THE USE OF FUNGICEDAL SPRAYS TO
REDUCE MOLD COUNTS EN BLACK
RASPBERRIES

“unis for 5h. chm of M. S.
MICHEGAN' STATE COLLEGE
William Justin Young

1955

THES}S

This is to certifg that the

thesis entitled

The Use of I‘ungicidal Sprays to Reduce Mold

Date

0-169

Cofints in Black Raspberries

presented by

William Justin Young

has been accepted towards fulfillment
of the requirements for

iii.— degree in M Mt PathOJ-Ogy

 

Major professor

E. S. Beneke

May 26. 1955

 

 

THE USE OF FUNGICIDAL SPRA YS TO REDUCE MOLD COUNTS
IN BLACK RASPBERRIES

A 881' RACT

Mold count. tolerances for processed black raSpberry fruit are
difficult, if not impossible to maintain. during hot, humid weather.
Because of this fact fungicidal treatments were applied to fruit while
still on the bush to ascertain whether or not the mold counts could be
kept below the tolerance

Five fungicides, ferbam, nabam, dichlone, glyodin and actidione,
were applied to both Cumberland and Logan variety black raspberries
at varying intervals before harvest time. These fungicides were .
used sineg and in combination with a spreader-sticker Triton B-1956.
Fruit was harvested from these plots at normal picking times and
mold counts were made from it to check possible differences in the
mold counts.

Growth studies were carried out using these five fungicides and
four of the fungi which are most frequently isolated from black
raspberry fruit with high mold counts. The fungicides were incor- ,
porated into media at field concentrations and at three other con-
centrations above and below this level. The fungi were grown upon
these media and their growth, as indicated by colony diameter
increase, was measured and compared against that of a check. An

indication of the inhibiting power of the fungicides was thus obtained.

The results of the field studies and growth studies were then
compared to see which fungicides, if any, were effective in reducing
the growth of the fungi which cause the high mold counts in black
respberries. It was found that the results of the growth studies
follow quite closely the results of the field studies. Of the fungicides
used, however, only ferbam seemed to have merit. It inhibited all
fungal growth at. all concentrations in the growth studies and it
reduced the mold counts in the fruits obtained from the ferbam
Sprayed plots.

It is believed that a program of preharvest Sprays to lower
mold counts in black raSpberries could be effective but further
investigations into the practicality of such a program are necessary.

William Justin Young

Department of Botany
and Plant Pathology

THE USE OF FUNGICIDAL SPRA YS TO REDUCE MOLD COUNTS

IN BLACK RA SPBERRIES

by

William Justin Y__oung

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant. Pathology

1955

THESlS

.1: VJ at... I.- ndulu. Elsi

N

J.

..
. _

 

TABLE OF CONTENTS

Chapter Page
I. _ Introduction. . . . . . . . . . . . ....... 1
II. Review of Literature .......... . . . . 3
111. Field Methods .................. 7

IV. Field Results ..... . . . . . ..... . . .10
V. Laboratory Methods .............. 15

VI. Laboratory Results .......... . . . .17
VIL. Discussion .................... 34
VIII. Summary .................... 41
Bibliography .................. 42

354776

LIST OF TA BLES

 

 

Table Page

I. Effect of fungicides on black raspberry

moldcounts. . . .. ........... . . . . 11
2. Official weather bureau data . . . . . . . . . . 12
3. Effect of fungicides on growth rates

of Alternaria humicola . . . . . . . ..... . _ 18
4. Effect of fungicides on growth rates

of Botrytis cinerea ........... . . . . 20
5. Effect of fungicides on growth rates

of Cladosporium Sp. . . . . ....... . . . 22

 

6. Effect of fungicides on growth rates
of Fusarium Sp. ................ 24

 

 

Figux'

LIST OF FIGURES

Figure Page

I. Fungal colony growth at 96 hours with
various concentrations of ferbam ...... . . 26

II. Fungal colony growth at 96 hours with
various concentrations of nabam ........ 27

III. Fungal colony growth at 96 hours with
various concentrations of glyodin ....... 29

IV. Fungal colony growth at 96 hours with
various concentrations of actidione ...... 31

V. Fungal colony growth at 96 hours with
various concentrations of dichlone ....... 33

ACKNOWLEDGMENT

The writer wishes to express his sincere thanks to Dr. E. S.
Beneke for his assistance and constructive criticism throughout the
course of the research and during the preparation of the thesis.

Many thanks are also due my good friend Robert H. Fulton for
the many hours he Spent aiding me in applying fungicides and
harvesting fruit.

The writer appreciates the aid of the Michigan Fruit Canners,
Inc. who provided the services of a technician in making the mold
counts and who provided the financial assistance which enabled this
work to be carried out.

Finally, the writer wishes to eXpress his appreciation to his

wife, Dorothy, for her inSpiration and for her assistance in the

preparation of the manuscript.

CHAPTER I

INTRODUCTION

The Federal Food and Drug Administration in its efforts to keep
food products free from decomposition and spoilage has set certain
standards for canned and frozen black raspberries. One of these states
that the mold count in the packaged product must be less than 40 per
cent of the microsc0pic fields of a standard Howard test chamber. This
standard, while reasonable in normal weather, becomes extremely
difficult, if not impossible, to maintain during periods of hot, humid
weather.

In the fall of 1949 a considerable quantity of canned and frozen
black raspberries was seized by the Federal Food and Drug Adminis-

tration. Their claim was that these berries had excessive mold counts
and thereby were spoiled and unfit for human consumption. In the
summer of 1950, during the processing period, a hot, humid Spell
caused mold counts to go so high that the processors were forced to
suSpend operations. These actions naturally caused worry to both
Processor and grower alike, for Michigan packs a sizeable portion of
the frozen and 80 per cent of the canned black raspberries in the United
States.

It was the hope of the processor that some means could be
devised for treating the berry at. harvest time to reduce the mold count.

I.

2.
Accordingly, field dips were tried as the berries were picked, but these
had little apparent effect on the increase in mold count. It was dis-
covered at this time that under the conditions of hot and humid weather
the fungal hyphae were present in the ripe berry even while still on the
bush (3). As a result it was deemed advisable to see if applications of
fungicides to the berries a short time before harvest would have any
effect on germination of the fungal Spores and their subsequent growth
into the raSpberry. The writer selected several representatives from
the various classes of organic fungicides for application to the
raspberry plants. These were applied to the raSpberry plants a short
time before harvest and their effect on subsequent mold counts was .

studied.

CHAPTER 11

REVIEW OF LITERATURE

The use of the mold count as a criterium for judging food products
dates back to 1911. In that year Howard (8) suggested a mold counting
method to aid in the judging of quality in tomato products. Briefly he
suggested the following procedure. A Thoma-Zeiss blood counting
chamber was used and the microsc0pe was adjusted so that each field
of the chamber covered 1. 5 millimeters. Sufficient pulp was placed in
the chamber to cover it to a depth of 0.1 millimeters. This gave a
volume of one and one-half cubic millimeters for each field. If an
aggregate of not more than three mycelial fragments covered one-sixth
or more of the diameter of a field, the field was considered positive.
When 25 to 50 microsc0pic fields were counted and 25 per cent or more
of the fields were positive, the canned product could be condemned. In
1917 Howard and Stevenson (9) revised these tolerances to-allow a
maximum of 66 per cent positive microsc0pic fields in the total fields
counted. A change was made to the Howard mold counting chamber at
this time.

In 1924 Needham and Fellers (13) applied the Howard method of
mold counting to the detection of Spoilage in canned berry products.
They worked with canned strawberry and blackberry products and found
that from five to ten per cent of decayed or moldy berries in a lot could

3.

4.
give mold counts of 40 per cent and higher. They also found that soft
overripe fruit could increase mold counts even though no visible mold
was present.

In 1939 the mold counting standard was changed to allow only a 50
per cent mold count and in 1938 this was decreased to 25 per cent for
tomato juice. In 1940 the standards were changed again, this time a 15
per cent mold count was allowed for tomato juice and 40 per cent for
other tomato products. These are the standards that were in effect for
tomato products at the time of this work. However, at the same time
the indicated range for seizure of canned black raspberry fruits was
considered somewhere around 20 to 40 per cent although no definite
tolerance was established by the Food and Drug Administration.

Although the mold count was originally intended for use with tomato
products its use has been broadened so that all types of canned and
frozen berry products are subjected to it. In the fall and winter of 1949
a considerable quantity of black raSpberries was condemned on the
basis of this application. The Federal Food and Drug Administration
claimed that the mold counts were excessive and that the berries were
unfit for human consumption. Beneke (2) in 1950 noted, however, that
in canned raSpberry products when the mold count ran to 100 per cent
of the microsc0pic fields of the test chamber, the mlume occupied was

only . 00162 per cent.

As a result of these excessive mold counts Fabian 231. (3) in
1950 began a series of investigations concerning the factors that cause
high mold counts. Some possible control measures also were investi-
gated. Using ferbam, captan, COpper 3-phen, Dowicide A, and
dehydroacetic acid as the fungicides, numerous combinations of dips
were tried. Flats of fruit were dipped into the fungicide just after
picking and after the fruit had been brought to the cannery. None of
these treatments significantly reduced the percentage of mold count.
They found that. under weather conditions of high heat and humidity the
mold was already growing in the berry at picking time. This naturally
meant that an original high mold count was present in the berries no
matter how carefully they were handled. Beneke (3) found the fungi
primarily responsible for the high mold counts to be Alternaria

humicola, and CladOSporium sp. Other fungi frequently isolated were

 

 

Pullularia pullulans, BOthiiS cinerea, Penicillium Sp. , Fusarium Sp. ,

Rhizopus sp. , Trichoderma sp. , Aspergillus sp. , Oospora Sp. ,

 

Monilia sp. , and Mucor Sp.

Since it was known that the fungus may be present in the ripe berry
it was thought that perhaps a fungicide applied to the green fruit, before
the fungus had a chance to penetrate the berry, might provide a deposit
which would prevent Spore germination or inhibit mycelial growth. In

the tomato industry field Sprays have been used with success to keep

6.
down fungus growth which leads to high mold counts. In 1944 McNew(10)
used preharvest fungicidal sprays in an attempt to reduce losses and
trim costs on canned tomato products because of high mold counts

caused by Colletotrichum phomoides. He found ferbam to be the most

 

effective fungicide used. In 1948 Fulton (4) found that tomatoes were

relatively resistant to Colletotrichum phomoides when they were in the

 

green stage but that susceptibility began ten days before harvest time
and increased until the harvest time. In 1949 Schroeder (14) found that

zerlate, nabam and ferbam were very effective in reducing Colletotrichum

 

phomoides infections. In 1950 Greenspan and McKellar (6) were able to

 

reduce mold counts by preharvest sprays of peracetic acid.

These results tend to show that high mold counts can be reduced
by preharvest Sprays on the green and semi~ripe fruit, at least in the
tomato industry. This present work was carried out in the summer of
1951 to ascertain if preharvest field sprays might be applicable to the
raspberry as well as the tomato industry as a means of reducing mold

counts.

C HA PTER III

FIELD METHODS

A grower was selected in the Benton Harbor area who grew both
the Cumberland and the Logan varieties of black raSpberry, these being
the varieties in current commercial use in Michigan. Identical Spray
plots were set up in plantings of each variety and consisted of single
row strips 100 feet in length. In the case of the Cumberland variety, a
second spray was applied to the southern 50 feet of the plots. The
actidione plots of the Cumberland variety received three sprays with
the third Spray having been applied to the southern 25 feet of the plot.
The Logan plots received one Spray each of all the fungicides twelve
days before harvest and a second spray on the actidione plots four days
before harvest. The Cumberland plots received two sprays of all
fungicides, one sixteen and the other eight days before harvest. A
third spray of actidione was applied four days before harvest. The
berries were harvested at normal picking times, brought to the cannery,
and frozen for storage until mold counts could be made.

The five fungicides used in these tests were ferbam which is the
ferric salt of dimethyldithiocarbamate, nabam which is the disodium
salt of ethylene bisdithiocarbamate, dichlone which is 2:3-dichloro -l:4-
nalphthoquinone, glyodin which is 2-heptadecylglyoxalidine, and actidione
which is the antibiotic cyclohexamide. These were used singly and in

7..

8.
combination with a Spreader sticker Triton- B-1956 which is a phthalic
glycerol alkyd resin. The dosages were as follows: ferbam, one and
one -half pounds per 100 gallons; nabam, two quarts per 100 gallons;
plus one -half pound of ZnSO4; glyodin, one and one-half quarts per 100
gallons plus one -half pound of lime; dichlone. one-eighth pound per 100
gallons and actidione two parts per million or 0, 76 grams. These were
all recommended dosages except for dichlone where a lower rate was
applied to avoid possible injury.

The materials were applied with a John Bean Sprayer with a
seven and one -half gallon pump at about 250 pounds pressure per square
inch. A Friend brand Spraygun was used for application. The bushes
were sprayed until the run-off point was reached. This amounted to
about 400 gallons per ac re.

The picking of the raspberry frnits .on the plots was carried on at
the same time that the grower picked his planting. One and one -half
quarts were picked at random from each Spray plot. The berries were
placed in polyethylene bags and taken immediately to the processor.
Here they were held for several hours to Simulate actual processing

time and then frozen for storage until the mold counts could be made..
The counts were made at the Benton Harbor plant of the Michigan Fruit
C anners, Incorporated and were made by the inspector employed by the

COmpany. A Cefaly power pulper with a O. 027 inch screen was used to

9.
grind the berries for a puree and the counts were made with the Howard
mold count chamber in accordance with official Association of Official

A gricultural Chemists methods .

CHAPTER IV

FIE LD RESULTS

When all the mold counts had been made from the berry samples
taken from the various fungicide plots it. was noticed that there was wide
variation in the mold counts between plots. Some of these variations
in the mold counts may be due to weather conditions at the time of
picking and differences in fungicides, but sometimes they may be due
to variation in samples used for mold counts. Fabian st 31. (3) in 1950
found mold counts ranging from 8 to 52 per cent in various samples
taken from a Single 30 pound tin,of frozen black raSpberries.

The mold counts, which were made from the Logan variety of
raSpberries picked July 6, 1951, ran from zero to twelve per cent 3
(Table 1). This showed very little variation between plots at that time
and was no doubt due to: (1) the lack of rainfall before picking and the
resultant lack of high humidity (Table 2), and (2) the fact that this was
the first picking reduced the possibility of overripe berries being in—
cluded. A few overripe berries can noticeably increase the per cent
of mold count in a given quantity of berries. The mold counts, as
shown in Table 1, made on the Logan variety raspberries which were
Picked on July 10, 1951 began to Show wider differences. There had
been one and one -half inches of rain during July eighth and ninth
(Table 2) and this provided better growing conditions for the fungi.

10.

11.

TABLE 1.

EFFECT OF FUNGICIDES ON BLACK RASPBERRY

MOLD COUNTS

 

 

 

Fungicidesl Logan [ Cumberland
— July6 1 July 10 J July 13

Ferbam 41' l 8 4 4 C 16 24
Ferbam -B 1956 6 4 10 10 10 4

Actidione 0 4 2 14 18 14 -- 10 22 -- 10 6

Actidione - B1956 10 4 O 8 4 24 10 18 6 6 28 28

Nabam 12 8 16 8 38 20"
Nabam - B 1956 0 14 26 16 34 42
Glyodin 6 44 10 10 23 44
Glyodin - B 1956 12 8 14 16 84 8
Dichlone 2 24 6 16 64 35
Dichlone - B 1956 4 26 13 4 4 92
Control 6 20 4 18

1' First number appearing is the mold count for one spray, the second
for two Sprays, and the third for three Sprays.

12.
TABLE 2.

OFFICIAL WEATHER BUREAU DATA

United States Weather Bureau
Benton Harbor, Michigan

Date Median High Low Rainfall Time
June 25 64 79 52

26 73 83 89 Trace

27 76 83 60 Trace

28 63 76 60

29 72 74 50

30 72 74 52

July 1 71 73 54

2 76 79 51

3 79 82 79

4 58 79 55 Trace

5 61 66 54 Trace

6

7 84 86 45

1:00 - 6 a.m.

6 69 84 63 .55" 4:00 - 6 p. m.
9 81 82 63 1. 00" 35:33 : 1‘15““;
10 69 82 58 Trace
11 67 73 62

12 66 71 58

13 73 76 51

13.

Also this was the second picking of these plots and the chances for
overripe berries to be included was greater. Table 1 shows that the
mold counts made from the fruit in the ferbam and nabam Sprayed

plots were quite low while the fruit from the actidione and glyodin
sprayed plots gave mold counts which showed wide variation. The mold
counts from the actidione treated berries ranged from 0 to 24 and those
on the glyodin treated fruit ranged from 6 to 44. The dichlone treated
berries gave moderately high counts of 24 and 26 per cent.

The first picking of the Cumberland variety plots was also made
on July 10, 1951. Again as with the Logans (see Table 1) there was
wide variation between Spray plots. When the Cumberlands had two
Sprays of fungicide before picking there did not seem to be any indication
that a second spray had been any more effective in reducing mold counts
than just one Spray. The ferbam Sprayed plots continued to show the
loweSt mold counts. The nabam Sprayed plots still gave low mold
counts although somewhat higher than the counts:made on the Logans.
The glyodin and dichlone Sprayed plots gave considerably lower mold
counts than those on Similarly tested Logans picked on the same date.
The mold counts for the actidione plots remained about the same on the
Cumberlands as they had on the Logans. One would normally eXpect
slightly lower mold counts on the Cumberlands picked on July 10, 1951
because it was the first picking for the Cumberlands and the last

picking for the Logans.

14.

The mold counts made from the berries picked on July 13, 1951
showed an increase in percentage (see Table 1). This was probably due
to the favorable conditions for good fungal growth after the rains of
July eighth and ninth and the fact that this was the last picking. Also
there may have been washing off of the fungicide during the rainy period.
The ferbam sprayed fruit again showed the lowest mold counts. These
were somewhat higher than the preceding counts for the ferbam plots
but not enough to make a significant difference. Mold counts made from
nabam Sprayed fruit showed a substantial increase at this time. It is
thought that this may have resulted from washing off of the fungicide.
Mold counts made from the actidione Sprayed fruit continued to be
erratic while both the glyodin and the dichlone Sprayed plots yielded
fruit which had higher mold counts.

The results obtained from the use of the Spreader sticker were
inconclusive. In some cases the mold count showed a reduction when
the Spreader was incorporated with the fungicide and in other cases the
mold count was higher. The Spreader sticker was, however, of
considerable help in wetting the green fruit and therefore would seem

to have a place in the Spray program.

CHAPTER V

LA BORATORY METHODS

This project was initiated late in the fruiting season of the black
raspberry and consequently no time was available to check the effect of
the fungicides chosen on the fungi known to occur at high frequencies in

the black raspberry. AS it was desirable to know the effect of the

 

fungicide on the organisms £1 vitro, a series of growth rate comparisons
.were run later in which varying amounts of the fungicides used were
incorporated into an agar medium. The rate of colony growth on the
fungicide was compared with that of untreated checks.

The organisms used in this experiment were Alternaria humicola

 

Oudemans, Cladosporium Sp. , Botrytis cinerea Persoon, and Fusarium

 

 

 

Sp. _A_. humicola and Cladosporium. Sp. are the most frequently

 

isolated fungi from black raspberry fruits according to Beneke (3).

I}. cinerea and Fusarium Sp. are also quite regularly found in black

 

raspberry fruit.

The fungicides used were nabam, ferbam, glyodin, dichlone and
actidione. These were tested at concentrations which were equivalent
to those used in the field tests and at concentrations above and below
this amount. In the case of nabam concentrations of 1250, 2500, 5000,
and 7500 parts per million were used, 5000 being the normal field
ConCent ration. Dichlone was used at concentrations of 150, 250, 500

15.

16.
and 1000 parts per million. One hundred and fifty parts per million was
the concentration used in the field. Glyodin was used at concentrations
or 500, 1000, 3700 and 5000 parts per million, 3700 being the concen- i
trat ion used in the field. Ferbam was used at 5000, 1000, 1800, and
2500 parts per million. Eighteen hundred parts per million was the
concentration used in the field. Actidione was used at 2, 5, 10, and 20
parts per million. Two parts per million was the concentration used in
the field.

A two per cent malt extract agar was selected for the growth
medium. This was sterilized in an autoclave at 15 pounds pressure for
l 5 minutes and the fungicide added aseptically to the cooled medium
just before it was poured into Petri plates. The plates were inoculated
with five and one ~half millimeter agar plugs cut with a flame sterilized
cork borer from the periphery of five-day old cultures of the fungi used.
These tests were run at 24 and 28 degrees Centigrade and the daily
increase in the diameter of the colony was measured for four days.
Three replications were made of each combination and the final

figures are an average of these.

C HA PTER VI

LABORATORY RESULTS

The results obtained from the growth studies when the fungicides
were incorporated into the media proved to be quite interesting. These
results are shown in Tables 3, 4, 5 and 6. The fungicides which had
been used on the Spray plots whose fruit gave the lowest. mold counts
usually showed the most inhibition of fungal growth when used for the
studies.

Ferbam was quite inhibitory to fungus growth when incorporated
into an agar medium. The fruit from the plots which were Sprayed
with ferbam also had low mold counts. The concentration used in the
field test was 1800 parts per million or one and one-half pounds per
100 gallons while the concentrations used in the growth studies were
500, 1000, 1800, and 2500 parts per million. No fungal growth was
observed on any of the plates during the period of observation and
measurement, as Shown in Figure I. Some Sparse aerial mycelium
was noted on the surface of the inoculating plug but no growth was
observed on the surface of the agar containing the fungicide.

Nabam (see Figure 11) which showed results in the field similar to
those of ferbam also inhibited all fungi tested at all concentrations used.
These were 1250, 2500, 5000, and 7500 parts per million. Apparently
the Cause of the high mold counts obtained from the fruit on the last

17.

18.

TABLE 3.

EFFECT OF FUNGICIDES ON GROWTH RATES
OF ALTERNARIA HUMICOLA

 

Chemical Hours Temp. Control Concentration in parts per million

1 150 250 500 1000
24 24° 11' 12 11 11 -
28° 12 12 13 12 7
48 24° 17 19 18 17 8
28° 18 20 21 19 10
Dichlone
72 24° 26 27 27 25 14.5
28° 27 29 29 26 16
96 24° ‘34 37 34 32 20
28° 35 38 36 34 22
500 1000 3200 5000
24° 13 8 7 p - _
24 26° 14 10 7 - -
24° 21 12 9 8 -
43 28° 21 13.5 9 8 6
Glyodin
24° 32 17 12 11 8
72 28° 31 19 11 11.5 9
24° 40 24 15 13 11
96 28° 40 25 14 14 13
1250 2500 5000 7500
24° 9 - - - -
24 28° 11 - — - -
240 13.5 - - - -
43 28° 19 - - - -
Nabam
240 21 - - - -
72 280 25 - - - -
24° 31 - - - -
95 280 37 a - - -

1’ Figures represent diameter of colony in millimeters.

19.
TA BLE 3. (continued)

Chemical Hours Temp. Control Concentration in parts per million

0 500 1000 1800 2500
24 240 ll - - - -
28 14 - - - —
o
48 33° 33 ‘ ' ‘ ’
Ferbam o
72 240 30 - - - -
28 32 - - - -
o
96 240 40 - - - -
28 41 - - - -
O 2 5 10 20
24 24o 6 - ' ' "
28 7 - - - -
48 24: 14 18 15 14 14
28 15 16 16 13 . 14
Actidione o
72 240 22 25 24 24 2'1
28 28 25 25. 5 23 23
o
96 240 29 31 32 33 29

28 35 34 34 32 32

20.

TABLE 4.

EFFECT OF FUNGICIDES ON GROWTH RATES
OF BOTRYTIS CINEREA

 

C? hemical Hours Temp. Control Concentration in parts per million

0 150 250 500 1000
24 240 15 ' 12 11 9 -
28 10 _ 9 9 8 -
O
48 240 35 27 26 23 -
28 28 20 20 20 6
chhlone o
72 240 56 38 38 35 11
28 36 31 29 27 10
96 24: 78 47 46 40 13
28 43 39 36 34 12
o 500 1000 3700 5000
24 16 8 7 6 -
24 28° 10 - - - .-
O
48 240 34 14 11.5 10 8
28 19 10 8 7.5 —
Glyodin o ,
7 240 60 28 16 14 10
2 28 28 17 13 10 -
O
24 , 77 39 25 18 13
96 28° 40 22 20 12 -
0 1250 2500 5000 7500
24 14 - - - -
24 28° 10 - - - -
24° 35 - - - -
43 28° 24 - - - -
Nabam '
24° 58 - - — -
72 28 ° 33 - - - -
24° 80 - - - -
96 28° 41 - - - -

Figures represent diameter of colony in millimeters.

21.
TABLE 4. (continued)

Chemical Hours Temp. Control Concentration in parts per million

0 500 1000 1800 2500
24 17 - - - -
2
4 28° 11 - - - -
O
24 37 - - - -
48 28° 20 - - - -
Fe rbam o
72 240 62 - - - -
28 34 - - - -
O
24 80 - - - -
9° 28° 46 - - - -
2 5 10 20
24 24° 16 13.5 10 9 -
28° 13 10 10 - -
48 24° 36 30 24 19 11
28° 24 19 19 11
Actidione
72 24° 59 49 40.5 30.5 24
28° 34 30 29 20 12
24° 80 61 52 51 36

96 28° 45 40 37 27 17

22.

TABLE 5.

EFFECT OF FUNGICIDES ON GROWTH RATES
OF CLADOSPORIUM SP.

 

(,‘hemical Hours Temp. Control Concentration in parts per million
0 1 150 250 500 1000
24 240 6.5 ' 7 7 6 -
28 5.5 - - - _
0
48 28° 2 5 1g 2 g :
Dichlone o
72 240 13 13 12 10 -
28 10 10 7 7 -
0
96 240 16 16 14 12 -
28 12 11 9 10 -
0 500 1000 3700 5000
24 7 - - - -
24 280 _ _ _ _
24° 9 6 5. - —
48 280 _ __ _ _ _
Glyodin
72 24° 11 8 7 6. -
28° 7 6 - - -
6 24° 14 11 9 8 -
9 28° 8 8 7 6 -
0 1250 2500 5000 7500
24 6 - - - -
24 280 _ _ _ _ _
24° 9 - - - -
48 28° _ _ _ _ _
Nabam
24° 12 - - - -
72 28° 6 - - - -
24° 14 - - - _
96 28° 7 _ _ _ _

1 ' FiEUres

represent diameter of colony in millimeters.

23.
TABLE 5. (continued)

Chemical Hours Temp. Control Concentration in parts per million

0 500 1000 1800 2500
24 24o 6 F ’ ' ’
28 - — - - -
O
48 240 10 - - — -
28 6 - - _ _
Fe I'bam o
72 240 14 - - - -
28 8 - - - -
O
96 240 16 - - - -
28 9 - - - -
O 2 5 10 20
24 24o 7 - _ - ‘
28 - - — — -
O
24 '9 8 7 7 6
48 28° 6 6 6 - —
A ctidione 0
72 24 18 15 12 10 9
28° 7 8 8 7 6
O
96 24 21 18 15 13 11

28° 10 10 9.5 8 7.

24.

TABLE 6.

EFFECT OF FUNGICIDES ON GROWTH RATES
OF FUSARIUM SP.

 

C hemical Hours Temp. Control Concentration in parts per million

0 1 150 250 500 1000
24 - ' 7 6 — —
24 28° 6 8 6.5 - -
48 24: 6 11 10 6 -
28 13 15 11 7 —
Di 0 hlone
72 24° 15 24 22 10 -
28° 20 28 25 16 -
O
96 240 24 33 30 14 -
28 30 39 32 23 -
500 1000 3700 5000
24° 13 10 8 7 6.
24 28° 18 11 9 8 7
24° 29 23 16 14 14
43 28° 34 23 20 15 14
Glyodin
24° 42 35 25 20 18
72 28° 49 37 32 24 19
24° 60 45 33 24 21
96 280 64 50 45 30 23
1250 2500 5000 7500
24° 9 - - - -
24 28° 11 - - - -
24° 18 - - - -
48 28° 20 - - - -
Nabam
24° 29 - - — -
72 28° 31 - - - -
240 42 - - - -
95 28 ° 44 - - - -

1. - . . . .
FlgUreS represent d1ameter of colony in m1111meters.

25.
TABLE 6. (continued)

Chemical Hours Temp. Control Concentration in parts per million

0 500 1000 1800 2500
24 - - - - -
24 o
28 9 - - - -
o
48 32° if ' ‘ ‘ ‘
Ferbam 0
72 240 24 - - — —
28 36 - - - -
o
96 240 35 - - - -
28 44 - - - -
o . 2 5 10 20
24 24o — - - ' -
28 ‘ 9 - - - -
o
24 9 6. 5 6 - -
48
28° 14 8 7 8 -
Actidione o
28 25 14 9 7 -
o
96 24 35 19 11 7 -

28° 39 22 13 10 -

Colony Diameter (mm)

75

70

65

60

55

50

45

40

 

 

A. humicola — - .1 .....
B. cinerea
Cladosporium Sp.-— - _
Fusarium Sp. — —— __ _.

 

 

 

 

 

 

 

 

 

500 1000 1800 2500

Concentration (parts per million)

Figure I.

Fungal colony growth at 96 hours with
various concentrations of ferbam

Colony Diameter (mm)

 

 

A. humicola-- -- ---‘
B. cinerea

 

 

 

Cladosporium spr— - -
Fusarium Sp.-—- — — —

 

 

 

ck 1250 2500 '5000 7500
Concentration (parts per million)
Figure 11. Fungal colony growth at 96 hours with

various concentrations of nabam

27

28.
picking date was due to the fungicide being washed from the fruit by
rain or from natural breakdown of the fungicide rather than its in-
ability to inhibit fungal growth.

Glyodin (as shown in Figure 111) reduced the growth of all

organisms tested but inhibited only Cladosporium Sp. at 5000 parts per

 

million, a concentration above the 3700 parts per million used in the

field. At 3700 parts per million the growth of CladOSporium Sp. was

 

reduced by 50 per cent while the growth of A. humicola was reduced by
66 per cent. There was still some growth at 5000 parts per million
however. Wellman and McCallan (16) in 1947 reported that the ED 50

of glyodin for Spore germination of A. solani and _S_. fructicola was -

 

twelve parts per million. Generally, however, the concentration of a
compound needed to inhibit mycelial growth is greater than the con-
cent ration needed to inhibit Spore germination. Also there may be
absorption effects occurring between the.:compound and the media.

The growth of Fusarium sp. was reduced by over 50 per cent at

 

3700 parts per million but there was still considerable growth at 5000
part 8 per million, which was over one -third the amount of growth in the
cont r01. The growth of _B__. cinerea was greatly reduced at 500 parts per
milliOn. Concentrations of 1000 and 3700 parts per million did not
reduce growth much more than 500 parts per million. However, at

5000 parts per million there was another great reduction in the rate of

Colony Diameter (mm)

29.

 

 

 

75
70.
A. humicola —————— a.
B. cinerea ____.
65 ' CladOSporium sp__. . __

 

Fusarium sp.—-— — -— —

 

 

-___.\‘

 

Ck 500 1000 3700 5000
Concentration (parts per million)

Figure 111. Fungal colony growth at 96 hours with
various concentrations of glyodin

30.

growth. The effect of different concentrations was also noticed when
dichlone and glyodin were used in the growth studies. It is interesting
to note that these three compounds Showed similar sharp reduction in
the growth of B cinerea at the lowest concentrations used, with a
leveling of growth rates through the intermediate concentrations and
then a further sharp reduction at the highest levels.

Actidione gave very little inhibition at. two parts per million (see
Figure IV) which was also the concentration used in the field. The
field results would indicate that actidione was somewhat better than
indicated 19. vi_t_1;9_ so there may be an absorption of the compound by the
agar medium. Gottlieb gt a_l. (5) reported adsorption of actidione by
soil and in agar there may be a similar type action. Thanos (15)
reported stimulation effects of actidione between one-half and ten parts

per million on the germination of spores of C. cucumerinum and B.

 

cinerea. In these studies the growth of A. humicola was stimulated to
some extent by actidione. This stimulation was noticed at two, five,
and ten parts per million. Above ten parts per million there was a
decrease in growth. The growth of l_3. cinerea was reduced by 18 per
cent at two parts per million but at 20 parts per million there was only
a 50 per cent reduction in growth as compared to the control. The

growth of Fusarium sp. was reduced by half at two parts per million

 

and Completely inhibited at twenty parts per million. Growth of

Colony Diameter (mm)

75

70

65

60

55

50

45

4O

35

 

31.

A. humicola ________
B. cinerea
Cladosporium sp,—_—
Fusarium Sp.——~ _ _ ..-

 

 

 

 

 

 

 

i A
\ ,... \
[Ar—W", \\
V’V \‘
\
\
\
L \
r \
\
\A‘
\\~
1 \ k\
\ \-.\
\ k-
\ \~\
\ ‘0
\
\
\
\k\
:3:
ck 2 '5 ‘10 20

Concentration (parts per million)

Figure IV. Fungal colony growth at 96 hours with

various concentrations of actidione

32.

Cladosgorium Sp. was slightly reduced at two parts per million but at

 

twenty there was a 50 per cent reduction.

Dichlone (Figure V) stimulated both Fusarium Sp. and A. humicola
at 150 parts per million which was equivalent to the concentration used
in the field. At 1000 parts per million Fusarium Sp. was inhibited
completely and the growth of A humicola had been reduced by 50 per
cent. McNew and Burchfield (11) reported the ED 95 of dichlone against

A. solani on tomato foliage to be 100 parts per million. They also

 

regard dichlone as a AAA compound which means that the ED 50 values
for Spore germination range from O. 001 to 0. 01 parts per million.

The growth of CladOSporium Sp. was not reduced at 150 parts per

 

million of dichlone but with increasing concentrations of the fungicide
showed gradually decreasing rates of growth until complete inhibition
was reached at 1000 parts per million. B. cinerea showed sharp
reduction in growth at 150 parts per million. At 250 and 500 parts per
million there was only slightly more reduction in the amount of growth,
however, between 500 and 1000 parts per million there was another

Sharp reduction in growth.

Colony Diameter (mm)

75

70

65

 

 

33..

A. humicola .........
B. cinerea
Cladosporium Sp.—-—
Fusarium sp,_ _ __ __

 

 

 

 

 

 

10,L'"““‘""\~\ \.
.\.\~\ \
5_ "\E \
\.\
\\
0 a.
ck 150 250 500 1000

Concentration (parts per million)

Figure V. Fungal colony growth at 96 hours with

various concentrations of dichlone

CHAPTER VII

DISCUSSION

Throughout all experiments the fungicide ferbam gave good results.
The fruit which was harvested from the ferbam Sprayed plots had very
uniform, and for the most part, low mold counts (Table l). The mold
counts made on the fruit picked July thirteenth were Slightly higher but
this no doubt was due to the more Optimum conditions for mold growth
brought about. by the rains of July eighth and ninth. In addition there
probably was some washing of the fungicide although the fruit from the
plots where a Spreader was used had counts that were just as low as
those on any other day. Results of i_n_ 3111:) studies (as shown in Figure
I) in which ferbam was incorporated in various concentrations into a
two per cent malt extract agar to measure inhibitory action against
various fungi, were highly satisfactory. Ferbam completely inhibited
the growth of all four fungi against which it was tested at all concen-
trations used.

The consistently low mold counts obtained from the fruit Sprayed
with ferbam and the complete inhibition of fungi in i_n_ X_i_t_1_‘9 studies would
seem to indicate that ferbam may be a suitable fungicide for use on
fruit to reduce mold counts. Other considerations such as residue and
effect on the canned product were not studied and will not be discussed

here.

34.

35.

When used during 19. v_it_r_o studies, nabam (Figure 11) also gave
complete inhibition of all fungi tested and at all concentrations. It
would appear thus to have good potentialities, although in the field, the
results were somewhat erratic (Table l). The mold counts made from
fruit harvested from plots Sprayed with nabam only we re low, with the
exception on mold counts made from fruit harvested on July thirteenth.
This can be accounted for by the rains of July eighth and ninth which
may have washed the fungicide deposit from the fruit. Also there may
have been a compound breakdown although nabam is considered to have
a good residual action, at least for five days. The mold counts made
from the fruit harvested from the plots which were Sprayed with nabam
plus B 1956 were higher on both the tenth and the thirteenth of July.
This may, however, be due to some interaction between the fungicide
and the Spreader which reduces the effectiveness of the fungicide.
Since these high mold counts were from the Cumberland fruit only,
there may be the possibility of variation in varietal susceptibility,
although more likely eXplanation would be that the conditions we re less
favorable for fungal growth during the period when the Logan variety
was maturing and being harvested.

On the basis of mold counts made from harvested fruit it would
not seem that nabam would be a desirable chemical to use to reduce

raspberry fruit mold counts. This would not be due to the fact that the

36.

compound is ineffective against fungi, as can be seen by i_n_ m9 studies,
but due rather to a lack of ability 1.0 maintain a lethal residue or an
inability to act under these particular circumstances.

Mold counts made from the fruit harvested from the actidione
(as Shown in Table 1) Sprayed plots Showed considerable variation.
The highest count obtained, which was 28 per cent of the microscOpic
fields, was not extreme but the wide variation on any one day would
indicate that the compound is not completely effective. Natural
variation in the sampling method for the mold count, of course, would
enter into this to some extent. The number of Sprays seemed to have
had little effect on the sample taken for the final mold count. Fruit
which had received only one Spray often had a lower mold count than
fruit which had had two or three Sprays. In general, one would assume
that at the concentration used, two parts per million, actidione was not
as effective as could be desired. The 12 112:9 studies (Figure IV)
showed that at two parts per million there was little inhibition of

Cladosporium sp. and a stimulation of Alternaria humicola. These two

 

organisms are the ones isolated most frequently from black raspberry
fruit and if we can assume that they are chiefly reSponsible for causing
high mold counts then the erratic mold counts are explained. Botrytis
cinerea also Showed much growth at two parts per million, and while

found less frequently, its great volume of growth should also contribute

37.

to the mold count total. The i_n_ 31139 results are lower than the ill 2132
studies would indicate, but an undetermined amount. of adsorbtion of
actidione by the agar medium must be allowed for. This adsorbtion
would reduce the effective amount of compound present in the medium
and thereby allow greater growth than an actual concentration of two
parts per million. There was considerable variation in the mold count
between actidione and actidione plus B 1956 Sprayed fruit. There may
be an interaction between these two chemical compounds which reduces
the effective amount of actidione present.

On the basis of the present tests actidione would not seem a
desirable compound for the reduction of high mold counts on black
raSpberry fruits. However, since it is apparently adsorbed into plant
tissue according to Hamilton (7), it thereby would not be as likely to be
washed off by rain. If a reasonably higher dosage could be used without
undue toxicity and expense, it might be a desirable compound to use in
reducing black raspberry mold counts.

The results obtained with the use of dichlone were unsatisfactory.
In the laboratory growth studies (Figure V) 150 parts per million, equal
to the field concentration, had no effect on the growth of Cladosporium
sp. and it stimulated the growth of Alternaria humicola. At this con-
cent ration the growth of Botrytis cineria was reduced and the growth of

Fusarium Sp. was stimulated. Since Cladosporium Sp. and Alternaria

 

38.

humicola are isolated about 70 per cent of the time from raspberry

fruits with high mold counts, one would eXpect that fruit harvested from

dichlone Sprayed plots might have higher mold counts. Such was the

case. When the weather conditions were adverse, that is hot and humid,
the mold counts tended to run high; when normal weather conditions
were present the mold counts were also normal (see Table 1).

It is probable that dichlone would produce better results at higher
concentrations although in the growth studies there was considerable

fungal growth present at 500 parts per million which was very close to

a normal field concentration.

Glyodin performed very much the same as dichlone. Although it
did not stimulate the growth of any fungus tested it allowed growth of
all of them at a concentration equivalent to that used in the field tests.
In the field tests (Figure 111) it apparently failed to inhibit the (growth of
the fungi which are reSponsible for high mold counts brought about by
hot, humid weather. Low mold counts were obtained from glyodin
sprayed fruit only when it was picked during periods when conditions
were unfavorable for fungus growth and when one would normally expect
low mold counts (Table 1). On the basis of this eXperiment it would
Seem that glyodin is not a suitable chemical for inhibiting the growth of

the fungi causing high mold counts in black raSpberries.

39.

Of the five fungicides ferbam seems to offer some possibility of
reducing the fungi which are causing the high mold counts in black
raspberries. It inhibited the growth of all fungi tested in_ v_it_1;o and the
fruit harvested from ferbam Sprayed plots Showed consistently low mold
counts.

There are many other factors to be considered, however, when
discussing the feasibility of applying preharvest Sprays to control high
mold counts in black raSpberry fruits. The primary consideration to
the grower would of course be eXpense; would the use of a preharvest
Spray be insurance against the closing of canneries during the periods
when high mold counts occur and would the extra cost of such 3 Spray
be returned. Another consideration is that of moving Spray equipment
through the planting so close to harvest time. Would the berries be
bruised or broken from the bushes thus lowering quality of fruit and
yield.

Of primary interest to the canner is the problem of residue. ‘Will
the normal washing procedures remove the chemical so that legal
tolerances may be met. Another important factor is whether the
fungicide would have any effect on the canned product. Adams e131. (1)
working with canned black currants found that zinc nabam, glyodin,
dichlone, ziram and thiram residues stimulated hydrogen production in

the canned product even at very low concentrations and that these

40.
chemicals may adversely affect the flavor of the canned product.
Moore (12) however stated that canned sour cherries which had been
Sprayed with ferbam had been kept for two years with no adverse affect

on flavor or keeping quality. 16“}

5.x"- 1

The results obtained here are merely indications. The work
Should be repeated under more adverse conditions to see if comparable
results are obtained. Extensive tests are also required to determine
whether residue tolerances could be met. 'If these problems are
resolved one remaining is economic feasibility. However, if by the
use of preharvest Sprays fruit mold counts can be held to a level which
would forestall closing the canneries. then such Sprays would no doubt

be extremely important.

CHAPTER VIII

SUMMARY

Five fungicides were applied on black raspberry bushes as pre-

harvest Sprays in an attempt to lessen or prevent the fungal growth

which causes high mold counts in the harvested fruits. Mold counts

were made with fruit harvested from the Spray plots to determine the
effectiveness of the Spray.

Growth studies were carried out in which varying concentrations

of the fungicides were incorporated into media. Upon these media

were grown two fungi which are most frequently isolated from raspberry
fruits with high mold counts and two others which are commonly
isolated. The amount of increase in colony diameter was used as an
index of fungicide effectiveness.

The results of field studies and growth studies were compared to
see which fungicides, if any, were effective in reducing the growth of
the fungi which cause high mold counts in black raSpberries.

Ferbam, ferric dimethyldithiocarbamate, was found to inhibit all
fungal growth in i_n_ v_it_rg studies and to bring about a reduction in black
raSpberry mold counts by inhibiting or killing the fungi which cause
the higher counts.

It is suggested that further eXperiments be conducted under more

adverse conditions. Explorations Should be made to determine the

economic necessity and practical possibilities of such 8 Spray program.

41..

10.

11.

BIBLIOGRAPHY

Adams, W. B. , W. M. Dowson and R. W. Marsh. Effect of
organic Spray residues on canned black currents. Rep. Agric.
Res. Sta. Bristol 1947: 163-171, 1948.

Beneke, E. S. The volume occupied by fungus hyphae in raspberry ’3
fruits. Mich. Agric. EXpt. Sta. Quarterly Bull., 33: 124-126,
1950.

Fabian, F. W., E. S. Beneke, F. J. Erickson and R. Floate.
What is a reasonable mold count for black raspberries in the
Great Lakes region? The Canner 112 (18): 14-16, (19): 16-20,
(20):16-20, 1951.

‘3-4- As:
i

r"

Fulton, J. P. Infection of tomato fruits by Colletotrichum
phomoides. PhytOpath. 38: 235-247, 1948.

 

 

Gottlieb, David, Paul Siminoff and M. Martin. The production and
role of antibiotics in soil. IV. Actidione and Clavacin.
PhytOpath. 42: 493-496, 1952.

Greenspan, F. P. and D. G. MacKellar. Peracetic acid cuts
mold on tomatoes. Food Packer 31 (7): 24-25, 1950.

Hamilton, J. M. and M. Szkolnik. Factors involved in the per-
formance of Cyclohexamide (actidione) against Coccomyces
hiemalis. Abstract PhytOpath. 43: 109, 1953.

 

Howard, B. J. Tomato ketchup under the microsc0pe. U. S.
Dept. Agric. Bur. of Chem. Circular 68, 1911.

Howard, B. J. and C. H. Stephenson. Microsc0pica1 studies on
tomato products. U. S. Dept. Agric. Bull. 581, 1917.

McNew. G. L. Control of anthracnose on cannery tomatoes.
Canner 98 (25): 21-22, 46, 48, 50, 1944.

McNew, G. L. and H. P. Burchfield. _Fungitoxicity and biological
activity of quinones. Contrib. Boyce Thompson Inst. 16;: 357-374,
1951.

42.

12.

13.

14.

15.

16.

43..
Moore, J. D. oral communication,

Needham, G. H. and C. R. Fellers. An application of the Howard
method to the detection of Spoilage in berry products. Assoc. of
Official Agric. Chem. 8: 312-327, 1925.

Schroeder, W. T. Control of, tomato diseases by spraying. Bull.
N. Y. Agric. Expt. Sta. 724. 28 pp. 1847.

Thanos, A. Unpublished Ph. D. thesis, Michigan State College,
134 pp. 1952.

Wellman, R. H. and S. E. A. McCallan. Glyoxalidine derivatives
as foliage fungicides. 1. Laboratory Studies. Contrib. Boyce
Thompson Inst. 14: 151-160, 1946.

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