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thesis entitled
EPIDEMIOLOGY AND CHEMICAL CONTROL OF POST-
HARVEST DECAYS OF BLUEBERRY FRUIT CAUSED BY ALTERNARIA
SPP. AND COLLETOTRICHUM GLOEOSPORIOIDES

 

presented by

John Stephen Hartung

has been accepted towards fulfillment
of the requirements for

M. S. degree in Botany and

 

Plant Pathology

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EPIDEMIOLOGY AND CHEMICAL CONTROL OF POSTHARVEST
DECAYS 0F BLUEBERRY FRUIT CAUSED BY ALTERNARIA

 

SPP. AND COLLETOTRICHUM GLOEOSPORIOIDES

 

By

John Stephen Hartung

A THESIS

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

MASTER OF SCIENCE

Botany and Plant Pathology

l980

ABSTRACT

EPIDEMIOLOGY AND CHEMICAL CONTROL OF POSTHARVEST
DECAYS 0F BLUEBERRY FRUIT CAUSED BY ALTERNARIA
SPP. AND COLLETOTRICHUM GLOEOSPORIOIDES

 

By
John Stephen Hartung

In a two year study in a commercial blueberry field, conidia

of Alternaria spp., which could cause blueberry fruit decays, were not

 

trapped from air in large numbers until fruit began to ripen. Inocu-
lation experiments did not provide evidence that latent infections of

blueberry fruits by Alternaria spp. played a significant role in the

 

disease cycle. Other experiments showed that temperature and moisture
conditions such as are found in the Michigan harvest season, as well
as damage to fruit such as is caused by mechanical harvesting opera-

tions, are favorable to Alternaria and other blueberry rotting fungi.

 

Captofol fungicide treatments applied during the growing season con-

trolled Alternaria decays after harvest.

 

Conidia of Colletotrichum gloeosporioides, causal agent of
blueberry anthacnose disease, were trapped from rainwater washings of
infested commercial blueberry bushes from the Bud Swell through Ripe
Fruit growth stages of the blueberry bush. Inoculation studies showed

that g, gJoeosporioides incited a blossom blight and that latent

 

infections of fruit established during the bloom period could play a

major role in the disease cycle.

ACKNOWLEDGMENTS

I express my gratitude and appreciation to Dr. D. C. Ramsdell
and Dr. C. L. Burton who provided guidance and the opportunity to
learn and grow throughout this course of study. I also acknowledge
the advice and effort of Dr. A. L. Jones who served on my guidance
committee.

Special thanks are due to Dr. B. J. Dyko for assistance in
fungal identifications and to Mr. S. P. Eisensmith who provided
invaluable and patient assistance with the computerized analysis and
plotting of the data.

The support of the Michigan Blueberry Growers Association

and of Mr. John Nelson are gratefully acknowledged.

ii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES

Section

I. EPIDEMIOLOGY AND CHEMICAL CONTROL OF POSTHARVEST
DECAYS 0F BLUEBERRY FRUIT CAUSED BY ALTERNARIA SPP.

 

INTRODUCTION

The Genus Alternaria (Nees) Fries .
History and Occurrence of Alternaria Decays of
Blueberry Fruit

 

 

MATERIALS AND METHODS

The Alternaria Isolate Used in This Study

Effect of Temperature on Fungal Growth . .

The Effects of Free Water, 100% Relative Humidity,
and Temperature on Spore Germination

Field Locations for Alternaria Studies

 

 

Monitoring of Environmental Parameters in the. Field .

Trapping of Airborne Alternaria Conidia .

In Vitro Fungicide Tests fbr Control of
—Aitern naria alternata

Field Evaluation of FUngicides for Control of
Alternaria Decays of Blueberry Fruit

Inoculat1on Studies on Mature Blueberry Bushes in
the Field and at East Lansing .

Isolation from Host Tissues to Determine the Over-
wintering Site of the Pathogen

 

 

 

RESULTS

Effect of Temperature on Radial Growth of Alternaria

 

alternata .

The Effects of Free Water, lD0% Relative Humidity,
and Temperature on Conidia] Germination of
Alternaria alternata . . . .

 

iii

Page
vi

viii

A

COLON \JO‘ 0‘ 4:. N N

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\IU'IUON—i

l7

l7

Section

II.

Preliminary Screening of Fungicides for Activity
Against Alternaria alternata . . .

Results of Fungicide Testing in the Field for
Control of Alternaria alternata Decays of
Blueberry Fru1ts .

 

 

Inoculation Studies in the Field and at East Lansing.

Using Alternaria alternata

Results of Trapping of Air Dispersed Conidia of
Alternaria alternata in a Commercial Blueberry
Planting in l978 and 1979 . .

Isolation from Host Tissues to Determine the Over-
wintering Site of the Pathogen . . .

 

 

DISCUSSION

Postharvest Decays Caused by Alternaria alternata

 

EPIDEMIOLOGY AND CHEMICAL CONTROL OF POSTHARVEST
DECAYS 0F BLUEBERRY FRUIT CAUSED BY COLLETOTRICHUM
GLOEOSPORIOIDES

 

 

INTRODUCTION

The Genera Gloeosporium Desm. and Mont. and
Colletotrichum (Penz. ) Sacc. .

History anleccurrence of Gloeospgrium Decays of
Blueberry . . . . . .

 

 

 

MATERIALS AND METHODS

Colletotrichum gloeosporioides Isolate Used in
This Study . .

Effect of Temperature on Fungal Growth and Spore
Germination in Free Water and in Air at 100%
Relative Humidity .

Field Location for Colletotrichum gloeosporioides.
Studies and Monitor1ng of Environmental
Parameters in the Field

 

 

Trapping of Rain Dispersed Cenidia of Colletotrichum.

glpeosporioides

In Vitro and Field Testing of Fungicides for
—Control of Postharvest Decays of Blueberries
Caused by C. gloeosporioides . .

Inoculation Studies of Mature Blueberry Bushes in.
the Field and at East Lansing .

Isolations from Host Tissues to Determine the
Overwintering Site of the Pathogen .

 

 

 

iv

Page

23

26
3l

37
53
54
54

57
58

58
60
63

63

66

66
66

68
68
68

Section
RESULTS

Effect of Temperature on Radial Growth of
Colletotrichum gloeosporioides .

The Effects of Free Water.l0% Relative Humidity,
and Various Temperatures on the Germination of
Colletotrichum gloeosporioides Conidia . .

Preliminary Screening of Fungicides for Activity
Against Colletotrichum glgeosporioides .

Fungicide Testing in the Field for Control of
Colletotrichum gloeosporioides Decays of
Blueberry FFUits . .

Inoculation Studies in the Field and at East
Lansing Using Colletotrichum gloeosporioides

Trapping of Rain Dispersed Conidia of
Colletotrichum gloeosporioides .

Isolation from Host Tissues to Determine the Over-
wintering Site of the Pathogen

 

 

 

 

 

 

DISCUSSION

Appendix

EXPERIMENTS TO EVALUATE THE ROLE OF MECHANICAL
HARVESTING AND SORTING METHODS IN THE EPIDEMIOLOGY
0F POSTHARVEST DECAYS OF BLUEBERRY FRUIT

MATERIALS AND METHODS

Purpose and Location of Harvesting Experiments

The Effects of Harvesting Methods and Bruising
on Postharvest Decay Incidence . .

Mechanical vs. Hand Harvesting and Sorting Methods
of Commercially Raised Fruit .

Isolation of Pathogens from Blueberry Branches
Broken by a Mechanical Harvester .

RESULTS

The Effects of Harvesting Methods and Bruising
on Decay Incidence Postharvest

Mechanical vs. Hand Harvesting and Sorting Methods
of Commercially Raised Fruit .

Isolation of Pathogens from Blueberry Twigs Broken
by a Mechanical Harvester . . . . .

DISCUSSION
LITERATURE CITED

Page
70

7O

70
73

73
76
80
85
87

92
93
93
93
95
96
97

97
100
103

105
107

Table

10.

LIST OF TABLES

SECTION I

Conidial germination of Alternaria alternata on glass
slides at 100% relative humidity and various
temperatures . . . . . . .

 

Conidial germination of Alternaria alternata on glass
slides in free water at various temperatures

 

Fungicide trial for control of Alternaria decays of
blueberry fruit at Grand Junction, MI in 1978

 

Fungicide trial for control of Alternaria decays of
blueberry fruit at Grand Junction, MI in 1979

 

Data from field inoculations of blueberry bushes with
Alternaria alternata at Grand Junction, MI in 1978

 

Data from field inoculations of blueberry bushes with
Alternaria alternata at Grand Junction, MI in 1979

Data from inoculations of potted blueberry bushes with
Alternaria alternata at East Lansing, MI in 1979

Correlation of hourly catches of Alternaria spp.
conidia with environmental parameters for seven
episodes of spore release at Grand Junction, MI

 

Chi square analysis of hourly catches of Alternaria
spp. conidia and environmental parameters in a
commercial blueberry planting at Grand Junction,
MI in 1978 . . . . . . . . . .

 

Chi square analysis of hourly catches of Alternaria
spp. conidia and environmental parameters in a
commercial blueberry planting at Grand Junction,
MI in 1979 . . . . . . .

 

vi

Page

21

22

27

29

32

33

34

42

44

46

Table
11.

12.

13.

Chi square analysis of hourly catches of Alternaria
spp. conidia and leaf wetness in a commercial
blueberry planting at Grand Junction MI in 1978
and 1979 . . . . . . . . . . . . .

 

Results of regression of hourly spore catches on four
environmental parameters for seven spisodes of spore
release of Alternaria spp. at Grand Junction, MI
in 1978 and 1979 . . .

 

Results of regression of hourly spore catches of
Alternaria spp. on four environmental parameters
for two years data from a commercial blueberry
planting at Grand Junction, MI

 

SECTION II

Conidial germination of Colletotrichum gloeosporioides
on glass s1ides in glass distilled water at various
temperatures

Data from field inoculations of blueberry bushes with
Colletotrichum gloeosporioides at Grand Junction,
MI in 1978 . . .

 

Data from field inoculations of blueberry bushes with
Colletotrichum gloeosporioides at Grand Junction,
MI in 1979 . . .

Data from field inoculations of blueberry bushes with
Colletotrichum gloeosporioides at East Lansing, MI
in 1979 . . . . . . . . . . .

 

APPENDIX

Effect of harvesting method and bruising on blueberry
fruit rot at Grand Junction, MI in 1978 .

Effect of harvesting method and bruising on blueberry
fruit rot at Grand Junction, MI in 1979 .

Effect of harvest and sorting methods on blueberry
fruit rots at Grand Junction, MI in l978 .

Effect of harvest and sorting methods on blueberry
fruit rots at Grand Junction, MI in 1979 .

vii

Page

48

50

51

72

77

78

79

98

99

101

102

Figure

—J
0

LIST OF FIGURES

SECTION I

(A) Alternaria alternata conidia.
(B) AlternaFia alternata conidia borne in chains,

 

 

Alternaria alternata sporulating on blueberry fruit

 

Mycelial growth of Alternaria alternata on PDA at
several temperatures after 7 days

 

Growth inhibition of Alternaria alternata mycelium at
96 hr by fungicides incorporated into PDA

(A) Alternaria glternata leafspot 96 hr after inoculation
with 7 x 10 conidia/m1 sterile distilled water.

(B) Alternaria alternata leafspot disappearing after
an additional 10 days . . . . .

Relationship between Alternaria spp. conidia trapped
per hour and various environmental parameters at
Grand Junction, MI, from September 11 to
September 16, 1978 . . . . .

Relationship between Alternaria spp. conidia trapped per
hour and various environmental parameters at Grand
Junction, MI from July 28 to July 31, 1978 .

 

Relationship between Alternaria spp. conidia trapped per
hour and various enviFonmental parameters at Grand
Junction, MI from August 2 to August 4, 1979

SECTION II

Blighted blossom cluster and rotting fruit caused by
inoculation of blueberry bush with 106
Colletotrichum gloeosporioides conida per m1
sterile distilled water at the pink bud growth
stage . .

viii

Page

18
18

20

24

35

39

40

41

64

Figure

2. Colletotrichum gloeosporioides entering fruiting wood
via blighted blossoms . . . . .

 

3. Spore masses of Colletotrichum gloeosporioides on a
blighted blossom after 48 hr in a moist chamber

 

4. Colletotrichum gloeosporioides conida (x 400)

 

5. Mycelial growth of Colletotrichum gloeosporioides on
PDA at several temperatures after 10 days

 

6. Growth inhibition of Colletotrichum gloeosporioides
mycelium at 96 hr by fungicides incorporated
into PDA . . . . . .

 

7. weekly trapping of Colletotrichum gloeosporioides
conidia in a commercial blueberry planting at
Grant Junction, MI in 1978 . . .

 

8. Weekly trapping of Colletotrichum gloeosporioides
conida in a commerdial Blueberry planting at
Grand Junction, MI in 1979 . .

ix

Page

64

64
64

71

74

81

83

SECTION I

EPIDEMIOLOGY AND CHEMICAL CONTROL OF POSTHARVEST
DECAYS 0F BLUEBERRY FRUIT CAUSED BY
ALTERNARIA spp.

 

INTRODUCTION

The Genus Alternaria (Nees) Fries

The genus Alternaria was established by C. G. Nees von

 

Esenbeck and E. M. Fries in the first quarter of the nineteenth
century. The genus was subsequently studied by many mycologists

along with other phaeodictyosporic genera, including Stemphyllium,

 

Macrosporhmn and Ulocladium (23, 28). Taxonomic chaos resulted by

 

 

the middle of this century owing to poorly preserved type specimens,
inadequate descriptions and illustrations, and an over-reliance on
variable mycological characters, including spore shape and dimen-
sions, number and kind of septations in the conidia, number of
conidia in a chain, and the host from which a specimen originated

(28). Particular confusion arose over the genera Alternaria and

 

Macrosporium. Wiltshire stated that "both genera are used almost

 

indiscriminately for manifestly congeneric species" (38). He

declared that the two genera were really one, Alternaria, in the

 

concept of Elliot, Bolle, Mason and Nees (38).

Alternaria can be distinguished from Ulocladium and

 

 

Stemphy11ium as follows (23): Alternaria conidia have an ovoid

 

 

shape and a clear beak at the distal end, while Ulocladium conidia

 

are obovoid and lack a beak. when Alternaria conidia are borne

 

singly or in chains, the proximal end of each conidium is wider

than the distal end; the reverse is true for Ulocladium conidia.

 

2

 

Conidia of the genus Stemphyllium are elliptical with a slight
constriction of the center of the spore.

The genus Alternaria was subdivided by Wiltshire into two

 

forms (38). One section included those species which formed conidial
chains with short beaks on the individual conidia. The other
included those species which formed conidial chains only rarely

and whose conidia had long, filiform beaks (28). Neergaard refined
this concept by forming sections based on the number of conidia in

a chain (23). The Longicatenatae, with conidia in chains of ten or
more, were one section. This section included A, 332213. The
Brevicatenatae with conidia in chains of three to five were another

section. Alternaria tenuissima was included in the Brevicatenatae

 

although Neergaard acknowledged it as intermediate in type between
the two sections (23).

Alternaria conidia are porospores borne at the tip of

 

specialized conidiophores which are morphologically and functionally
distinct from the vegetative hyphae. The conidia mature as dictyo-
spores, although the production of longitudinal septa may be delayed
in some species or even absent in some individual conidia (28). The
size of conidia, length of conidial beaks, number of conidia in a
chain, conidial color and segmentation can be variable, even among
cultures derived from single spore isolates. These characters are
known to be affected by lighting conditions, nutrient content of the
substrate, and the age of the particular culture (28).

Alternaria alternata was described by Simmons from a neotype

 

specimen (28). Conidia are ovoid, usually with a basal pore. The

beak represents one-quarter to one-third of the total conidial
length.. The conidial size ranges from 18-47Lm1long by 7-18 pm wide,
averaging 31-13 pm. There are three to eight transverse and one to
two longitudinal septa per conidium. The conidial wall can be

smooth or minutely roughened. Alternaria alternata includes A,

 

tenuis, and is very closely related phylogenetically to A, tenuissima.

 

History and Occurrence of Alternaria
*Decays of Berberry Fruit

 

Although blueberries (Vaccinium corymbosum L.) have been

 

grown commercially and marketed for several decades, only in the past

decade have post harvest decays caused by Alternaria sp. become a

 

problem. Decays caused by Botrytis cinerea Pers. ex Fr. always

 

dominated in the past. Alternaria tenuissima (Fries) Nitts was

 

identified as the principle cause of storage decay of North

Carolina blueberries in 1971 (18). New Jersey blueberries in 1970

 

were decayed by Alternaria tenuis Auct. in storage tests but decays

caused by Botrytis cinerea and Gloeosporium fructigenum were more

 

 

prevalent (8). In storage areas and retail sites, Alternaria was

 

continuously present in New Jersey blueberries from 1970-1972. This

trend led Ceponis 35 31. to suspect that Alternaria decays were of

 

increasing importance to the New Jersey blueberry industry (12).

In the years 1973-1976 Alternaria became the most common

 

cause of blueberry decays in New Jersey. This coincided with the
beginning of the use of mechanical harvesting for fresh market fruit.

The explanation given at the time for the phenomenon was that

mechanical harvesters damaged the fruit and enabled Alternaria,

 

normally a weak pathogen and saprophyte, to invade the fruit (10).
Other factors cited which may have contributed to the change in
pathogen profile were restrictions on field burning of prunings,
inoculum buildup on fruit left on the ground by the harvesters,
different blueberry varieties, improper fungicide use, and possible

changes in virulence of Alternaria alternata (10). The introduction

 

and widespread usage of benomyl fungicide also coincided with the

increase in Alternaria decays of blueberries.

 

As mechanical harvesting of fresh market fruit was expected

to expand rapidly, it was feared that Alternaria decays would become

 

increasingly important. This was especially worrisome because a

fungicide which was truly effective against Alternaria was not

 

known. For these reasons development of post harvest methods of
disease control were urged (10).

This is the first study of the etiology and epidemiology of
post harvest decays of Michigan blueberries. Thus, the alteration
of the pathogen profile in Michigan decays cannot be documented,

although the shift from predominantly Botrytis decays to Alternaria

 

and Gloeosporium decays is thought to have occurred as it did in

 

North Carolina and New Jersey. In a four year study of Michigan
bludberry cankers reported in 1974, Neingartner and K105 reported

Alternaria spp. to be present in 48% of blighted or cankered blue-

 

berry stems (37). Even though the etiology of blueberry decays had
not been studied in Michigan, the pathogen was clearly established

in Michigan blueberry fields by 1974.

MATERIALS AND METHODS

The Alternaria Isolate Used in This Study

Several single spore isolates of Alternaria spp. were made

 

from rotting blueberry fruits. Two of these isolates were used for
further study, and were sent to Dr. Emory Simmons, University of
Massachusetts, Amherst, Massachusetts, for species identification.

These were identified as Alternaria alternata (29).

 

The average length and greatest width of 50 conidia of each
isolate grown on PDA with continuous fluorescent lighting were
measured by me and found to be consistent with A, alternata as
described by Simmons (28). The conidial color, number and kind of
septations, and size of the conidial beak were also consistent with
A, alternata when observed on conidia grown under the same conditions.

Figure 1 is a micrograph of A, alternaria conida and conidial chains

 

from the isolates used in this study when grown on V-8 agar (36).

On rotting blueberry fruits A, alternata appears as a dense
green to black velvety mat on the surface of the ripe fruit (Figure
2). The appearance of the fungus in culture was greatly affected by
the light regimen used. With continuous darkness the mycelium was
olive colored and hirsute. With continuous soft fluorescent lighting
the culture became black due to greatly increased sporulation.
Sporulation was also greatly favored by growth on V-8 agar as com-

pared to potato dextrose agar.

Effect of Temperature on Fungal Growth
The effect of temperature on the mycelial radial growth rate
was studied by incubating cultures at controlled temperatures and
measuring colony diameter after seven days. A 7-mm cork borer was
used to cut mycelial plugs from the advancing margin of a culture of
A, alternata growing on potato dextrose agar (PDA-—Difco Products
Co., Detroit, Michigan 48201). The plugs were placed aseptically in
the center of petri plates containing PDA and incubated at 5, 10,
15, 20, 27, or 30C. Five replicates per temperature tested were
used. The experiment was conducted to determine the optimum
temperature for fungus growth and development in the field and in
storage. ‘
The Effects of Free Water, 100% Relative

Humidity, andllemperature on
Spore Germination

 

Spore germination studies were carried out in the laboratory
to gain information about how environmental parameters influence
spore germination and fruit infection. Spore suspensions were
prepared by flooding the surface of PDA grown cultures of an A,
alternata isolate, scraping the surface with a bent glass rod, and
decanting the resulting spore suspension through four layers of
cheesecloth.

Spore germination in free water was studied by placing the
spore suspensions in the wells of depression slides and incubating
the slides over water at various temperatures. Four replicates per

temperature tested were used. At intervals, the slides were removed

from the incubation chambers and the mean percent germination and
the mean germ tube length was determined. In each replicate the
first 100 conidia encountered were scored as germinated or not
germinated and the first 25 germ tubes encountered were measured.
This was done at the meniscus of the spore suspension since at the
bottom of the depression the spore concentration was too great to
count and germination was inhibited. A spore was considered as
germinated if a germ tube was visible which was at least as long as
the spore was wide. The temperatures tested were 5, 10, 15, 20, 25,
and 30C, and the lengths of incubation were 8, 16, 24, 32 and 40
hours at each temperature tested.

To determine if spores could germinate in air at 100% rela-
tive humidity spore suspensions prepared as described were sprayed
onto glass coverslips with a DeVilbiss No. 15 atomizer. The cover
slips were air dried at room temperature. After the spore suspen-
sion had dried on the coverslips the coverslips were placed on micro-
scope slides which were in turn placed inside petri dishes lines with
wet paper towels. Two petri plates each containing one microscope
slide with two cover slips on it were prepared for each temperature/
time combination to be studied. This was important because a slide
was taken out of its container only once for study. Because of this
it did not matter if water condensed on the coverslips after they
were removed from the petri plates, as happened with the cover slips

incubated at low temperatures.

Field Locations for Alternaria Studies
Epidemiological studies of A, alternata were carried out at
a commercial blueberry farm at Grand Junction, Michigan which had a
history of postharvest decay problems caused by A, alternata and

Colletotrichum gloeosporioides. Weather parameters were monitored,

 

airborne Alternaria spores were trapped, and inoculation studies

 

were carried out in the northeast corner of the 16-hectare (40-acre)
field. This area was approximately 0.06 hectare (1/7 acre) in size
and contained 18 mature Jersey blueberry bushes in each of six rows.
No fungicides were applied in this area during the course of the
study.

Monitoring of Environmental Parameters
in the Field

 

 

Solar radiation intensity was measured with a solar radia-
tion monitor, rainfall was measured with a tipping bucket rain gauge
accurate to 0.25-mm (0.01-inch), and wind speed was monitored using
a contact anemometer accurate to 0.1 km/hr (0.16 mile/hr)

(Weather Measure Co., Sacramento, California 95841). Duration of
leaf wetness was recorded using a leaf wetness meter (M. DeWitt Co.,
Hengelo, The Netherlands). Relative humidity and air temperature
were monitored using a hygrothermograph (Belfort Instrument Co.,
Belfort, Maryland 21224). All instruments were 7-day continuously
recording types and were installed within the rows of the blueberry
bushes. All charts were changed at weekly intervals. By comparing
the leaf wetness and rainfall charts for the same time periods,

the leaf wetness periods were attributed to rainfall or to dew.

lU

Trapping of Airborne Alternaria Conidia

 

A Burkard volumetric air spore trap (Burkard Scientific
Sales Ltd., Rickmansworth, Herts., England) was installed in the
field and run continuously from a 12V DC battery power source
from April 18, 1978 to September 19, 1978 and from April 18, 1979
to August 28, 1979. The starting date for spore trapping was before
bud break and the stopping date was after harvesting was completed
in both seasons. The intake orifice was 1 m above the ground and
the spore trap tapes upon which the spores were impacted were
changed at weekly intervals. The machine was adjusted weekly to
draw 8-10 liters (2.1-2.6 gallons) of air per minute.

The spore trap tapes were prepared by mounting the clear
Mellinex® tape supplied by the manufacturer on a clock drum with
double sided tape and coating it with a Gelvatol® (Monsanto
Chemical Co., St. Louis, Missouri 63101) water mixture as per
directions supplied by the manufacturer (1). After drying overnight,
the tapes were recoated with a viscous mixture of vaseline, parafin,
and toluene (l) which was the trapping medium. Spores were trapped
for one week in the field and the resulting tape was cut into 48
mm (1.9 inch) sections corresponding to single 24 hr days. Hourly

counts of Alternaria spp. conidia were made using a compound light

 

microscope with 10x oculars and a 10x objective lens. No stains

were used.

11

_Ig Vitro Fungicide Tests for Control of
Alternaria alternata

 

A preliminary experiment was conducted to select promising
fungicides for field testing by testing their effect on radial

growth of the Alternaria alternata isolate obtained from rotted

 

fruit jg_yjt§9, The fungicides selected for screening were:
Bayleton 50% wettable powder (triadimefon), ferbam 65% wettable
powder, Difolatan (captafol) 0.5 kg/l (4 lb/gallon) flowable, captan
50% wettable powder, Benlate (benomyl) 50% wettable powder, Phaltan
(folpet) 5 % wettable powder, Dithane M-45 (mancozeb) 80% wettable
powder, Funginex (triforine) 20% emulsifiable concentrate, and
Bravo (chlorothalonil) 75% wettable powder.

The experiment was done by mixing the various fungicides
into PDA at concentrations of 0.1, l, 10, and 100 ug/ml (active
ingredient basis). Mycelial plugs were taken from the advancing
edge of the PDA-grown fungus culture to be tested with a 7-mm cork
borer and placed in the center of each fungicide amended PDA test
plate. The cultures were incubated at room temperature under con-
tinuous cool white fluorescent light and after four days the mycelial
radial growth was measured. Data were recorded as percent inhibition
of radial growth by the fungicide as compared to radial growth on

unamended PDA plates. Four replicates per treatment were used.

12

Field Evaluation of Fungicides for Control of
Alternaria Decays of Blueberry Fruit

 

Fungicide testing was carried out at the same commercial
field described previously. The study was carried out to determine

what fungicides, if any, could control Alternaria decays post-

 

harvest when applied at various times during the growing season.

The fungicides were applied using a Tecnoma 3-point hitch
air blast sprayer (Tecnoma Co., Eppernay, France). One hundred
eighty-seven liters/hectare (20 gal/acre) of fungicidal spray was
applied at a hydraulic pressure of 10.5 kg/cm2 (150 lbs/inz) and a
ground speed of 5.8 km/hr (3.6 mph). The experiment was carried out
in 1978 using a completely randomized design with four replicates
per treatment. Two control treatments consisted of water only from
the spray rig. Each replicate consisted of seven bushes in a row.

The fungicides used and the application schedule for 1978
are given in Table 3. The fungicides were applied either throughout
the season as eight applications from May 2 (green tip) through
July 12 (early blue fruit) or as four late season applications only,
from June 2 (5% petal fall) through July 12 (early blue fruit).

The fungicide testing methodology was the same in 1979
except that fewer treatments were applied, and five replicates per
treatment were used. The fungicides were applied either throughout
the season as seven applications from May 1 (green tip) to July 6
(first blue fruit) or as a single massive application (SAT) on

May 1, 1979 (Table 4).

13

The three center bushes of each replicate of the fungicide
trial were harvested as a group with hand-held electrical vibrators
(Blueberry Equipment Co., South Haven, Michigan 49090). The weight
of the ripe fruit in each replicate was recorded as well as the
weight of one level cup of fruit and the number of berries in one
level cup. A subsample of 100 berries was taken from each replicate
and incubated on moist paper towels in plastic boxes so as not to
touch one another and with the stem end upwards. After four days
at room temperature the fruit was evaluated based on the presence
or absence of sporulating fungus. The fruit was scored as healthy

(no Sporulation) or rotted by Alternaria. The data were recorded

 

as the percent of fruit in each category on a count basis.

Inoculation Studies on Mature Blueberry Bushes
in the Field and at East Lansing

 

 

Two single spore isolates of Alternaria alternata isolated

 

from rotting blueberries were used for inoculation studies. The
isolates were stored over the winter on half strength PDA (IPDA)
at 5C. The cultures for inoculation were grown on V-8 agar (36)
in plastic petri dishes under continuous fluorescent light at room
temperature for four to eight days. Spore suspensions were made
by flooding the cultures with sterile distilled water, scraping
with a sterile bent glass rod, and decanting the resulting spore
suspension through four layers of cheesecloth. The concentration
of A, alternata conidia was adjusted to 7x105 conidia/m1 sterile
distilled water in 1978 and to 3x105 conidia/ml sterile distilled

water in 1979 using a hemacytometer. The two single spore

14

isolates contributed equally to each spore suspension used for
inoculations.

Inoculation studies were carried out at two locations to
determine at which phenotypic stage(s) the blueberry bush was
susceptible to infection by A, alternata. At Grand Junction,
Michigan, the mature blueberry bushes were of the cv. Jersey. At
East Lansing potted 3-4 year old bushes of the cvs. Bluecrop and
Berkley were used.

The spore suspension was sprayed onto unwounded plant
tissue until runoff occurred. The inoculated branch was then placed
in a clear polyethylene bag containing a wet paper towel to main-
tain free water since preliminary experiments showed that free
water stimulated conidial germination. Four replicate branches
were inoculated with A, alternata and four with water only, as con-
trols on each inoculation date. The plastic bag covering the
inoculated branch was removed after 18-24 hours. Because it was
thought that high daytime temperature would interfere with the
infection process, the inoculated bushes at East Lansing were kept
in a growth chamber (Sherer-Gillette Co., Marshall, Michigan 49068)
at 21C while the bag covered the bush.

Inoculation dates and growth stages for the 1978 and 1979
seasons are given in Tables 5, 6, and 7.

The ripe fruit from each inoculated bush or branch was
harvested by hand by replicate and placed into separate polyethylene

pint bags. In order to prevent cross contamination of the replicates

15

and treatments separate disposable plastic gloves were used to pick
each replicate. Fruit with visible Sporulation was discarded in
order to prevent mass inoculation of the replicates.

The fruit was placed by replicate on a l-cm wire mesh screen
over water in plastic buckets so as not to touch one another. The
buckets were covered to maintain high relative humidity. After four
days incubation the fruit was scored as healthy (no Sporulation) or
decayed by Alternaria (visible Sporulation). The data were recorded
as the percent fruit in each category on a count basis.

Isolation from Host Tissues to Determine the
Overwintering Site of the Pathogen

 

 

Experiments were done to determine where Alternaria spp.
overwintered in blueberry bushes. In late October of 1977, 35
blighted fruit stems were collected. They were surface disinfested
by immersing in 0.25% sodium hypochlorite for five minutes followed
by rinsing with sterile distilled water. Tissue transfers were
aseptically made to petri plates containing IPDA. In the same
manner 129 apparently healthy flower buds were used for tissue
transfers.

In the spring of 1978 more blighted fruiting wood was col-
lected from which to make isolations. Twigs were surface disinfested
in 1% sodium hypochlorite with a few drops of Tween-80 added as a
wetting agent for 10 minutes with stirring under bench vacuum.
Other lots of twigs were isolated from directly without surface

disinfestation to determine if the pathogen was located deep within

16

the twig or at or on its surface. Tissue transfers were made onto
PDA and the resulting fungal cultures were identified after four to

eight days.

RESULTS

Effect of Temperature on Radial Growth
of Alternariaalternata

 

The optimal temperature tested for mycelial growth of

Alternaria alternata in this study was 27C (Figure 3). Alternaria

 

 

alternata grew more than twice as fast at 200 as compared to 15C,
and twice as fast at 15C as compared to 10C. Very little growth
occurred after seven days at 5C. The results indicate that

Alternaria decays would be favored at high temperatures such as

 

are common during the blueberry harvest season.

The Effects of Free Water, 100% Relative Humidity,
and Temperature on Conidial Germination of
Alternaria alternata

 

 

Conidia of the A, alternata isolate used germinated readily
at temperatures of 100 or greater at 100% relative humidity (Table
1). Free water was, however, very stimulatory for both conidial
germination and germ tube elongation at all temperatures tested,
particularly at the lower and higher temperatures (Table 2).
Conidial germination was greatest at 20C in free water and at 25C
at 100% relative humidity.

Alternaria alternata conidia produced several germ tubes

 

each in this experiment. This created a wide variation in germ
tube lengths, which is reflected by the standard deviations about
the mean of germ tube length in Tables 1 and 2.

17

18

Figure 1-A.--Alternaria alternata
conidia

 

Figure 1-B.--Alternaria alternata
conidia borne in chains.

 

Figure 2.--Alternaria alternata
sporulating on
blueberry fruit.

 

 

20

 

80"

70"

60"

Colony Diameter (mm)

10)-

Inltlal colony Diameter

 

1 1 J 1 1 4
10 15 20 25 30

Temperature (C)

 

Figure 3.--Mycelial growth of Alternaria alternata on PDA at
several temperatures after 7 days.

 

 

21

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23

Since free water was not required for germination, infection
of fruit could occur at 100% relative humidity, although the infec-
tion rate would be increased in the presence of free water.
Temperatures of 15-25C would be optimal for fruit infection.

Preliminary Screening of Fungicides for Activity
Against Alternaria alternata

 

 

Radial growth inhibition of A, alternata on fungicide
amended PDA as compared to non-amended PDA was determined for nine
fungicides and the results for eight of those fungicides are shown
in Figure 4. The ninth fungicide tested, Bravo, showed almost no
activity against A, alternata.

The fungicides which showed the greatest activity against
A, alternata in this study were Funginex (triforine), ferbam, and
Difolatan (captafol). Triforine caused more than 90% growth
inhibition of A, alternata at a concentration of 100 ug/ml.

Captan and Bayleton caused 65% and 55% inhibition, respectively,
at the same concentration. Benlate (benomyl) was ineffective
against A, alternata in this test.

Difolatan was clearly the most effective fungicide at con-

centrations lower than 100 ug/ml. The results of this 1g Vitro

 

screening of fungicides were used to select fungicides for field
testing for control of postharvest decays of blueberries caused by

A, alternata.

24

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Difolatan Phaltan

 

Figure 4.--Growth inhibition of Alternaria alternata mycelium
at 96 hr by fungicides incorporated into PDA.

 

25

 

 

 

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Figure 4.--Continued.

26

Results of Fungicide Testing in the Field for
Control of Alternaria alternata Decays
of’BTUeberry‘Fruits

 

 

The results of the 1978 and 1979 fungicide trials for

control of Alternaria decays of blueberries are shown in Tables 3

 

and 4, respectively.
In both seasons the four treatments which gave the greatest

Alternaria decay control all involved Difolatan (captafol). Because

 

of the low decay incidence and variability among the replicates in

1978, the differences in fungicidal control of Alternaria were not

 

judged to be significant (p_= .05) by Duncan's Multiple Range (DMR)

test. The differences in fungicidal control of Alternaria decays

 

were found to be significant (p_= .05) by the DMR test in 1979, but
not by the Student-Newman-Keul (SNK) procedure. Difolatan 4F at

the rates of 9.4 1/ha (4 qt/acre) throughout the season and at

28.2 l/ha (12 qt/acre) on the first application date only (SAT) gave

significantly better control of Alternaria decays than any of the

 

other fungicides tested in the_field in 1979.

Difolatan 4F at the rate of 9.4 l/ha throughout the season
did cause a significant reduction (p_= .05, DMR) in the yield of
the bushes harvested for fungicide evaluation in 1979, but not in
1978. Whether this represented a true yield reduction or merely a
delay in ripening was not determined.

None of the fungicides tested had any effect (p_= .05) on
the average berry weight or size in either year as determined by

DMR test in 1978 and SNK test in 1979.

27

TABLE 3.--Fungicide trial for control of Alternaria deca

 

.blueberry fruit at Grand Junction, MI in 1978.

ys of

 

 

 

% Alternaria Yieldv
Treatment Decay Grams
Difolatan 4Fw 9.4 L/ha (4 qt/acre) 0.12 a 11991 ab
E+Lx
Difolatan 4F 4.7 L/ha (2 qt/acre) 0.21 ab 11331 ab
L
Difolatan 4F 9.4 L/ha (4 qt/acre) 0.23 ab 10182 b
L
Difolatan 4F 4.7 L/ha (2 qt/acre) 0.38 abc 11205 ab
E+L
Funginex (CME 74770) ljl7L/ha (16 oz/acre) 0.42 abcd 14676 a
20% ECY
E+L
Captan 50% WPZ 5.6 kg/ha (5 1b/acre) 0.43 abcd 10992 ab
+
Benlate 50% WP 1.1 kg/ha (1 lb/acre)
E+L
Control (untreated) 0.45 abcd 14634 a
That Liquid Sulfur 4.7 L/ha (2 qt/acre) 0.53 abcd 10437 ab
E+L
0.79 bcd 11205 ab

Captan 50% WP 5.6 kg/ha (5 1b/acre)
+

Benlate 50% WP 1.1 kg/ha (1.1b/acre)
L

28

TABLE 3.--Continued.

 

 

% Alteggariau Yieldv
Treatment Decay Grams
Captan 50% WP 5.6 kg/ha (5 1b/acre) 0.82 cd 7347 c
+
Ferbam 76? WP 6.8 kg/ha (6 1b/acre)
E+L
Funginex (CME 74770) 20% EC 1.75 L/ha 0.85 Cd 10779 ab
(24 fl oz/acre)
L
Captan 50% WP 5.6 kg/ha (5 lb/acre) 0.88 cd 10650 ab
4.
Ferbam 76% WP 6.8 kg/ha (6 lb/acre)
L
Control (untreated) 0.90 cd 12780 ab
Funginex (CME 74770) 20% EC 1.75 L/ha 0.96 d 11121 ab
(24 f1 oz/acre)
E+L

 

TAll fungicides applied on dates and at rates iven with a
Tecnoma airblast sprayer run at 12.6 kg/cm2 (180 1b/in ). All com-
pounds applied in 187 liters of solution per hectare (20 gallon/acre).

UBerries were hand harvested and incubated without touching
for four days in moist chambers. Mean percent decay of four repli-
cates of 100 randomly selected berries each. Means with letter follow-
ing in common judged to be not significantly different (p = .05) by
Duncan's Multiple Range test. "

VFruit was harvested on 8/10, 8/11, 1978. Mean yield of four
replicates of three bushes each. Represents total crop yield. Means
with letters following in common judged to be not significantly dif-
ferent (p = .05) by Duncan's Multiple Range test.

WFlowable.

xE+L = Early + Late applications. Dates of early applications
were 5/2 (1/16 inch green tip). 5/19 (early pink bud), 5/10 (1/4 inch
green tip). and 5/26/78 (l-5% bloom). Dates of late applications were
6/2 (5% petal fall), 6/13-15 (small green fruit), and 6/28 (late green
fruit), and 7/12/78 (first blue fruit). L = Late applications only.

YEC = Emulsifiable Concentrate

ZWP = Wettable Powder

29

TABLE 4.--Fungicide trial for control of Alternaria decays of
blueberry fruit at Grand Junction, MI in 1979.

 

 

% AlternariaU

 

 

T W Yield
Treatment ’ Decay Grams
Difolatan 4Fx 9.4 L/ha (4 qt/acre) 5.2 a 4992 a
Through Season
Difolatan 4F 28.2 L/ha (12 qt/acre) 7.6 a 6628.4 abc
First Application Only
Difolatan 4F 4.7 L/ha (2 qt/acre) 11.2 ab 6350.2 abc
Through Season
Benlate 50% WP 1.1 kg/ha (1 lb/acre 13.5 abc 5862.0 abc

+
Difolatan 4F 4.7 L/ha (2 qt/acre)
Through Season
That Sulfur F 4.7 L/ha (2 qt/acre) 18.4 abc 5600.6 ab
Through Season
Lime Sulfur 9.4 L/ha (1 gal/acre) 18.4 abc 6089.2 abc
First Application Only
Funginex 20% ECY 1.75 L/ha (24 oz/acre) 20.8 abc 5009.8 a
Through Season
Control (no sprays) 22.8 bc 6997.7 bc
Procymidone 50% WP2 1.1 kg/ha (1 lb/acre) 25.4 bc 7423.8 c
Through Season
Lime Sulfur 9.4 L/ha (4 qt/acre) 28.8 c 6384.2 abc

.g.
Captan 50% WP 5.6 kg/ha (5 lb/acre)
Lime Sulfur First Application Only;
Captan Only through Season

30

TABLE 4.--Continued.

 

 

 

% AlternariaU Yieldv
Treatment Decay Grams
Lime Sulfur 9.4 L/ha (4 qt/acre) 43.4 d 5055.2 a

+

That Sulfur F 4.7 L/ha (2 qt/acre)
Lime Sulfur First Application Only;
That Sulfur Only Through Season

 

TA11 fungicides applied on dates and at rates gi en with a
TecnomaDair blast sprayer run at 12.6 kg/cm2 (180 lbs/in ). All
compgunds applied in 187 liters of solution per hectare (20 gallons/
acre .

UBerries were hand harvested and incubated without touching
for four days in moist chambers. Mean percent decay of five
replicates of 100 randomly selected berries each. Means with letter
followin in common judged to be not significantly different
(p = .051 by Duncan's Multiple Range test.

vFruit was harvested on 8/15/79. Mean yield of five repli-
cates of three bushes each. Represents about 1/3 of total crop
yield. Means with letter following in common judged to be not
significantly different (p = .05) by Duncan's Multiple Range test.

wFirst application - 5/ 1/79 - Green Tip

Second application - 5/11/79 - Pre-Bloom

Third application - 5/16/79 - 10% Bloom

Fourth application - 5/23/79 - 40% Bloom

Fifth application - 6/ 5/79 - Petal Fall

Sixth application - 6/21/79 - Green Fruit
Seventh application - 7/ 6/79 - First Blue Fruit
xF = Flowable

YEC = Emulsifiable Concentrate

ZWP = Wettable Powder

31

Tables 3 and 4 show that Alternaria decay incidence was ten

 

or more times greater in 1979 than in 1978. This reflects a differ-
ence in the criteria used to characterize a blueberry as rotted by

Alternaria in each year. In 1978 only berries with abundant green

 

to black velvety mycelium on them were classified as rotted by

Alternaria. In 1979 the criteria were expanded to include berries

 

with a small grayish white mycelium at the stem end scar. Isola—

tions showed that these mycelia were also Alternaria, although they

 

were non-sporulating at the time of evaluation.

Inoculation Studies in the Field and at East
Lansing Using Alternaria alternata

 

Inoculations with A, alternata did not consistently increase
the incidence of A, alternata decays postharvest above the
inoculated control levels in either season in the field or in the
potted bush inoculations at East Lansing (Tables 5, 6 and 7). The
data from the potted bush inoculation experiment at East Lansing in
1978 were unusable due to problems with insect and avian pests.

The results imply that Alternaria decays of fruit are not the result

 

of latent infections established during the growing season.

The isolates of A, alternata used in this study did cause a
transient leafspot on succulent blueberry leaves when inoculated on
them early in the season. The symptoms consisted of small red spots
on the inoculated succulent leaves which faded as the season pro-
gressed (Figure 5). The symptom is very similar to one reported

by Milholland in North Carolina caused by A, tenuissima (20), and

 

32

TABLE 5.--Data from field inoculations of blueberry bushes with
Alternaria alternata at Grand Junction, MI in 1978.3,

 

 

 

 

 

Mean Percent Fruit c Mean Percent Fruit
Inoculation Date Rotted by Alternaria Rotted by Alternaria
and Growth Stage (Inoculated) (Control)
5/2/78
Bud Swell 4.2 i 1.7 1.5 i 1.0
5/16/78
Bud Break 8.6 i 5.5 8.8 i 2.6
5/23/78
Pink Bud 10.3 i 1.9 6.5 i 2.8
5/30/78
Full Bloom 1.5 t 1.0 0.0
6/6/78
Petal Fall 1.1 t 0.9 17.7 t 11.2
6/21/78
Green Fruit 0.0 0.0
7/16/78
Early Blue Fruit 0.0 2.6 i 1.1

 

aBushesswere inoculated on dates given with a spore sus-
pension of 7x10 conidia/ml sterile distilled water or with sterile
distilled water only.

bFruit was hand harvested on 8/1/78 and held for four days
at room temperature in moist chambers.

CPercent visibly sporulating Alternaria rots on a count
basis. Numbers given are the means of four replicates t the
standard deviations of the means.

 

33

TABLE 6.--Data from field inoculations of blueberry bushes with
Alternaria alternata at Grand Junction, MI in 1979.3,b

 

 

 

 

 

Mean Percent Fruit c Mean Percent Fruit
Inoculation Date Rotted by Alternaria Rotted by Alternaria
and Growth Stage (Inoculated) (Control)
5/2/79
Bud Swell 10.5 i 2.5 9.8 i 2.5
5/9/79 d
Bud Break 9.8 i 2.2 ---
5/16/79
Pink Bud 20.0 i 4.2 10.3 i 1.8
5/23/79
50% Bloom 13.3 i 2.1 36.3 i 3.3
6/13/79
Early Green Fruit 13.3 i 4.2 14.0 i 4.2
7/5/79
Late Green Fruit 24.5 i 2.9 18.0 i 1.1
7/18/79
First Blue Fruit 16.8 i 2.2 2.8 i 0.7
8/1/79
First Ripe Fruit 13.5 i 1.0 9.8 i 2.1

 

aBushes were inoculated on dates given with a spore sus-
pension of 3x105 conidia/ml sterile distilled water or with
sterile distilled water only.

bFruit was hand harvested on 8/15/79 and held at room
temperature for four days in moist chambers.

cPercent visibly sporulating Alternaria rots on a count
basis. Numbers given are the means of four replicates t the
standard deviations of the means.

d

 

Missing data.

34

TABLE 7.--Data from inoculations of potted blueberry bushes with
Alternaria alternata at East Lansing, MI in 1979.a’b

 

 

 

 

 

Mean Percent Fruit c Mean Percent Fruit
Inoculation Date Rotted by Alternaria Rotted by Alternaria
and Growth Stage (Inoculatedl (Control)
4/28/79
Bud Swell 15.4 i 5.5 13.8 i 3.7
5/10/79
Bud Break 19.4 i 6.4 21.2 i 5.7
5/17/79
Early Pink Bud 22.5 1 5.5 14.4 i 2.9
5/24/79
Full Bloom 20.6 i 6.6 19.9 i 3.6
6/14/79
Green Fruit 19.7 i 0.7 12.7 i 3.2
7/19/79
First Blue Fruit 3.3 t 2.9 2.3 i 1.5

 

aPotted bushes were inoculated on dates given with a spore
suspension of 3x105 conidia/m1 sterile distilled water or with
sterile distilled water only.

bFruit was hand harvested on 7/31/79 and held 6 days at
room temperature in moist chambers.

cPercent visibly sporulating Alternaria rots on a count
basis. Numbers given are the means of four replicates i the
standard deviation of means.

 

35

Figure 5-A.--Alternaria alternata leafspot 96 hr.
after’inoculation with 7 x 105
conidia/m1 sterile distilled water.

 

Figure 5-B.--Alternaria alternata leafspot
disappearing after an additional
10 days.

 

.36

 

37

implies that the virulence of the isolates was not attenuated in
culture. The fungus reisolated from these spots was indistinguish-
able in culture from the isolates which were used to inoculate the
bushes.

Results of Trapping of Air Dispersed Conidia of

AlternariaAALternata in a Commercial
Blueberry PlantingAjn 1978 and 1979

 

 

In 1978 the number of Alternaria conidia trapped per hour

 

increased rapidly in the last week of June. The daily peak catches
of conidia remained relatively high until trapping was discontinued
on September 17, 1978. The greatest catch of conidia occurred on
September 15, when 909 conidia were recorded in a single hour.
These conidia were probably produced on berries which had been left
in the field by the mechanical harvester either on the bushes or on
the ground, as the field had been mechanically harvested one month
earlier. Some may have been produced on weeds or saprophytically
on other plant debris.

In 1979 a similar general pattern was observed. The daily
peak catches of conidia remained high until trapping was discontinued
on August 28 after rising in early July. The greatest catch of
conidia occurred on July 18 when 334 were recorded in one hour. In
both seasons spore catches never exceeded 50 per hour in May and
June and were considered negligible.

The relationships between release of Alternaria conidia and
environmental parameters monitored simultaneously were investigated

by analyzing seven individual episodes of spore release by

38

statistical methods. Four of these episodes occurred during 1978,
and theremminder occurred during 1979. These episodes ranged from
three to five days in length and included time periods with both
very high and very low numbers of spores trapped per hour. Three
of these periods were analyzed graphically and are shown in
Figures 6, 7, and 8. The results show that spore release tended to
occur at times with higher air temperatures, lower relative humidi-
ties, dry leaf surfaces, sunshine, and wind.

Simple correlation coefficients (r) were calculated which
related spore release to the various environmental parameters.

These data are presented in Table 8. Release of Alternaria conidia

 

was consistently positively correlated with air temperature, solar
radiation intensity and wind speed. Release of conidia was
negatively correlated with relative humidity.

Chi square analysis of the spore release patterns for the
same episodes was also carried out to determine the nature and
strength of the associations between low and high rates of conidial
production and various environmental parameters. High rates of
conidia release were associated consistently with higher air
temperatures, low relative humidity, high solar radiation intensity,
and higher wind speed, and the absence of water on leaf surfaces.
Low rates of conidial release were consistently associated with
lower air temperatures, high relative humidity, lower solar radia-
tion intensities and darkness, low wind speeds, and the presence of

water on leaf surfaces. These associations were consistently

39

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43

significant (p < .01) throughout the season, but in two of the seven
episodes solar radiation intensity was not associated with spore
release and for one episode each there was no association between
wind speed and spore release and leaf wetness and spore release.

The same associations between the various environmental parameters
monitored and spore release were found in chi-square analyses of

the data from the entire seasons of 1978 and 1979. These data are
presented in Tables 9 and 10, respectively. The chi square analyses
of spore release and leaf wetness for 1978 and 1979 are shown in
Table 11. The null hypotheses (Ho) tested by the chi square
analyses of spore release and environmental parameters shown in
Tables 9-11 were that the magnitude of the spore release was not
related to (a) air temperature, (b) wind speed, (c) percent relative
humidity, (d) solar radiation intensity, and (e) the presence or
absence of free water on the leaves. As a result of these analyses,
all of the null hypotheses were rejected.

Standard multiple regression analyses were performed using
the spore trapping and environmental data which had been simul-
taneously recorded to determine which, if any of the environmental
parameters consistently contributed the greatest amount to the
variance explained by the regression equations. The analyses were
carried out using the data from the same seven episodes for which
simple correlation coefficients had been calculated and chi square
analyses performed. All independent variables were entered into the

equation simultaneously and the contribution to the explained

44

TABLE 9.--Chi square analysis of hourly catches of Alternaria spp.
~ conidia and environmental parameters in a commercial
blueberry planting at Grand Junction, MI in 1978.

 

 

Alternaria spp. spores A1r Temperature (C)

trapped from the air ROW

Less than 21 22-32 Total

 

 

 

Less than 50/hr: Count 710 504 1214
Row Percent 58.5 41.5 74.6
Column Percent 92.3 58.6
Total Percent 43.6 31.0
51-909/hr: Count 59 354 413
Row Percent 14.3 85.7 25.4
Column Percent 7.7 41.3
Total Percent 3.6 21.8
Column 769 858 1627
Total 47.3 52.7 100.0
Corrected chi square = 239.8 with 1 degree
of freedom
£_= 0.0000.

 

Alternaria spp. spores Wind SPEEd km/hr

trapped from the air

 

Row
Less than 4.0 4.1-22.0 Total

 

 

Less than 50/hr: Count 1095 256 1351
Row Percent 81.1 18.9 75.0
Column Percent 77.9 64.6
Total Percent 60.8 14.2
51-909/hr: Count 310 140 450
Row Percent 68.9 31.1 25.0
Column Percent 22.1 35.4
Total Percent 17.2 7.8
Column 1405 396 1801
Total 78.0 22.0 100.0

Corrected chi square = 28.4 with 1 degree
of freedom
A_= 0.00001

 

TABLE 9.--Continued.

45

 

Alternaria spp. spores

 

Relative Humidity (%)

 

 

trapped from the air Less than Row
73 74-97 98-100 Total
Less than 50/hr: Count 241 247 566 1054
Row Percent 22.9 23.4 53.7 74.9
Column Percent 54.8 68.4 93.4
Total Percent 17.1 17.6 40.2
51-909/hr: Count 199 114 40 353
Row Percent 56.4 32.3 11.3 25.1
Column Percent 45.2 31.6 6.6
Total Percent 14.1 8.1 2.8
Column 440 361 606 1407
Total 31.3 25.7 43.1 100.0

Raw chi square = 213.2 with 2 degrees of freedom

3 = 0.00001

 

Alternaria spp. spores
trapped from the air

 

Solar Radi tion
(gm-cal/cm /min)

 

0.01- 0.57- Row

 

None 0.56 1.12 Total
Less than 50/hr: Count 819 314 218 1351
Row Percent 60.6 23.2 16.1 75.0
Column Percent 83.3 68.7 60.4
Total Percent 45.5 17.4 12.1
51-909 hr: Count 164 143 143
Row Percent 36.4 31.8 31.8 25.0
Column Percent 16.7 31.3 39.6
Total Percent 9.1 7.9 7.9
Column 983 457 361 1801
Total 54.6 25.4 20.0 100.0
Raw chi square 87.0 with 2 degrees of freedom.

3 = 0.00001

 

46

TABLE 10.--Chi square analysis of hourly catches of Alternaria spp.
conidia and environmental parameters in a commercial

blueberry planting at Grand Junction, MI in 1979.

 

 

Alternaria spp. spores

 

 

Air Temperature (°C)

 

 

 

 

 

. Row
trapped from the a1r Less than 21 22-32 Total
Less than 50/hr: Count 820 559 1379

Row Percent 59.5 40.5 94.4

Column Percent 97.7 89.9

Total Percent 56.1 38.3
51-334/hr: Count 19 63 82

Row Percent 23.2 76.8 5.6

Column Percent 2.3 10.1

Total Percent 1.3 4.3

Column 839 622 1461

Total 57.4 42.6 100.0

Corrected chi square = 40.2 with 1 degree

of freedom

.3 = 0.00001
Alternaria spp. spores ”1nd Speed km/hr Row
trapped fr°m the a‘r Less than 4.0 4.1-22.0 Total
Less than 50/hr: Count 1232 185 1417

Row Percent 86.9 13.1 94.3

Column Percent 95.1 89.8

Total Percent 82.0 12.3
51-334/hr: Count 64 21 f 85

Row Percent 75.3 24.7 5.7

Column Percent 4.9 10.2

Total Percent 4.3 1.4

Co1umn 1296 206 1502

Total 86.3 13.7 100.0

Corrected chi square = 8.2 with 1 degree

of freedom

.3 = 0.0041

 

47

TABLE 10.--Continued.

 

Relative Humidity (%)

 

Alternaria spp. spores

 

 

trapped from the air Less than Row
73 74-97 98-100 Total
Less than 50/hr: Count 330 317 716 1363
Row Percent 24.2 23.3 52.5 94.3
Co1umn Percent 86.2 95.2 98.2
Total Percent 22.8 21.9 49.6
51-334/hr: Count 53 16 13 82
Row Percent 64.6 19.6 15.9 5.2
Column Percent 13.8 4.8 1.8
Total Percent 3.7 1.1 0.9
Column 383 333 729 1445
Total 26.5 23.0 50.4 100.0

Raw chi square = 68.8 with 2 degrees of freedom
E_= 0.00001

 

Solar Radi tion
Alternaria spp. spores (gm-cal/cm /min)
trapped—from the air
0.01- 0.57- Row

 

 

 

None 0.56 1.12 Total
Less than 50/hr: Count 658 407 352 1417
Row Percent 46.4 28.7 24.8 94.3
Column Percent 99.1 93.1 87.8
Total Percent 43.8 27.1 23.4
51-334/hr: Count 6 30 49 85
Row Percent 7.1 35.3 57.6 5.7
Column Percent 9.9 6.9 12.2
Total Percent 0.4 2.0 3.3
Column 664 437 401 1502
Total 44.2 29.1 26.7 100.0

Raw chi square = 61.6 with 2 degrees of freedom.
f_= 0.00001

 

48

TABLE ll.--Chi square analysis of hourly catches of Alternaria spp.
conidia and leaf wetness in a commercial Blueberry p1ant-
ing at Grand Junction, MI in 1978 and 1979.

 

 

Alternaria spp. spores Leaf Netness - l978

 

 

 

. Row
trapped from the a1r Dry Net Tota1
Less than 50/hr: Count 764 588 1351

Row Percent 56.5 43.5 75.0

Column Percent 65.4 92.6

Total Percent 42.4 32.6
51-909/hr: Count 403 47 450

Row Percent 89.6 10.4 25.0

Column Percent 34.6 7.4

Total Percent 22.4 2.6

Column 1166 635 1801

Total 64.7 35.3 100.0

Corrected chi square = 160.4 with 1 degree of

freedom

.3 = 0.00001

 

Alternaria spp. spores Leaf Wetness ' ‘979

 

 

 

. Row
trapped from the a1r Dry Net Total
Less than 50/hr: Count 711 706 1417

Row Percent 50.2 49.8 94.3

Column Percent 90.7 98.3

Total Percent 47.3 47.0
51-334/hr: Count 73 12 85

Row Percent 85.9 14.1 5.7

Column Percent 9.3 1.7

Total Percent 4.9 0.8

Column 784 718 1502

Total 52.2 47.8 100.0

Corrected chi square = 39.6 with 1 degree of
freedom
P.= 0.00001

 

49

variance (r2) of each variable was calculated with all the other
variables entered into the equation. The dependent variable used
was the number of Alternaria spp. conidia caught per hour by the
Burkard spore trap. The independent variables used were air
temperature (C), wind speed (km/hr), solar radiation intensity

(gm cal/cmZ/min), and percent relative humidity. The results of
the regression analyses for the seven episodes of spore release are
shown in Table 12. The same analyses were performed on data from
the entire 1978 and 1979 seasons, starting from the date when more
than 50 conidia were trapped in one hour and continuing until spore
trapping was discontinued. The results of the multiple regression
analyses of yearly data are shown in Table 13.

In six of the seven episodes analyzed, the percent relative
humidity contributed the greatest amount toward the variance
explained by the regression. For the duration of the episode of
7/29-8/01 1978 the wind speed was very high, nearly twice the
seasonal average shown in Table 13 and in this case it contributed
the greatest amount to the variance explained by the four environ-
mental parameters.

In the analyses of the seven episodes summarized in Table 12,
each of the parameters contributed substantially toward the explained
variability on several occasions. A ranking of the variables in
order of decreasing contributions to the explained variance was
impossible, however, since each variable at times contributed

substantially, but at other times only made negligible contributions.

50

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52

In the regression analysis of seasonal data summarized in
Table 13, air temperature and relative humidity made the greatest
contributions toward the explained variance in 1978. In 1979,
however, solar radiation made a contribution nearly equal to that
of air temperature and relative humidity.

The low values of r2 obtained in the seasonal regression
analysis summarized in Table 13 as compared to the relatively high
values of r2 found in Table 12 show the importance of applying
regression analysis to individual episodes of spore release.

Parameters not included in the regression analysis probably
contributed to the unexplained variance. For example, wet leaves
were shown by the chi square analysis to be associated with low
levels of spore catches, and dry leaves with high levels of spore
catches. This parameter was not included in the regression analysis,
however, since it was not measured as a continuous variable. Rain-
fall only rarely coincided with spore release, and so was not
included in the analysis. No attempt was made to measure biological
parameters associated with spore release such as the amount of
Alternaria-bearing substrate in the field. Knowledge of this

parameter might greatly increase the value of r2.

 

The statistical tests made confirmed observations made by
plotting spore catches and environmental parameters against time for

three of the episodes. The results are shown in Figures 6, 7, and 8.

53

Isolation from Host Tissues to Determine the
Overwintering Site of the Pathogen

 

 

Of the blighted fruiting wood twigs brought to the laboratory
in November 1977, 19/35 (54%) yielded Alternaria spp. when tissue

 

isolations were made onto PDA. 0f the apparently healthy flower
buds brought to the laboratory at the same time, only 1/129 (0.7%)

yielded Alternaria spp. when tissue isolations were made onto PDA.

 

These results suggest that Alternaria may overwinter in blighted

 

fruiting wood, but not flower buds.
In the spring of 1978, similar blighted wood was brought to
the laboratory, and 5/36 (14%) of the blighted twigs yielded

Alternaria spp. when tissue isolations were made after surface

 

disinfestation. When tissue isolations were made without surface

disinfestation, 19/32 (59%) of the twigs yielded Alternaria spp.,

 

indicating that Alternaria spp. had overwintered and were located

 

predominantly at or near the surface of the blighted wood. The

Alternaria isolates found in wood at this time of year were not

 

identical to the A, alternata isolate from fruit in the same field,
but they were members of the "Alternaria alternata group" (30)
members of which have been reported to cause decays of blueberry
fruit (10, 18). The virulence of these wood isolates was not
determined. However, it is likely that they could cause a decay of
blueberry fruit.

Other fungi isolated from the stems included Trichoderma

 

spp., Penicillium spp. and Colletotrichum gloeosporioides.

 

DISCUSSION

Postharvest Decays Caused by Alternaria alternata

The detrimental effects of A. alternata on blueberries were
shown to be limited to the harvest season. Only relatively insig-
nificant numbers of A, alternata conidia were trapped from air in
a commercial blueberry field before the first blue fruit stage of
blueberry bush development in both 1978 and 1979. Inoculations of
mature blueberry bushes with A, alternata throughout both growing
seasons from the bud swell stage through the ripe fruit stage failed

to induce Alternaria postharvest decays above natural uninoculated

 

background levels in both seasons. The fact that the inoculations
did induce a transient spotting of succulent leaves in both seasons
implies that the virulence of the isolates used had not been
attenuated in culture. It seems likely that the increase in spore
catches reflect sporulating decays of damaged fruit which were

infected by Alternaria spp. and immediately decayed. However, it

 

is possible that infection does occur early in the season and that
the great increase in spores trapped late in the season is due to
Sporulation on fruits infected early in the season.

The relationships between A, alternata spore release and
meteorlogical parameters monitored in this study are consistent
with those observed by others for related species (15, 16, 27, 32)
and for A. alternata (26). Spore release was positively correlated

54

55

with air temperature, solar radiation intensity, and wind speed and
negatively correlated with relative humidity. Large spore catches
were very strongly associated with dry leaves, and low spore catches
with wet leaves. These patterns are consistent with the hypothesis
that A, alternata conidia are violently discharged into air in
response to changes in relative dumidity and light intensity as has
been demonstrated for A, tgflgj§_(l7).

Although the general pattern of Alternaria spore release had

 

been previously established, the strength of the correlations between
the meteorological factors and spore release had not been determined.
Multiple regression analysis of the spore trapping and environmental
data of this study revealed that relative humidity was the factor
which had the greatest influence on the magnitude of spore release.
Wind speed, solar radiation intensity, and air temperature also
contributed substantially toward explaining the observed magnitude
of spore release in several of the episodes studied. The variance
unexplained by the regression analyses was probably due to the
contributions of variables not entered into the regression analyses,
such as leaf wetness duration preceding spore liberation and the
amount of spore bearing substrate in the field.

Since A, alternata causes only a postharvest decay problem
and is not very active in the field prior to the blue fruit stage
of fruit maturity, application of fungicides early in the season to

control Alternaria decays postharvest would likely be of little

 

value. Application of fungicide only late in the season would have

a good chance of controlling Alternaria decays. Difolatan was

 

56

clearly the best of the fungicides tested for controlling Alternaria

 

decays in 1979, when a single massive application of Difolatan
(28.2 1/ha) at the green tip growth stage of the blueberry bush in

1979 controlled Alternaria decays after harvest. It is likely that

 

a single application of Difolatan (captafol) made 21 days preharvest

(the maximum legal interval) would control Alternaria postharvest

 

decays.
The cause of the observed association between mechanical

harvesting and increased Alternaria decay incidence (10) is in part

 

due to the greater damage occurring to fruits when harvested by

machine as compared to hand. This damage increases Alternaria

 

decays (Appendix, p. ). Growers using mechanical harvesting
equipment often try to harvest the crop in as few passes over the
bushes as possible. Because of this the fruits tend to become soft

and over-ripe which can predispose them to decays by Alternaria

 

spp. which are generally considered to be non-aggressive pathogens

(10).

SECTION II

EPIDEMIOLOGY AND CHEMICAL CONTROL OF POSTHARVEST
DECAYS OF BLUEBERRY FRUIT CAUSED BY
COLLETOTRICHUM GLOEOSPORIOIDES

 

57

INTRODUCTION

The Genera Gloeos orium Desm. and Mont.
and Colletotr1c5um (Penz.) Sacc.

Many diseases of fruits, vegetables, and ornamentals share a
similar etiology and symptomatology, and are commonly known as
"anthracnoses." Typical symptoms include fruit or vegetable rot,
and blossom or foliage blight (22). When anthracnose decays of
blueberries were first studied and reported, the workers reported

the causal fungus to be Gloeosporium (8, 10, 12, 33, 34), the

 

imperfect form of Glomerella cingulata (Stonem.) Spaulding and

 

Schrenk.

The genus Gloeosporium was erected by Desmaziers and

 

Montagne in 1849 with g. castagnei Desm. and Mont. as the type
species. According to von Arx (2), Saccardo used the genus

Gloeosporium to include Melanconiaceous fungi growing on leaves

 

with continuous, hyaline conidia. This treatment was inconsistent

with the type specimen (2). Gloeosporium was separated from

 

Colletotrichum by Saccardo because Gloeosporium had glabrous

 

 

acervuli whereas Colletotrichum had setose acervuli (2).

 

Glomerella cingulata was thoroughly reviewed by von Arx in

 

order to resolve taxonomic confusion arising from a multiplicity of
synonyms for both the perfect and imperfect stages of the fungus (2).

Von Arx dismembered the genus Gloeosporium entirely because it was

 

too heterogeneous and reassigned the species to several genera, one

58

59

of which was Colletotrichum. In the present study the nomenclature

 

of von Arx (2) has been followed, and the fungus causing anthracnose

decays of blueberries is called Colletotrichum gloeosporioides

 

(Penz.) Sacc. in place of Gloeosporium fructigenum.

 

Acervuli of C, gloeosporioides are found on necrotic regions

 

of clearly defined lesions, vary from setose to glabrous, are
irregular in shape, and can be as large as 500 micrometers in
diameter. The acervular spore mass is salmon colored (22). Conidia
are cylindrical with obtuse ends, hyaline, aseptate, uninucleate,

and range in size from 9 to 24 micrometers long by 3 to 6 micrometers
wide. They are borne on unicellular, hyaline or faintly brown
phialidic conidiophores. Conidia are dispersed by splashing water
(22).

On PDA, colonies of C. gloeosporioides are grayish-white to

 

dark gray with aerial mycelium varying from sparse to uniform and
mat-like. The reverse side of the colony is unevenly white to gray
(22).

A closely related species, 9, acutatum Simmonds, differs

from Q. gloeosporioides in colony color which is orange to pink and

 

in its fusiform conidia which are slightly smaller, especially in

width, than conidia of Q. gloeosporioides. Perithecia have not

 

been observed for this taxon. It has been reported only once in
North America (13).

In the review of the literature on post harvest decays of

 

blueberries caused by g, gloeosporioides which follows, C, gloeo-

sporioides will be referred to as Gloeosporium sp. as it was

 

 

originally reported.

60

History and Occurrence of Gloeosporium
Decays of Blueberry

 

 

In spite of the commonly held belief that the most important

fungus involved with blueberry (Vaccinium corymbosum L.) decays was

 

Botrytis cinerea Pers. ex Fr., the incidence of decays caused by a

 

Gloeosporium sp. was five times greater than that of B, cinerea in

 

New Jersey studies conducted from 1970 to 1972 (12). Decays caused

by this Gloeosporium were greatly favored by extremely wet weather

 

in 1972. As would be expected, decay incidence increased from the
field to storage and to retail outlets (12).
In another New Jersey study carried out in 1971 the fre-

quency of Gloeosporium fructigenum rots was greater than the fre-

 

quencies of either Botrytis cinerea rots or Alternaria tenuis rots

 

 

for the season as a whole (4.0, 3.0 and 2.4%, respectively). Sig-

nificantly, the ratio of Gloeosporium decays to Alternaria decays

 

 

and Gloeosporium decays to Botrytis decays shifted from approxi-

 

mately 2:1 in early season to 6:1 in mid-season to 45:1 in late
season. This trend was consistent with field observations that

Gloeosporium decays became more common as the season progressed (8).

 

Glomerella cingulata was shown to cause both a blossom

 

blight and a postharvest decay of cranberry (Vaccinium macrocarpon

 

L.) fruit in New Jersey (35). Blossom blight of blueberry and
latent fruit infection were reported from New Jersey but no data
were presented (34).

Regular application of fungicides to control Gloeosporium

decays had not been practiced in New Jersey because the disease had

61

been considered to be of minor importance. However, in 1970

Gloeosporium spp. caused the decay of five percent of the blueberries

 

in storage and marketing channels (12). Field fungicidal spray pro—

grams were initiated, on the assumption that control of Gloeosporium

 

in the field would diminish its destructive potential in the
marketplace (10). A spray program using captan was preferred. A
ferbam-captan experimental spray program was ineffective as were
post harvest dips with benomyl (10). The ineffectiveness of the
benomyl dips is interesting because benomyl is generally effective

against Gloeosporium spp. (25). Very promising results were obtained

 

using captafol (Difolatan) in field tests (10).
In one study, very soft, leaky berries accounted for 31.5%
of all decayed fruit, but no visible sign of a pathogen was present.

Isolation onto PDA revealed Gloeosporium in 20%, Alternaria in 22%,

 

 

and Botrytis in 35% of these fruits (lO).
Milholland has described disorders of blueberry bushes

caused by Gloeosporium minus Shear in North Carolina (19, 21).

 

Leaf lesions resulted from inoculation of succulent blueberry leaves
(cv. Croatan) with conidial suspensions of 106/ml. Spore germination
and appressorial production on inoculated leaves was increased 2.5-
fold when the inoculum was suspended in 50% glucose as compared to
distilled water. This may account for the presence of large A, miggs
lesions at blueberry hydathodes because of the sugars present in

solution there (19).

62

The leaf lesion isolate was shown to be identical myco-
logically and equal in pathogenicity to isolates causing severe
cankering and dieback on blueberry stems (21). Cankers were
observed to begin at the leaf scar or flower buds. Acervuli
were produced over the entire canker. It was not clear whether
the fungus penetrated the leaf scar directly or via attached

infected petioles. Gloeosporium minus hyphae grew intracellularly

 

in the cortex, xylem and phloem of infected stems (21).

MATERIALS AND METHODS

Colletotrichum gloeosporioides Isolate
Used in This Study

 

A single spore isolate of the causal organism of anthracnose
decay was made from rotting blueberry fruit and used for all further
studies. On rotting fruit the fungus was macroscopically visible
only as salmon colored spore masses (Figure 1). Similar salmon
colored spore masses were found on blighted blossom clusters
(Figures 2 and 3). Hyaline setae were microscopically visible in
the acervuli on rotting blueberry fruit (14).

On PDA the colonies produced numerous acervuli with salmon
colored spore masses. The mycelium was generally dense. Colony
morphology and sporulation was apparently unaffected by constant
soft fluorescent lighting or constant darkness. The fungus produced
a large amount of a pink-purple pigment in culture on PDA, which was
especially visible from the reverse side of the petri plates. No
setae were observed in acervuli produced on PDA (14).

Conidia grown on PDA measured 18.0 um long by 7.8 pm wide
(mean of 50 conidia measured). They were cylindrical to slightly
elliptical and hyaline with granular appearing inclusions (Figure 4).

The mycelial characters were consistent with those described

for Colletotrichum gloeosporioides (Penz.) Sacc. (22). The pink to

 

63

64

Figure l.--Blighted blossom cluster
and rotting fruit caused
by inoculation of blueberry
bush with 106 Colletotrichum
gloeosporioides conidia per
m1 sterile distilled water at
the pink bud growth stage.

Figure 2.--Colletotrichum
gloeosporioides entering
fruiting wood via
blighted blossoms.

 

Figure 3.--Spore masses of Colletotrichum
gloeosporioides on agblighted
blossom after 48 hr in a moist
chamber.

Figure 4.--Colletotrichum
gloeosporioides conidia
(x 400)

 

66

purple pigment produced on PDA has been reported for g, acutatum

Simmonds ex Simmonds (13) previously, and for Glomerella cingulata

 

from blueberry (33). Since conidial measurements clearly favored

C, gloeosporioides rather than C. acutatum, the fungus was identified

 

as C, gloeosporioides (Penz.) Sacc.

 

Effect of Temperature on Fungal Growth and Spore
Germination in Free Water and in Air at
100% Relative Humidity

 

 

 

These experiments were carried out in exactly the same

manner with the 9, gloeosporioides isolate used as described in

 

Section I with the Alternaria alternata isolate except that radial

 

growth of the fungus was measured after 10 days instead of after 7
days. These experiments were performed to identify environmental
conditions which in the field would lead to infection and decay of
fruit by C, gloeosporioides.

Field Location for Colletotrichum loeos orioides
Studies and Monitoring of Environmental

Parameters in the Field

 

 

 

The 9, gloeosporioides studies were carried out in the same

 

field simultaneously with the A, alternata studies described in

Section I.

Trapping of Rain Dispersed Conidia of
Colletotrichum,gloeosporioides

 

In April 1978, ten water traps were placed in bushes which
contained dead fruiting wood similar to the kind from which E,
gloeosporioides had been isolated previously. A water trap con-

sisted of a 15-cm (6 inch) plastic funnel anchored in the bush

67

with epoxy glue and connected by a length of Tygon tubing to a
plastic 3.79 liter (1 gallon) bottle anchored in the ground. Rain-
water washing over the branches above the funnel carried any

spores present into the bottles. Approximately 25 ml of a preserva-
tive solution containing 50% ethanol, 5% glacial acetic acid, 10%
formaldehyde, and 35% water was added to each bottle each week.

Experiments showed that conidia of C, gloeosporioides failed to

 

germinate in this solution when it was diluted as much as 15:1.
In June of 1978 three additional water traps were installed

in bushes which earlier had been inoculated with C, gloeosporioides

 

and which had blighted blossoms. These traps collected spores from
the blighted blossoms and in one case from rotted fruit.
In 1979 eight bushes which had been inoculated in 1978

with C, gloeosporioides had water traps installed in them. The

 

pathogen had been reisolated from six of these bushes. In order to
further increase the consistency of spore capture, the traps were
"baited" with diseased plant material in 1979. Blighted twigs,
flower parts and rooted fruits were wrapped in cheesecloth and
placed in equal amounts in the funnels as the season progressed.
The bottles were emptied weekly in 1978 and early in 1979
and then after individual rains, starting on June 27, 1979. The

concentration of g, gloeosporioides conidia in each bottle was

 

estimated by taking a subsample of water from the bottle after
recording the total volume of rainwater in the trap. Approximately
35 ml of this subsample was filtered through two layers of cheese-

cloth and centrifuged at 7000xg in a refrigerated centrifuge

68

(International Equipment Co., Needham Heights, Massachusetts 02194).
The pellet containing spores was resuspended in one-fourth the
original volume and the spores in the suspension were counted

using a hemacytometer. Three counts were made on each subsample
and averaged. The total number of conidia collected and the con-
centration of conidia in the rainwater was then estimated.

Ig_Vitro and Field Testing of Fungicides for
Contro o ost arvest ecays o ue err1es

Caused’by’g, gloeosporioides

 

All jfl_vitro and field testing of fungicides for control of

 

C, gloeosporioides was done simultaneously with and by the same

 

methodology described in Section I for A, alternata.

Inoculation Studies of Mature Blueberry Bushes
in the Field and at East Lansing

 

The inoculation studies using one single spore isolate of

g, gloeosporioides were done simultaneously with and by the same

 

methodology described in Section 1 for A, alternata. The only

difference was that the concentration of Q, gloeosporioides

 

conidia used for inoculation studies was 106/m1 sterile distilled
water. The inoculation dates and phenotypic growth stages are
given in Tables 2, 3 and 4.

Isolations from Host Tissues to Determine
the Overwintering75ite of the Pathogen

 

These isolations were done as described in Section I for
A,, alternata. In addition to the isolations described in Section I,

in the fall of 1978 and spring of 1979 isolations were made from

69

blighted twigs which resulted from inoculations made during the
1978 season. Isolations were made onto half strength PDA (QPDA)
after surface disinfesting the twigs by soaking them in 1% sodium

hypochlorite plus Tween-80 for 10 minutes with stirring.

RESULTS

Effect of Temperature on Radial Growth of
Colletotrichum_gloeosporioides

 

The optimal temperature tested for radial mycelial growth

of C, gloeosporioides was 20C. Growth was slightly slower at 27C

 

but almost no growth occurred at 30C (Figure 5). At 30C the colony
morphology was greatly distorted and the characteristic pink/purple
pigment was lacking. The fungus grew twice as fast at 20C than at
25C and almost no growth was found after ten days at 5 and 10C.
The results indicate that anthracnose decays are favored by tempera-
tures of 15 to 27C, but that higher temperatures would adversely
affect the pathogen.

The Effects of Free Water 100% Relative Humidity,

and Various Temperatures on the Germination
of Colletotrichum gloeosporioides Conidia

 

 

Germination of g, gloeosporioides conidia in free water was

 

slow at all temperatures tested (Table 1). Conidia failed to
germinate at 5C and 10C after 40 hours in water. The optimal
temperature tested for percent conidial germination in free water
was 30C. However, germ tube elongation was much slower at this
temperature than at 20C, which was consistent with the mycelial
growth abnormalities observed at higher temperatures.

Conidia failed to germinate at any temperature in air at
100% relative humidity. The results indicate that free water

70

71

 

 

 

 

so 1-
70 '-
50 1—
.5:. so -
E
.2
c:
E ‘° "
30 II-
20 '-
10 -
\ lnltlol Colony Diameter
1 1 .11 1 1 1
5 10 15 20 25 3O
Tompontun (C)

Figure 5.--Mycelia1 growth of Colletotrichum gloeosporioides on
PDA at several temperatures after 10 days.

 

72

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73

and temperatures of 15 to 25C may be required for about 12 or more

hours in order for g, gloeosporioides to infect the fruit. Such

 

conditions were provided after the inoculations which were success-

fully made in the present study.

Preliminary Screenigg of Fungicides for Activity
Against Colletotrichum gloeosporioides

 

The compounds which showed the greatest activity against

E, gloeosporioides were Funginex (triforine), Bayleton, captan,

 

ferbam, and captafol (Difolatan). These compounds all caused 90%

or greater inhibition of radial mycelial growth of C, gloeosporioides
in this test at concentrations of 100 pg/ml (Figure 6). Bayleton

and captafol (Difolatan) caused 80% or greater radial growth
inhibition at a concentration of 10 ug/ml and benomyl was moderately

effective against 9, gloeosporioides at all concentrations tested

 

in this experiment.

Fungicide Testing in the Field for Control of
Colletotrichum gloeosporioides Decays
o ueberry ruits

 

 

The incidence of C, gloeosporioides decay was too low in

 

both 1978 and 1979 to give any meaningful data about fungicidal

control of g, gloeosporioides decays. This was surprising since

 

spore trapping data indicated that 9, gloeosporioides conidia were
being produced throughout both seasons in a portion of the same
field in which the fungicides were tested. Decay incidence was
determined by the same procedure which induced abundant sporulation

of C. gloeosporioides on fruit harvested for evaluation in the

 

74

 

 

 

 

 

 

 

 

 

 

 

 

 

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3 7’ 90 b 3 E 00 H
g g . g a L
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5'3 3'!
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pg/ml pg/ml
Benlate Triforine
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00 00 '—

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Forum Inhlbmon of
mycelial radial growth
8 :9
Forum Inhibition of
mycelial radial growth
8 1S
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10 10
1'1 1'1 [—1 g
0.1 1 10 100 0.1 1 > 10 100
11 g/ml palm!
Dithane M-45 Bayleton

 

 

 

Figure 6.--Growth inhibition of Colletotrichum gloeosporioides
mycelium at 96 hr by fungicides incorporated into
PDA.

 

75

 

 

100

 

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Figure 6.--Continued.

 

76

inoculation experiments (Tables 2, 3 and 4), so that this procedure
was not responsible for the low observed decay incidence.

Workers in New Jersey found that the incidence of anthrac-
nose decays increased greatly as the harvest season progressed (8).
In both seasons of the present study, harvesting was limited to
the early part of the harvest season. Perhaps if the harvesting of
the fungicide treated plots had been extended into the late season
a higher incidence of anthracnose decays would have been found.

Inoculation Studies in the Field and at East
Lansing Using Colletotrichum gloeosporioides

 

The isolate of g, gloeosporioides used in this study was

 

shown to cause a fruit decay after harvest in both seasons when
inoculated during the growing season onto mature bushes in a com-
mercial blueberry field (Tables 2 and 3). Fruit developed anthrac-
nose decays postharvest when bushes were inoculated with the patho-
gen as early as the bud swell growth stage in both seasons. No
differences in phenotypic growth stage susceptibility to C,

gloeosporioides were apparent in either season. The data show

 

that anthracnose decays after harvest can be the result of latent
infections which are established during the growing season.

Colletotrichum gloeosporioides was also shown to cause a

 

severe blossom blight when bushes were inoculated before or during
the bloom period (Figure 2). This blossom blight can be distinguished

from blossom blight caused by Monilinia vaccinii-corymbosi because

 

C. gloeosporioides rapidly enters the fruiting wood (Figure 2)

77

TABLE 2.--Data from field inoculations of blueberry bushes with
Colletotrichum gloeosporioides at Grand Junction, MI
in 1978.8;5

 

 

 

 

 

Mean Percent Fruit Mean Percent Fruit

Rotted by Rotted by
Inoculation Date C, gloeosporioides Q, gloeosporioides
and Growth Stage ‘(inoculated) (Control)
5/2/78
Bud Swell 16.6 i 6.8 0.0
5/16/78
Bud Break 13.4 i 9.1 0.2 i 0.16
5/23/78
Pink Bud 33.4 i 9.3 0.0
5/30/78
Full Bloom 5.5 i 2.5 3.0 i 2.0
6/6/78
Early Petal Fall 7.7 i 6.4 0.0
6/13/78
Early Green Fruit 35.3 i 16.2 0.0
6/22/78
Green Fruit 10.7 t 2.2 0.5 i .57
7/16/78
Early Blue Fruit 7.5 i 4.2 0.0

 

aBushes were inoculated on the dates given with a spore sus-
pension of 1x10 conidia/m1 sterile distilled water or with sterile
distilled water only.

bFruit was hand harvested on 8/1/78 and held for 4 days at
room temperature in moist chambers.

cPercent of fruit with visibly sporulating g,
.gloeospgrioides on a count basis. Numbers given are the means of
four replicates i the standard deviations of the means.

 

78

TABLE 3.--Data from field inoculations of blueberry bushes with
Colletotrichum gloeosporioides at Grand Junction, MI
in 1979.335

 

 

 

 

 

Mean Percent Fruit Mean Percent Fruit

Rotted by c Rotted by
Inoculation Date .9. loeosporioides g, gloeosporioides
and Growth Stage (Nioculated) (Control)
5/2/79
Bud Swell 31.0 i 12.0 0.0
5/9/79 d
Bud Break 37.5 i 9.2 ---
5/16/79
Pink Bud 1.5 i O 4 8.5 i 5 2
5/23/79
50% Bloom 6.8 i 1.9 14.5 i 8.6
6/13/79
Early Green Fruit 18.8 i 5.9 0.0
7/5/79
Late Green Fruit 29.0 i 7.2 0.0
7/18/79
First Blue Fruit 12.6 i 6.5 1.3 i 0.6
8/1/79
First Ripe Fruit 23.0 i 8.5 0.5 i 0.2

 

aBushes were inoculated on the dates given with a spore sus-
pension of 1x106 conidia/m1 sterile distilled water or with sterile
distilled water only.

bFruit was hand harvested on 8/15/79 and held for 4 days at
room temperature in moist chambers.

cPercent of fruit with visibly sporulating C,
loeospgrioides on a count basis. Numbers given are the means of
our replicates i the standard deviations of the means.

d

 

Missing data.

79

TABLE 4.--Data from field inoculations of blueberry bushes with
Colletotrichum gloeosporioides at East Lansing, MI, in

 

 

 

 

 

1979.31D
Mean Percent Fruit Mean Percent Fruit
Rotted by Rotted by
Inoculation Date 9, gloeosporioides Q, gloeosporioides
and Growth Stage (Inoculatedli (Control)
4/28/79
Bud Swell 5.1 i 2.9 0.0
5/10/79
Bud Break 2.8 i 1.1 0.0
5/17/79
Early Pink Bud 0.0 0.0
5/24/79
Full Bloom 7.1 i 1.1 1.7 i 1.1
6/14/79
Green Fruit 21.3 t 3.5 0.0
7/19/79
First Blue Fruit 11.3 i 4.8 0.0

 

aPotted 3 or 4-year-old bushes were inoculated on the dates
given with a spore suspension of 1x106 conidia/ml sterile distilled
water or with sterile distilled water only.

bFruit was hand harvested on 7/31/79 and held for 6 days
at room temperature in moist chambers.

cPercent of fruits with visibly sporulating Q.
gloeosporioides on a count basis. Numbers given are the means of
four replicates i the standard deviations of the means.

 

80

and M. vaccinii-corymbosi does not. Monilinia vaccinnii-corymbosi

 

 

also produces characteristic cream-colored spore masses 0n the

blighted tissue in the field. Colletotrichum gloeosporioides

 

produces characteristic salmon colored spore masses in the field,
and particularly if incubated in a moist chamber for 48 hours
(Figure 3). Koch's postulates were completed for both the blossom
blight and the postharvest fruit decay phase of blueberry anthrac-
nose disease when the fungus reisolated from diseased tissue was
indistinguishable in culture from the isolate used to inoculate the
bushes.

The data from the potted bush experiment at East Lansing
are in complete agreement with the field studies even though the
cultivar Berkley was used at East Lansing in 1979 (Table 4). The
results of the 1978 potted bush experiment at East Lansing were
unuseable due to problems with insect and avian pests.

Trapping of Rain Dispersed Conidia of
Colletotrichum gloeosporioides

 

The trends of the spore trapping data of 1978 and 1979 are
very consistent (Figures 7 and 8). However, because of differences
in the experimental procedures used in 1978 and 1979 the data in
Figures 7 and 8 are qualitatively but not quantitatively comparable.

The proportion of water traps which yielded C, gloeosporioides in

 

1979 was greater than in 1978 due to the relocation and "baiting"
of the traps in 1979. High concentrations of conidia were found
in rainwater collected during the bud swell and bud break growth

stages of the blueberry bushes. Because no new growth was present

 

81

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85

on the bushes at this time, these conidia were being produced on
blighted fruiting wood from which the pathogen was readily isolated.
Large numbers of conidia were trapped during the bloom period in

1979 although there was no rain at all during this period in 1978
from which to trap spores. Particularly large numbers of conidia
were trapped during the early green fruit stage in both seasons in
nearly all of the traps in the field. Following the green fruit

stage the number of conidia per m1 of rainwater collected and the

number of traps which collected 9, gloeosporioides decreased in

 

1978 in the non-inoculated bushes. This phenomenon was observed
to a lesser extent using the "baited" traps in 1979. Conidial
production in inoculated symptom-bearing bushes was high as the
fruit ripened in 1978. Large spore catches were made with the
"baited" traps in 1979 at this stage. Conidial concentrations
greater than 30,000/m1 were observed during the blue fruit stages
of both seasons.

Isolation from Host Tissues to Determine the
Overwintering Site of the Pathogen

 

 

Among the blighted fruiting twigs from which tissue isola-
tions were made in November 1977, only 1/35 (3%) yielded

Colletotrichum gloeosporioides. Many more yielded Alternaria spp.

 

 

None of the 129 healthy flower buds from which tissue transfers

were made yielded g, gloeosporioides on PDA.

 

Among the blighted twigs from which tissue isolations were

made after surface disinfestation in the spring of 1978, 3/36 (12%)

86

yielded 9, gloeosporioides; only 1/32 of the non-surface dis-

 

infested twigs yielded 9, gloeosporioides.

0f ten blighted twigs originating from g, gloeogporioides
inoculations made in 1978, six yielded 9, gloeosporioides in the
spring of 1979 upon isolation onto PDA. This comparatively high
rate of overwintering in these twigs implies that the pathogen over-
winters in such wood. The lower rates of g, gloeosporioides
isolated from other blighted twigs may imply that those twigs were
killed by severe winter weather and not by g, gloeosporioides or

that Alternaria spp. can replace C, gloeosporioides in some wood

 

since such twigs yielded predominantly Alternaria spp. (Section 1).
Other fungi isolated from blighted twigs in these experi-

ments included Trichoderma spp. and Penicillium spp.

 

DISCUSSION

In contrast to Alternaria alternata which was shown in this

 

study to be a problem for blueberry growers mainly at harvest time,

Colletotrichum gloeosporioides was shown to be a problem for blue-

 

berry growers from the bud swell stage in the spring through the
end of the harvest season. In both seasons large numbers of C,

gloeosporioides conidia were trapped in rainwater runoff from

 

bushes from the bud swell stage of the blueberry bush through harvest
time. Inoculations of fruit buds, blossoms, and fruits with Q.

gloeosporioides caused high rates of anthracnose decay postharvest.

 

Fruit which was apparently healthy rapidly developed anthracnose

decay postharvest even if the inoculation with Q. gloeosporioides

 

had been made during the pre-bloom stages of the host.

The fact that C, gloeosporioides is both present and

 

destructive to blueberries from the bud swell stage through the
harvest season dictates that a control program should be begun at

budbreak and continued through harvest. Because 9, gloeosporioides

 

overwinters in fruiting wood killed by the pathogen, such material
should ideally be pruned out of the bushes and removed from the
field.

The fungicide testing experiments for control of anthracnose
decays carried out in 1978 and 1979 were disappointing because they
revealed very low disease pressure at harvest time in 1978 and 1979.

87

88

The disease pressure was so low that no useful information about the
relative efficacy of the various fungicides for control of anthrac-
nose decays was gained.

The verylow postharvest anthracnose decay incidence was sur-
prising since spore trapping data indicated that the pathogen was
present and active in the field throughout the season. Anthracnose
blossom blight was observed in the field in both seasons in non-
inoculated bushes. The incubation method for the fruit in the
fungicide trials was the same as that used in the inoculation trials,
and so cannot be blamed for preventing detection of Q, glpgpn

sporioides infection.

 

The incidence of anthracnose decays of blueberries has been
reported to increase dramatically late in the harvest season (12).
Since in both seasons our fruit was harvested relatively early in
the season at the growers' request, the period of maximum disease
pressure was probably missed. Nonetheless, it is still curious that
the observed decay incidence was so low when the pathogen was
present and active in the field.

Colletotrichum gloeosporioides has been shown to cause

 

 

latent infections on citrus, avocado, and papaya fruit (3, 4, 31).
The results of this study clearly show a similar phenomenon with
blueberry fruit since inoculation of blueberry bushes throughout
the season resulted in apparently healthy fruit which developed

anthracnose decays postharvest.

89

With citrus as a host, Q. gloeosporioides developed

 

appresoria on the surface of the fruit. Infection hyphae remained
dormant in the cuticle and in the outer three or four cell layers
of the flavedo. Growth of these dormant infection hyphae was very
rapid in cv. Robinson tangerines in response to degreening with

50 ppm ethylene (4). The amount of anthracnose decay in inoculated
cv. Robinson tangerines was also proportional to the amount of
ethylene added to the incubation chambers (5).

The number of dormant appressoria and infection hyphae/mm2
of fruit surface was also shown to be proportional to subsequent
antracnose decay. If 99 or fewer appressoria/mm2 of fruit surface
were present, no anthracnose decay developed even in response to
50 ppm ethylene for five days (5).

The concept of latent infection, and the requirement of a
certain minimum density of latent infections to cause disease can

explain why 9, gloeosporioides is more prevalent as a cause of decay

 

late in the harvest season. Only late in the harvest season have
the blueberry fruits acquired a large enough number of latent

infections of p. gloeosporioides that ethylene produced as a part

 

of the senescence process activates enough of the latent infections
to cause anthracnose decays. Fruits harvested earlier in the
season, as for example those used to evaluate fungicides in the
course of this research, have not accumulated enough latent infec-

tions to develop postharvest anthracnose decays.

90

The fact that flower buds and blossoms inoculated with C,

gloeosporioides before and during the bloom period produced

 

apparently healthy fruit which rapidly decayed postharvest, may

imply that these latent infections remained viable even after an
entire growing season. It would be dangerous to assert, however,
that the fruit rot observed postharvest on fruit obtained from

bushes inoculated before or during bloom was entirely due to latent
infections, all of which were established on the date of inoculation.
Secondary spread of the pathogen from blighted blossoms to fruit was
probably observed, as shown in Figure 4. However, inoculations made
after petal fall consistently caused latent infections which resulted hi
decay postharvest without causing any other visible symptoms in

the bushes. The latent infections were therefore viable for at least
six to eight weeks, and quite likely for the full twelve weeks of

the growing season.

The accumulation of viable latent infections throughout the
course of the growing season is an interesting phenomenon which
could lend itself to further studies. Knowledge of the precise
requirements for fruit infection and the critical number of
appressoria/mm2 of blueberry fruit surface required for anthracnose
decay development could lead to the development of mathematical
models to guide fungicide applications. The use of fungicides with
eradicant action for control of anthracnose decays is a very
attractive possibility which deserves study. The nature of the

interaction between the fruit, ethylene, and the latent infection

91

also deserves further study to determine if the ethylene acts
directly on the dormant appressoria and hyphae to revive them, or
if this response is mediated by other physiological changes in the
host. The use of hot water dips after harvesting fruit may be

effective in controlling decay caused by C, gloeosporioides (6. 7)

 

because the high water temperature is capable of eradicating super-
ficial, latent infections which otherwise deve10p into anthracnose

decays.

APPENDIX

EXPERIMENTS TO EVALUATE THE ROLE OF MECHANICAL
HARVESTING AND SORTING METHODS IN THE
EPIDEMIOLOGY OF POSTHARVEST DECAYS
OF BLUEBERRY FRUIT

92

MATERIALS AND METHODS

Purpose and Location of Harvesting Experiments
It has been observed that the recent increase in relative

importance of Alternaria and Colletotrichum (Gloeosporium ) post-

 

 

harvest decays of blueberry fruits coincided with the mechanization
of the blueberry harvest (10), but no attempt to document the effects
of mechanical harvesting and sorting methods on blueberry decay has
been reported. This was attempted in the present study. All
harvesting experiments were carried out in the commercial field
previously described during the first harvest of the field in each
season.

The Effects of Harvesting Methods and Bruising
on Postharvest Decay Incidence

 

An experiment was carried out in 1978 to determine if
bruising of hand harvested fruit would cause a decay incidence
pattern different from that of hand harvested fruit and comparable
to that of mechanically harvested fruit. Some bushesin the field
were expected to have a higher decay incidence than others, due to
higher pathogen p0pulations (particularly of C, glgeosporioides) in
those bushes than in others, so the experiment was carried out with
a factorial design of three harvesting treatments at each of five
locations in the field with four replicates per treatment-location

combination. This gave a total of sixty experimental units.

93

94

Five groups of three bushes each were marked with colored
ribbon and eight cups of fruit were harvested by hand at each
location. Four cups of fruit from each location received no further
treatment; the other four cups were bruised by dropping them two
times from a height of one meter into a plastic container lined
with paper towels. Disposable plastic gloves were used to pick the
fruit at each location and the paper towels lining the plastic con-
tainer were changed after the fruit from each location had been
bruised in order to avoid cross contaminating fruit from different
locations in the field.

Immediately after the fruit was hand picked at each location,
four cups of fruit were collected from the same bushes from an over-
the-row mechanical blueberry harvester (Blueberry Equipment Co.,
South Haven, Michigan 49090). The three harvesting treatments were:
mechanical harvesting only, hand harvesting only, and hand harvesting
followed by bruising.

Each replicate of fruit weighing approximately 200 gm, was
incubated for four days in ventilated plastic containers at room
temperature. The replicates were then scored for the presence of
visibly sporulating Alternaria, Colletotrichum, or other fungi.

The data were recorded as the percent rotted fruit in each category
on a fresh weight basis. The same experiment was repeated in 1979
except that four instead of five locations in the field were used

in 1979 giving only 48 experimental units.

95

Mechanical vs. Hand Harvesting and Sorting
Methods of Commercially Raised Fruit

 

 

An experiment was done in 1978 and repeated in 1979 in order
to determine what effects, if any, hand vs. mechanical harvesting,
hand vs. mechanical sorting, and the location of origin of the fruit
in the field had on the incidence of postharvest decay. The treat-
ments were arranged in a 2 by 2 by 4 factorial design, with
four replicates per treatment combination, giving a total of 64
experimental units.

The experiment was carried out by marking with ribbons four
groups of five bushes each from which the fruit was to be harvested.
These were located at different places in the field and were con-
sidered as the location treatment. Eight one-half liter (1 pint)
lots of fruit were harvested by hand at each location using dis-
posable plastic gloves to avoid cross contamination of the replicates.
Eight one-half liter (1 pint) lots of fruit were taken from the
mechanical harvester at the same locations immediately after it
harvested the bushes. Four of the hand harvested lots from each
location, and four of the mechanically harvested lots from each
location were sorted mechanically using a commercial pneumatic
sorter (Blueberry Equipment Co., South Haven, Michigan 49090) and
the remaining lots were sorted by hand. After receiving their
various harvesting and sorting treatments, the fruits were incubated
by replicates of about 200 gm in ventilated plastic containers for
four days at room temperature. The fruits were then scored as

healthy (firm marketable fruit), rotted by g, gloeosporioides,

96

rotted by Alternaria spp. or rotted by other fungi (visible

 

sporulation), or as "leakers" (soft, watery, unusable fruit without
visible sporulation). The results were recorded as the percent of
each replicate in each category on a fresh weight basis.

Isolation of Pathpgens from Blueberry Branches
Broken by a Mechanical Harvester

 

 

The possible role of the commercial over the row mechanical
blueberry harvester in providing overwintering sites for decay
causing fungi was investigated. Immediately after the commercial
blueberry field had been harvested in l978, numerous twigs which
had been broken by the harvester were tagged with ribbon so that
they could be found at a later date. In November of 1978, three
months later, 79 of these broken twigs were collected and tissue
transfers were made onto iPDA after surface disinfestation in 1%
sodium hypochlorite plus Tween-80 for 10 minutes. At the same time,
54 apparently healthy, unbroken twigs were brought to the lab and
tissue isolations were made from them in the same manner. In each
case four isolations were made from each branch onto each plate.

The percentage of branches of each type yielding Alternaria spp.

 

of Q. gloeosporioides was recorded.

 

RESULTS

The Effects of Harvesting Methods and Bruising
on Decay Incidence Postharvest

 

 

The results of these experiments carried out in 1978 and
1979 are shown in Tables 1 and 2, respectively.
In 1978, the incidence of visibly sporulating p,

gloeosporioides decays was significantly increased by both

 

mechanical harvesting and bruising. Colletotrichum gloeosporioides
decay incidence was also dependent on the location in the field
from which the fruit originated in 1978. These effects were not
reproduced in 1979.

In 1978 and 1979 the incidence of visibly sporulating

Alternaria decays was significantly increased by bruising the fruit

 

prior to incubation. Mechanical harvesting also increased Alternaria
decays slightly in 1978, but decreased them in 1979. This was
probably due to the high incidence of "leakers" (watery, unusable
fruit whose decay was of unknown etiology) in 1979 which was not
recorded for this experiment.

The total decay incidence (Alternaria + Colletotrichum) was
increased in the 1978 season by bruising the fruit as compared to
either hand harvesting only and mechanical harvesting only. In
1979 mechanical harvesting appeared to reduce total decay incidence.
This observation is very misleading, however, since the number of

"leakers" was not recorded for this experiment.

97

98

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99

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100

Mechanical vs. Hand Harvesting and Sorting
Methods of CommerciallyiRaised Fruit

 

 

The results of these experiments carried out in 1978 and
1979 are shown in Tables 3 and 4, respectively.

The incidence of Alternaria decay was not significantly

 

effected by harvesting method, sorting method, or location in the
field from which the fruit originated in 1978. The results were

similar in 1979 except that the incidence of Alternaria decays was

 

actually reduced by mechanical as compared to hand harvesting of the
fruit. These results are misleading because they do not take into
account the incidence of "leakers" which was not recorded in 1978
but which was probably very high.

The incidence of p, gloeosporioides decays was significantly

 

increased by mechanical as compared to hand harvesting and was
dependent on the location in the field from which the fruit origi-
nated in 1978. These results were not reproduced in 1979. In
both seasons, however, hand vs. pneumatic sorting of blueberry
fruit had no influence on subsequent decay incidence.

The incidence of "leakers” was very high in 1979, and com-
prised 75% of all unusable fruit in the experiment in which it was
measured. The incidence of "leakers" was significantly increased
by both mechanical harvesting and sorting as compared to hand
methods, and was also dependent on the location of origin of the
fruit in the field.

Because "leaker" incidence was not recorded in 1978, the

percent total decay in 1978 is not directly comparable to the

lOl

 

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102

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.Hz .neeeenee enono eo ween ewenw xenonoewn no meeneoe mnwenem eno emo>wom we eeowwm11.e uem<w

103

percent total decay in 1979. In both years, however, total decay
incidence was increased by mechanical harvesting and was dependent
on the location in the field from which the fruit originated. The

sorting methods used did not significantly affect Alternaria or

 

Colletotrichum decay incidence in either year.

 

Isolation of Pathogens from Blueberry Twigs
Broken by a Mechanical Harvester

 

 

Three months after being broken by the mechanical blueberry
harvester 34/79 (43%) of the blueberry twigs yielded coolonies of

Alternaria spp. after tissue isolations were made onto PDA as

 

compared to 12/54 (22%) from unbroken twigs. Usually all four of
the individual tissue isolations made from a broken twig on to PDA

yielded Alternaria spp. if one of them did, while often only one of

 

the tissue transfers from unbroken twigs yielded Alternaria spp.

 

on PDA. The pathogenicity of these Alternaria spp. isolates from

 

wood was not determined. However, it was determined that the

isolates were not the same Alternaria alternata which was isolated

 

from decayed blueberry fruit, although the wood isolates were

members of the "Alternaria alternata group" (30). Other members

 

of this group have been reported to cause decays of blueberry
fruit, so it is possible that these isolates could cause decay as

well (10, 18). It is clear that Alternaria spp. invaded twigs

 

broken by the harvester, and damage to bushes caused by the
mechanical harvester may provide additional overwintering sites

for Alternaria spp. in the blueberry field.

 

104

Colletotrichum gloeosporioides was not found in the

 

unwounded stems, and in only 1/79 (1.2%) of the harvester broken

twigs.

DISCUSSION

The role of mechanical harvesting and sorting methods in
the incidence of postharvest decays of blueberry fruit is complex.
In 1978 and in 1979 bruising of fruit increased the incidence of

visibly sporulating Alternaria decays as compared to unbruised

 

hand harvested fruit. Mechanical harvesting had little effect on

sporulating Alternaria decays in 1978 and decreased their incidence

 

in 1979. This reduction in sporulating Alternaria decay incidence

 

in mechanically harvested fruit is misleading since the proportion
of "leakers” was not recorded in either year in this experiment
but they comprised 75% of all unusable fruit in the 1979 harvest
and sorting methods experiment. The effect which bruising had on

Colletotrichum decay incidence was also obscured by the incidence

 

of leakers in the experiment.

The incidence of visibly sporulating Alternaria decays was

 

little effected by either methanical harvesting or sorting as com-
pared to hand methods in 1978 or in 1979. The incidence of
"leakers" was greatly increased by both mechanical harvesting and
sorting in 1979. The incidence of "leakers" was not determined in
1978, but "leakers" were present.

Mechanical harvesting and sorting methods favor a non-
sporulating form of deterioration, the magnitude of which far

exceeds that of Alternaria and Colletotrichum decays. This

 

105

106

deterioration may be caused by bacteria or yeasts or may be of
fungal etiology since the common blueberry rotting fungi have been
isolated from such fruit (10). The etiology of these "leaker"
decays needs to be determined, as well as the mechanism by which
mechanical harvesting and sorting methods increase their incidence.
The shift in pathogen expression from sporulating decay on
hand harvested fruit to non-sporulating decay on mechanically
harvested fruit cannot be attributed to simple bruising of the
fruit, since in both seasons simple bruising increased the amount

of sporulating Alternaria decays, while mechanical harvesting had

 

no effect or decreased the incidence of such decays.

This study documents an increase in unusable fruit after
harvesting by machine as compared to hand harvesting. In several
of the experiments it was shown that the location in the field
from which the fruit originated had an effect on the decay incidence
of the fruit after harvest, implying that fungicidal spray programs
in the field before harvest could contribute to postharvest decay
control. Further work should be done to modify harvesting and
sorting methods to reduce decay incidence. The use of hot water
and fungicide dips in processing mechanically harvested fruits
would be of value in controlling decays caused by damage to the

fruits as has been demonstrated (6, 7).

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