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ABSTRACT

FUNGICIDE REDISTRIBUTION
ON POTATO LEAVES

BY

Chong—Jin Kim

Redistribution of fungicide from the point of ap-
plication on a potato leaf was determined by electron probe
analysis, direct observation of deposits on leaf cuticle,
and by bioassay using the late blight fungus, Phytophthora

infestans. Because of suitability for electron probe an—

 

alysis, metallic fungicides, Kocide 101 containing cupric
hydroxide and Du-ter containing triphenyl tin hydroxide,
were used throughout.

Using electron microprobe analysis, there was no
detectable fungicide migration from 0.001 ml fungicide on
the potato leaf surfaces before or after weathering. Direct
Optical examination also showed clearly defined rims at the
margin of the droplets of measured or sprayed copper fung-
icides, but those of tin fungicides were less well defined.
There was no evidence of movement of fungicide from the ori—
ginal droplet.

Sporangial germination of Phytophthora infestans
on the potato leaves was inhibited within 5 mm of measured

fungicide drOplets of Kocide 101 and Du-ter. Toxicant had

diffused none or only slightly from the original droplet

as measured by spore germination. However, infection of the
fungus was conspicuously inhibited 15-20 mm or more from

the fungicide drOplets. Infection hyphae were apparently
more sensitive to different toxicants than were sporangio-
Spores. The detectable level of c0pper and tin in these bio-
assay trials was apparently below the level of sensitivity

of the electron probe.

The initial effluents from fungicide sprayed leaves
markedly reduced germination of sporangiospores. Effluents
collected after 5 or more hours weathering were low or lacking in
toxicity. Biofilm and Pinolene added to technical compounds
of tin and copper were slightly less effective than the

commercial fungicieds.

FUNGICIDE REDISTRIBUTION

ON POTATO LEAVES

BY

Chong—Jin Kim

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1973

.fl.§e/

ACKNOWLEDGEMENTS

fihfiifii‘“:

a? The author is greatly indebted to Dr. W. J. Hooker
for his guidance, encouragement, and help during the course
of this work and in the preparation of the manuscript.

The author would also like to thank the other members
of his committee, Drs. H. S. Potter, D. J. Dezeeuw, R. W.
Chase, and W. G. Fields for their real interest and assis—
tance.

Greatful recognition is extended to Mr. Leon Alwood
for his help in green house, and to Mr. T. C. Yang for his
kindness and help.

Acknowledgement is also made to the Kennecott Copper
Corp., Thompson-Hayward Chemical Co., Miller Chemical and
Fertilizer Corp., and Colloidal Products Division, Kalo

Laboratories, Inc. for partial support of this study.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS . . . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . . . .
LIST OF FIGURES. . . . . . . . . . . . . .
INTRODUCTION. . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . .
MATERIALS AND METHODS. . . . . . . . . . . .
RESULTS . . . . . . . . . . . . . . . .

Electron probe examination of fungicide drops . .
Sensitivity of the electron probe . . . . . .
Microscope examination of fungicide drops . . .
Germination of Phytophthora infestans sporangio—
spores at distances from the fungicide drOp
Infection of Phxtophthora infestans near the
fungicide drOp on potato leaf. . . . .
Germination of Phytophthora infestans spores in
fungicide at different concentrations . . .
Germination of PhytOphthora infestans in effluents

 

 

 

 

from fungicide sprayed leaves. . . . . . .
DISCUSSION . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . . .

iii

Page

ii

iv

12
12
18
23
25
31
36
38
43
47

49

 

Table

LIST OF TABLES

 

 

 

 

 

 

Page
Fungicide and surfactant preparations. . . . . 6
Germination of P. infestans sporangiospore at dis—
tances from the Kocide lOl drop on potato leaf. . 29
Germination of P. infestans sporangiospore at dis—
tances from Du-ter drOp on potato leaf . . . . 30
Infectivity of P. infestans at distance from a
fungicide drOp . . . . . . . . . . . . 35
Toxicity of fungicides in water suspension to
Phytophthora infestans sporangiospores . . . . 37
Toxicity to Phytophthora infestans of effluents
from potato leaves sprayed with cupric hydrox-
ide + different surfactants at time intervals
during weathering . . . . . . . . . . . 40
Toxicity to Phyt0phthora infestans of effluents
from potato leaves sprayed with triphenyl tin
hydroxide + different surfactants at time in-
tervals during weathering. . . . . . . . . 41

iv

Figure

10

11

12

LIST OF FIGURES

 

 

Page
Piece of interveinal leaf tissue prepared for
electron probe analysis. . . . . . . . . 8
Device for collecting sporangiOSpores of Phy—
tophthora infestans free from decayed leaf
tissue . . . . . . . . . . . . . . 11
Electron probe analysis through drop of Kocide
101 on potato leaf surface before weathering. . 13
Electron probe analysis from within drop of
Kocide 101 on potato leaf surface after 4
hours weathering . . . . . . . . . . . 14
Electron probe analysis through drop of
Du-ter on potato leaf surface before
weathering . . . . . . . . . . . . . 15
Electron probe analysis through drop of Du-ter
on potato leaf surface after 4 hours weather—
ing . . . . . . . . . . . 16
Electron probe analysis from within drop of
cupric hydroxide with Biofilm on potato leaf
surface before weathering . . . . . . . . 19
Electron probe analysis from within drop Of
cupric hydroxide‘with Pinolene on potato
leaf surface after 4 hours weathering . 20
Electron probe aruilysis through drop of tri-
phenyl tln hydroxide with Biofilm on potato 21
leaf surface aftfitr 4 hours weathering - - °
Electron probe arlalysis through drop of tri-
Phenyl tln hydrOXide with Pinolene on potato 22
leaf surface aftEEr 4 hours weathering .

24

Fungicide deposit; on potato leaf epidermis

Plan for fungiciéhe application on potato leaflet

surface, separathgn of segments, and sporangio—
spore applicatiorl. . . . . . .

26

INTRODUCT ION

Several workers have suggested that poor coverage
of plant foliage by fungicide may be somewhat compensated
by redistribution of initial deposits. It is generally
recognized that most fungicides are redistributed to some
extent following the initial application. The extent to
which this takes place is not completely understood.

When deposits of Bordeaux mixture on the potato
leaves were washed, redistribution was demonstrated by
Bj51ing and Sellgren (1957). Since 1965, Hilep has re-
ported a series of papers on 'the redistribution of fungi—
cide on plants', in which he demonstrated fungicide redistri-
bution in the laboratory and field. Hislop and Cox (1970)
demonstrated redistribution of various fungicides applied
to detached broad—bean leaflets by laboratory bioassay with

Botrytis fabae. The results showed that the protected areas

 

on the leaves inoculated after washing were often much
greater than on unwashed leaves.

Complete coverage of fungicide on the leaf surface
is difficult to accomplish in field application, and re—
distribution of initial deposits is necessary for sufficient
control of disease. Concentrated low volume application is
economically important in the field, and since coverage is
not complete, redistribution becomes important in disease
control. Therefore, information concerning the extent to

whcih redistribution takes place is highly desirable.

The object of the present work was to measure re-
distribution of copper and tin on the potato leaf surface,
and to verify local redistribution (Bj6rling and Sellgren,
1957; Hislop and Cox, 1970). To accomplish this, direct
observation with Optical microscope, electron probe analysis,

and bioassay with Phytophthora infestans were employed.

 

LITERATURE REVIEW

Perhaps the first observation of fungicide redistri—
bution was made by Millardet in 1882 and published in 1885.
Copper sulfate and lime were dissolved in water and applied
with a brush on grape leaves to prevent pilfering. Re-
duction of mildew of the vine on treated leaves followed.
Quoting from the translation by F. J. Schneiderhan, "it
is not necessary that the leaves be wholly covered by the
mixture. I believe it safe to assert that a single spot of
it on each leaf is sufficient". Ricaud and Paulin, 1884
(Large, 1940) found that grapes growing on poles treated with
copper sulfate to prevent wood decay showed less downy mildew
than neighboring grapes on untreated supports. Hamilton
‘32 21. (1943) showed that rains can wash a protectant from
a heavily covered area and drop it onto unprotected areas
below. Hilep (1965, 1966a, 1967, 1969) has described
distant redistribution of fungicide experimentally in the
laboratory and in the field.

In contrast to the concept of distant redistribution,
Bj6rling and Sellgren (1957) used the term '1ocal redistri-
bution'. They found that best protection against late
blight was obtained by the smallest droplets of Bordeaux
mixture, and control was interpreted as the result of a local
redistribution of Bordeaux particles to unprotected leaf

areas by dew or rain. Morgan and Russell (1965) suggested

redistribution of active fungicide from initial deposits

of dodineanricaptan in a trial of diluted fungicides applied
in form of much smaller droplets for control of apple scab.
Hislop and Cox (1970) worked on local redistribution of
various fungicides by bioassay with broad bean leaflets and
by leaf printing techniques. They observed that fungicidal
particulate and soluble material will spread in water from
the initial deposit and that the protected areas on leaves
inoculated after washing were often much greater than on
unwashed leaves.

The extent of redistribution was related to the in—
herent toxicity, physical form, duration of weathering, and
tenacity of fungicides (Hislop and Cox, 1970), and wettable
powder formulations were to some extent resuspended in washing
water, and frequently redeposited in previously unprotected
areas.

Goossen (1958) presents data to show that copper con-
centration is higher on lower leaf surfaces of Sprayed leaves
after a rain than before. He suggests that fungicide should
not be highly tenacious as fungicide transfer from one part
of the plant to another is an important aspect of control.
Hislop and Cox (1970) confirmed Goossen's suggestion, and
reported that most fungicides tested were probably moved
over the leaf surface in particulate form. The effect of
time of washing on redistribution was examined by varying
lengths of time before drying and inoculation. The results
showed that protection from redistributed fungicide decreased

the longer leaves were washed.

 

Differences in protection from particle size fractions
may be related to tenacity or toxicity effects (Hislop and

Cox, 1970). Assay with Alternaria tenuis confirmed that

 

small particles were more toxic from large particles. They
also found that redistribution from large fungicide drops
applied with a microliter syringe was better from the more
concentrated suspensions than from dilute suSpensions.
Rich (1954) found that during weathering of dried Bordeaux
deposits, the larger the initial deposit, the smaller was
the percentage lost, but the larger the deposit of zineb,
the greater was the proportion of its mass lost by weathering.

None of the wetting agents representing non—ionic,
anionic, and cationic types, enhanced redistribution (Hislop
and Cox, 1970). At low concentrations (Somers, 1957» rep-
resentative wetting agents of anionic, cationic, and non-
ionic type slightly lowered the amount of copper spray de—
posit at 'run-off', but in the absence of supplements, spray
retention increased with increasing spray concentration and
with increasing wettability of the surface.

High concentrations of leaf exudates sometimes found
in rain and dew were inhibitory to germination of Botrytis
cinerea spores (Kovacs and Szedke, 1956). Germination of

Alternaria tenuis was slightly inhibited by the presence of

 

leaf exudates (Hislop, 1966a), and soluble copper in leaf
washing was relatively non-toxic. Biedermann and Mfiller

(1952) observed inactivation of copper by leaf excretions.

MATERIALS AND METHODS

Preliminary studies of fungicide deposition were made
with smooth leaf surfaces of pepper and sugar beet and with
the hirsute leaf surface of potato. No marked differences
existed in drOp distribution nor tenacity on leaves of the
plants tested. For this reason subsequent tests were made

with leaves of potato, Solanum tuberosum L.

 

Bioassay for fungicidal action was made with the

fungus, Phytophthora infestans (Mont.) deBary, which is the

 

incitant of the late blight disease of potato.

Spray materials were prepared in distilled water as
described (Table 1).

Distribution of copper and tin fungicides was deter-
mined by electron probe analysis of potato leaf surfaces.
DrOpS of fungicide, 0.001 ml volume, were placed between the
lateral veins on the upper surface of potato leaflets using
an Hamilton microliter syringe. The drop location was identi—
fied by placing two drops of ink one on each side of the
fungicide drop. After drying, leaves were subjected to:

1. No further treatment

2. Weathering for varying periods in a moist chamber
using an atomizer which delivered to the leaf
surface 0.25 to 1.5 mm of water per hour. During
weathering the leaflet was arranged so that the tip
pointed downward at approximately 30 degrees.

Later, interveinal pieces were cut from the area
between the lateral veins of the leaflet. One end of the

leaf piece was cut diagonally to indicate clearly the upper

surface where the fungicide had been applied (Fig. 11 In order

Table l.--Fungicide and surfactant preparations.

 

 

 

Product, manufacturer, and description Amount/liter
Fungicides -
Kocide 101, 2 lbs/25 gal, cupric hydrox- 9.576 gm

ide (86%) plus surfactant, Kennecott
Copper Corp., Houston, Texas

Cupric hydroxide (Technical), 2 lbs/25 9.576 gm
gal, no surfactant, 100% active, Ken-
necott Copper Corp., Houston, Texas

Du-ter, 0.6 lbs/25 gal, triphenyl tin 3.051 gm
hydroxide (47.5%) plus surfactant,

Thompson-Hayward Chem. Co., Kansas

City, Kansas.

Triphenyl tin hydroxide (98%), no sur- 1.525 gm
factant, Thompson-Hayward Chem. Co.,
Kansas City, Kansas

Surfactants

Pinolene (Nu-Film-17), 2 pints/100 gal, 10.000 gm
nonionic, Miller Chemical and Fertilizer
Corp., Hanover, Pennsylvania

Biofilm, 6 02/100 gal, anionic-nonionic 0.466 gm
blend, Colloidal Products Division,

Kalo Laboratories, In., Petaluma, Cal-

ifornia

h 23 mm lg direction of
slope

T
a ,3, ’
l

 

 

 

 

 

 

s— \\ ~—-~-35 m >1

ink drop fungicide
drop

Figure l.--Piece of interveinal leaf tissue prepared for
electron probe analysis.

to protect the surface, each piece was separately placed

in a small piece of Kleenex tissue, then into flat paper
towels. For obtaining dry, flat sections a glass plate was
put on the paper towels for several days.

Analysis of copper was made with an electron probe
(Applied Research Laboratories Model EMX-Sna) set for K
alpha 1.537 A, 0.05 micro amperes, 25 KV. Tin was deter-
mined at L alpha 3.595 A, 0.1 micro amperes, 25 KV.

Measured droplets as well as droplets on leaf sur-
faces following spraying were examined optically. A glass
microsc0pe slide covered with an adhesive film was adpressed
to the leaf surface. The epidermis of the sprayed leaf sur-
face remained fastened to the glass slide as the leaf was

stripped away.

Preparation of leaf epidermal surfaces for micro—

scopic examination:

 

(l) A small amount of Duco cement or Elmer's Clear
Cement was drOpped on a slide glass and immediately spread
with a glass rod or wooden stick to make a thin film on the
surface. After approximately 1 minute a small amount of
rubber cement was spread on the top of the film of Duco
cement using the same method as described earlier. It was
important that the rubber cement be smeared before the Duco
cement dried out. These two kinds of cements must not be
mixed. Neither rubber cement nor Duco cement alone will
serve. Duco cement and Elmer's clear cement seemed to be
equally good. It was better to make the film of the rubber
cement slightly thicker than the film of Duco cement.

This composite film may be used up to several months after
preparation.

(2) The sample leaf was placed on the slide, pre-
pared with adhesive and the leaf surface was pressed on the
film with the thumb. The pressing was done so the leaf
surface was completely in contact with the film. Then the
leaf was peeled from the slide leaving the epidermal layer
attached to the Duco film.

(3) Permanent preparations lasting a few months
at least may be made by dropping Duco cement on the epider-

mis and covering this with a cover glass.

10

Preparation of sporangiospores of P. infestans:
Fresh young sporangiospores were obtained as follows:

(1) Matured leaves of the potato cultivar, 41956,
were placed on damp paper towels on moistened Sphagnum moss
in a wooden flat.

(2) One drop of the sporangiospore suspension was
inoculated each leaflet. The flat with inoculated leaflets
was kept in a polyvinyl bag to maintain high moisture and
placed in a growth chamber at l9-20°C.

(3) After 2 days the inoculated leaflets were
soaked briefly in distilled water, and shaken gently. There—
after, they were treated similarly every one or two days.

(4) Four to five days after inoculation, typical
whitish masses of sporania appeared on the lower surface of
the leaflets. Thus, sporangiospores were less than one or
two days old, since the most recent rinsing in distilled
water had removed the mature spores.

Device for collecting sporagia of P. infestans:

 

Spores were collected from infected leaves in a moist chamber
using vacuum from an aspirator with the device illustrated (Fig.
2). Initially a few ml of water were placed in the collector

to serve as a spore trap. Solutes from diseased leaf

tissue were avoided in the collected Spore sample as

spores were moved directly into the reservoir of water

by air current from the place of formation, the sporangio-
spores, which are well above the substrate. By this means

desiccation was avoided as spores were immediately suspended

ll

 

<——to vacuum

glass tube with
polished tip

water suspension of
sporangiospores

 

late blight
lesion

 

Figure 2.--Device for collecting sporangiospores of Phytophthora
infestans free from decayed leaf tissue.

 

 

in a liquid after removal from the sporangiospores. spor-
angiospores in suSpension were transferred to the test sys-

tems promptly after collecting.

RESULTS

Electron probe examination of fungicide drOps: Dis—
tribution of fungicides as measured by the electron probe
was essentially similar on either smooth leaved plants, pepper
and sugar beet or on an hirsute leaf such as that of potato.

Sprays of Kocide 101 and Du—ter were prepared in
concentrations of 2 lbs and 0.6 lbs commercial fungicide
per 25 gal respectively. Individual droplets of 0.001 ml
volume contained approximately 0.00001 gm of copper fungi—
cide and 0.000003 gm of tin fungicide respectively. These
amounts of copper and tin were well within the level of sen—
sitivity of the electron probe. Concentrations of copper
and tin are shown in counts per second on the accompanying
graphs as 3,000 and 300 for Cu and 100 for Sn. Each unit on
the horizontal (X) axis indicates 0.1 mm along the surface
of the leaf. Recorder sensitivity for c0pper was changed
during the analysis. Where copper was apparently lacking on
the leaf surface and the recorder pen was at or near the
base line, sensitivity was set at 300 counts per full scale
and where concentrations were high, sensitivity was changed
to 3,000 counts per full scale.

Electron probe determinations were usually begun
within the drop and quantitative measurement made in a straight
line along the leaf sample between the larger lateral veins
(Fig. 4). Analysis of certain drOps (Figs. 3, 5, 6) were made

starting outside the drOp and extended through the drOp.

12

l3

 

 

 

 

 

Intensity
3000 counts per full scale
300 counts per full scale

 

 

 

 

 

 

 

 

 

 

 

n A n . . I I I 1%

Distance between unit marks (0.1 mm)

Figure 3.—-Electron probe analysis through drop of Kocide 101 on
potato leaf surface before weathering. Where copper
concentration was high 3000 counts per full scale was
used. Where counts approached the base line sensi-
tivity was changed to 300 counts per full scale.

l4

oaaom Haze nod munsoo com

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

oaaom Hazy non muosoo ooom
zpfimcoweH

Distance between unit marks (0.1 mm)

101 on potato leaf surface after 4 hours weathering.

Figure 4.—-Electron probe analysis from within drop of Kocide

Intensity

l5

 

 

 

 

 

 

 

 

 

4100 counts per full scale

 

 

 

 

 

 

 

 

 

  

! A I I I I l 1 I

Distance between unit marksV(0.l mm)

 

Figure 5.--Electron probe analysis through drop of Du-ter on
potato leaf surface before weathering.

 

Intensity
100 counts per full scale

16

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.L I n l L I I l I

Distance between unit marks (0.1 mm)

Figure 6.—-Electron probe analysis through drop of Du-ter on
potato leaf surface after 4 hours weathering.

17

Copper and tin deposits of commercially available
Kocide 101 (Figs. 3, 4) and Du—ter (Figs. 5,6) respectively,
were characterized on leaves by clearly defined margins.
These margins were distinct on the electron probe with or
without weathering. Concentrations of metal abruptly fell
to the base level at the drop edge. The edge of the drOp
could be visually distinguished in the optical microscope
of the electron probe, and the border identified simultaneously
on the electron probe graph. Apparently from or around a
single drOp of commercial fungicide there was no well de-
fined spreading over the leaf in quantities which were de—
tectable by the electron probe.

Both fungicides frequently formed a dense rim around
or in portions of the perimeter of the drop. Within such a
drop the central area had metal in varying concentrations and
the concentration on the rim was higher than that near the
center. This rim was not consistently present nor was it
entire and continuous. Evidence of such a heavy rim is shown
on the right margin of the droplet in Figs. 5, 6 and to a
lesser extent in Fig. 3.

Occasionally specks of copper were present near the
drOp. These are believed to have been bounced from the de-
posit by the electron beam during electron probe examination.
This was evident by microscopic observation of the specimen
during migration of the specimen through the electron beam.

Drops of either fungicide with Biofilm or Pinolene

again had clearly defined margins butthe marginal rim was not

18

quite so evident. There apparently was some movement from the
point of original application to surrounding leaf surface.
Possibly the movement could have been described as a crawling
type of surfactant action. COpper or tin so moved from the
original drop did not adhere well to the surface and was
apparently not as effective as measured in bioassay.

Droplets with Biofilm and cupric hydroxide (Fig. 7)
had a well defined margin but copper had migrated outward in
concentrations sufficient to have been recorded in the probe.
Droplets of Pinolene with cupric hydroxide (Fig. 8) were
similar. Droplets of triphenyl tin with Biofilm (Fig. 9) or
with Pinolene (Fig. 10) had a more gradual slope at the
margin than those with the commercial Du—ter fungicide and
also there was apparently a low level deposition outside the
droplet.

Sensitivity of the electron probe: Parallel bioassay

 

trials suggested that active toxicant existed immediately
outside the drOplet. This level of copper or tin may have

been below the level of sensitivity of the electron probe

and, therefore, may not have been detected in the pre—

vious tests. To determine the sensitivity of the electron
probe, serial dilutions were prepared with Kocide 101 and
Du-ter. Droplets (0.001 ml volume) were placed on potato
leaves as previously described. Kocide 101 in the drOplet as in
the previous studies (2.265 gm commercial fungicide/235 m1;

2 lbs/25 gal) could be detected at dilutions of 1/1000 but

not a 1/10,000. Du-ter (0.72 gm commercial fungicide/236 ml;

m

- F. r;- *fl‘vm-

._...._-a- -
C

’ ‘0’ ”217'."

 

 

 

 

 

 

 

Intensity

 

 

3000 counts per full scale

19

 

 

 

 

 

 

 

 

 

 

 

 

-———-—-‘—-———-—-

 

 

 

J

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

i} J W. “J {Nikki L

l l J_ .L 4

Distance between unit marks (0.1 mm)

#

Figure 7.--Electron probe analysis from within drop of cupric

hydroxide with Biofilm on potato leaf surface be-
fore weathering.

Intensity

3000 counts per full scale

20

 

 

-.--.A -..-~— a.

 

.-_...._-..._—

 

 

 

a.---.—..-____...._.-__._-- ..... .

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

300 counts per full scale

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\. Lt)
I I l L IL'Ji l a l '

 

'Distance between unit marks (0.1 mm)

Figure 8.--Electron probe analysis from within drop of cupric
hydroxide with Pinolene on potato leaf surface
after 4 hours weathering.

 

i. . oil: unit-1...! .a;
i . ‘ (Ii. 4

Intensity
100 counts per full scale

21

 

 

 

 

 

 

 

 

 

 

 

L I _ 1

Distance between unit marks (0.1 mm)

Figure 9.--Electron probe analysis through drop of triphenyl
tin hydroxide with Biofilm on potato leaf surface
after 4 hours weathering.

A l l l ,

22

 

 

 

Intensity
100 counts per full scale

 

 

 

 

 

 

.l 1 L I 1

Distance between unit marks (0.1 mm)

Figure lO.--Electron probe analysis through drop of triphenyl
tin hydroxide with Pinolene on potato leaf surface
after 4 hours weathering.

23

0.6 lbs/25 gal) was evident at 1/10 dilution on one trial

and at l/100 dilution in a second trial. The original drOp-
let before dilution contained 0.0095975 mg and 0.0030508 mg
of Kocide 101 and of Du-ter respectively. It was concluded
that the concentration of toxicant determined by bioassay was
below the level of sensitivity of the electron probe.

Microscope examination of fungicide drops: Direct

 

optical examination of spray deposits was accomplished by
stripping away the upper leaf surface with the adhesive film
described earlier. A number of droplets from each prep-
aration were observed and attempts made to assess variation
between them. Photographs of representative deposits are
shown (Figs. 11A, B).

Copper droplets could be examined with relative
ease. Triphenyl tin hydroxide was more difficult to observe
on the leaf surface due probably to an initial concentration
considerably less than that of copper and because it was
some what less Opaque. Measured droplets of tin compounds
(0.001 ml) could be seen but observations of Sprayed drop—
lets were not satisfactory

A rim of heavy Spray concentration at the margin of
the measured drop was present with commercial Kocide 101 and
Du-ter (Fig. 11 A). This rim was not completely continuous,
but in general, was clearly defined, and the rim was not lost
after mild weathering. Sprayed droplets of copper compounds
frequently had a heavy rim on at least one Side and a less

conspicuous margin elsewhere (Fig. 11 B).

24

 

Figure ll.-—Fungicide deposit on potato leaf epidermis.
A: Du-ter measured droplet, and B: Kocide 101
spray. (Magnification 210 X).

25

With both Spreaders, Biofilm and Pinolene, technical
compounds lacked the clearly defined rim and the drops became
optically less dense on weathering. These Spreaders did not
perform better than the Spreaders in the commercial fungi—
cides under test. There was a noticeable loss of fungi-
cide following a few hours of gentle mist. Droplet margins
were not as well defined and were often identified only with
difficulty.

These observations support those of the electron
probe but were not considered as precise.

Germination of Phytophthora infestans sporangiospores

 

at distances from the fungicide drop: Drops of fungicide

 

(0.001 ml volumes) were placed on potato leaf surfaces.
Kocide 101 was prepared at 2 lbs/25 gal so that a drop (0.001
ml) contained 0.0095975 mg Kocide 101. Du—ter was prepared
0.6 lbs/25 gal to contain in 0.001 ml 0.0030508 mg commercial
fungicide.

Fungicide droplets were dried on the leaf surface
and exposed to mild weathering in a mist chamber for 5 hrs.
The amount of mist was approximately 0.36 mm per hr. Leaf
surfaces on which the fungicide drOp was placed were oriented
at a 30° angle so that the drainage during weathering would
run down the surface to be examined. The leaf was then
surface dried in air and a strip approximately 1 cm wide and
2.5 cm long was cut so that the fungicide drop was at one end
of the strip. The strip was then cut into segments 5 mm wide

but left attached as shown (Fig. 12). This was done to preclude

26

 

 

 

 

 

 

K 20 mm 3,1
T l : I
E : : : ——->
I t u ,
O ' i ' direction of
H M E O i 7,0 if 70 slope
I\ ' ”I ,/”i
"R ““““ x/IR, ‘./ '
AL ;R\\i /T//;;I1::::HJR\
T \/,/-— ““\\\
fungicide drOps of line of .
drop spore separatlon
suspension

Figure 12.-—Plan for fungicide application on potato leaflet
surface, separation of segments, and sporangio-
Spore application.

washing of toxicant across the leaf during application of
Spore suspensions. The spore suspension was prepared using
the spore collector shown (Fig. 2), and sporangiospore
concentration was set at 500 or more spores per 0.001 ml.
A drop of spore suspension, 0.001 ml, was then placed at 0,
5, 10, and 15 mm from the original fungicide drop. The
segments were then incubated in a moist chamber for 48 hrs.

The leaf surface with the spores was air dried after
which the epidermis with the attached spores was stripped
from the leaf using the procedure previously described. The
strip was then stained with Ziehl's carbol fuchsin.

Spore germination counts were made to determine the
percentages of spore germination near the original fungi-
cide droplet and at 5, 10, and 15 mm from it. At each

location 50 Sporangiospores were counted and the percent

 

 

27

germination determined. Empty Sporangial cases or those

with germ tubes were considered nahave germinated (Fig. 13A).

 

Figure 13A: Germinated Phytophthora infestans sporangio-
spores showing empty spore cases. These spores
were removed from the leaf surface using ad-
hesive.

 

B: Nongerminated spores. Note the shrivelled pro-
toplast adhering to the inside surface of the
spore wall. (Magnification, 840 X).

28

The prot0plast of nongerminated sporangiospores adhered
closely to the walls on one side (Fig. 13B) and accurate
counting was difficult. Data presented are the combined
average results from 3 trials and data from each trial con-
sisted of two fields of 50 sporangiospores. Results with
both fungicides as well as the copper and tin compounds with
surfactants, Biofilm and Pinolene, are presented for Kocide
101 (Table 2) and Du-ter (Table 3).

Germination was consistently low with both fungi-
cides applied on or very close to the drOpS (0 mm distance
from drop). Similar results were obtained with cupric
hydroxide and triphenyl tin hydroxide with either spreader.
Kocide 101 was relatively nontoxic at the 0 hour time 5-15
mm from the drop. Toxicity increased with 5 to 24 hours of
weathering. Cupric hydroxide was relatively nontoxic
5—15 mm from the point of application with either Biofilm
or Pinolene.

Du-ter was effective at the 0 hour 5-15 mm from the
drop. It was relatively nontoxic 5-15 mm from the point
of application after 5-24 hours weathering. Triphenyl tin
hydroxide with Pinolene was toxic at 5—15 mm at 0 hour. How—
ever at later times it was relatively nontoxic away from
the drop. Triphenyl tin hydroxide with Biofilm somewhat less
toxic at 5-15 mm before weathering and was relatively nontoxic

after weathering.

29

Table. 2.--Germination of g. infestans sporangiospore at distances
from the Kocide lOI drop E7on potato leaf.

 

 

Treatment and

 

duration of Germination at various distances from
weathering fungicide drop-
0 mm 5 mm 10 mm 15 mm
0 hour
No fungicide 33% 27% 29% 22%
Kocide 101 l 20 18 21
Biofilm + cupric hydroxide 0 22 23 26
Pinolene + cupric hydroxide l 30 24 22
5 hours
No fungicide 34 44 34 42
Kocide 101 2 8 19 20
Biofilm + cupric hydroxide 0 16 15 ll
Pinolene + cupric hydroxide l 21 20 23
24 hours
No fungicide 34 34 35 33
Kocide 101 1 2 8 8
Biofilm + cupric hydroxide o 13 12 12
Pinolene + cupric hydroxide o 22 22 3o

 

lFungicide drop of 0.001 ml contained 0.009575 mg of Kocide 101 (2 lbs/
25 gal).

2/Combined Spore germination data from counting 2 fields of 50 spor-
angiospores each for each of 3 trials.

 

30

Table 3.--Germination of P. infestans sporangiospore at distances
from Du-ter cropl/on potato leaf.

Treatment and

 

duration of Germination at various distances from
weathering fungicide drop
0 mm 5 mm 10 mm 15 mm

0 hour
No fungicide 14% 17% 12% 14%
Du-ter 0 9 7 3
Biofilm + triphenyl tin
hydroxide 0 15 15 9
Pinolene + triphenyl tin
hydroxide 3 9 11 8

5 hours
No fungicide 19 23 19 19
Du—ter l 15 16 16
Biofilm + triphenyl tin
hydroxide l 21 24 26
Pinolene + triphenyl tin
hydroxide 3 18 15 18

24 hours

No fungicide 25 27 20 20
Du-ter 2 ll 13 17

Biofilm + triphenyl tin
hydroxide 1 16 24 14

Pinolene + triphenyl tin
hydroxide 4 ll 13 16

 

l-/Fungicide drop of 0.001 ml contained 0.0030508 mg Du-ter (47.5% active)
(0.6 lbs/25 gal).

2/

— Combined spore germination data from counting 2 fields of 50 spor—
angiOSpores each for each of 3 trials.

 

 

31

Infection of Phytophthora infestans near the fungicide

 

drop on potato leaf: Distribution of fungicide around the

 

point of application was determined by demonstrating infection
sites in a cleared potato leaf.

A potato terminal leaflet was cut along the midrib
into two parts. A drop (0.001 ml) of fungicide was placed
on the center of the upper surface of one of the half leaflets.
The opposite half leaf recieved no fungicide. After surface
drying, both half leaves were weathered for 30 minutes by
atomizer Spraying with approximately 0.36 mm of water per
hour. Both half leaflets were surface dried in air, and
sprayed with a sporangiospore suspension. The Spore sus—
pension was obtained by suspending spores from diseased leaves
in glass distilled water. The half leaflets were kept in a
dish with moistened filter papers, and incubated for 48 hours
in a growth chamber at l9-20°C.

After incubation half leaflets were boiled in water
for 5 minutes, then heated in 95% ethyl alcohol until they
were clear. Infection sites as indicated by numerous small
necrotic spots then become visible. In cross section of the
potato leaf, necrotic areas appeared as shown in Fig. 14.

Incidence of infection was determined by placing a
grid (Fig. 15) on the surface of the half leaflets and
counting infection points within each of the concentric
areas with under a bacterial colony counter.

The protected area of the leaf surface was usually
more or less circular around the point of application sug-

gesting diffusion from the droplet (Fig. 16). The circular

32

 

Figure l4.--Cross section of potato leaf showing the extent
of necrosis after 48 hours of incubation in
these trials. (Magnification, 210 X).

pattern was not a constant feature presumably because drain—
age patterns distributed the toxicant irregularly over the
surface.

The number and frequency of infection sites is pre—
sented (Table 4). Each number is the average infection of

at least 10 half leaves. Infectivity was usually precluded

 

 

 

33

   

 

I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
_I

-—--——-——— 20 mm

III IIIIIIIIIIIIII II IIIIIIIIIIII IIIIIIL

Figure 15.--Grid pattern used in quantitatively determining

incidence of infection at distances from the

p
O
r
d
e
d
.l
C
.l
g
n
u
f

 

 

 

 

34

 

Figure l6.——Phypophthora infestans infection sites on potato
leaves after 48 hours incubation and clearing
in ethanol. Fungicide (0.001 ml) was placed on
the leaf at the black circle, weathered 0.5
hrS, surface dried, and Sprayed with spore sus-
pension A, Kocide and B, Du—ter. (Magnification
l X).

 

35

1/

Table 4.—-Infectivity of P. infestans at distance from a fungicide drop— .

 

 

 

 

 

‘ 2/ ‘ Infection points%{ i
Tox1cant— at distances from droplet application Control
and date 0—5 mm 6—10 mm 11-15 mm 16-20 mm 0-20 mm
of test (A) (B) (c) ID).5./ A+B+C+D
No. CFAi/ No. CFA No. CFA No. CFA
Kocide
7/24/71 0.5 8 6 33 14 44 34 78 74
7/25/71 0.1 2 2 8 7 22 15 36 68
7/26/71 0.0 o 2 10 8 24 19 43 108
Du-ter
6/8/71 0.0 0 3 l6 9 30 22 51 137
6/9/71 0.5 7 13 67 37 117 50 - 114 208
6/10/71 0.7 11 6 27 21 55 ' 9 21 101
6/11/71 0.3 4 4 23 17 54 42 96 288

 

i/Fungicide (1) applied in 0.001 ml droplet to half leaf of potato, (2) surface
dried, (3) weathered, 30 minutes in mist chamber (0.36 mm water/hr), (4)
surface dried, (5) sporangiospores of Phytophthora infestans sprayed on leaf,
(6) incubated in an illuminated mist chamber approximately 48 hrs, (7) leaf
cleared by boiling in 95% ethyl alcohol.

E/Kocide 101 droplet (2 lbs/25 gal); 0.0095975 mg applied in 0.001 ml volume.

Du-ter droplet (0.6 lbs commercial (47.5% active) /25 gal); 0.0030508 mg
(47.5% active) applied in 0.001 ml volume.

E/Brown spots indicating early infection counted in a circular area a distance
shown from the point of application. Area of A is 20 mmz; B, 59 mm ; C,
98 mmZ} D, 137 mm2 respectively. These total 314 mmz, the area of the control
and comprise 6, 19, 32, and 43% respectively of the total area surveyed.

i/CFA: Correction for area differences between the area of observation and the
area of the control.
Factors are for: A, 16.0; B, 5.3; C, 3.2; and D, 2.3.

é/In certain instances leaflets were not sufficiently large to permit observa-
tion in area D. Average values in this column are calculated only from
leaves of sufficient size. Values for the Control were corrected also for
this area difference.

 

36

in circle A at the point of fungicide application. A few
infection sites were counted in this area for both of the
fungicides. Positive evidence was obtained of fungicidal
action preventing infection by P. infestans some distance
from the point of application. Usually infectivity was pro—
gressively reduced from the 16—20 mm, zone D, towards the
zones, C, B, and A. Occasionally infection in the control
area was not as high as that of the outer zones near the

fungicide drOp. The exact reason for this is not apparent,

 

however, it is generally difficult to obtain a consistently

high level of uniformity of infection with P. infestans.

 

A single drOp of fungicide protected a considerable
area of the leaf surface. Quite frequently infection was very
low on the entire half leaf treated with a single drop of
fungicide. This suggested that for practical purposes pro-
tection would have been adequate under field conditions where
inoculum load per leaf was not excessive.

Germination of PhytOphthora infestans spores in
fungicide at different concentrations: Information was dee
sired concerning the toxicity of the fungicides, Kocide 101
and Du-ter, in dilute solutions to sporangiospores. This
information should give some indication of the concentration
on the leaf surface capable of retarding germination and
possibly infection.

Fungicides were serially diluted (Table 5). A drop
of fungicide (0.01 ml) was placed in a van Tieghem cell.

Sporangiospores less than 48 hours old, obtained from infected

 

 

 

37

Table 5.--Toxicity of fungicides in water suspension to
Phytophthora infestans sporangiospores.

 

 

 

distilled % germination of sporangiospores

 

water at concentrations (ppm active)
Du-ter control indicated
3/ 475 47.5 4.75 0.475
test 4/10/71 33, 54— 0, 0 0, 0 9, 9 22, 34
test 4/18/71 44, 57 0, 0 2, 3 20, 9 44, 70
test 5/1/71 71, 59 0, 0 l, 4 4, 5 8, l3
. 2/

KOClde— control 1000 100 10 1
test 5/2/71 39, ll 0, 0 l4, 6 9 50, 54
test 5/3/71 75, 86 0, 0 0, 0 0, 0 79, 71
test 5/8/71 2, l3 0, 0 0, 0 0, 0 ll, 0
test 5/9/71 81, 85 0, 0 0, 0 0, 0 67, 72

 

l/Commercial Du-ter (47.5% active).

_2_/
_3_/

Kocide 101 100% active in the preparation.

Paired numbers indicate percent germination in 100 spore sample
from 2 separate van Tieghem cells.

 

 

38

potato leaves were transferred directly by using a bevelled
wooden applicator stick. The bevelled edge was dampened with
glass distilled water and touched to the mass of Sporangio-
spores on the surface of the diseased leaves. Since spores
over a constant area were thus harvested the number of
spores transferred was relatively constant. Over 1000
sporangiospores were present in each van Tieghem cell. By
this method of transferring spores, the concentration of tox—
icant was not modified by adding inoculum in water suspension.

The sporangiospores in a van Tieghem cell were incu—
bated in a refrigerator at 6-7 C for one hour, then removed
to a growth chamber at 19—20 C for 24 hours. After incu-
bation, 100 random spores were counted to calculate the per-
cent germination. Empty sporangial cases and sporangiospores
which had germinated with germ tube were considered to have
been germinated spores (Table 5).

The percent germination of spores obtained by this
method was extremely variable and was slightly higher than
with the suction tube method described (Fig. 2).

Germination of Phytophthora infestans in effluents from

 

fungicide sprayed leaves: Estimation of toxicity of ef—

 

fluents from fungicide sprayed leaves was done by determining
percentage of germination of sporangiospores. Leaves from
green house grown plants were sprayed with fungicide to ap-
proximate good field coverage and then surface dried. They
were placed in a moist chamber and exposed to fine water

drOplet precipitation of approximately 0.35 mm per hour.

 

 

39

Effluents dripping from the leaflet tips were collected

in small vials at intervals during a 96 hour weathering
period. 0 hour indicates the time of collecting the first
few drops of effluent at the beginning of the weathering
period, and the other effluents were collected at the several
times indicated in (Tables 6, 7).

Toxicity of leaf effluents was determined using
sporangiospores obtained from the surface of potato leaves
infected with late blight using the spore collector (Fig. 2).
A concentrated suspension of more than 1,000 sporangio-
spores per 0.001 ml were obtained as counted with a stand-
ard blood haemocytometer.

DrOps of effluent, 0.009 ml, were transferred to a
cover slip. An 0.001 ml drOp of spore suspension was added
and the droplet was mixed with a small pointed strip of
Parafilm M manufactured by the American Can Co. The cover
slip was then inverted over a van Tieghem cell. Empty
Sporangial cases or those with germ tube were counted after
incubation in the refrigerator at 7-8 C. Results of tests
with Kocide 101 (Table 6)and with Du-ter (Table 7) are shown.
Controls involving no fungicide were prepared using effluents
from unsprayed leaves collected in a manner similar to that
described for the fungicide trials.

The initial effluent of both copper (Table 6) and tin
(Table 7) was toxic but after 5 hrs., effluents were low in
toxicity and lacking in toxicity after longer periods. Neither
surfactant modified the behavior of either toxicant in these

trials.

 

 

40

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42

Inconclusive evidence in these trials suggested that
a toxic substance may have been eluted from untreated potato
leaves at the 0 hr. time. The initial effluent (0 hour)
caused some reduction in germination. After a few hours,

however, the effluent was no longer toxic.

 

 

 

DISCUSSION

The extent to which fungicide redistribution is im-
portant in potato disease control has not been accurately
measured. It is generally accepted that redistribution takes
place somehow. The two fungicides studied, Du~ter (tri—
phenyl tin hydroxide) and Kocide lOl (cupric hydroxide),
were selected because of suitability for electron probe
analysis.

Direct observation of the fungicide drop by visual
observation and electron probe analysis suggest that the
nature of the droplet margin and appearance of the drOplet
was not appreciably changed by surface water action on the
leaf. DrOplet margins were clearly defined and discrete, and
no detectable fungicide had migrated. Optical examination
supported the electron microprobe analysis, and showed
clearly defined rims in the margins. Kocide 101 and Du—ter
seemed to be very tenacious, and the deposit did not resus—
pend in mild weathering.

By direct observation and bioassay of washed deposit,
Hislop and Cox (1970) found that wettable powder formulations
were resuspended and frequently redeposited in previously un—
protected areas. They also pointed out that Bordeaux and
Burgundy mixtures were very tenacious and were not resuspended.
Redistribution in my observations must have been below the

level of sensitivity of the electron probe because protective

43

 

 

 

44

action away from the fungicide drOp was demonstrated by sub—

sequent bioassay with P. infestans.

 

Sporangial germination on the leaves close to the
fungicide drop was very low and 5 mm or more from the drOp,
germination approximated that of the control. This suggests
that toxicant has little effect a short distance from the fun~
gicide droplets. This also supports the electron micro-
probe analysis and optical observation. Again the level of

toxicant redistributed from the drOp of fungicide was ap-

 

parently very low and not sufficient to prevent germination
of sporangiospores.

Lower concentrations of toxicant redistributed from
the fungicide drOp were demonstrated using infection as the
criterion for protection. Infection tests around the funi-
cide droplets (Table 4), demonstrated that protected areas
were considerably larger than the area occupied by the ori—
ginal drop of fungicide. Infection was inhibited 15—20 mm
from the fungicide drOplet. Thus,high fungicidal protection
seems to be due to greater sensitivity of germ tubes and in—
fective hyphae than that of sporangiospores. There are a
number of reports comparing mycelial and spore sensitivities
to fungicides. Diphenyl, for example, does not inhibit spore
germination of several species of fungi, but it is inhibitory
to hyphal growth of those organisms (Ramsey et al., 1944;
Horsfall, 1956). The process of infection by germ tubes from
fungus Spores is very complex, and many requirements are

needed to complete infection.

 

45

The possibility exists that fungicide droplets may
have been redistributed by spraying the spore suspension,
over the leaf surface and the fungicide may have migrated
more extensively. Hislop and Cox (1970) reported that two
cycles of wetting followed by drying increased protection.

If redistribution was accomplished by the spray application
of spore suSpension, in my tests it was not possible to dif-
ferentiate between redistribution following the initial
weathering and that following application of spore suspension.
Under field conditions, however effective protection should
be anticipated by distribution on either or both occasions.

The protected area in the infection test was not
always circular around the point of application. This was
presumably due to leaf orientation and to drainage patterns
distributing the toxicant irregularly over the surface.
Irregular leaf surface topography associated with the leaf
hairs, stomata, vascular networks,and wax deposits etc. may
have been involved.

The initial effluents from fungicide sprayed leaves
markedly reduced germination of sporangiospores. After a
few hours, however, the effluents were no longer toxic. Thus,
readily soluble portions of these two fungicides were washed—
off the leaves during the first few hours. The fact that
effluent from untreated leaves showed some toxicity suggests
that a toxic substance is present on the leaf surface of the
potato. Others have also reported toxic materials in leaf

exudates. High concentrations of leaf exudate sometimes found

 

 

in rain and dew were inhibitory to spores of Botrytis,
Ascochyta, and Puccinia (Kovacs and Sze6ke, 1956); and

germination of Alternaria tenuis conidia was slightly in—

 

hibited by the presence of leaf exudates (Hislop, 1966a).

In addition to toxicity of the effluent from fungi-
cide-treated leaves, complex interactions between leaf ex—
udates and the fungicide should be anticipated. In the
study of sporangiospore germination at distances from the
fungicide drop, the percent spore germination on untreated
leaves without weathering was less than those weathered for
5 hours. These results also suggest the presence of a
toxic substance on the potato leaf surface.

With the Spreaders, Biofilm and Pinolene, technical
compounds of tin and copper showed less redistribution than
did the commercial fungicide, Kocide 101 or Du—ter at
5 mm distance from the fungicide drop following 5 and 24 hours
weathering (Tables 2, 3). Electron microprobe analysis did
not show marked differences in redistribution comparing
the tin and copper compounds with commercial Kocide 101 and
Du-ter. The effluent from the leaf sprayed with tin plus
Pinolene showed high germination following 5 hours weathering.
These observations suggest that the Spreaders Pinolene
or Biofilm were not more efficient than the surfactants form-
ulated in the commerical fungicides.

As Hislop and Cox (1970) has pointed out, redistri-
bution of copper was improved after washing. In my trials
tin was also redistributed as was copper. The results also
confirm Goossen's suggestion (1958) that tenacity of most “

fungicides is sufficient for maximum redistribution.

SUMMARY

Redistribution of copper and tin on the potato leaf
surface was investigated by electron microprobe analysis,

direct microscope observation, and bioassay with PhytOphthora

 

infestans.

 

Electron micrOprobe analysis of fungicide drOplets

containing copper or tin fungicides on the potato leaf showed

 

that droplet margins were clearly defined and often had rim
of heavy fungicide concentration. These margins were clearly
defined and fungicide had not migrated in detectable quanti"
ties with or without weathering. DrOps of either fungicide
with Biofilm or Pinolene had clearly defined margins but the
marginal rim was not as evident. Bioassay demonstrated that
active toxicant existed immediatly outside the drOplet, but
the levels of c0pper or tin were apparently below the level
of sensitivity of the electron probe.

Microscope examination of measured droplets and sprayed
deposits showed clearly defined marginal rims, but the rim of
heavy spray concentration at the margin of the drops of
Kocide 101 and Du-ter was not completely continuous. Techm
nical compounds of c0pper or tin with either Biofilm and
Pinolene lacked the clearly defined rims.

Sporangial germination on leaves close to the fungi"
cide drop was very low (0 mm distance from drop). Toxicity

of Kocide 101 increased with 5 to 24 hours of weathering.

47

 

48

Reduced suppression of germination was obtained with cupric
hydroxide plus either spreader.

Spore germination was suppressed by Du—ter up to
15 mm from the point of application immediately after apv
plication. After weathering spore germination was reduced
only at the site of the fungicide drop. Results were es-
sentially similar with either spreader.

Inhibition of infection around the fungicide drops
after 30 min of weathering was evident up to 20 mm from the
point of fungicide application. The area of fungicide dis—
tribution from the point of application was much larger when
determined by frequency of infection sites than it was when
estimated by prevention of spore germination. Perhaps the
toxicants were more effective against zoospores, or germ tubes,
or infection hyphae than against infection p35 pg.

Spore germination was effectively prevented by 10 ppm
of Kocide 101 but not by 1 ppm. Du—ter at 47.5 ppm prevented
spore germination of most spores but not all spores.

The first effluents from fungicide sprayed leaves
prevented sporangiospore germination. After 5 hours of
gentle weathering, there was some evidence of low toxicity.

Toxicity was not evident in effluents after 24 hrs or longer.

 

LITERATURE CITED

 

 

LITERATURE CITED

Birdermann, W. Und E. Mfiller. 1952. Die Inaktivierung
des gelBsten Kupfers (II) in Fungiziden. Phytopathologische
Zeitschrift 18: 335—337.

Bj6rlong, K. and K. A. Sellgren. 1957. Protection and
its connection with redistribution of different drOplet
sizes in sprays against Phytophthora infestans. Kungliga
Lantbrukshogskfilans Annaler 23: 291—308.

 

Burchfield, H. P. 1959. Physical chemistry of fungi-
cidal action: Physical properties and chemical reactivities
in relation to the effectiveness of fungicides. In C. S.
Holton et al. (ed.) Plant pathology, Problems and progress
1908-1958.—_pp. 293-303. University of Wisconsin Press.

Goossen, H. 1958. Importance of rain—resistant prop-
erty of fungicides to the control of Phytophthora infestans
de Bary. Hofchen-Briefe Bayer Pflanzenschutz—Nachrichten
(English edition) 11: 70—80.

 

Hamilton, J. M., G. L. Mack and D. H. Palmiter. 1943.
Redistribution of fungicides on apple foliage. Phytopathology
(abstract) 33: 5.

Hislop, E. C. 1965. The redistribution of fungicides
on plants. I. Redistribution by Splash in the laboratory.
Annual Report of the Agricultural and Horticultural Research
Station Long Ashton for 1964. pp. 175—180.

Hislop, E. C. 1966a. The redistribution of fungicides
on plants. II. Solution of copper fungicides. Annals of
Applied Biology 57: 475-489.

Hislop, E. C. 1966b. The redistribution of fungicides
on plants. III. Redistribution in the field. The Annual
Report of the Agricultural and Horticultural Research Station
Long Ashton for 1965. pp. 193—200.

Hislop, E. C. 1967. The redistribution of fungicides on
plants. IV. Redistribution on apple trees by rain splash.
The Annual Report of the Agricultural and Horticultural
Research Stateion Long Ashton for 1966. pp. 184-189.

Hislop, E. C. 1969. Splash dispersal of fungus spores

and fungicides in the laboratory and greenhouse. Annals of
Applied Biology 63: 71—80.

50

51

Hislop E. C. and T. W. Cox. 1970. Local redistribution
of fungicides on leaves by water. Annals of Applied Biology
66: 89-101.

Kovacs, A. und E. Szeoke. 1956. Die phytopathologishe
Bedeutung der kutikularen Exkretion. Phytopathologische
Zeitschrift 27: 335—349.

Large, E. C. 1940. The advance of the fungi. Henry
Holt. New York. p. 227.

Millardet, P. M. A. 1885. Traitement du Mildiou et
du rot. Journal d'Agric. Pratique 1885 (2): 513—16. Trans-
lation by F. J. Schniederhan. Phytopathological Classic No.
3. pp. 7-11. 1933.

Morgan, N. G. and J. H. Russell. 1966. Spray application
problems: LXXIV. The biological efficiency of small—volume
spray applications of finely—atomized dilute fungicides for
the control of apple scab. Annual Report of the Agricultural
and Horticultural Research Station Long Ashton for 1965.
pp. 206—208.

Rich, S. 1954. Dynamics of deposition and tenacity of
fungicides. Phytopathology 44: 203—213.

Somers, E. 1957. Studies of spray deposits. III.
Factors influencing the level of 'run—off' deposits of copper
fungicides. Journal of the Science of Food and Agriculture
8: 520—526.

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