
inaequalis to dodine in Nova Scotia. Can. Plant Dis.
Surv. 57:57-60.

 

Solel, Z., and L. V. Edgington. 1973. Transcuticular movement
of fungicides. Phytopathology 63:505-510.

Staub, T., F. Schwinn, and P. Urech. ’1979. CGA-64251, a
new broad-spectrum fungicide. Phytopathology 69:1046.

Szkolnik, Michael. 1978. Techniques involved in greenhouse
evaluation of deciduous tree fruit fungicides. Ann. Rev.
Phytopathol. 16-103-129.

Van Gestel, J., and J. Van Reempts. 1979. Scanning electron
microscopy of Penicillium italicum on oranges treated with
R-23979, an imidazole derivative. Plant Dis. Rep. 63:
283-287.

PART I

POSTINFECTION CONTROL OF APPLE SCAB WITH
CGA-64251, BITERTANOL, AND PHENAPRONIL

INTRODUCTION

In the development of pest management programs for apple

scab, caused by Venturia inaequalis (Cke.) Wint., highly effective

 

fungicides are needed to control disease after the identification of
apple scab infection periods. Currently, the availability of fungi-
cides for after-infection control is limited because of fungicide
resistance, because some fungicides can only be used early in the
growing season to avoid phytotoxicity problems, and because public
agencies have cancelled or delayed registrations. During the past
12 yr, several fungicides which inhibit ergosterol biosynthesis and
membrane function have been tested for apple scab control. The
pyrimidine fungicides triarimol, fenarimol and nuarimol and the
piperazine fungicide triforine have been studied most extensively,
and were found to control apple scab after the onset of infection
(6, 8, 12, 14, 15). Recently, certain triazole and imidazole fungi-
cides, possessing protective and curative activities against many
Ascomycetous fungi, and which inhibit ergosterol biosynthesis in a
manner similar to the pyrimidine fungicides, have become available
for testing (4, 5, 16).

Two triazole fungicides: CGA-64251 (l-((2-(2,4-Dichloro-
phenyl)-4-ethyl-l,3-dioxolan-2-y1)methy1)-1fl-1,2,4-triazole) and

bitertanol, and the imidazole fungicide phenapronil were studied.

The objectives were to determine the extent of their postinfection
control abilities, to examine their effects on apple scab lesion
and conidial development and morphology, and to establish how these

new fungicides might be incorporated into apple scab control pro-

grams.

MATERIALS AND METHODS

Laboratory,$tudies

 

Monoconidial isolations of V. inaequalis were made by
rubbing detached lesions across the surface of potato dextrose agar
(PDA) and transferring single germinating conidia to fresh PDA in
petri plates. Conidia,produced on cheesecloth wicks saturated with
4% malt extract broth (19), were removed by adding sterile water to
drained culture bottles and shaking vigorously. Sterile Millipore
filter discs (13 mm diameter) were placed on PDA and 0.3 ml of a
fungicide suspension was applied to each disc. Sterile water was
applied to control discs. A spore suspension (0.3 ml) was applied
to each disc and germination of the conidia was observed 3, 6, and

21 days later.

Greenhouse Studies

Actively growing single shooted McIntosh apple (Malus pumila

 

Mill.) trees in pots were inoculated with a suspension of conidia

of y, inaequalis (3 X 105/m1). The suspension was made by washing

 

infected apple leaves with distilled water, and was atomized onto
the trees 1 hr before placing them in a mist chamber at 20 C for
47 hr. The youngest leaf on each shoot was tagged for later

reference.

10

After inoculation, trees were placed on a cheesecloth
enclosed bench in the greenhouse. The cheesecloth was wetted daily
to maintain relative humidities of 80 to 100%. At different inter-
vals after inoculation, four plants for each fungicide treatment
were removed from the enclosure, sprayed with fungicide and, after
the deposit dried, returned to the enclosure. Eight inoculated
plants were left unsprayed as controls.

Data were recorded 18 days after inoculation by visually
estimating (i) the leaf area covered with sporulating lesions and
(ii) the leaf area covered with both normal and non-sporulating
(chlorotic) lesions, using a standard diagram for comparison (18).
The four leaves below the tagged leaf were evaluated. Fungal
development within the chlorotic lesions was determined by removing
with ethanol, chlorophyll from several leaves with chlorosis and
staining the fungus with basic fuchsin (13). The experiment was

done twice.

Field Studies

 

In 1979 an orchard of 3-yr-old McIntosh apple trees on
M26 rootstock was sprayed to runoff with a handgun at 500 psi.
Treatments were arranged in a randomized complete block design
with four blocks and three trees per replicate. Data were taken
at about 2-wk-intervals from mid-June to mid-September on 20
terminals per replicate.

In 1980 an orchard of 5-yr-old McIntosh apple trees on M7

rootstock was used. Treatments were replicated four times in a

11

completely randomized design using single tree plots. Tags were
tied to the youngest expanded leaf after critical infection periods
to identify susceptible leaves exposed to infection. Data were
taken on 30 fruit spurs and 20 terminals per replicate.

Infection periods were predicted with a microprocessor-based
instrument placed between two apple trees and about 1.5 m above
ground level. This instrument, a modification of a unit described
by Jones et a1. (9), monitored temperature, rainfall, leaf wetness,
and relative humidity in the orchard. Incubation periods were esti-
mated based on the average temperature during the infection periods
by using the Mills table (11).

Possible effects of the fungicides on conidial morphology
and development were determined by examining lesions with an ISI
Super III scanning electron microscope (International Scientific
Instrument Corp., Santa Clara, CA 95050). Leaf disks with indi-
vidual lesions were cut from randomly collected leaves with a cork
borer, fixed in phosphate buffered 4% gluteraldehyde for 24 hr.
washed twice in 0.1 M_phosphate buffer (pH 7.3), and post-fixed
in a 1:1 mixture of 0.2 M_phosphate buffer and 2% 0504 for 24 hr.
Samples were washed again in phosphate buffer, dehydrated in an
ethanol series and critical point dried. Dried specimens were
mounted on stubs and coated wtih a 20 angstroms thickness of gold
in a sputter coater. In addition, lesions were rubbed across the
surface of PDA to remove the conidia and germination was evaluated

after 24 and 48 hr.

12

To determine the viability of the fungus within chlorotic
lesions, isolations were attempted using the procedures of Hoch and
Szkolnik (8). Leaves were collected on 3 June from each replicate
of treatments showing chlorotic lesions, and washed for 5 hr in
running tap water. Twenty chlorotic lesions per treatment were
removed with a cork borer, dipped briefly in 70% ethanol and cut
into quarters. Each set of quarters was placed in a petri dish
containing 3% malt extract agar amended with 250 ppm streptomycin.

Plates were examined for growth of v, inaequalis after 20 days at

 

20 C. Unsprayed controls were included for comparison.

RESULTS

Laboratory Studies

 

Phenapronil (1235.8 pg active ingredient (a.i.)lml),
bitertanol (599.2 pg a.i.lml) and CGA-64251 (37.4 pg a.i.lml) were
applied to Millipore filters. Conidia on all filters germinated,
but germ tubes of fungicide-treated conidia were short, distorted,
swollen, and branched, often with a dark swollen structure at each

hyphal tip (Figure 1).

Greenhouse Studies

 

Phenapronil (617 pg active ingredient (a.i.)/ml), bitertanol
(149.8 and 299.6 pg a.i.lml) and CGA-64251 (18.7 pg a.i./m1) were
applied to trees 2, 3, 4, 4.5, 5, and 5.5 days after inoculation.
Two fungicides: (i) fenarimol (EL-222 12.5% EC) at 42 pg a.i./m1
from Elanco Products Co., Indianapolis, IN 46206 and (ii) phenyl
mercuri triethanol ammonium lactate (Puritized Agricultural Spray
(PAS) 7.5% liquid) at 93.8 pg a.i./ml from Niagara Chemical Division
of FMC Corp., Middleport, NY 14105, which have exhibited good
eradicative properties in the past, were included for comparison.

In several treatments, particularly those where fungicide
was applied 4 days or more after inoculation, both normal sporulat-
ing lesions and chlorotic flecks or yellow nonexpanding and non-

sporulating lesions were present (Table l). CGA-64251, bitertanol

13

14

Figure 1.--Germinated conidium of Venturia inaequalis on filter
paper saturated with 12.5 pg of CGA-64251. Arrows
indicate swollen areas at hyphal tips.

 

 

15

 

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17

at 299.6 pg a.i.lml, and phenapronil were similar in effectiveness
to fenarimol and PAS when applied 2 and 3 days after inoculation.
At 4 days, the percent leaf area covered wtih chlorotic flecks was
high for all fungicides except phenapronil and CGA-64251, but only
leaves sprayed with PAS and bitertanol at 149.8 pg a.i./m1 exhibited
normal lesions. At 4.5, 5, and 5.5 days, all treatments continued
to suppress the development of normal lesions to some extent com-
pared to unsprayed trees.

Leaves with chlorotic flecks retained the basic fuchsin
stain in the chlorotic areas of the leaves, indicating the lesions

contained fungal growth (Figure 2).

Field Studies, 1979

 

CGA-64251 10% W (18.7 pg a.i./ml) was evaluated in five
postinfection spray schedules as follows: (i) an after-infection
eradication program with treatment applied within 3 days from the
beginning of wetting periods predicted to give infection; (ii) two
presymptom eradicant programs, the first with treatments applied
2 days before lesions were predicted to appear, and a second pro-
gram where the initial spray was repeated 1 wk later; and (iii)
two postsymptom eradicant programs, the first with treatment applied
2 days after lesions were visible and a second program where the
initial spray was repeated 1 wk later. A protective spray schedule
was included for comparison. Timing of the sprays in relation to

predicted infection periods is shown in Figure 3.

18

Figure 2.--Apple leaf, with chlorophyll removed, stained with
basic fuchsia to detect subcuticular hyphae (A) and
occasional surface growth (8) of Venturia inaequalis

 

 

in chlorotic lesions.

19

 

20

 

 

 

 

 

 

 

 

 

 

 

 

—

WES. m PROTECTIVE pgmoo I979
UGHT
MEDIUM____ I POST INFECTION PERIOD
HEAVY """""" FIRST LESIONS
MAY 12
‘59flfilnflJfli’ I ll 3
PROTECTIVE
I II =
POST INFECTION =
AFTERINE MiiiZfl NEED
PRESYMPTOM ' :1 §
1 .
I SPRAY I "‘1 IIMiZED p:iIZZ]
2 SPRAY F (Tm-Fm
POST SYMPTOM ' ' l 5
N” .
I SPRAY Fl rT—‘iiZB E
2 SPRAY r”’” rTwfliiijian
I 1 1 I
y I I TIIT'T‘IT'ITITI IITU‘UIVITYIY'IIIII'Y'II‘

TTTI TI 1"! IIIIFTTIIYTIITUTTITIIT111]!IITYTITT TI

202530 51015 202530 4 91419 2529 4 91419
APRIL MAY JUNE JULY

Figure 3.--Schedule Of sprays applied in 1979 in relation to pre-
dicted infection periods. The date for each spray
corresponds to the right margin of the black squares.
For purposes of interpreting the possible effect Of
each spray, a 3-day postinfection period and a 5-day
protective period are illustrated. The 3-day post-
infection period is from greenhouse studies (Table l),
but the 5-day period for protection is an estimated
time. Arrows designate initial infection period for
which spray or spray sequence was applied.

21

The pre- and postsymptom eradicant programs with a repeat
spray 1 wk after the first, resulted in significantly more control
than the one-spray eradicant programs, and were not Significantly
different from the protective schedule in effectiveness, even
though three fewer sprays were applied (Table 2). The presymptom
sprays on 7 and 14 May were applied for infection periods beginning
24, 26, and 27 April and 2 and 12 May; sprays on 15 and 22 June
were for infection periods beginning 7, 10, and 11 June; and sprays
on 4 and 11 July were for infection periods on 27 and 29 June.

The postsymptom eradication sprays for these infection periods
were applied 14 and 21 May, 19 and 26 June, and 9 and 16 July.
NO susceptible tissue was present during the light infection
period starting 21 April.

The after-infection schedule was not significantly differ-
ent in effectiveness from the protective schedule on 11 July, but
significantly more scab was recorded on 1 and 27 August (Table 2).
However, the last after-infection spray was applied on 29 June,

2 to 3 wk earlier than the last pre- and postsymptom sprays.

Field Studies, 1980
Bitertanol 50% W (299.6 pg a.i.lml) and CGA-64251 10% W
(18.7 pg a.i./ml) were evaluated in three postinfection spray
schedules as follows: (i) an after-infection eradication program
with treatment applied within 3 days from the start Of wetting
periods predicted to give infection; (ii) a presymptom eradicant

program with one spray applied 2 days before lesions were predicted

£22

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23

to appear and a second spray 1 wk later; and (iii) a postsymptom
eradicant program with a spray applied 2 days after lesions were
visible and a second spray 1 wk later. Two protective spray
schedules, one at 7-day and the other at 14-day intervals, were
included for comparison. Timing Of the sprays in relation to
predicted infection periods is shown in Figure 4.

Data taken on 22 May indicated the control Of scab from
infection periods beginning 27 and 28 April. After-infection
schedules with both compounds were as effective as protective
schedules in controlling scab from these infection periods.

Pre- and postsymptom schedules, regardless of chemical, were not
significantly different from each other or from the untreated
trees. Since the first scab lesions were Observed on 13 May,
the presymptom spray Of 12 May was applied too late to be con-
sidered truly presymptom. Data taken on 5 June indicated a
slight increase in scab on leaves Of fruit spurs, but no major
changes in the effectiveness Of the treatments.

The severity of the 13 and 17 May infection periods is
illustrated by the data taken from terminal shoots on 6 June,
since the buds that gave rise to these shoots were dormant during
the infection periods in April. Data for this date indicate that
bitertanol gave better control when applied as a protectant or if
applied soon after infection, while CGA-64251 gave better control
when applied later in the incubation period. Chlorotic nonsporu-
lating lesions were also noted at this time, and examination of

tagged leaves indicated these lesions were from the heavy infection

24

 

I F 11 PIno ,
, PROTECTIVE PERIOO
LIGHT —-——. 1980
MEDIUM N I _ _. I POST INFECTION PERIOO
HEAVY --------- FIRST LESIONS
W
PROTECTIVE
1 DAY
I4 DAY

POST INFECTION
AFTER INF.

 

  

PRESYMPTOM

 

 

POST S YMPTOH

 

 

 

 

l
_ I

I i 1111 I I
l - 1

 

 

 

; ll 1 1 -
Ivvvvlv VIlvvvleIIIInnlvuvlnixl n I" ulvtrvInvrlvvvyltrTvltvrvlvvnlIIITIII'II IIIIY'VI ""I""I""I"
202530510152025304 9141924294 9141924293813
APRIL MAY JUNE JULY AUGUST

Figure 4.--Schedule of sprays applied in 1980 in relation to
predicted infection periods. The date for each spray
corresponds to the right margin Of the black squares.
For purposes of interpreting the possible effect Of
each spray, a 3-day postinfection period and a 5-day
protective period are illustrated. The 3-day post-
infection period is from greenhouse studies (Table 1),
but the 5-day period for protection is an estimated
time. Arrows designate original infection period fOr
which spray or spray sequence was applied.

25

period Of 17 May when 3.02 cm rain, 30 hr wetting, and a mean
temperature Of 13.3 C were recorded. Similarly, chlorotic lesions
were noted on 24 June following a series Of five infection periods
between 30 May and 7 June. Except where specified, chlorotic
lesions were included in the data. After-infection and presymptom
schedules showed little increase in percent leaves infected between
6 June and 28 July, while disease development on postsymptom treat-
ments was steady, but significantly less than for unsprayed con-
trols. Data for lesions per terminal, taken on 28 July, included
an additional factor to correct for chlorotic lesions (Table 3).
A comparison Of total lesions per terminal with normal lesions per
terminal showed that more lesions on bitertanol treated trees were
chlorotic compared tO CGA-64251 treatments.
Effect of Fungicides on Lesion Development
and Isolation Recovery in 1980

Lesions from the 27 and 28 April infection periods were
collected for scanning electron microscope examination on 21 May,
2 days after the second presymptom treatments on 19 May; on 18 May,
2 days after the first postsymptom treatments on 16 May; and on
25 May, 2 days after the second postsymptom treatments on 23 May.

The density of conidia in lesions from trees sprayed after
lesions appeared (Figure 5C) was less than in lesions from unsprayed
trees (Figure 5A). In addition, conidia in lesions from the pre-
symptom treatments were ampulliform and appeared immature (Figure 50),
while conidia in lesions from unsprayed trees were mature in the

lesion center (Figure 5B) with a narrow zone of ampulliform conidia

26

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27

Figure 5.--Scanning electron micrographs Of typical lesions
and conidia Of Venturia inaequalis from apple
trees sprayed with bitertanol 50% W 299.6 pg

a.i.

(A)

 

/m1 and CGA-64251 10% W 18.7 pg a.i./m1.

Lesion from unsprayed tree with dense
sporulation (X390).

(B) Obpyriform conidia in lesion from unsprayed

(C)

tree (arrow) (X4800).

Reduced sporulation in a lesion sprayed 2
days earlier with bitertanol (X390).

(0) Ampulliform immature conidia in a lesion

(E)

sprayed twice with bitertanol and sampled
2 days after the second spray (X2840).

Subcuticular (a) and infrequent deformed
surface growth (b) in a chlorotic lesion
from a tree sprayed with CGA-64251 on a
l4-day schedule (X390).

Surface growth in a chlorotic lesion from
a tree sprayed with bitertanol on a l4-day
schedule (X2045).

28

 

29

at the lesion margins. Lesions taken from postsymptom sprayed
trees, either after the first or second spray, had normal Obpyri-
form spores in the lesion centers but a wider zone of ampulliform
spores than lesions from unsprayed trees.

When lesions from pre- and postsymptom sprayed trees were
rubbed across PDA, the conidia were difficult to remove and did
not germinate, while large numbers Of viable conidia were Obtained
from unsprayed lesions.

Chlorotic lesions on leaves exposed to the 13 and 17 May
infection periods were taken on 10 June from the l4-day and post-
infection schedules Of CGA-64251 and bitertanol, and from pre-
symptom and postsymptom treatments of bitertanol. Subcuticular
fungal growth was visible in most chlorotic lesions (Figure 5E).
Sporulation, with many ampulliform conidia, was Observed in lesions
from the after-infection schedules for both chemicals, but only
scattered abnormal surface growth was observed for all other treat-
ments (Figures 5E and SF). The ratio of lesions with significant
fungal growth to total number examined was 1/6 and 5/5 for the
l4-day and after-infection treatments, respectively, Of CGA-64251;
and 3/6, 1/5, 6/7, and 5/5 for the l4-day, presymptom, after-
infection,and postsymptom treatments, respectively, of bitertanol.

Isolations were attempted from chlorotic lesions collected
3 June from terminal leaves exposed to the infection periods Of

13 and 17 May. The ratio Of successful isolations Of V, inaequalis

 

to the total number Of lesions examined were 5/11 (45.5%) for

lesions from the l4-day schedule Of CGA-64251 and 4/8 (50%) and

30

7/10 (70%) for lesions from the l4-day and postinfection treatments
of bitertanol. The ratio Of isolation was 11/11 (100%) for lesions

from unsprayed leaves.

Effects Of Tree Growth

Modified growth patterns were noted with both compounds in
the 1979 and 1980 field studies. The elongation Of lateral shoots
was retarded and leaves were smaller, thicker, puckered, and darker
green than leaves on unsprayed trees. The effects were more pro-
nounced with CGA-64251 than bitertanol, and were most severe on
trees treated with a 7-day schedule. Transmission electron micro-
scope examination Of leaf samples taken from trees sprayed on a
7-day schedule with CGA-64251 showed no abnormalities in cuticle
structure or in organelles and internal cell structure. However,
light microscope examination Of several leaf sections revealed an
increase in the number of palisade layers in leaves from sprayed
trees (Figure 6B) compared to leaves from unsprayed trees

(Figure 6A).

31

Figure 6A.--Cross-sections Of apple leaves taken from unsprayed
trees. Palisade cells are two to three layers deep.

32

 

33

Figure 6B.--Cross-sections of apple leaves taken from trees
sprayed with CGA-64251 on a 7-day schedule.
Palisade cells are three or more layers deep.

34

 

DISCUSSION

Results from the greenhouse studies indicated that with
CGA-6425l, phenapronil, and high rates of bitertanol, infections
were controlled when the chemicals were applied within 3 days after
inoculation. Similar results were Obtained in field trials with
CGA-64251 and bitertanol. Therefore, all three compounds should
be effective as after-infection controls in apple scab pest manage-
ment schemes where dilute sprays are applied up to 3 days after the
beginning of infection periods.

Field data from 1979 and 1980 indicate that CGA-64251 and
bitertanol are capable of inactivating and controlling apple scab
when applied several days after infection has occurred. If the
compound is applied within 3 days after an infection period, one
spray is sufficient to control infection from that infection period.
However, if sprays are applied later in the infection period or
after lesions are visible control is still possible but two sprays,
1 wk apart, must be applied.

In 1980 two infection period sequences were Of particular
importance; a heavy infection on 17 May when 3.02 cm Of rain was
recorded, and a series Of four moderate and one light infection
periods occurring in the first week Of June. Both primary and

secondary inoculum were present during these infection periods,

35

36

and many heavily-infected unsprayed trees were present in the
orchard. As a result of high inoculum pressure and ideal weather
for infection, many treatments developed chlorotic lesions, indi-
cating partial control. These lesions failed to develop further,
but did contribute to the lesion count data for these treatments.
Analysis Of disease development on the after-infection
treatments throughout the season indicated that disease was con-
trolled for the primary infection periods in late April; however,
the assumption of effective protective control for the heavy
infection period in mid-May was probably not justified, and a
certain amount of disease became established. This was, however,
brought under control, and successfully controlled throughout the
season, as shown by the lack Of significant increase in disease
for this schedule. While a disease increase was noted for the
presymptom schedules between 6 June and 24 June, due primarily to
chlorotic lesion development, control was also achieved on these
treatments as shown by the lack Of increase in percent leaves
infected on 28 July. Disease increased steadily on postsymptom
treatments. This is not surprising, as spray was not applied in
these schedules until lesions were Observed. An important feature
of this increase is its controlled incremental nature, indicating
that even though lesions were allowed to occur after each infection
period, two sprays brought the disease under control each time.
The scab which Occurred on the presymptom treatments was probably
the result Of inaccuracy in estimating the number Of days between

infection and sporulation; for example, the first presymptom

37

spray was applied on 12 May and the first lesions were Observed
on 13 May.

Scanning electron micrographs showed a reduction in conidial
density within sprayed lesions, and many conidia were immature. The
percentage Of immature forms was higher for treatments applied soon
after an infection period. Various researchers (1, 3, 7, 10, 17)
have reported reductions in conidia with postsymptom applications
of benomyl and dodine, particularly if more than one spray was
applied. Although attempts to germinate conidia from treated
lesions were unsuccessful, isolation from some chlorotic lesions
was possible. Thus, the action of these fungicides on subcuticular
growth may be fungistatic, and failure to apply the second spray
or to continue the spray program into the season could result in
renewed activity Of previously suppressed lesions. This failure
to affect subcuticular growth has been previously reported for
dodine, captan, zineb, and lime sulfur (2), however, the possi-
bility that a cumulative effect from repeated spraying through the
season is likely. The possibility that the fungus in suppressed
lesions may be capable Of ascocarp production during the winter,
and thus contribute to primary inoculum in the following year,

should be examined.

LITERATURE CITED

Albert, J. J., and F. H. Lewis. 1962. Effect Of repeated
applications of dodine and Of captan on apple scab
foliage lesions. Plant Dis. Rep. 46:163-167.

Albert, J. J., and J. W. Heuberger. 1961. Apple scab I.
Effect of one application of fungicides on leaf lesions
on previously unsprayed trees. Plant Dis. Rep. 45:
759-763.

Alexander, S. A., and F. H. Lewis. 1975. Reduction of apple
scab fungus inoculum with fungicides. Plant Dis. Rep.
59:890-894.

Buchel, K. H., W. Kramer, W. Meiser, W. Brandes, P. E.
Frahberger, and H. Kaspers. 1979. Chemistry and
properties of phenyl-triazole-methanes, a highly active
class Of new azole fungicides. Abstract 475 in Abstracts
Of Papers, IXth Int. Congr. Of Plant Protection,
Washington, D.C.

Carley, H. E. 1979. A new multiple use fungicide:
o-butyl-o-phenyl-lH—imidazole-l-propanenitrile.
Phytopathology 69TT023.

Fuchs, A., and W. Ost. 1976. Translocation, distribution and
metabolism Of triforine in plants. Archives of Environ.
Contam. and Toxicol. 4:30-43.

Heuberger, J. W., and R. K. Jones. 1962. Apple scab II.
Effect Of serial applications of fungicides on leaf
lesions on previously unsprayed trees. Plant Dis.
Rep. 46:159-162.

Hoch, H. C., and M. Szkolnik. l979. Viability Of Venturia
inaequalis in chlorotic flecks resulting from fungicide
applicatiOn to infected Malus leaves. Phytopathology
69:456-462.

 

Jones, A. L., S. L. Lillevik, P. 0. Fisher, and T. C. Stebbins.
1980. A microcomputer-based instrument to predict
primary apple scab infection periods. Plant Dis. 64:
69-72.

38

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

39

Jones, R. K., J. W. Heuberger, and J. D. Bates. 1963. Apple
scab 111. Effect of serial applications Of fungicides on
leaf lesions on previously unsprayed trees--inhibition
Of conidial germination, removal (suppression) Of the
organism, and subsequent development of late terminal
infection. Plant Dis. Rep. 47:420-424.

Mills, W. D., and A. A. LaPlante. 1951. Control Of diseases
and insects in the orchard. N.Y. Agric. Exp. Stn.
(Ithaca) Ext. Bull. 711, pp. 18-22.

Pearson, R. C., M. Szkolnik, and F. W. Meyer. 1978. Suppres-
sion of cedar apple rust pycnia on apple leaves following
postinfection applications of fenarimol and triforine.
Phytopathology 68:1805-1809.

Preece, T. F. 1959. A staining method for the study Of apple
scab infections. Plant Pathol. 8:127-129.

Prusky, 0., Benjamin Jacoby, and A. Dinoor. 1979. Is the
accumulation Of a systemic fungicide at the infection
sight related to its eradicative action? Pest. Biochem.
and Phys. 12:75-78.

Ragsdale, Nancy N. 1975. Specific effects Of triarimol on
sterol biosynthesis in Ustilago maydis. Biochemica et
Biophysica Acta 380:81-96.

 

Staub, T., F. Schwinn, and P. Urech. 1979. CGA-64251, a new
broad-spectrum fungicide. Phytopathology 69:1046.

Szkolnik, M., J. R. Nevill, and L. M. Henecke. 1973.
Fungicidal inhibition Of production Of new conidia from

established foliar apple scab lesions. Phytopathology
63:208.

Tehon, L. R., and G. L. Stout. 1930. Epidemic diseases of
fruit trees in Illinois: 1922-1928. 111. Nat. Hist.
Surv. Bull. 18:415-502.

Tuite, John. 1969. Plant pathological methods: fungi and
bacteria. Burgess Publishing Co., Minneapolis, MN.,
239 pp.

PART II

VOLATILITY AND SYSTEMIC PROPERTIES IN APPLE
OF CGA-64251, BITERTANOL, AND
PHENAPRONIL FUNGICIDES

4O

INTRODUCTION

Before a fungicide can be effectively incorporated into a
pest management program, not only its disease control abilities, but
also the characteristics of its movement and localization within the
plant must be determined. The ability of a systemic fungicide to
enter the plant, and the extent and speed of its movement after
entry, determine the application conditions which are optimally
effective and the level and type of control which can be expected.
Solel and Edgington (4) studied the transcuticular movement of
fungicides using isolated apple cuticles, and Edgington et al. (2)
have used washings Of treated leaves to determine the amount and
rate of uptake. Also, the volatility Of a compound may seriously
affect its control characteristics. In the case of fungicides for
the control Of apple scab, volatilization in the orchard after
application could result in a reduction in the period of effective
protective control of disease and a reduction in the amount of
fungicide available for uptake by the plant. When used in an
enclosed environment, such as the greenhouse, however, an increase
in control may be noted (1, 3, 5).

In this study, the uptake and distribution properties in
apple of three experimental compounds which are characteristics Of

a family of ergosterol inhibiting fungicides presently being

41

42

developed, were examined by using leaf washings and by oxidation
of the plant parts after application of radioactive labeled com-
pounds. In addition, autoradiographs were made to further

visualize distribution patterns.

MATERIALS AND METHODS

Volatility of Fungicides

 

A 10 pl droplet of radioactive fungicide was applied with
a syringe, fitted with a 10 pl calibrated disposable capillary
pipet, to round microscope cover glasses held in the dark at 20 C
and at a relative humidity Of 5%. At various times after applica-
tion, five cover glasses for each fungicide were each dropped into
15 m1 scintillation fluid (Aqueous Counting Scintillant, Amersham
Corp., Arlington Heights, IL 60005), shaken thoroughly, and counted
in a Searle, Model 6868 ISOCAP/300 liquid scintillation counter
(Searle Analytic Inc., Des Plaines, IL 60018). Each sample was
counted for 10 min, and disintegrations per minute (0PM) calculated

from a quenched external standard curve.

Uptake of Fungicides
Thirty-six single-shooted McIntosh apple (Malus pumila Mill.)

 

trees growing in pots in the greenhouse were used. Ten 10 pl drop-
lets of radioactive fungicide were applied to the youngest fully
expanded leaf of each plant with a calibrated capillary pipet. At
various times after application, the shoots and leaves were cut
from three trees for analysis. Each cut was made with a new razor
blade to avoid contamination. The two leaves below and the two

leaves above the leaf to which fungicide was applied, the growing

43

44

tip cluster, and the fungicide treated leaf were each oxidized at
900 C in an 0X-200 Biological Material 0xidizer (R. J. Harvey

Instrument Corp., Hillsdale, NJ 97642). The 14

CO2 was trapped in
a 2:1 mixture of Permafluor V Scintillant and Carbosorb 11 carbon
dioxide absorber (Packard Instrument Co., Downers Grove, IL 60515)
and each sample was counted in the scintillation counter. Before
oxidizing, the fungicide treated leaf was washed in 10 m1 of 95%
ethanol to remove fungicide remaining on the leaf surface. In a
preliminary study, it was determined that more than 90% of the
radioactivity was removed in the first wash and very little
fungicide was removed by additional washings. The wash was added
to 10 ml scintillation fluid and counted. Zero-time values were

determined by applying ten 10 pl droplets to leafsized pieces of

analytical filter paper, oxidizing, and counting.

Autoradiography

 

Nine equal-sized single-shooted, McIntosh apple trees
growing in pots in the greenhouse were separated into three groups,
with each group of three trees receiving a different compound.
Three 10 pl droplets Of radioactive fungicide were applied with a
calibrated capillary pipet to the youngest fully expanded leaf Of
each plant. The plants were held in the greenhouse for 7 days at
about 18 to 25 C, after which the shoots were removed, placed on
Fuji NO-Screen X-ray film, and pressed in a plant press. The
pressed plants were stored at -10 C for 21 days and the films

developed in Kodak Liquid X-ray DevelOper (number 146 5335).

RESULTS

Volatility Of Fungicides

 

Radioactive bitertanol ((UL-byphenyl-14C)-Baycor, specific
activity 41.72 pCi/mg) was supplied by Mobay Chemical Corp., Kansas
City, MO 64120. Radioactive CGA-64521 ((U-triazole ring-14C)-
CGA-6425l, specific activity 61.1 pCi/mg) was supplied by Ciba-
Geigy Corp., Greensboro, NC 27409. Radioactive phenapronil ((ring-
14C)-Sisthane, specific activity 8.93 p1 Ci/mg) was supplied by
Rohm and Haas Corp., Philadelphia, PA 19105. All fungicides were
initially dissolved in 1 ml of ethanol and diluted to 10X final
test strength (6170 pg active ingredient (a.i.)/m1, 2996 pg
a.i./ml, and 187 pg a.i./m1 for phenapronil, bitertanol, and
CGA-64251, respectively) with distilled water. Dilutions to test
strength were made by adding portions of this stock solution to
distilled water.

Cover glasses with fungicide were placed in scintillation
vials at 0.5, l, 2, 4, 8, 24, 48, 72, and 120 hr after application.
Phenapronil and bitertanol samples showed no reduction in radio-
activity in 120 hr (Figure 1). When these data were subjected to
regression analysis, the best line fit to each data set had a
slope not significantly different from zero (P=0.01). The radio-
active CGA-64251 dropped by about 50% in 72 hr, and a multiple

regression analysis of the data yielded the equation:

45

46

 

PHENRPRONIL

OPHIIO'
110 120

100

25

 

BITERTRNOL

DPHIIO'
21 23

19

17

 

20

COR-64251

18

18

12

DISINTEGRHTIONS PER MINUTE-10'
10 14

 

 

 

 

abfir'rrf'rfrfrfr'rrl'I'Tfiv'r'
10 20 30 4O 50 60 70 80 90 100 110 120 130
HOURS FROM STRRT

Figure l.--Radioactive fungicide remaining on cover glasses at
various times after application.

47

v = Ke('°°°°62 X) (r2 = 0.8216)

where K is the amount of radioactivity applied at time zero in DPM,
X is hours after application, e is the irrational constant 2.71828,

and Y is the amount Of radioactivity remaining at time X.

Uptake of Fungicides

 

Radioactive phenapronil (617 pg a.i./m1), bitertanol
(299.6 pg a.i./ml), and CGA-64251 (18.7 pg a.i./m1) were applied
to apple leaves and samples were taken at 0.5, l, 3, and 7 days
for analysis. Radioactivity was detected in all samples. The
average amount Of fungicide that moved out of the treated leaf
was 5.7, 27, and 2.3% for CGA-64251, bitertanol, and phenapronil,
respectively. All chemicals moved both upward and downward, and
less radioactivity was detected, after 7 days, in the leaves
immediately above or below the treated leaf than in the uppermost
leaves or in the second leaf below the treated leaf (Figure 2).
At 12 hr, 90% Of the radioactive CGA-64251 had entered the leaf,
after which equilibrium was maintained (Figure 3A). The other
fungicides moved into the leaves more slowly (Figures 38 and 3C).
Equilibrium was reached at 24 hr for bitertanol and within 3 days
for phenapronil, with about 40 and 60% of the radioactivity,

respectively, crossing the cuticle.

Autoradiggraphy

 

Radioactive phenapronil (6170 pg a.i./m1), bitertanol

(2996 pg a.i./ml), and CGA-64251 (187 pg a.i./m1) were applied

48

 

 

 

 

 

 

APPLE LEAF '73 OF MOBILE FRACTION
POSHWON BHERTANOL GOA-64251 PHENAPRONH.
26 24 37
4O 17 19
£31————- 3 16 5

@——— TREATED LEAF

fi—T 5 12 3
<EEEESN____——— 25 31 36

 

 

Figure 2.--Distribution Of the portion of radioactive fungicide
which had moved out Of treated leaves, 7 days after
application.

.mo>no_ o_aam one? onwowmcnm o>NuonoNnnc No unoEm>oE anaowpnomcncpiI.m ocsmwm

=5» 5:: «:3 hzchm Kuhn—z “:3

5:3...“ zu—mc at;

 

 

449

 

 

 

I

 

 

 

 

 

 

 

‘

o NzcoN z.=..a n M Nzcoa 2.:N_a n L

.zcoa 2.x..z a . . .
uocceaa ccuo a a

33.5» as: a .m m H 33.5» .33 a <
0 c I
v
.n oochNuN_o Lo Nzu=u>oc «coao.Naoaz¢.N . a. I z z on use .
o_zo¢e¢zoze Lo Nzo=o>oc guano—NaomzcxN : u . «go no N ocu>o cs o.~:o «N .

 

 

IOIIBIONIH 83¢! SNOIIUUOBINISIO

 

 

 

 

50

to plants. Autoradiographs confirmed the results of the uptake
studies, but showed distinct differences in distribution patterns
between the three fungicides. A large amount Of labeled CGA-64251
and phenapronil was distributed throughout the leaves to which they
were applied (Figures 4A and 4B). Accumulation of labeled chemical
was also noted in the shoots and, with phenapronil and CGA-64251,
labeled chemical was Observed in the growing tip and the lower
leaves. By contrast, most of the labeled bitertanol that remained
in the treated leaf did not move from the spots where it was applied
(Figure 4C). There was also accumulation in the major veins, but
little or no movement into the interveinal areas. Small amounts

Of labeled chemical were also noted in leaves above and below

the treated leaf.

51

Figure 4A.--X-ray film autoradiograph showing the pattern Of
movement and distribution Of radioactive fungicides
in apple shoots 7 days after application Of CGA-64251
to a single leaf (the darkest leaf).

 

52

 

53

Figure 4B.--X-ray film autoradiograph showing the pattern of
movement and distribution Of radioactive fungicides
in apple shoots 7 days after application Of phena-
pronil to a single leaf (the darkest leaf).

54

 

55

Figure 4C.--X-ray film autoradiograph showing the pattern of
movement and distribution of radioactive fungicides
in apple shoots 7 days after application of
bitertanol to a single leaf (the darkest leaf).

 

56

 

DISCUSSION

Analysis of the potential for volatilization Of these
three fungicides from cover glasses indicated that a significant
portion of CGA-64251 is volatilized within 24 hr after application,
which supports previous research by Szkolnik (5). In orchard
applications this may not be of concern, as uptake studies showed
that a very high percentage of the compound crosses the cuticle
and enters the leaf within 12 hr after application; however,
volatilization may be an important source of control in greenhouse
situations. In addition, the ability to enter the plant quickly
reduces the length of time the chemical is exposed to the possi-
bility Of being washed from the plant by rain. The entry rates
Of bitertanol and phenapronil were somewhat slower, and thus they
would be exposed on the leaf surface for a longer time; however,
they showed no volatilization within 120 hr.

The movement and distribution of the fungicides varied
considerably between plants. However, a pattern was evident.
Fungicide moved out Of the leaf to which it was applied much more
rapidly if the plant was actively growing. If the shoot failed to
elongate, or elongated only slightly, translocation was slow and
the amount moved was less. The highest concentration of the mobile

fraction seemed to be associated with the leaves closest to the

57

58

fungicide treated leaf at 3 days, but at 7 days, the primary sites
of concentration were the growing tip and the lower leaves on the
stem.

Autoradiographs showed a pattern Of distribution similar to
the scintillation studies. Only a very small fraction of the applied
fungicide is moved out of the treated leaf and into the growing tip.
This may not be enough fungicide to achieve control Of scab on the

new tissue, and this possibility Should be investigated further.

LITERATURE CITED

Clark, Terence, Derek R. Clifford, Adrian H. B. Deas, Peter
Gendle, and David A. M. Watkins. 1978. Photolysis,
metabolism and other factors influencing the performance
Of triadimefon as a powdery mildew fungicide. Pesticide
Science 9:497-506.

Edgington, L. V., P. P. 0. de Wildt, K. Jacques, and J. Psutka.
1976. The study Of transcuticular movement Of fungicides,
in Proceedings: Apple and pear scab workshop, pp. 32-35,
A. L. Jones and J. D. Gilpatrick, eds., N.Y. Agric. Exp.
Stn., Special Report 28, 38 pp.

Scheinpflug, H., and V. Paul. 1977. On the mode of action Of
triadimefon. Neth. J. P1. Path. 83 (suppl. l):105-lll.

Solel, Z., and L. V. Edgington. 1973. Transcuticular movement
of fungicides. Phytopathology 63:505-510.

Szkolnik, M. 1980. Control Of apple powdery mildew by vapor
from a new triazole fungicide. Phytopathology 70:469.

59

APPENDICES

60

APPENDIX A

SCANNING ELECTRON MICROGRAPHS OF LESIONS
SPRAYED WITH CGA-64251 OR BITERTANOL

61

62

Figure Al.--Lesion (top, X390) of Venturia inaequalis from
untreated trees, showing dense sporulation and
normal mature conidia (bottom, X4800).

 

 

63

 

64

 

Figure A2.--Lesion (top, X390) of Venturia inaequalis from
presymptom treatment Of CGA-64251, taken 2 days
after second spray was applied. Conidial density
is less than for unsprayed lesions and conidia .
(bottom, X1390) are immature and ampulliform. '

 

 

 

 

65

 

66

Figure A3.--Lesion (top, X390) Of Venturia inaequalis from
presymptom treatment Of bitertanol, taken 2 days
after second spray was applied. Conidial density
is less than for unsprayed lesions and many
conidia throughout the lesion (bottom, X2840)
are immature and ampulliform.

 

67

 

68

Figure A4.--Lesion (top, X440) of Venturia inaequalis from
postsymptom treatment Of CGA-64251, taken 2 days
after first spray was applied. Conidial density
is less than for unsprayed lesions with many
immature conidia at the lesion margins. Conidia
at lesion center are normal and mature (bottom,

X950).

 

-_.-_—~ ._.-..-v

w ___'-. A _._

 

 

 

69

 

70

Figure A5.--Lesion (top, X390) of Venturia inaequalis from
postsymptom treatment of bitertanol, taken 2 days
after first spray was applied. Conidial density
is less than for unsprayed lesions with many
immature conidia at the lesion margins. Conidia
at lesion center are normal and mature (bottom,
X950 . .

 

71

 

72

Figure A6.--Lesion (top, X390) Of Venturia inaequalis from
postsymptom treatment of CGA-64251, taken 2 days
after second spray was applied. Conidial density
is less than for unsprayed lesions with many
immature conidia at the lesion margins. Conidia
at lesion center are normal and mature (bottom,
X4800). -

 

73

 

74

Figure A7.--Lesion (top, X390) of Venturia inaequalis from
postsymptom treatment of bitertanol, taken 2 days
after second spray was applied. Conidial density
is less than for unsprayed lesions with many
immature conidia at the lesion margins. Conidia
at lesion center are normal and mature (bottom,
X1890).

 

75

 

APPENDIX B

SCANNING ELECTRON MICROGRAPHS OF CHLOROTIC LESIONS
SPRAYED WITH CGA-64251 OR BITERTANOL

76

77

Figure Bl.--Chlorotic lesion (top, X390) and deformed surface
growth (bottom, X3040) Of Venturia inaequalis from
l4-day treatment of CGA-64251, taken on 10 June.

Subcuticular growth is evident in the lesion
center.

 

 

78

 

79

Figure BZ.--Chlorotic lesion (top, X270) of Venturia inaequalis

 

from l4-day treatment Of bitertanol, taken 10 June.
Most conidia are ampulliform and immature (bottom,
X2045 .

80

 

 

 

81

 

Figure 83.--Chlorotic lesion (top, X390) of Venturia inaequalis
from after-infection treatment Of CGA-64251, taken

10 June. Most conidia are ampulliform and immature
(bottom, X2890).

 

 

82

   

83

Figure B4.--Chlorotic lesion (top, X270) of Venturia inaequalis

 

from after-infection treatment of bitertanol, taken
10 June. About 50% Of conidia (bottom, X2060) are
ampulliform and immature.

 

 

84

 

 

85

Figure B5.--Chlorotic lesion (top, X390) and deformed surface
growth (bottom, X1890) of Venturia inaequalis,
from presymptom treatment of bitertanol, taken
on 10 June.

 

87

Figure B6.--Chlorotic lesion (top, X400) of Venturia inaequalis
from postsymptom treatment of bitertanol, taken
10 June. Some subcuticular growth is evident at
the lesion center. Sparse surface growth is
abnormal and a few immature conidia are present
(bottom, X3040).

88

 

APPENDIX C

TRANSMISSION ELECTRON MICROGRAPHS 0F
PALISADE CELLS OF APPLE

89

90

Figure Cl.--Transmission electron micrographs of longitudinal
section of apple palisade cells taken from leaves
of 7-day schedule of CGA-64251. No membrane or
organelle abnormalities are evident.

91

 

92

Figure C2.--Transmission electron micrographs Of longitudinal
section of apple palisade cells taken from leaves
of unsprayed trees.

93