1:-

r;

EI“
'13"?!
31

..u

.1 ,5.

i...v§a
.ntv.
r

.. 33.
15.3}.
7‘ o .I .

 

as

E 3-;

yank
2‘ ’1‘?“
‘4‘;‘-‘.
. .‘,I .
‘hfii’flfi
“I "I

2;
.’§ ‘
I‘Ifi .\

;\fi‘
... .£..

2..

.1. u: .1

u.‘ \. .u
.‘ ab

.
3::

.q. ,$i.
,. E: .xer
e: ”3

anus

. 1:

”uni

'0
a!
7

if...

ht:

firm.”

 

bull}.

 

 

 

 

NIVERSITY LIBFARIES

ll ‘l‘i‘gl ll ll

MICHIG

l \lilillillm

3 12930

 

 

 

 

 

 

 

 

 

 

 

 

 

 

l

 

 

 

 

This is to certify that the
thesis entitled
Epidemiology and management of

Colletotrichum oocoodes
on processing tomatoes

presented by

Janet Merrick Byrne

has been accepted towards fulfillment
of the requirements for

 

Master degree in m

Department of Botany and Plant Pathology

/ Major professor

Date_Decanhen_L3.,_l995

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

LIBRARY

Michigan State
University

 

 

 

PLACE N RETURN BOX to roman this checkout from your mead.
TO AVOID FINES Mun on or baton duo duo.

DATE DUE DATE DUE DATE DUE

:5 r2. «1.2 2595

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

usu I. An Afflnnotlvo Action/Emu Opportunity lnotltulon
W

””1

_i—* 7- , . _

EPIDEMIOLOGY AND MANAGEMENT OF
COLLETO TRICHUM COCCODES
ON PROCESSING TOMATOES
By

Janet Merrick Byme

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1996

ABSTRACT
EPIDEMIOLOGY AND MANAGEMENT OF
COLLETO TRICHUM COCCODES
ON PROCESSING TOMATOES
By

Janet Merrick Byme

Factors influencing the epidemiology and management of tomato anthracnose in
processing tomatoes caused by Colletotrichum coccodes were studied in field and
laboratory studies. The effectiveness of the fungicide Bravo 720 (chlorothalonil) at 3.0
pt/A for control of fruit rot on processing tomatoes was assessed in field plots during
1993-1995. Fungicide treatments included: 1) 8 sprays at 7—day intervals; 2) 6 sprays at
10-day intervals; 3) 4 sprays at 14-day intervals; 4) 4, 3, and 6 sprays respectively,
scheduled by an early blight forecaster (TOM-CAST) using a threshold of 20 disease
severity values; and 5) unsprayed. All treatments significantly reduced fruit rot compared
to the control. An economic assessment revealed that for two years the 7 day application
regime provided the greatest benefit per acre. A foliar leaf disk assay was evaluated as a
tool for screening breeding lines for anthracnose fruit rot resistance. Results using
explants from inoculated foliage of four cultivars did not reflect resistance levels of fruit
to anthracnose. Conidial germination and infection processes on tomato foliage were
observed and quantified over 24 hours in continuous leaf wetness, in two hour intervals at
25 °C. Conidial germination and melanized appressoria formation followed a linear
trend. Infection vesicles were present 22 hours after inoculation. Results from this study

will help future researchers investigate the role of foliar infections in fruit rot incidence.

DEDICATION

This manuscript is dedicated to four people who have always been an integral part Of my

life:

To my parents, who encouraged my curiosity and interest in science from a very young
age, and who instilled in me a great love for learning. Their encouragement and their
belief in me, not to mention the many sacrifices they made to financially support many

years of my education, are gratefully acknowledged.

To my grandma and grandpa Boynton, who showed me the hard work, unpredictability

and rewarding nature of agriculture. Little did they know what they started when they

first paid me to help pick berries for market.

iii

ACKNOWLEDGEMENTS

I owe a great deal of my success at Michigan State to my major advisor, Dr. Mary
Hausbeck who has greatly enriched my professional development. Her encouragement,
enthusiasm, patience and financial support has made my time here most productive and
enjoyable. The suggestions and support of my committee members, Dr. Ray
Hammerschmidt and Dr. David Jacobson, is also greatly appreciated.

The trials and tribulations I experienced were made easier by the friendship and
words of encouragement from many people in the lab and the department, I thank you
all. I owe special thanks to John Kusnier who spent many hours working in my field
plots before, during and after sunrise.

I am grateful for the support (and many care packages) I received from my family.
Finally, my husband's confidence and faith in my pursuits, from over the miles, for the

past few years have been a great blessing to me.

iv

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................... vii
LIST OF FIGURES ..................................................................................... ix
LITERATURE REVIEW ............................................................................ 1
Introduction 1
Life Cycle of C olletotrichum coccodes 2
Foliar Infection 4
Histology of Infection 6
Commercial Disease Control Strategies 8
Effectiveness of Cultivar Resistance 9
Literature Cited 10
I. EVALUATION OF A LEAF DISK ASSAY FOR DETECTING
RESISTANCE TO ANTHRACNOSE FRUIT ROT .............................. 13
Abstract 14
Introduction 1 5
Materials and Methods 16
Results 18
Discussion 22
Literature Cited 24
II. BENEFITS ASSESSMENT OF THE USE OF FUNGICIDES TO
CONTROL FRUIT ROT IN PROCESSING TOMATOES .................. 26
Abstract 27
Introduction 28
Materials and Methods 32
Results 38
Discussion 49
Literature Cited 52

TABLE OF CONTENTS (Continued)

III. FOLIAR INFECTION PROCESSES OF COLLETOTRICHUM

C CC C ODES ........................................................................................ 54

Abstract

Introduction

Materials and Methods
Results

Discussion

Literature Cited

vi

55
56
58
66
7O
75

II.

LIST OF TABLES

Rafi:

EVALUATION OF A LEAF DISK ASSAY FOR DETECTING RESISTANCE
TO ANTHRACNOSE FRUIT ROT

Summary of analysis of variance for average number of colonies
of Colletotrichum coccodes per leaf disk. 20

Effect of cultivar on mean number of Colletotrichum coccodes
colonies per leaf disk. 21

Effect of leaf position on mean number of colonies of
Colletotrichum coccodes per leaf disk. 21

BENEFITS ASSESSMENT OF THE USE OF FUNGICIDES TO CONTROL

' FRUIT ROT IN PROCESSING TOMATOES

Disease Severity Values (DSVs) calculated by counting the
hours of leaf wetness and averaging the temperature during
the wetness period. 31

Weather data for the trial site during the growing seasons of
1993, 1994, and 1995.33

Key to equations used to calculate benefit per acre (BPA) and
return per fungicide dollar (RPF D). 37

The effect of foliar fungicide application on area under the disease
progress curve (AUDPC) for foliar blight in 1993 and 1994. 39

Summary of analysis of variance for AUDPC representing foliar
blight over time and percent healthy fruit for 'Ohio 7814' and
'Ohio 8245' tomatoes with various fungicide treatments in 1995. 41

vii

I321:

10.

11.

The effect of foliar fungicide application on AUDPC
representing foliar blight and percent healthy fruit for
'Ohio 7814' and 'Ohio 8245' tomatoes in 1995.

The effect of foliar fungicide application on percent healthy
fruit in 1993 and 1994.

Benefit per acre (BPA) and return per fungicide dollar
(RPF D) of foliar fungicide application schedules to control
fungal fruit rots in tomato production valued at $1,600 per
acre in 1993, 1994, and 1995.

Benefit per acre (BPA) and return per fungicide dollar
(RPF D) of foliar fungicide application schedules to control
fungal fruit rots in tomato production valued at $2,000 per
acre in 1993, 1994, and 1995.

Benefit per acre (BPA) and return per fungicide dollar
(RPFD) of foliar fungicide application schedules to control
fungal fruit rots in tomato production valued at $2,400 per
acre in 1993, 1994, and 1995.

Benefit per acre (BPA) and return per fungicide dollar
(RPF D) of foliar fungicide application schedules to control
fungal fruit rots in tomato production valued at $1,600 per

acre in 1993, 1994, and 1995 calculated with the modified model.

viii

42

43

45

46

47

48

LIST OF FIGURES

Fi re Rafi

1. EVALUATION OF A LEAF DISK ASSAY FOR DETECTING RESISTANCE
TO ANTHRACNOSE FRUIT ROT

1. Effect of cultivar (Heinz 7155, Ohio 8245, Ohio 7814,
Heinz 9033) and leaf position (top, middle, bottom) on
average number of colonies of C olletotrichum coccodes
per leaf disk. 19

III. FOLIAR INFECTION PROCESSES OF COLLETOTRICHUM C OCCODES

l. a)Ungerminated conidia of Colletotrichum coccodes 2 hours

after inculation. b)Conidia producing germ tubes 2 hours

after inoculation; scale bars = lOum. 61
2. a)Globose, unmelanized appressoria produced by a germinated

conidium of Colletotrichum coccodes, 4 hours after inoculation.
b)Appressorium produced by a germinated conidium beginning to
melanize, 6 hours after inoculation; scale bars = Nam. 63

3. a)Completely melanized appressoria produced by conidia of
Colletotrichum coccodes, 18 hours after inoculation. b)Hyaline
infection vesicle produced by a melanized appressorium, 24 hours

after inoculation; scale bars = lOum. 65
4. Percentage of Colletotrichum coccodes conidia germinated

at 25C, error bars represent standard error of the mean. 67
5. a)Appressorium produced terminally on an extended germ tube.

b)Infection by Colletotrichum coccodes through a stoma; scale
bars = lOum. 68

ix

F i gurg

Percentage of germinated Colletotrichum coccodes conidia
with unmelanized and melanized appressoria at 25C, bars
represent standard error of the mean.

71

LITERATURE REVIEW

INTRODUCTION

Processing tomato (Lycopersicon esculentum Mill.) production in Michigan
covers 4,600 acres and was valued at 10.6 million dollars in 1994 (MDA, 1995).
Neighboring states Of Indiana, Ohio, and the province of Ontario also have significant
acreage devoted to processing tomatoes.

Processing tomato production differs considerably from fresh market tomato
production. Processing tomatoes are left in the field to ripen, unlike fresh market fruit
which are harvested when still green. Before harvest, applications of ethrel are made to
the processing tomato crop to induce even ripening and kill the vines. The fruit are then
mechanically harvested and loaded into semi trucks for transport to the processing
facilities. A delay in harvesting or processing may allow development of several fruit
rots.

Anthracnose (C olletotrichum coccodes (Wallr) Hughes) is the major fungal
disease affecting processing tomato fruit in the Eastern and Midwestern United States
(Poysa et al., 1993; Barksdale, 1981). When fruit are ripe, disease symptoms develop
rapidly. Processors have very low tolerance for anthracnose fruit rot with as few as one
or two lesions per fruit making the fruit unusable (Precheur et al., 1992). Incidence of

anthracnose 23% in tomatoes intended for whole-peeled product reduces crop value by

2

25% (R. Latin, Purdue University, personal communication). Disease incidence 210%
results in total crop loss (R. Latin, Purdue University, personal communication).

Economically viable tomato production in the Midwest is becoming increasingly
difficult. Significant disease pressure, due to the conducive climate, combined with
limited effective fungicides has created a unique challenge to growers. Growers rely on
fungicide use and crop rotation to control anthracnose fruit rot. Despite regular
applications of fungicides, losses of up to 15% may occur (Pulling et al., 1995; Farley,
1976)

LIFE CYCLE OF COLLE T OTRICHUM COCCODES

A fungus causing tomato anthracnose was first described in 1897 (Sherf and
MacNab, 1986). Tomato anthracnose occurs worldwide throughout Asia, Europe, Africa,
the East Indies and North America (Sherf and MacNab, 1986). In North America it is an
econOmically important disease in the Northeast and Midwestern areas of the United
States (Gleason et al., 1995).

C. coccodes is classified in the form-subdivision Deuteromycotina, form-class
Coelomycetes, form-order Melanconiales (Moore-Landecker, 1990). C. coccodes is
referred to in older literature as C. phomoides (Sacc.) Chester and C. atramentarium
(Berk. and Br.) Tauben.

C. coccodes has a wide host range of plants in 14 families, mostly within the
Solanaceae, Cucurbitaceae, and Leguminosae. C. coccodes has been associated with
potato early dying complex and potato black dot disease (Otazu et al., 1978; Kotcon et
al., 1985). C. coccodes has been studied as a potential biological control agent for the

weed velvetleaf (Abutilon theophrasti Medik.) (Wymore and Watson, 1986).

3

C. coccodes grows readily in culture. Dillard (1988) found that conidia and
sclerotia have different optimum temperatures for growth. Conidia germination was
greatest at 22°C after 24 hours on agar media. Germination of conidia is inhibited at
temperatures lower than 10 and higher than 31°C. Isolates of C. coccodes vary in
pathogenicity, growth, sclerotia size, pigmentation, and are often observed sectoring in
culture.

C. coccodes infects green and red fruit. Infections of green fruit usually remain
latent until physiological changes occur in the fruit. Such changes may be triggered by
ripening of the fruit, or exposure to low temperatures (Illman et al., 1959). Fruit
symptoms begin as small, dark, sunken lesions with a water-soaked appearance that
increase in diameter and coalesce leaving a large, sunken, sofi area on the fruit (Tu,
1980). Under favorable temperatures, lesions on ripe fruit become visible within 5-6
days afier infection (Dillard, 1989). Black acervuli are produced just beneath the skin of
the infected fruit. As lesions mature, setose sclerotia develop from the stromata of the
acervuli within the infected fruit tissue (Tu, 1980).

C. coccodes may overwinter as sclerotia in soil and infested debris for a minimum
of one year (Farley, 1976). Acervuli form on infested debris in soil, producing conidia
which serve as the primary source of inoculum of tomato fruit (Illman, 1960; Pantidou
and Schroeder, 1955). Moisture is necessary for release of the conidia from the
gelatinous matrix in the acervulus. Conidia of C. coccodes are cylindrical with obtuse
ends, hyaline, aseptate, and 16-24 x 2.5-4.5 mm in size (Mordue, 1967). Conidia are

disseminated by splashing rain. Severe disease incidence is most often associated with

4

high rainfall during the growing season (Dillard, 1989). Disease incidence may also be
increased by use of overhead irrigation (Raniere and Crossan, 1959).

In addition to infecting fruit C. coccodes is capable of infecting root and stem
tissue. Kendrick and Walker (1948) grew seedlings in artificially infested sand and
Observed small necrotic flecks on the tap root and hypocotyl. Pantidou and Schroeder
(1955) found C. coccodes isolates varied in their ability to infect root tissue. Microtome
sections of stem tissue detected no fimgal growth in vascular tissue. Mature roots were
easily infected by C. coccodes, causing discoloration of the root system (Illman, 1960).
Sclerotia and active acervuli were produced on senescent root tissue.

FOLIAR INFECTION

C. coccodes is not known to cause a foliar blight on tomato although it can infect
foliage producing small necrotic spots with a chlorotic halo where sporulation may occur
(Younkin and Dimock, 1944). Histological studies have documented the ability of C.
coccodes to infect foliage 42 hours after inoculation (Illman, 1960). The significance of
foliar infection and inoculum production in fruit rot was studied by several groups of
researchers in the 1940's and 50's, however their conclusions are contradictory.

Foliar inoculation of tomato seedlings with C. coccodes resulted in necrotic
lesions and premature senescence of cotyledons (Kendrick and Walker, 1948). In attempt
to determine the significance of foliar inoculum in fruit infections, plants in the field were
inoculated with an albino, morphologically recognizable strain of C. coccodes. After
failing to recover the albino strain from infected fruit, it was concluded that sporulation of

C. coccodes in foliar lesions was not a significant source of secondary inoculum

5

(Kendrick and Walker, 1948). Results of this study have been criticized because of its
use of an albino isolate. Albino isolates produce non-melanized appressoria which are
typically not able to sucessfully infect intact host tissue.

In contrast, Pantidou and Schroeder (1955) concluded that C. coccodes can infect
foliage and subsequently sporulate providing significant inoculum for fruit infection.
Typical fruit lesions were reproduced when filtrates from washed, crushed leaves with L.
coccodes conidia were sprayed on fruit. Seedlings were inoculated with morphologically
recognizable C. coccodes strains and allowed to grow for three months. Once fruit had
developed, plants were incubated for 24 hours in a moist chamber. Foliar sporulation
ensued and fruit developed typical anthracnose lesions from which the recognizable C.
coccodes strains were isolated.

Pantidou and Schroeder (1955) observed sporulation in foliar lesions caused by
Alternaria and flea beetle feeding of greenhouse and field grown tomato plants.
Sporulation of C. coccodes was also abundant on the lower, more mature leaves. In
commercial production, growers may reduce their fungicide sprays once ethrel has been
applied in preparation for harvest. The time after ethrel application may in fact be very
important in anthracnose disease management given Pantidou and Schroeder's (1955) ‘
observations Of abundant sporulation on older foliage.

Hausbeck and Linderman (1992) used a semi-selective medium to quantify C.
coccodes colony development on inoculated foliage incubated at 15, 20, and 25 °C with
varying dew periods. At 15 °C, a minimum of 16 hours of leaf wetness were necessary

for foliar infection, whereas only 12 hours were necessary at 20 and 25 °C. Additionally,

6

bottom leaves were noted to be significantly more susceptible to infection than top and
middle leaves.
HISTOLOGY OF INFECTION

Histological studies of C. coccodes infection on fruit, foliage, root and stem tissue
were conducted by lllman (1960). The most extensive studies were conducted on fruit
and indicated that infection occurs directly through the intact cuticle and is not dependent
on wounds for entry. Germination and infection of inoculated mature fruit by C.
coccodes conidia occurs rapidly. Using periclinal scalpings of inoculated fruit, hyaline,
unthickened germ pores were observed emerging from germinating conidia. Appressoria
formed on the surface of tomato fruit inoculated with a conidia] suspension of C.
coccodes when maintained in a high relative humidity. Well developed primary
infection pegs were Observed pushing through the cuticle on inoculated fruit kept in a
moist chamber for two days.

The appressoria wall is strengthened by melanin production, allowing a build up
of high internal hydrostatic pressure necessary for cuticle penetration (Kubo and
F urusawa, 1986). Polysaccharides presumably play a role in adhesion of the appressoria
tO the cuticle (Hamer et al., 1988). lllman (1960) hypothesized that adhesion was
necessary for mechanical penetration by the infection peg. In addition to the mechanical
pressure exerted by the appressoria, cuticle penetration may involve secretion of cutin
degrading enzymes. (Dickman et al., 1982).

Once the cuticlehas been penetrated, the tip of the infection papilla expands

(Illman, 1960) . lllman (1960) observed cell wall degredation of the fruit tissue

7

surrounding the tip of the infection peg after three to four days of incubation. The degree
Of cell wall degredation was dependent upon fruit maturity. While degredation in
immature fruit was minimal, erosion in mature fruit resulted in a crater as large as 10am
in diameter around the infection peg. Once this degredation occurs, the penetration peg
expands to form an infection vesicle. Intracellular primary hyphae grow from the
infection vesicles into adjacent cells (Bailey, et al., 1992)

lllman (1960) investigated the histology of foliar infection making observations
on inoculated foliage of plants incubated in a moist chamber at room temperature for 42
hours. Samples were taken from inoculated foliage 19 and 42 hours after incubation.
Appressoria were observed 19 hours after inoculation, and occasionally an infection peg
was noted. After 42 hours, infection was established and hyphae were present in the
infected cell.

Pantidou and Schroeder (1955) made histological observations on inoculated
leaves incubated for 24 hours at 21-24°C. Twenty-four hours afier inoculatiOn, conidia
had germinated and germ tubes present on the epidermis had formed appressoria.
Appressoria were usually located in the depression between two adjacent epidermal cells.
Host cells with appressoria on their surface appeared darkly stained. Acervuli were
Observed in sections made 6 days afier inoculation.

Histological studies on root, hypocotyl and stem tissue found these tissues
significantly more resistant to infection than mature fruit. Infections of hypocotyls of
inoculated plants were restricted to superficial cell layers. lllman (1960) observed

darkened papillae in association with appressoria formation on hypocotyl and root tissue.

8

The papillae impeded or prevented infection of the tissue. lllman (1960) described brown
flecking observed on inoculated petioles as the result of a hypersensitive-like reaction
which limited the progress of infection. C. coccodes is capable of remaining viable for
months after hypersensitive responses occur in the plant tissue, demonstrated by
reisolation from the hypocotyl lesion.
COMMERCIAL DISEASE CONTROL STRATEGIES

In most years, it is economically impossible to produce a processing tomato crop I
in the Midwest without frequent fungicide applications (Precheur et al., 1992). The
products registered for control of fungal diseases in commercial tomato production are
limited to two active ingredients (chlorothalonil and Ethylenebisdithiocarbamate
(EBDC)). Some processing companies in the United States and Ontario refuse to buy
tomato products if EBDC fungicides have been used (Brammel, 1993; Hausbeck and
Latin, personal communication). Thus, growers rely almost exclusively on chlorothalonil
for disease control on processing tomatoes.

Traditionally growers have applied fungicide every 7-10 days, resulting in as
many as 10-12 sprays each growing season (Precheur et al., 1992; Gleason et al., 1995).
An increasing number of growers are using a disease forecaster called TOM-CAST
developed to prompt fungicide applications to control Alternaria solani. TOM-CAST is
a simplified version of the forecaster "FAST" (Madden et al., 1978). TOM-CAST
utilizes the duration of leaf wetness and average air temperature during the wetness
periods to calculate a daily disease severity value (DSV) of 0 to 4, corresponding to

conditions unfavorable to highly favorable for A. solani conidial formation (Pitblado,

 

V2

1a

in:
of
brc

cul

IIO‘
ma:
fling

resu

9

1988; Pitblado, 1992). When DSV's accumulate to a predetermined threshold,
fungicides are applied and the DSV is reset. TOM-CAST may reduce the number Of
applications needed by as much as 50% without compromising fruit quality or yield
(Gleason et al., 1995).

Processing companies are encouraging their contracted growers to use the TOM-
CAST system. Individual states in cooperation with processing companies have begun to
set up automated regional weather monitoring equipment. Growers access the daily DSV
values through a "1-800" phone number. This has alleviated the need for individual
growers to purchase and maintain their own weather monitoring equipment and has
facilitated the adoption Of the TOM-CAST system by many growers.

EFFECTIVENESS OF CULTIVAR RESISTANCE

Cultivars with varying amounts of resistance are commercially available. With
increased host resistance, fungicides use can be decreased while maintaining a high level
of fruit quality (Fry, 1977). Barksdale and Stoner (1981) estimated the resistance in their
breeding lines to be worth three to four fungicide applications compared to two standard
cultivars.

Levels of resistance currently available are not sufficient to replace fungicide use.
However, resistance may be used in combination with a disease forecasting system to
maximize fungicide use. When Fulling et al. (1995) integrated TOM-CAST based
fungicide sprays with anthracnose resistant cultivars, a more liberal DSV could be used

resulting in three to four fewer fungicide applications compared to susceptible cultivars.

 

Glt‘
195
we

Han
altat

10

LITERATURE CITED

Bailey, J .A., O'Connell, R.J., Ring, R.J., Nash, C. 1992. Infection Strategies of
Colletotrichum Species. In: Bailey, J .A. and Jeger, M.J. (eds), Colletotrichum: Biology,
Pathology and Control, CAB International, UK, pp. 88-120.

Barksdale, TH, and Stoner, AK. 1981. Levels of tomato anthracnose resistance
measured by reduction of fungicide use. Plant Disease 65:71-72.

Brammall, RA. 1993. Effect of foliar fungicide treatment on early blight and yield of
fresh market tomato in Ontario. Plant Disease 77:484-488.

Dickman, M.B., Patil, S.S.a nd Kolattukudy, PE. 1982. Purification, characterization
and role in infection of an extracellular cutinolytic enzyme from Colletotrichum
gloeosporioides Penz. on Carica papaya L. Physiological Plant Pathology 20:333-347.

Dillard, HR. 1988. Influence of temperature, pH, osmotic potential, and fungicide
sensitivity on germination of conidia and growth from sclerotia of Colletotrichum
coccodes in vitro. Phytopathology 78:1357-1361.

Dillard, J .R. 1989. Effect of temperature, wetness duration, and inoculum density on
infection and lesion development of Colletotrichum coccodes on tomato fruit.
Phytopathology 79: 1063-1066. '

Farley, JD. 1976. Survival Of Colletotrichum coccodes in soil. Phytopathology 66:640-
641.

Fedewa, DJ. and Pscodna, S.J. (eds) 1995. Michigan Agricultural Statistics. MDA,
Michigan pp. 32-33.

Fulling, B.A., Tigchelaar, EC, and Latin, R. 1995. Integration of host resistance and
weather-based fungicide scheduling for control of anthracnose of tomato fruit. Plant
Disease 79:228-233.

Fry, W.E. 1977. Integrated control of potato late blight - effects of polygenic resistance
and techniques of timing fungicide applications. Phytopathology 67:415-420.

Gleason, M.L., MacNab, A.A., Pitblado, R.E., Ticker, M.D., East, D.E., Latin, R.X.
1995. Disease-warning systems for processing tomatoes in eastern North America: are
we there yet? Plant Disease 79:113-121.

Hamer, J .E., Howard, R.J., Chumley, F.G., Valent, B. 1988. A mechanism for surface
attachment in spores of a plant pathogenic fungus. Science 239:288-290.

ll

Hausbeck, MK. and Linderman, SD. 1992. Influence of dew period and temperature on
infection of tomato foliage by Colletotrichum coccodes. Phytopathology 82: 1091.

Homby, D. 1968. Studies on Colletotrichum coccodes III. Some properties of the
fungus in soil and in tomato roots. Transactions of the British Mycological Society 51,
541-553.

lllman, W.I., Ludwig, RA. and Farmer J. 1959. Anthracnose Of canning tomatoes in
Ontario. Canadian Journal of Botany 37: 1237-1246.

lllman, W.I., 1960. Anthracnose Disease Of Tomato, PhD. Thesis, University Of Western
Ontario, London, Ontario, Canada.

Kendrick, J .B. and Walker, J.C. 1948. Anthracnose of tomato. Phytopathology 38:247-
260.

Kotcon, J.B., Rouse, DJ. and Mitchell, J.E. 1985. Interactions of Verticillium dahliae,
Colletotrichum coccodes, Rhizoctonia solani, and Pratylenchus penetrans in the early
dying syndrome of Russet Burbank potatoes. Phytopathology 75:68-74.

Kubo,Y. and Furusawa, I. 1986. Localization of melanin in appresoria Of C olletotrichum
lagenarium. Candian Journal of Microbiology 32:280-282.

Madden, L., Pennypacker, SP. and MacNab, AA. 1978. FAST, a forecast system for
Alternaria solani on tomato. Phytopathology 68:1354-1358.

Mordue, J.E.M. 1967. C olletotrichum coccodes. Descriptions of Pathogenic Fungi and
Bacteria. No. 131. Commonwealth Mycological Institute. Kew, Surrey, England.

Moore-Landecker, E. 1990. Fundamentals of the Fungi. Prentice Hall, NJ, 56lpp.

Otazu, V., Gudmestad, NC. and link, RT. 1978. The role of Colletotrichum
atramentarium in the potato wilt complex in North Dakota. Plant Disease Reporter 62,
847-851.

Pantidou, ME. and Schroeder, W.T. 1955. Foliage as a source of secondary inoculum
for tomato anthracnose. Phytopathology 45:338-345.

Pitblado, RE. 1988. Development of a weather timed fungicide spray program for field
tomatoes. Canadian Journal of Plant Pathology 10:371.

Pitblado, RE. 1992. Development and implementation of TOM-CAST. Ontario
Ministry of Agriculture and Food Publications p18.

12

Precheur, R.J., Bennett, M.A., Tiede., R.M., Wiesse, K.L., and Dudek, J. 1992.
Management of fungicide residues on processing tomatoes. Plant Disease 76:700-702.

Raniere, LC. and Crossan, D.F. 1959. The influence of overhead irrigation and
microclimate on Colletotrichum phomoides. Phytopathology 49:72-74.

Sherf, AF. and MacNab, AA. 1986. Vegetable Diseases and Their Control. John
Wiley & Sons, NY.

Tu, J .C. 1980. The ontogeny of the sclerotia of Colletotrichum coccodes. Canadian
Journal of Botany 58:631-636.

Wymore, LA. and Watson, AK. 1986. An adjuvant increases survival and efficacy of
Colletotrichum coccodes , a mycoherbicide for velvetleaf (Abutilon theophrasti).
Phytopathology 76:11 15-1 1 16.

Younkin, SB. and Dimock, AW. 1944. Foliage infections of Lycopersicon esculentum
by Colletotrichum phomoides. Phytopathology 34:976-977.

SECTION I

EVALUATION OF A LEAF DISK ASSAY FOR DETECTING RESISTANCE TO

ANTHRACNOSE FRUIT ROT

ABSTRACT

A leaf disk assay system was evaluated as a tool for screening tomato cultivars to
anthracnose fruit rot. Foliage of four green-house grown tomato cultivars (Ohio 7814,
Ohio 8245, Heinz 9033, Heinz 7155) was inoculated with conidia of C. coccodes and
exposed in a dew chamber to 24 hours of wetting at 25 °C. One week after inoculation,
the severity of infection was quantified by placing explants from leaves on the surface Of
a medium consisting of the following ingredients / L: 300 m1 filtered V-8 juice, 700 ml
double-distilled water; 20 g Difco agar; and the following antimicrobial agents added
afier autoclaving: 0.13 g pentachloronitrobenzene; 0.14 g benomyl; 0.10 g streptomycin
sulfate; 0.10 g tetracycline HCl, and 0.1 g chloramphenicol. On this medium, colonies
of C. coccodes were compact and easily identified and quantified after 4 days. Explants
removed from Ohio 8245 and Ohio 7814 had significantly more colonies than cultivars
Heinz 9033 and Heinz 7155. Number of colonies were significantly higher on explants
removed from bottom than from middle and top leaves. These results indicate that this

assay using tomato foliage does not reflect resistance levels of fruit to anthracnose.

14

15

INTRODUCTION

Anthracnose (Colletotrichum coccodes (Wallr) Hughes) is the most serious
fungal disease affecting tomato (Lycopersicon esculentum Mill.) fruit grown for
processing in the Eastern and Midwestern United States (Poysa et al., 1993; Barksdale,
1981). Processing fruit are especially susceptible to infection by C. coccodes, an
otherwise weak pathogen, since the crop is harvested when fully ripe (Homby, 1968;
Fulton, 1948; Kendrick and Walker, 1948). Fruit symptoms begin as small, dark, sunken
lesions with a water-soaked appearance that increase in diameter and coalesce leaving a
large, sunken, soft area on the fruit (Tu, 1980). Under favorable temperatures, lesions
on ripe fruit become visible within 5-6 days after infection (Dillard, 1989).

C. coccodes is not known to cause a foliar blight on tomato although it can infect
foliage producing small, necrotic spots with a chlorotic halo where sporulation may occur
(Younkin and Dimock, 1944). F Oliar infection occurs readily on plants subjected to 24
hours of leaf wetness in continuous darkness at 25 °C (Hausbeck and Linderman, 1992).
The significance of foliar infection and inoculum production in fruit rOt is not currently
known.

Anthracnose fruit rot is currently controlled by rotation and fungicide application.
Traditionally, growers apply fungicides every 7-10 days, resulting in as many as 10-12
sprays each growing season (Precheur et al., 1992; Gleason et al., 1995). Cultivars
resistant or highly tolerant to anthracnose reduce the need for fungicide treatment

(Barksdale and Stoner, 1981). When genetic resistance is integrated with an

l6

environmentally-based forecasting system for fungicide application, increased action
thresholds can be used requiring three to four fewer fungicide applications per season
compared to the same forecasting program using susceptible cultivars (Fulling et al.,
1995; Fry, 1977).

Currently, cultivars under development must be grown to maturity in the field to
evaluate their resistance to anthracnose fruit rot. Environmental conditions that cannot be
controlled in the field play an important role in disease development, and may effect
evaluation results from year to year. The development of a tomato foliage assay to
evaluate resistance of fruit to C. coccodes in developing tomato lines would eliminate the
need for mature fruit, thereby, decreasing the evaluation time. Furthermore, a laboratory
assay could be conducted under controlled environmental conditions known to be
conducive to disease development. Detached leaf assays have been used to detect early
blight resistance in potato cultivars . Disease development on inoculated leaf disks
treated with NAA or BAP is highly correlated with disease incidence in the field (Bussey
and Stevenson, 1991).

The Objective Of this study was to evaluate a leaf disk assay system as a tool for
screening tomato cultivars for resistance to anthracnose fruit rot.

MATERIALS AND METHODS
Plant Culture. Tomato seedlings of four tomato cultivars (Ohio 7814, Ohio 8245, Heinz
9033 and Heinz 7155) were grown in a research greenhouse on the campus of Michigan
State University in 163 cm3 cell packs containing Baccto soilless media (Michigan Peat

Company, Houston, TX) for 4-5 weeks prior to transplanting to 15.2 cm clay pots. Plants

17

were watered individually with drip tubes to prevent leaf wetness and fertilized with 6
grams of 15-10-12 slow release fertilizer (Sierra Chemical Company, Milpitas, CA) at
transplanting. Plants with 12-14 nodes were used for this experiment.

Inoculation. C. coccodes was isolated from an infected tomato fruit and grown on
medium containing V8 juice (30%) and agar (2%) (DIFCO, Detroit, MI) in 100 x 15 mm
petri dishes at 20°C under continuous cool-white fluorescent light. Prior to inoculation,
10-14 day Old cultures were flooded with 10 ml of sterile distilled water and gently
scraped with a glass rod. The conidial suspension was adjusted to a concentration of 1 x
105 conidia/ml using a hemacytometer.

A top, middle, and bottom leaf was tagged on each of the three plants of each
cultivar used. Tagged leaves were spray inoculated with the conidial suspension until
runoff using a Preval pressurized sprayer (Precision Valve Corporation, Yonkers, NY).
Plants were incubated in a dew chamber (Percival Manufacturing Co., Boone, IA) in
continuous darkness for 24 hours at 25 °C. Following incubation, plants were removed
from the dew chamber and placed at 22°C under continuous light.

Using a 1.3-cm-diameter cork borer, 6 leaf disks, were excised from each of the 3
marked leaves per plant seven days following inoculation. Leaf disks were surface
sterilized in a .525% sodium hypochlorite solution for 1 minute, triple rinsed in sterile
distilled water and blotted dry. Leaf disks were plated onto semi-selective V8 media (300
ml V8 juice, 700 ml dde, 20 g Difco agar, 0.13 g pentachloronitrobenzene, 0.14 g
benomyl, 0.1 g streptomycin sulfate, 0.1 g tetracycline HCI, 0.1 g chlorarnphenicol)

developed to support discreet colonies of fungal growth that can be quantified (Hausbeck

l8

and Linderman, 1992). Three leaf disks were placed in a 100 x 15 mm petri dish and
incubated at 22°C under 12 hours of continuous cool-white florescent light. Colonies of
C. coccodes were counted on leaf disks 4, 6, and 8 days after plating with the aid Of a
compound dissecting microscope (x16).

This experiment was arranged in a split block design with cultivar as the main
plot and leaf position as the sub-plot. Mean number Of colonies per disk were analyzed
using the analysis of variance (ANOVA) procedure of the Statistical Analysis System
(SAS Institute, Cory, NC). The least significant difference (LSD) P=0.05 was calculated
to separate the mean cultivar and mean position values. This experiment was repeated
once.

RESULTS

In the second experiment, leaves in the bottom position on plants of cultivar
Heinz 9033 prematurely senesced and dehisced, precluding excision and plating of leaf
disks. C. coccodes is known to cause premature senescence of infected foliage (Illman et
al., 1955; Kendrick and Walker, 1948). For this reason the leaf disks that would have
been excised from those leaves were assigned a mean number Of colonies per disk of 4,
equal to results in the first experiment (Figure 1).

Number Of colonies per leaf disk were significantly affected by cultivar (Table l).
Cultivars Ohio 8245 and Ohio 7814 had 3.5 and 2.9 colonies per leaf disk, respectively,
significantly more than cultivars Heinz 9033 and Heinz 7155 which had 1.8 and 1.7
colonies per leaf disk, respectively (Table 2).

Leaf position had a significant effect on number of colonies per leaf disk (Table

19

 

 

 

§§
///fl

 

 

 

 

 

 

E TOp Leaf
Middle Leaf
\\\\\\\ Bottom Leaf

 

 

 

 

V/////////////////a

 

 

 

 

 

 

 

V///////////////

 

Experiment 1

 

 

 

 

S
7//////////

 

 

 

 

 

 

 

_ _ _ _ _
6 5 4 3 2 1 O

962 Log mo_co_oo ho 39:5: 3903».

 

8245 7814 9033

7155

Cultivar

Experiment 2

 

 

 

V
r

N\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\.
////////////a

 

 

 

[:1 Top Leaf
m Middle Leaf
s\\\\\\V Bottom Leaf

 

 

 

 

 

 

 

 

 

 

 

 

N\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\R
7////////////////////////////////,

 

 

 

 

 

 

 

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\R
7////////

 

 

 

 

 

 

 

_ _ _ r _
6 5 4 3 2 1 0

Cam. Ema mo_co_oo no ConEac mmmam><

 

8245 7814 9033

7155

Cultivar
Figure 1. Effect of cultivar (Heinz 7155, Ohio 8245, Ohio 7814, Heinz 9033) and
leaf position (top, middle, bottom) on average number of colonies of Colletotrichum

coccodes per leaf disk.

zzxiésetc v .3 12:315.... ...JLE2£ 9.4.5.15: .5. ou::.2=>.3 m._.r._».:::....: >..:..::::.A . ~ 323.!

 

20

 

 

o2 3.8 mm Eta
Zomd 3N mwé N Egg—tonne x .858.—
mvomd omd 3.3 0 308.535 n 32:3 a .852:—
Eomo a? N3 e 33...: a 858..
a a. Seed o _ . _ m om.mo_ N .558.—

vodom K .5525 you 33h.

34 2.8 N. mate
Kmmd cm.m cod m 2.2:...an a .3223
temood 3.3 2.? m .23an
wms v 9.83 62.583

:.3 mm .5516 .5.— .33.

2; ~ «eon—tongue .8.— .55.

.mAaoum m2 mm a: 3.55

 

.xfln b8— uoq $38.98

5532.339 .3 wows—co .«o 538:: omflog 8m 85%? Co Ema—ace mo .Caegm . _ 935.

21

Table 2. Effect of cultivar on mean number of
Colletotrichum coccodes colonies per leaf disk.

 

 

Cultivar Colonies per leaf disk
7155 1.75az
9033 1.8la
7814 2.94 b
8245 3.55 b

 

zMeans followed by the same letter are not
significantly different according to Fisher's least
significant difference test (P=0.05).

Table 3. Effect of leaf position on mean number of
colonies of Colletotrichum coccodes per leaf disk.

 

 

Position Colonies per leaf disk
Top 1.33az
Middle 2.05a
Bottom 4.14 b

 

’Means followed by the same letter are not
significantly different according to Fisher's least
significant difference test (P=0.05).

22

3). Disks from the bottom leaf had an average of 4.1 colonies, significantly more than

disks removed from the middle and top leaves which had 2.1 and 1.3 colonies per disk,

respectively. Numbers of colonies on the middle and top leaves were not significantly

different. There was not an interaction between cultivar and leaf position.
DISCUSSION

Results of this study indicate that the leaf disk assay does not reflect resistance of
cultivars to anthracnose fruit rot. Fewer colonies formed on leaf disks of 'Ohio 7814' than
'Ohio 8245'. 'Ohio 7814' is a highly susceptible cultivar, against which resistance indexes
are calculated for other cultivars (Fulling et al., 1995). However, 'Ohio 8245' is
considered to have the greatest amount of resistance available to growers in the
Midwestern United States (M. Ricker, Heinz USA, personal communication). The means
of resistance is largely due to the late maturing nature of this cultivar, and therefore is not
likely to be reflected in a resistance assay using leaf tissue. 'Heinz 9033' is considered to
have moderate resistance or tolerance, while Heinz 7155 has little resistance. Colony
numbers observed for these cultivars were nearly equal, but significantly less than the
number Of colonies Observed on 'Ohio 7814' and 'Ohio 8245'.

Leaf disks removed from bottom leaves contained significantly higher numbers of
colonies than leaf disks removed from top and middle leaves. Hausbeck and Linderman
(1992) observed higher numbers of colonies on tomato leaf disks removed from bottom
than from mid to top leaves of plants that received 24 hours of leaf wetness at 20 or
25 °C. Pantidou and Schroeder (1955) Observed more abundant sporulation on lower,

more mature leaves. Not all plant parts are equally susceptible to infection by

23

Colletotrichum spp., resistance of some plant parts is due to failure of the pathogen to
penetrate the tissue (Esquerré-Tugayé et al., 1992). Resistance of the top and middle
leaves most likely results from the failure of the pathogen to penetrate the younger,
healthier tissue. Mechanical force is a major component of tissue penetration and
subsequent infection by C Olletotrichum spp. (Kubo and Furusawa, 1986). It is suspected
that in this study the senescent tissue Of bottom leaves is more easily penetrated by C.
coccodes than the younger tissue of middle and top leaves, thus allowing the significantly '
higher number of colonies Observed on the bottom leaf disks.

Colony numbers Observed in this study were lower than those Observed in similar
studies (Hausbeck and Linderman, 1992). Low infection rate is suspected to be due to
inadequate wetting of the foliage surface for the entire duration of incubation. Results of
this study may have better reflected commercial resistance levels had infection been

greater.

24

LITERATURE CITED

Bailey, J.A., O'Connell, R.J., Ring, R.J., Nash, C. 1992. Infection Strategies Of
Colletotrichum Species. In: Bailey, J .A. and Jeger, M.J. (eds), Colletotrichum : Biologi,
Pathology and Control, CAB International, UK, pp.88-120.

Barksdale, T.H. and Stoner, AK. 1981. Levels Of tomato anthracnose resistance. Plant
Disease Reporter 59:648-652.

Bussey, M.J., and Stevenson, W.R. 1991. A leaf disk assay for detecting resistance to
early blight caused by Alternaria solani in juvenile potato plants. Plant Disease 75:385-
390.

Dillard, HR. 1989. Effect of temperature, wetness duration, and inoculum density of
infection and lesion development Of Colletotrichum coccodes on tomato fruit.
Phytopathology 79: 1063-1066.

Esquerré-Tugayé, M.R., Mazua, D., Barthe, J .P., Lafitte, C., and Touzé, A. 1992.
Infection Strategies of Colletotrichum Species. In: Bailey, J .A. and Jeger, M.J. (eds),
Colletotrichum .' Biology, Pathology and Control, CAB International, UK, pp.121-133.

Fulling, B.A., Tigchelaar, EC, and Latin, R. 1995. Integration of host resistance and
weather-based fungicide scheduling for control of anthracnose of tomato fruit. Plant
Disease 79:228-233.

Fry, W.E. 1977. Integrated control of potato late blight - effects of polygenic resistance
and techniques of timing fungicide applications. Phytopathology 67:415-420.

Kubo, Y. and Furusawa, I. 1986. Localization of melanin in appressoria of C.
Iagenarium. Canadian Journal of Microbiology 32:280-282.

Fulton, J .P. 1948. Infection of tomato fruits by Colletotrichum coccodes in soil.
Phytopathology 62, 1288-1293.

Gleason, M.L., MacNab, A.A., Pitblado, R.E., Ticker, M.D., East, D.E., Latin, R.X.
1995. Disease-warning systems for processing tomatoes in eastern North America: are
we there yet? Plant Disease 79:113-121.

Hausbeck, M.K. and Linderman, SD. 1992. Influence of dew period and temperature on
infection of tomato foliage by Colletotrichum coccodes. Phytopathology 8221091.

25

Homby, D. 1968. Studies on Colletotrichum coccodes III. Some properties of the
fungus in soil and in tomato roots. Transactions of the British Mycological Society 51,
541-553.

lllman, WI. 1960. Anthracnose Disease of Tomato, PhD. Thesis, University of Western
Ontario, Canada.

Kendrick, J .B. and Walker, J .C. 1948. Anthracnose of tomato. Phytopathology 38:247-
260.

Mercer, P.C., Wood, R.K.S. and Greenwood, AD. (1971) Initial infection Of Phaseolus
vulgaris by Colletotrichum lindemuthianum. In: Preece, T.F. and Dickinson, C.H. (eds),
Ecology of Leaf Surface Micro-organisms, Academic Press, London, pp.381-3 89.

Pantidou, M.B., and Schroeder, W.T. 1955. Foliage as a source of secondary inoculum
for tomato anthracnose. Phytopathology 452338-345.

Precheur, R.J., Bennett, M.A., Tiedel, R.M., Wiesse, K.L., and Dudek, J. 1992.
Management of fungicide residues on processing tomatoes. Plant Disease 76:700-702

Tu, J .C. 1980. The ontogeny of the sclerotia of Colletotrichum coccodes. Canadian
Journal of Botany 58:631-636.

Younkin, SB and Dimock, AW. 1944. Foliage infections of Lycopersicon esculentum
by Colletotrichum phomoides. Phytopathology 342976-977.

SECTION II

BENEFITS ASSESSMENT OF THE USE OF F UNGICIDES

TO CONTROL FRUIT ROT IN PROCESSING TOMATOES

ABSTRACT

The effectiveness of the fungicide Bravo 720 (chlorothalonil) at 2.53 kg a.i./ha for control
of fruit rot on processing tomatoes was assessed in field plots in Michigan during 1993 -
1995. F ungicide programs included: 1) 8 sprays at 7-day intervals; 2) 6 sprays at 10-day
intervals; 3) 4 sprays at 14-day intervals; 4) 4, 3, and 6 sprays respectively, scheduled by
an early blight forecaster (TOM-CAST) using a threshold of 20 disease severity values;
and 5) unsprayed. In all years, all treatments significantly reduced fruit rot compared to
the control. The primary fruit rot in 1993 was late blight (Phytophthora infestans)
resulting in only 8.2% healthy fruit in the unsprayed plot. Chlorothalonil applied every 7
to 10 days resulted in 90.8% and 87.0% healthy fruit, respectively. Chlorothalonil
applied every 14 days or according to TOM-CAST, resulted in 82.0% healthy fruit. In
1994, the primary fruit rot was anthracnose (Colletotrichum coccodes) resulting in 69.3%
healthy fruit in the unsprayed plot. Chlorothalonil applied at 7- or 10-day intervals
resulted in 97.8% and 93.8% healthy fruit, respectively. In 1995, cultivar ('Ohio 7814' vs.
'Ohio 8245') did not affect percentage of healthy fruit or foliar blight progression. The
primary fruit rot was anthracnose resulting in 59.9% healthy fruit. There were no
significant differences in percentage of healthy fruit among the fungicide treated plots.
An economic assessment revealed that for two years the 7 day application regime

provided the greatest benefit per acre.

27

28

INTRODUCTION

Processing tomato (Lycopersicon esculentum Mill.) production in Michigan
covers 4,600 acres and was valued at 10.6 million dollars in 1994 (MDA, 1995).
Neighboring states of Indiana, Ohio, and the province of Ontario also have significant
acreage devoted to processing tomatoes.

Anthracnose (C alletotrichum coccodes (Wallr.) Hughes) is the major fungal
disease affecting tomato fruit in the Eastern and Midwestern United States (Poysa et al., I
1993; Barksdale, 1981). It is an especially serious disease in tomatoes grown for
processing because the crop is harvested when fully ripe. At this ripe stage, fruit are most
susceptible to infection by C. coccodes, an otherwise weak pathogen (Hornby, 1968;
Fulton, 1948; Kendrick and Walker, 1948). Fruit symptoms begin as small, dark, sunken
lesions with a water-soaked appearance that increase in diameter and coalesce leaving a
large, sunken, soft area on the fruit (Tu, 1980). Black acervuli are produced just beneath
the skin of the infected fruit. As lesions mature, setose sclerotia develop from the
acervuli within the infected fruit tissue and may remain viable in the soil for years
(Pantidou and Schroeder, 1955; Farley, 1976).

Processors have very low tolerance for anthracnose fruit rot with as few as one or
two lesions per fruit making the fruit unusable (Precheur et al., 1992). Incidence of
anthracnose 23% in tomatoes intended for whole-peeled product reduces crop value by
25% (R. Latin, Purdue University, personal communication). Disease incidence 210%
results in total crop loss (R. Latin, Purdue University, personal communication). At the

processing plant tomatoes must undergo a mold count test that includes fungal spores of

29

all pathogens (Dillard, 1988; Gould, 1983). Tomato loads with a mold count higher than
the US. Food and Drug Administration's acceptable limit are rejected by the processor
(Gleason et al., 1995). Infected fruit have an increased pH, also unacceptable to
processors (Sapers et al., 1978).

Economically viable tomato production in the Midwest is becoming increasingly
difficult. Significant disease pressure combined with limited effective fungicides has
created a unique challenge to growers. The climate of high humidity, frequent rainfall,
and warm temperatures, provide conditions conducive for many fungal pathogens of
tomatoes including anthracnose, early blight (Alternaria solani (Sorauer)), late blight
((Phytophthora infestans) (Mont) de Bary), and septoria leaf spot (Septoria lycopersici
(Spegazzini)). Despite regular applications of fungicides, losses of up to 15% may occur
(Fulling et al., 1995; Farley, 1976). In most years, it is economically impossible to
produce a processing tomato crop in the Midwest without frequent fungicide applications
(Precheur et al., 1992).

Fungicides registered for control of fungal diseases in commercial tomato
production are limited to two active ingredients (chlorothalonil and
Ethylenebisdithiocarbamate (EBDC)). In addition, some processing companies in the
United States and Ontario refuse to buy tomato products if EBDC fungicides have been
used (Brammall, 1993; Hausbeck and Latin, personal communication). Thus, growers
rely almost exclusively on chlorothalonil for disease control on processing tomatoes.

Traditionally, growers have applied fungicide every 7-10 days, resulting in as

many as 10-12 sprays each growing season (Precheur et al., 1992; Gleason et al., 1995).

30

An increasing number of growers are using a disease forecaster called TOM-CAST
developed to prompt fungicide applications to control A. solani . TOM-CAST is a
simplified version of the forecaster "FAST" (Madden et al., 1978). TOM-CAST utilizes
the duration of leaf wetness and average air temperature during the wetness periods to
calculate a daily disease severity value (DSV) of 0 to 4, corresponding to conditions
unfavorable to highly favorable for A. solani conidial formation (Pitblado, 1988;
Pitblado, 1992) (Table 1). When DSVs accumulate to a predeterInined threshold,
fungicides are applied and the DSV is reset. TOM-CAST may reduce the number of
applications needed by as much as 50% without compromising fruit quality or yield
(Gleason et al., 1995)

Although TOM-CAST was developed using data of A. solani, it is used to control
other fungal diseases of tomatoes including anthracnose fruit rot (Pitblado, 1992).
However, the epidemiology of C. coccodes differs significantly from that Of A. solani
(Dillard, 1989; Fulling et al., 1995). In certain years, fruit quality in FAST field plots
was reduced not by A. solani but by C. coccodes and other fruit rot pathogens
(Pennypacker et al., 1983). Gleason et al. (1995) concluded that additional research is
needed to improve the accuracy Of current tomato disease forecasting models used in .
eastern North America by including aspects Of the epidemiology of C. coccodes.

The objectives of this study were to compare the efficacy of calendar-based spray
schedules to a TOM-CAST based spray schedule and calculate an economic analysis of
the benefit and return of each fungicide schedule. This information will be a valuable aid

to growers trying to maximize the benefit of chemical control to maintain low disease

31

23503 .231— ue 9:5:

 

2s _ 33.3
NS _ 33.8
as 23.:
8-2 23.2

m N _
ADV ,,
Am>mnv mos—Er btogm 933.5 _ each. :32 _

 

 

 

 

 

 

 

 

 

 

 

 

rectum 36503 of wage 0528388 2: wEwfioE
one 82:25 .32 Co 950: 05 wcucsoo 3 Boom—:28 Am>me 82m> bto>om ammofln— .— 03:.

32

levels and produce a saleable product. Also, the documented benefits of fungicide use in
processing tomatoes may be needed to help maintain registration of chlorothalonil in the
future.
MATERIALS AND METHODS

Location and design of field experiments. Research plots were established in a loam
soil at the Botany and Plant Pathology Farm in East Lansing, Michigan in 1993-1995
using S-week old tomato seedlings grown in a commercial greenhouse in 288-cell flats. In
1993 and 1994, the plot was established on a site never planted to tomatoes. In 1995, the
plot was established on a site that had been planted to alfalfa for two years following 12
years of tomato production. Seedlings were transplanted on 11 June 1993, 27 June 1994,
and 1 June 1995. Seedlings were spaced 0.5 m apart (10 plants/ treatment row) on 1.8 m
centers, with 1.8 m spacing between blocks. In 1993 and 1994, a randomized complete
block design with six blocks was used. Each block was comprised of 5 treatment rows
('Ohio 7814') alternated with buffer rows ('Ohio 8645'). In 1995, the plot was arranged in
a split plot design with cultivar as the main plot ('Ohio 7814' and 'Ohio 8245') and
fungicide treatment as the sub-plot. Buffer rows were planted to 'Ohio 8704'. 'Ohio
8245' is more resistant to anthracnose fruit rot than 'Ohio 7814' (Fulling et al., 1995;
Gleason et al , 1995).

Hourly averages of leaf wetness and temperature data were collected with a digital
recorder (Omnidata DP223, Omnidata International, Inc, Logan , Utah) (Table 2). The

leaf wetness sensor was located approximately 30 cm above the ground in a buffer row.

33

 

 

 

SN fl: v.2 3.2 2.9“ 3mm 5535

2: m2 ”.2 32 mm: a? Essaom

3m 2: 0.8 $2 3: a: 3...»:

SN EN 3m 3m 32 2: 2....

2m we 93 m3 2:...

m2: 3.: 2.: m2: 3: .83 5.5:
$33 333.3%

 

.33 EB Jug— .moo_ .«o 2033 magnum 05 mats—u ozm E: of “8 83 8533 .N 03.5.

34

Fungicide Treatments. The fungicide chlorothalonil (Bravo 720 at 2.53 kg a.i./ha, ISK
Biotech, Mentor, OH) was used throughout the study. F ungicide applications were made
with a C02 powered backpack sprayer operated at 50 psi through two XR Tee-Jet 8004
flat-fan nozzles, spaced 55.25 cm apart, calibrated to deliver 345.8 L/ha. Fungicide
treatments were applied according to the following treatment regimes; 1) 8 sprays at 7-
day intervals, 2) 6 sprays at lO—day intervals, 3) 4 sprays at l4-day intervals, 4) sprays
applied according to TOM-CAST with a threshold of 20 DSVs and 5) control (no sprays).
The TOM-CAST treatment received 4 sprays in 1993, 3 sprays in 1994, and 6 sprays in
1995 based on the environmental data recorded in the field plot. Calendar based sprays
were initiated when crown fruit were 1/3 of the estimated final size (28 July 1993, 3
August 1994, and 25 July 1995). Treatments applied according to TOM-CAST were
initiated on 12 July (1993-1994) and 11 July (1995).

Cultural Practices. Each year the herbicide, Trifluralin (Treflan, Dow Elanco,
Indianapolis, IN) was preplant incorporated at a rate of 1.75 L/ha. Postplant weed
control was achieved with hand hoeing and cultivation. A side-dress of fertilizer at a rate
equivalent to 148:148: 148 (N :Psz) per hectare was applied on 6 July 1993, 18 July 1994,
and 26 July 1995. Overhead sprinkler irrigation was applied as needed.

Inoculation. Treatment rows were inoculated with C. coccodes-infested millet seed to
increase disease pressure. Sterile millet seed (400 ml) combined with distilled water
(160 ml) was infested with 6 1-cm disks of 10-14 day old C. coccodes grown on medium
containing V8 juice (30%) and agar (2%) (DIFCO, Detroit, MI). Inoculum was incubated

in 1 L flasks at 20°C with a 12-hour photoperiod for 3 weeks. Infested millet was dried

35

and bagged until used. Infested millet was spread to the side of all treatment rows at a

rate of 400 ml per 21.9 m of row.

Assessment of foliar disease. Percent defoliation due to early blight, late blight, and

septoria leaf spot was visually assessed 3-6 times each year during August and

September. Assessments were based on the inner 6 plants within each treatment row.
Area under the disease progress curve (AUDPC) was calculated to express the

cumulative incidence of leaf infection occurring over a l3-day (1993), 21-day (1994), and

36-day (1995) period according to the method of Shaner and Finney (1977):

A UDPC==: [(Y.

t+nl
i=1

+Y,)/2] [X,., -X,.]

in which Y, =percent foliar blight at the ith observation, X,=time (days) at the ith
observation, and n=total number of observations. AUDPC values were analyzed using
the analysis of variance (ANOVA) procedure of the Statistical Analysis System (SAS
Institute, Cory, NC). The least significant difference (LSD) a=0.05 was calculated to
separate the mean treatment values.

Tomato harvest and assessment of fruit mold incidence. One hundred fruit were
hand-harvested from the 5 innermost plants of the treatment row when at least 75% of the
fruit were ripe and evaluated for fruit rot. In 1993, late blight caused severe defoliation
and premature ripening of fruit in the unsprayed plot and this treatment was harvested on
13 September, whereas the remaining treatments were harvested on 17 September. In
1994 , cool weather delayed fruit ripening, and all treatments were harvested 4 October.

In 1995, plots were harvested when the later ripening cultivar Ohio 8245 was ripe on 5

36

October.

The percentage of healthy fruit was calculated for all treatments and analyzed
using the ANOVA procedure of the Statistical Analysis System (SAS Institute, Cory,
NC). The least significant difference (LSD) a=0.05 separated mean values.

Economic Benefit Analysis. Using a crop value of $80/ton, a benefits assessment was
determined for crop yields of 20 ($1,600), 25($2,000), and 30($2,400) tons/acre.
Assuming that chlorothalonil applied every 7 days provides acceptable disease control,
the yield loss percentage (YLP) resulting from anthracnose fruit rot was determined for
each treatment as the total fruit rot (%) (TFR) above that observed in the 7-day
application plot. A projected total yield for each treatment was expressed as a proportion
of the total yield potential assuming that the 7-day interval treatment resulted in no loss.
In treatments where YLP was greater than 2.9%, crop value was reduced by 25% to
represent the loss due to sorting and processing as paste rather than whole peeled
tomatoes.

The total cost (TCOST) of each fungicide program was calculated by multiplying
the number of applications by cost ($15.00/Acre/application) (Table 3). Net crop value
was determined by subtracting fungicide costs from the crop value. The benefit per acre
(BPA) represents the value of an unsprayed crop subtracted from the net value of a crop
treated with fungicides. The return per fungicide dollar (RPFD) was calculated by
dividing the BPA by the season-long fungicide cost to identify programs utilizing
fungicide dollars most efficiently.

A modified benefits assessment including value reductions typically incurred in

37

...m 9..., .0. 03015.... SM 000:

 

3:20 b0>0 08 EBB 3:20 05 .«o 033800050.— Axmamtmoov \ E0c0m M 8:20 30% 00a E020m A9 530% L
$220.50 8 .
8m «80 - 020>v - 3308300“ 020me :0 8m «moo - 0203 n 020me mama: 8.8m 0000 En E0c0m A8 «monom—

 

.mbwam 020350..“ We “moo - 02g QED A9 .80 - 023»

 

.Am09089 6202— 03:3 :05

 

 

.2058 8me mm wfimmgoa can 398 9 030 530.605 mud .3 02? no.8 baa—=8 =05 .aNAn—A.» .: Amy a.~A.—A> _
@822 cs 8.5 84.3 a 05 63382 3 8: 25$ .0 A2822 3 85 80. a ,

m 08 005800000 003 E08338 0.50:3 < A5613 CC-— 0 ectowm 0n 8 008300 0.03 02g coho A9 02.3
60.500: £000 .50 22» 008098 .89 .8 :oEomoa ugmficmm Cast—EC A ,

 

4:02:00: 320:: .3“; .«o «hr—ax. - Afib “8 mam "mu; 00:03
00:08:00: :0 Eu 0008000 :2: 80¢ 008858 003 mEEBE the“; E
003:3 92a 20: Eat 8.. SE mo 0w8=020a 05 £53650 38.8: .095 .8250 0.9.0006 03030000

 

 

 

 

 

m0E>oa 0.50 .E o.m “a 2020:: .984. 3 @0230 own 305 65 wfifismm< ...0w8:020m 3o: 20; 54> ..
32¢ 2.5 36026030 Eat 000.800.— szn 0:: EB 0mocomfiu§$u8m :8”. .38. make}

ABE 0803a Qm 05 an own 965 .8.“ 050% a 08303 .850 00a “moo .. 985m n hat—9.80

4:08:00: 5000 SM .8008 05 323325 00:30 38% .3 .098: Z 0.33m &

._88o.a £0308. 2 @5800 2.60:8 0E£w§m Eon—30...!

‘- ”8&5 3:8 863,50 3 83.215.203.838 52.3 82:28 2 nauusw0m1§ $013.?

38

commercial situations, due to anthracnose incidence, was determined for a crop value of
$80/ton producing 20 tons/acre ($1,600). Initial calculations were based on total fruit rot
(%) (TFR) rather than YLP used in the initial model. Projected total yield for each
treatment was expressed as a proportion of the total yield potential using the loss
resulting from each treatment. In treatments where TFR was greater than 2.9%, crop
value was reduced by 25% to represent loss due to sorting and processing as paste rather
than whole peeled tomatoes. In treatments where TFR was greater than 10%, crOp value
was reduced to O, to reflect the total loss that occurs in commercial processing at this
disease incidence level.

RESULTS
Management of foliar blight. In 1993, unsprayed plots were completely defoliated by
late blight. Fungicide applications limited final foliar disease severity to 18.5, 17.0, 21.1,
and 34.3% for sprays applied every 7, 10, and 14 days, and according to TOM-CAST,
respectively. Although late blight is not a common tomato disease in Michigan, the
widespread occurrence of this disease in potato fields elsewhere in the state apparently
provided adequate inoculum. In 1994, Septoria and early blight resulted in 19.2%
blighted foliage in the unsprayed plot at the last assessment date. Chlorothalonil limited
final foliar disease severity to 4.3, 6.3, 6.8, and 5.3% when applied every 7, 10, and 14
days, and according to TOM-CAST, respectively. According to AUDPC data, all
fungicide treatments significantly reduced foliar blight in comparison to the untreated
control in 1993-94 (Table 4). Fungicide treatments did not differ significantly in limiting

- foliar blight.

39

Table 4 . The effect of foliar fungicide application on area
under the disease progress curve (AUDPC) for foliar blight
in 1993 and 1994.

 

 

AUDPC
Foliar blight * day

Treatments 1993 1994
7-day 269.2 a 77.7 a
10-day 249.3 a 93.7 a
l4-day 283.7 a 101.0 a
TOM-CAST 383.0 a 122.7 a
Control 1154.0 b 227.8 b

 

Means within a column followed by the same letter are not
significantly different according to Fisher's least significant
difference test (P=0.05).

40

In 1995, when averaged across cultivars, final foliar disease severity was 19.9%
for the unsprayed control plot compared to 8.7, 9.2, 13.3, 12.1 and 19.6 % for sprays
applied every 7, 10, and 14 days, and according to TOM-CAST, respectively. Foliar
blight was not affected by cultivar (Table 5). AUDPC data (Table 6) indicated that all
fungicide treatments significantly limited foliar disease in comparison to the unsprayed
control. AUDPC data indicated that fungicide applications every 7 and 10 days and those
applied according to TOM-CAST were significantly more effective than applications
made every 14 days in limiting foliar blight.
Management of fruit rot. In 1993, late blight caused significant fruit rot leaving only
8.2% healthy fruit in the unsprayed plot (Table 7). All fungicide treatments reduced fruit
rot and produced significantly more healthy fruit in comparison to the unsprayed control.
In the fungicide treated plots, 82.0 to 90.8% of the fruit were healthy and did not differ
significantly among fungicide treatments. In 1994, anthracnose fruit rot resulted in
69.3% healthy fruit in the unsprayed control plot (Table 7). All fungicide treatments
reduced fruit rot and produced significantly more healthy fruit in comparison to the
unsprayed control. Applications of chlorothalonil every 7 days resulted in significantly
more healthy fruit than other fungicide treatments. The 10-day treatment had
significantly more healthy fruit than the 14-day, and TOM-CAST treatments. There was
no difference in healthy fruit between the 14-day and TOM-CAST treatments, despite the
TOM-CAST treatment having one less spray application.

In 1995, cultivar did not affect the incidence of healthy fruit (Table 5). All

fungicide treatments significantly reduced fruit rot in comparison to the unsprayed

41

 

 

 

 

5.5 oévvm _.m_mo_ fivmmm; ow fob—m
mommd ode méf omm_.o mamma— odgoh v EoE~aouhia>E=U
*Loood odma cfimmm. 32:56 @233 Ndvmm—m v «no—5:2?
NMoN 06mm: odewai 0m Eon—39:. .Su 33,—.
Q; Odom 06mg Won—mm m John.—
ocvmd omodw floov *Lcood mNonoo mdcammv m :3:—
ovwmd ode ode ovwmd odmoo odmoc _ 32:30
moms wdm _ Maw _ _ 233-50 .3.— 38h.
hAnP—A— m2 mm hAacum m2 mm m: 3.59m
Jamaal a\e dmaad

 

.32 E 9:258: 020323 98:9 5S, $onan .33 250. new .3: 020.
c8 E5 .358: :5on 28 2:: B>o :33 3:8 wEEoman UmQD< 8m 8519 mo $9398 .8 QEEzm .m 935—.

42

Table 6. The effect of foliar fungicide application on area
under the disease progress curve (AUDPC) representing
foliar blight and percent healthy fruit for 'Ohio 7814' and
'Ohio 8245' tomatoes in 1995.

 

 

Treatment AUDPC %Healthy Fruit
foliar blight‘day

7-day 167.6 a 96.7 a

10-day 196.6 a 96.2 a

14-day 297.2 b 93.2 a

TOM-CAST 202.7 a 95.7 a

Control 420.0 c 59.9 b

 

Means within a column followed by the same letter
are not significantly different according to F isher's least
significant difference test (P=0.05).

43

Table 7. The effect of foliar fungicide application on percent
healthy fruit in 1993 and 1994.

Hgalth! Emit 1°19!

 

 

Treatments 1993 1994
7-day 90.8 a ' 97.8 a
lO-day 87.0 a 93.8 b
14-day 82.0 a 90.5 c
TOM-CAST 82.0 a 90.2 c
Control 8.2 b 69.3 d

 

Means within a column followed by the same letter are not
significantly different according to Fisher's least significant
difference test (P=0.05).

44

control plot that had only 59.9% healthy fruit (Table 6). There were no differences
(P=0.05) in percentage of healthy fruit, among the fungicide treated plots.

Benefit and Return. In 1993-1994, applications of chlorothalonil every 7 days resulted
in the highest BPA, significantly different from all other treatments, due to the increased
incidence of healthy fruit (Tables 8-10). In 1995, applications of chlorothalonil every 7
days resulted in a significantly increased BPA compared to the 14-day treatment. BPA
did not differ significantly among the 10-day, 14-day, and TOM-CAST treatments in
1993 -95.

In 1993, the highest RPF D was associated with the 14-day and TOM-CAST
treatments, although these treatments resulted in a reduced incidence of healthy fruit. In
1994, the RPF D was similar for the TOM-CAST and 7-day treatments. In 1995, the 14-
day treatment resulted in a significantly higher RPFD than the 7-day treatment although
not significantly higher than the 10-day or TOM-CAST treatments. Long dew periods
during 1995 prompted 2-3 more fungicide applications according to the TOM-CAST
system compared to 1993-94, thereby reducing the RPFD.

Calculations using the modified model revealed that in 1993-1994 applications of
chlorothalonil every 7 and lO-days resulted in the highest BPA (Table 11). In 1995, BPA
of fungicide treatments did not differ significantly. In 1993-1995 RPFD from fungicide
treatments was significantly higher in comparison to the unsprayed control plot. In 1993-

1994 RPFD was similar for the 7-day and TOM-CAST treatments.

45

.c - mom: .m - va— .v - 32 ”358305 Hm<U-EOH E @233 magma go 3‘#852...
.Amodumv 38 853.8% 23083me
~32 {BEE 2 @6383 328,86 3:38:36 Ho: 03 3:2 2:3 05 .3 325:8 5:38 3 2323 £322

 

 

 

 

o ood n cod 0 cod 0 cod 0 cod 0 cod .2280

n3 cod 3:6 ”33.2 £3 86% n coda a 28mm 4Pm<Ué¢Oh

mood on EM 3%.3 n ooémm a 362 n 2.3% .25-:

an $8 3 RN 3 mod— 33 $.30 n ooéom n 3.53 .36-:—

n 5.0 an M26 9 ad— 3 CONN“. 3 codmc 3 8de 36$

30 ~ :5 _ m8 8 mag 33 mg _ 52:305.
Law 5308 . 3815qu

 

.33 E3 4&3 .33 E 23 3a 8on 3 ~52? 88:83 0389 E 38 :28 Ewc8 .838 3
833:8 53833 020358 3:8 .3 Sub: 3:8 “663:8 3a EBB 23 2mm: 23 3a Encom .w flash.

46

.o - 82 .m - $2 .v - 32 ”35832, $5.22 5 33% genie .3852...
Amodnmv 58 358$“. 280$in
ammo— m.8:mE 9 wEEooom East? bEmofiEwfi 8: 8a 5:2 08% 2.: 3 326:8 5:28 a £53, £802

 

 

 

o cod v cod 0 cod 0 cod 0 cod 0 cod .3280

p cm.» wows mmmdm an 8465 n oodmm a 3.22 «hm<U..—ZO.H

«8.: on _N.v «El: a $230 a emdmm a Emma: .96.:

an and o oofi n 3.2 an mwdww n OWEN n end—Q 53-3

a $6 an m5© a Oma— m 3.30 m 8.wa a oodmf .254.

m 00 _ woo _ maa _ mom: wag mg _ Became...
av EEMM 3V Emmom

 

.32 98 63— .32 E 0.8m “on 8on E was?» soaosooa 0889 E 38 3.5 .mde 35:8 8
3.36:8 cosmozmam 0239.3 3:8 90 2:5: 3:8 363:3 8m 532 93 2mm: Bum ha Ema—om .a 039—.

47

.o - 82 .m - g2 .v - 82 “3:08.35 55.28 a 33% msaflo .3852...

Amodnmv 38 3:28.“? Eggs—mi
~82 @5sz 8 $.65on 288:6 €585ch 5: 2m 8:2 2:3 05 3 635:8 5:33 a £53, £822

 

 

 

 

w cod n cod 0 cod 0 cod 0 cod 0 cod .3550
«8.2 «$6 «Slum an 8.2:: good? a 8.ch «Hm<U-ZO.—.
an 3.2 on mmd anmNm a 2:3 a 8.2m acqmv: :64.—
on 8.: o cod n eff nm 3.02: a 8.3m a 8.53 >51:
0 mmd an Rd 9 9&0— aoodv: meofioo «8.on >21.

30 _ go _ mom: 33 #03 mg _ 3253:.
Amy 530% 3v “modem

 

.mai 28 .voo. .32 E 28 5Q octmm 8 32? cocosuoa 99:2 5 32 :8.“ .mde .858 o.
833:8 cosmozqaa oflgwcé 3:8 «0 Emmy: 3:8 02233 “on 532 28 2%: Bow hum Egom .3 03:.

48

.o - 30: .m - vac: .v - moo: ”3:25.35 Hm<0¢2©h 5 823m 38% no 82822...
Amodumv EB oocobbfi Emu—mama
ammo— mzonmi 9 wEEoSm “saga bacmoEcwmm 8: 98 5:2 oEwm 2: 3 @3628 5:28 a :22? 232

 

 

 

o cod 3 cod 3 cod 9 cod 0 cod 0 cod .2280
n 8.: m T.H.: an ova a 362: n 3.x? on ooémm .rrm<U-—ZO.F
m :6: m 3.: n ow.m m cod? 9 oodoo 0 code: haw...“—
nm moN— a _m.: m a; m 3.3: an 8.08: a codes maul:
9 Rd m 91: pm cod a Omdn: a oo.mmm_ an 868 >275
woo: woo _ 82 m3 _ gm: moo _ 28832:.

Q,» 533: AH,» Egon

 

.388 BEES 05
£3, noun—:28 mo: 98 .32 .83 E Boa 5m cow. 5 3 32? souosuoa 92:2 5 98 E5 Ewan: 65:8 2
8.328 sauna—mam 022ch .828 :0 Emmy: 3:8 0229.3 Ba 522 98 2mg 28 “on Egom .: 03:.

49

DISCUSSION

This study indicates that economical production of commercially acceptable
processing tomatoes in Michigan is dependant upon fungicides. One objective of this
study was to compare calendar-based spray schedules to a TOM-CAST based spray
schedule. In two of the three years, percentage of healthy fruit in the TOM-CAST
treatment did not differ significantly from the 7-day interval treatment, and produced a
high RPF D, using both economic models. However the level of disease that occurred in
the TOM-CAST treatments, up to 18%, would not be commercially acceptable. The
TOM-CAST system was rigorously challenged by factors occurring in the research plots.
In 1993, significant disease pressure from P. infestans occurred, a pathogen that TOM-
CAST was not developed to predict. In addition, the TOM-CAST system is based only
on epidemiology of A. solani although some researchers believe it is effective against
other tomato pathogens (Pitblado, 1992). Treatment rows were inoculated with C.
coccodes each year to insure high inoculum levels in this study. Additional research is
needed to incorporate epidemiological aspects of C. coccodes into TOM-CAST to make
it a more inclusive forecaster (Gleason et al., 1995). Growers using the current TOM-
CAST system to schedule fungicide applications should be aware of the system's
limitations and be prepared to make additional fungicide applications when
environmental conditions conducive to disease, or significant disease pressure from
pathogens not included in the model occur. Efficacy of the TOM-CAST system may be

greater in other geographical areas, where weather conditions are less conducive to the

50

fruit rotting organisms, or in fresh market tomato production where fruit are harvested
while still green (Williamson and Hilty, 1988).

Cultivar ('Ohio 7814' vs. 'Ohio 8245') did not affect percentage of healthy fruit or
foliar blight progression. In contrast, when Fulling et al., (1995) integrated TOM-CAST
based fungicide sprays with anthracnose resistant cultivars, a more liberal DSV could be
used resulting in three to four fewer fungicide applications compared to susceptible
cultivars. Genetic resistance is not a substitute for chemical control, but may allow
growers to reduce fungicide sprays needed to achieve acceptable levels of disease control.
Gleason et al. (1995) suggested that resistance indices for tomato cultivars could be used
to adjust prescriptive TOM-CAST based fungicide application intervals to maximize
fungicide use. Resistance indexes (% of fruit anthracnose of a test cultivar / % fruit
anthracnose of the standard susceptible cultivar) calculated for 'Ohio 7814' and 'Ohio
8245' are 1.00 and 0.216, respectively (Gleason et al., 1995). 'Ohio 8245' is a later
maturing cultivar than 'Ohio 7814'. In this study, fruit were harvested when at least 75%
of the fruit were fully ripe and thereby more susceptible to infection and development of
anthracnose fruit rot symptoms. Due to uneven ripening of 'Ohio 8245' fruit, a portion of
the fruit may have been riper at harvest and evaluation than would typically occur
commercially thus masking the anthracnose fruit rot resistance expected with this
cultivar.

F ungicide applications at 7-day intervals provided the greatest BPA in two of the
three years, despite increased fungicide costs associated with this fiangicide schedule.

The magnitude of the BPA of this spray program varied from year to year. Data from the

51

initial model used in this study showed the economic benefit provided by a fungicide
spray program is increased in a season with severe disease pressure. In 1993, disease
pressure from P. infestans increased the benefit of all fungicide programs compared to
1994, a year with less disease pressure. Also, an increased crop value increases BPA and
RPFD. Without any fungicide applications one can expect a total crop failure of
processing tomatoes due to anthracnose, while an inadequate spray program can result in

significant fruit rot and financial loss despite fungicide expenditures.

52

LITERATURE CITED

Brammall, RA. 1993. Effect of foliar fungicide treatment on early blight and yield of
fresh market tomato in Ontario. Plant Disease 77:484-488.

Dillard, HR. 1988. Influence of temperature, pH, osmotic potential, and fimgicide
sensitivity on germination of conidia and growth from sclerotia of Colletotrichum
coccodes in vitro. Phytopathology. 78:1357—1361.

Dillard, HR. 1989. Effect of temperature, wetness duration, and inoculum, density on

infection and lesion development of C olletotrichum coccodes on tomato fruit.
Phytopathology. 79:1063-1066.

Farley, JD. 1976. Survival of Colletotrichum coccodes in soil. Phytopathology 66:640-
641.

F edewa, DJ. and Pscodna, SJ. (eds) 1995. Michigan Agricultural Statistics. MDA,
Michigan pp. 32-33.

Fulling, B.A., Tigchelaar, EC, and Latin, R. 1995. Integration of host resistance and
weather-based fungicide scheduling for control of anthracnose of tomato fruit. Plant
Disease 79:228-233.

Fulton, J .P. 1948. Infection of tomato fruits by Colletotrichum phomoides.
Phytopathology 38:235-246.

Gleason, M.L., MacNab, A.A., Pitblado, R.E., Ticker, M.D., East, D.E., Latin R.X.
1995. Disease-warning systems for processing tomatoes in eastern North America: are
we there yet? Plant Disease 79:113-121.

Gould, WA. 1983. Tomato Production, Processing, and Quality Evaluation. 2nd ed.
AVI Publishing Company, Westport, CT. 445 pp.

Homby, D. 1968. Studies on Colletotrichum coccodes 111. Some properties of the
fungus in soil and in tomato roots. Transactions of the British Mycological Society 51,
541-553.

Kendrick, IE. and Walker, J.C. 1948. Anthracnose of tomato. Phytopathology 38: 247-
260.

Madden, L., Pennypacker, SP. and MacNab, AA. 1978. FAST, a forecast system for
Alternaria solani on tomato. Phytopathology 68: 1354-1358.

53

Pantidou, ME. and Schroeder, W.T. 1955. The foliage susceptibility of some species of
cucurbitaceae to tomato anthracnose-inciting fimgi. Plant Disease Reporter 40(5):432-

436.

Pennypacker, S.P., Madden L.V., and MacNab, AA. 1983. Validation of an early blight
forecasting system for tomatoes. Plant Disease 67:287-289.

Pitblado, RE. 1988. Development of a weather timed fimgicide spray program for field
tomatoes. Canadian Journal of Plant Pathology 10:371.

Pitblado, RE. 1992. Development and implementation of TOM-CAST. Ontario
Ministry of Agriculture and Food Publications p18.

Poysa, V., Brammall, RA. and Pitblado, RE. 1993. Effects of foliar fungicide sprays on
disease and yield of processing tomatoes in Ontario. Canadian Journal of Plant Science
73:1209-1215.

Precheur, R.J., Bennett, M.A., Tiedel, R.M., Wiesse, K.L., and Dudek, J. 1992.
Management of fungicide residues on processing tomatoes. Plant Disease, 76:700-702.

Sapers, G. M., Phillips. J .G., Panasiuk, 0., Carre, J ., Stoner, A.K., and Barksdale, T.
1978. Factors affecting the acidity of tomatoes. HortScience 13:187-189.

Shaner, G., and Finney, RE. 1977. The effect of nitrogen fertilization on the expression
of slow-mildewing resistance in Knox wheat. Phytopathology 67:1051-1056.

Tu, J .C. 1980. The ontogeny of the sclerotia of Colletotrichum coccodes. Canadian
Journal of Botany 58:631-636.

Williamson, J .W. and Hilty, J .W. 1988. Comparison of a forecast generated system and
a weekly spray regime for early blight control in trellised tomatoes. Tennessee Farm and
Home Science (146) p. 8-12.

SECTION III

FOLIAR INFECTION PROCESSES OF

C OLLE TOTRICH UM C CC C ODES

ABSTRACT

Conidial germination and infection processes of C olletotrichum coccodes were
observed and quantified. The abaxial surface of two opposing terminal leaflets removed
from a fully expanded leaf at the 4-5th node were inoculated with lOuL droplets of
conidial suspension of C. coccodes. Leaflets were incubated for 2-24 hours in 2 hours
intervals at 25 °C under high relative humidity. Explants inclusive of the conidial droplet
were fixed, cleared and preserved for microscopic observation. Conidial germination
increased linearly with time (R2=0.73)(P=0.001), maximum germination (68.3%) was
reached 24 hours after inoculation. Gerrninated conidia with unmelanized appressoria
peaked 6 hours after inoculation (38.3%). Melanized appressoria formation followed a
linear trend (R2=0.74)(P=0.001), with the maximum (62.0%) occurring 24 hours afier
inoculation. Infection vesicles were produced by 22 hours, thus indicating successful

infection.

55

56

INTRODUCTION

Anthracnose (C olletotrichum coccodes (Wallr.) Hughes) is the most serious
fungal disease affecting tomato (Lycopersicon esculentum Mill.) fruit grown for
processing. Processing fruit are especially vulnerable to infection by C. coccodes, an
otherwise weak pathogen, since the crop is harvested when fully ripe and fruit most
susceptible (Homby, 1968; Fulton, 1948; Kendrick and Walker, 1948). Fruit symptoms
begin as small, dark, sunken lesions with a water-soaked appearance that increase in
diameter and coalesce leaving a large, sunken, soft area (Tu, 1980). Under favorable
temperatures, lesions on ripe fruit become visible within 5-6 days after infection (Dillard,
1989). Black acervuli are produced just beneath the skin of the infected fruit. As lesions
mature, setose sclerotia develop from the stromata of acervuli within the infected fruit
tissue (Tu, 1980).

C. coccodes may overwinter as sclerotia in soil and infested debris for a minimum
of one year (Farley, 1976). Acervuli form on infested debris in soil, producing conidia
which serve as the primary source of inoculum of tomato fruit (Illman, 1960; Pantidou
and Schroeder, 1955). Moisture is necessary for release of the conidia from the
gelatinous matrix in the acervulus. Conidia are disseminated by splashing rain. Severe
disease incidence is most often associated with high rainfall during the growing season
(Dillard, 1989).

C. coccodes is not known to cause a foliar blight on tomato although it can infect
foliage producing small necrotic spots with a chlorotic halo where sporulation may occur

(Younkin and Dimock, 1944). Foliar inoculation of tomato seedlings with C. coccodes

57

results in necrotic lesions and premature senescence of cotyledons (Kendrick and Walker,
1948).

The significance of foliar inoculum in fruit infections, was investigated by
Kendrick and Walker (1948) by inoculating plants with an albino, morphologically
recognizable strain of C. coccodes. After failing to recover the albino strain from
infected fruit, it was concluded that sporulation of C. coccodes in foliar lesions was not a
significant source of secondary inoculum for fruit infection (Kendrick and Walker, 1948).
In contrast, Pantidou and Schroeder (1955) concluded that C. coccodes can infect foliage
and subsequently sporulate providing significant inoculum for fruit infection. Typical
anthracnose lesions were reproduced when filtrates from washed, crushed leaves with C.
coccodes conidia were sprayed on fruit. Seedling were inoculated with morphologically
recognizable C. coccodes strains and allowed to grow for three months. Once fruit had
developed, plants were incubated in a moist chamber for 24 hours. F oliar sporulation
ensued and fruit developed typical anthracnose lesions from which the recognizable
strains of C. coccodes used to inoculate the foliage were isolated.

Histological studies of foliar infection by C. coccodes determined that
appressoria form 19 hours after inoculation although environmental conditions were not
specified (Illman, 1960). F orty-two hours after inoculation, a germ tube was present and
had infected a neighboring epidermal cell. The study conducted by lllman (1960)
included observations made only at these two time periods. Hausbeck and Linderman
(1992) determined that at 15 °C, a minimum of 16 hours of leaf wetness are necessary for

infection, whereas only 12 hours are necessary at 20 and 25°C. The objective of this

58

study was to observe conidial germination and infection processes of C. coccodes on
tomato foliage over time under controlled environmental conditions
MATERIALS AND METHODS

Plant Culture. Tomato seedlings (cv. 'Ohio 7814') were grown in a research greenhouse
on the campus of Michigan State University in 163 cm3 cell packs containing Baccto
soilless medium (Michigan Peat Company, Houston, TX) for 4-5 weeks then transplanted
to individual 15.2 cm clay pots. Plants were watered with drip tubes to prevent leaf
wetness and fertilized with 6 grams per pot of 15-10-12 slow release fertilizer (Sierra
Chemical Company, Milpitas, CA) at transplanting.
Inoculation. C. coccodes was isolated from an infected tomato fruit and grown on
medium containing V8 juice (30%) and agar (2%) (DIFCO, Detroit, MI) in 100 x 15 mm
petri dishes at 20°C under continuous cool-white fluorescent light. Prior to inoculation,
10-14 day old cultures were flooded with 10 ml of sterile distilled water and gently
scraped with a glass rod. The conidial suspension was adjusted to a concentration of 5 x
105 conidia/ml using a hemacytometer.

Plants with 7-9 nodes were chosen and 2 opposing terminal leaflets removed from
a fully expanded leaf at the 4th or 5th node. The two leaflets removed from each plant
were assigned to the same treatment with one plant used per treatment. Each leaflet was
placed abaxial surface side up on filter paper moistened with 2 ml of sterile distilled
water in an inverted 100 x 15 mm petri dish. Using a Hamilton syringe (Hamilton
Company, Reno, NE), 6-8 lOuL droplets of conidial suspension were placed on the

’ abaxial surface of each leaflet avoiding areas with pronounced venation. Petri dishes

59

were closed to maintain a high relative humidity and prevent evaporation of the inoculum
droplet during incubation.
Incubation. Inoculated leaflets were incubated in a dew chamber (Percival
Manufacturing Co., Boone, IA) in continuous darkness at 25°C, for 2, 4, 6, 8, 10, 12, 14,
l6, 18, 20, 22, and 24 hours. Following incubation, four leaf disks inclusive of the
conidial suspension droplets were excised from each incubated leaflet using a l-cm-
diameter cork borer. Leaf disks were fixed with a FAA solution (1 :18:1 v/v) for 2 hours
and cleared in a saturated solution of chloral hydrate (250 g/ 100 ml) for 5 days. Disks
were preserved in 1 dram vials of lactophenol solution (20 g phenol, 20 ml lactic acid, 40
g glycerin, 20 ml water) until microscopic observations were made.
Observations. Cleared leaf disks were stained with cotton blue stain in lactophenol
solution (100 ml lactophenol, 1 ml 1% aqueous cotton blue, 20 ml glacial acetic acid) and
mounted in glycerol for observation. All conidia within 4 arbitrarily chosen microscope
fields (x400) were counted on each of the 8 leaf disks comprising each treatment. The
percentage of germinated conidia, conidia with unmelanized appressoria, melanized
appressoria, and infection vesicles were determined for each disk. Fields including
pronounced venation where conidia were pooled in large concentrations were avoided.

A conidium was considered germinated if the germ tube was at least as long as the
width of the conidium (Lacy, 1994) (Figure 1). Appressoria were recognized by their
globose, sometimes slightly lobed structures formed terminally on a germ tube (Figure 2).

Melanized appressoria were distinguished by their darkened appearance (Figure 3).

60

Figure l. a) Ungerminated conidia of Colletotrichum coccodes 2 hours afier inoculation.
b) Conidia producing germ tubes 2 hours after inoculation; scale bars = 10 um.

61

 

62

Figure 2. a) Globose, unmelanized appressoria produced by a germinated conidium of
Colletotrichum coccodes, 4 hours alter inoculation. b) Appressorium produced by a
germinated conidium beginning to melanize, 6 hours after inoculation; scale bars = 10

um.

63

 

Figure 3. a) Completely melanized appressoria produced by conidia of Colletotrichum
coccodes, 18 hours after inoculation. b) Hyaline infection vesicle produced by a
melanized appressorium, 24 hours after inoculation; scale bars = 10 um.

65

 

66

Infection vesicles were observed as hyaline, globose structures without lobes, adjacent to
the melanized appressoria (Figure 3). Infection vesicles ranged in size from one half to
two-thirds that of the adjacent appressorium.

Replications consisted of the 8 leaf disks taken from the incubated leaflets in each
treatment. Each microscope field was considered one subsample of the leaf disk. The
experiment was repeated once. Data were subjected to an arcsine square root
transformation in order to normalize the variance (Little and Hill, 1978). Because
variance for both experiments was found to be similar using Bartlett's test at x2 of 0.95,
data were combined prior to analysis. Analysis of variance (AN OVA), analysis of trend
effect, and regression analysis were done using the ANOVA, GLM and REG procedures
of the Statistical Analysis System (SAS Institute, Cory, NC). The least significant
difference (LSD) a=0.05 separated mean values.

RESULTS

Conidial germination increased linearly with time following inoculation, R2 for
the regression of germination against time was 0.73 (P=0.0001). Two hours after
inoculation, 17.9% of conidia had germinated with maximum germination (68.3%)
occurring 24 hours after inoculation (Figure 4). Conidial germination was rarely
associated with trichomes or stomatal openings in this study (Figure 5). Penetration was
not associated with anticlinal cell walls as reported by Pantidou and Schroeder (195 5),
rather direct penetration through periclinal cell walls was observed. Germination and
appressoria formation also occurred on veins in the leaf tissue, although data were not

' taken from these sites.

Percentage of Germinated Conidia

100

90

8O

70

60

50

40

3O

20

10

67

 

 

”- l l l l l l l l l l 1 fl

 

 

 

 

24681012141618202224

Hours of Incubation

Figure 4. Percentage of Colletotrichum coccodes conidia
germinated at 25C, error bars represent standard error of
the mean.

68

Figure 5. a) Appressorium produced terminally on an extended germ tube. b) Infection
by Colletotrichum coccodes occurring through a stoma; scale bars = 10 um.

69

 

70

According to trend analysis, formation of unmelanized appressoria followed a cubic trend
(P=0.0001). Two hours after inoculation, only 1.5% of the germinated conidia had
unmelanized appressoria but within four hours 35.1% had these structures (Figure 6). Six
hours after inoculation, the percentage of germinated conidia with unmelanized
appressoria peaked (38.3 %) (Figure 4). The subsequent decline in unmelanized
appressoria corresponded to the increase in melanized appressoria. A percentage of
appressoria (l 1.5%) remained unmelanized for the duration of the study.

Development of melanized appressoria increased linearly, R2 for the regression of
melanized appressoria against hours of incubation was 0.74 (P=0.0001). Appressoria
melanization began 4 hours after inoculation (0.69%), and increased to 27.0% 6 hours
after inoculation (Figure 6). The maximum percentage of germinated conidia with
melanized appressoria occurred 24 hours after inoculation (62.0%).

Infection vesicles were produced in 2.7% and 1.8% of conidia with melanized
appressoria 22 and 24 hours after inoculation, respectively, indicating successful infection
by C. coccodes.

DISCUSSION

This study provides a chronological guide of the events involved in infection of
tomato foliage by C. coccodes under controlled environmental conditions. This study
was conducted on leaf tissue rather than on a synthetic surface due to reports in the
literature that the chemical nature of host surfaces influences both spore germination and
appressoria formation of C. musae (Swinburne, 1976). Temperature was controlled and

free water maintained throughout this study unlike earlier studies (Illman, 1960;

Percentage of Germinated Conidia

100

90

80

70

60

50

40

30

20

10

71

 

 

l l l l l l l l

+ unmelanized appressoria

 

 

F —I— melanized appressoria F
l— —l
_. / i‘ A
j ii/i i
l
i
_ / __._
/
_ /
l— ‘i/ L J l l l l l l l ‘1
2 4 6 8 10 12 14 16 18 20 22

Hours of Incubation

24

Figure 6. Percentage of germinated Colletotrichum coccodes
conidia with unmelanized and melanized appressoria
at 25C, bars represent standard error of the mean.

72

Pantidou and Schroeder, 1955).

In this study observations were begun 2 hours after incubation, previous
histological studies of foliar infection did not include observations prior to 19 hours after
incubation (Pantidou and Schroeder, 1955; lllman, 1960). Results of this study show

that much of the infection process is completed by that time. Numbers of germinated

conidia, unmelanized conidia, and melanized appressoria were counted at each incubation

interval. At 25 °C, conidia germinate within 2 hours of inoculation, followed by

I :_w1m-.

formation of unmelanized appressoria which peaked 6 hours after inoculation. In this
study, melanization of appressoria began four hours after inoculation. Although previous
researchers noted appressoria in their observations, the transition from unmelanized to
melanized was not documented.

Appressoria melanization is necessary for successful penetration of intact tissue
by many C olletotrichum species (Prusky and Plumbley, 1992). Melanin strengthens the
appressoria wall allowing a build up of high internal hydrostatic pressure necessary for
cuticle penetration (Kubo and F urusawa, 1986). Cuticle penetration may involve
secretion of cutin degrading enzymes, in addition to the mechanical pressure exerted by
the appressoria (Dickman et al., 1982). Germination and appressoria formation is not
host specific in C olletotrichum species (Young and Kauss, 1984). Colletotrichum species
conidia readily germinate and form appressoria on glass slides and other synthetic
surfaces (Emmett and Parbery, 1975; Lenné, 1978). The successful infection of tomato
foliage was complete 22 hours after inoculation in this study, documented by the presence

' of infection vesicles. Intracellular primary hyphae grow from the infection vesicles into

73

adjacent cells (Bailey et al., 1992).

Intracellular primary hyphae were not observed in this study, suggesting that
latency plays a role in foliar infection by C. coccodes. A significant amount of research
has been conducted on latent infections of C. gloeosporioides on unripe avocado fruit
(Prusky et al., 1991; Prusky et al., 1982; Prusky et al., 1983 ). Results of these studies
suggest that fungitoxic concentrations of an antifungal diene present in the peel and flesh
of the fruit inhibits development of subcuticular hyphae of the germinated appressoria.
Decrease in antifungal diene levels is associated with ripening of the fruit, and disease
development. Further time course studies should be pursued to determine the length of
the latency period, and factors involved in releasing latent infections of C. coccodes.

These results documenting the successful infection of tomato foliage indicate that
C. coccodes sporulation on foliage should be reexamined using controlled environmental
conditions. Previous researchers who concluded that foliar sporulation is an insignificant
source of inoculum for fruit infections may have used insufficient conditions to allow
infection or sporulation to occur. Using the conditions necessary for foliar infection
established in this study, a more conclusive study regarding the role of foliar sporulation
may be conducted. The role that foliar sporulation plays in fruit infection may be an
important aspect of disease control strategies.

An increasing number of growers are using a disease forecaster called TOM-
CAST, developed to prompt fungicide applications, to control Alternaria solani.
Although TOM-CAST was developed using data of A. solani, it is used to control other

fungal diseases of tomato including anthracnose fruit rot (Pitblado, 1992). However,

74

epidemiology of C. coccodes differs significantly from A. solani and the TOM-CAST
system does not always prompt adequate sprays to control anthracnose fruit rot (Gleason
et al., 1995). This inadequacy of the TOM-CAST system indicates a need for additional
information to improve the accuracy of the current forecasting model (Gleason et al.,
1995). Incorporation of C. coccodes epidemiological information of the infection process

may be needed to make the forecaster a more reliable disease management tool.

75

LITERATURE CITED

Bailey, J .A., O'Connell, R.J., Ring, R.J., Nash, C. 1992. Infection Strategies of
Colletotrichum Species. In: Bailey, J .A. and Jeger, M.J. (eds), Colletotrichum: Biology,
Pathology and Control, CAB International, UK, pp. 88-120.

Dickman, M.B., Patil, SS. and Kolattukudy, PE. 1982. Purification, characterization
and role in infection of an extracellular cutinolytic enzyme from Colletotrichum
gloeosporioides Penz. on Carica papaya L. Physiological Plant Pathology 20:333-347.

Dillard, HR. 1989. Effect of temperature, wetness duration, and inoculum density on
infection and lesion development of Colletotrichum coccodes on tomato fruit.
Phytopathology 79:1063-1066.

Emmett, R.W. and Parbery, D.G. 1975. Appressoria. Annual Review of Phytopathology
13, 147-167. ‘

Farley, J .D. 1976. Survival of Colletotrichum coccodes in soil. Phytopathology 66:640-
641.

Fulton, J .P. 1948. Infection of tomato fruits by Colletotrichum phomoides.
Phytopathology 38:235-246.

Gleason, M.L., MacNab, A.A., Pitblado, R.E., Ricker, M.D., East, D.E., Latin, R.X.

1995. Disease-warning systems for processing tomatoes in eastern North America: are
we there yet? Plant Disease 79:113-121.

Hausbeck, M.K. and Linderman, SD. 1992. Influence of dew period and temperature on
infection of tomato foliage by Colletotrichum coccodes. Phytopathology 82:1091.

Homby, D. 1968. Studies on Colletotrichum coccodes III. Some properties of the
fungus in soil and in tomato roots. Transactions of the British Mycological Society
51:541-553.

lllman, W.I., 1960. Anthracnose Disease of Tomato, PhD. Thesis, University of Western
Ontario, Canada.

Kendrick, 1.8. and Walker, J.C. 1948. Anthracnose of tomato. Phytopathology 38:247-
260.

Kubo, Y. and F urusawa, I. 1986. Localization of melanin in appressoria of
Colletotrichum Iagenarium. Canadian Journal of Microbiology 32, 280-282.

Lacy, ML. 1994. Influence of wetness periods on infection of celery by Septoria

76

apiicola and use in timing sprays for control. Plant Disease 78:975-979.

Lenné, J .M. 1978. Studies on the biology and taxonomy of Colletotrichum species. PhD
Thesis, University of Melbourne, Australia.

Little, TM. and Hills, F.J. 1978. Agricultural Experimentation Design and Analysis.
John Wiley and Sons, New York.

Pantidou, ME. and Schroeder, W.T. 1955. Foliage as a source of secondary inoculum
for tomato anthracnose. Phytopathology 452338-345.

Pitblado, RE. 1992. Development and implementation of TOM-CAST. Ontario
Ministry of Agriculture and Food Publications. p.18.

Prusky, D., Keen , N.T. and Eaks, LL. 1983. Further evidence for the involvement of a
preformed antifungal compound in the latency of Colletotrichum gloeosporioides in
unripe avocado fruits. Physiological Plant Pathology 22:189-198.

Prusky, D., Keen, N.T., Sims, J .J . and Midland, S. 1982. Possible involvement of an
antifungal compound in latency of C olletotrichum gloesporioides in unripe avocado
fruits. Phytopathology 72: 1578-1582.

Prusky, D., Plumbley, R.A., Kobiler, I. 1991. The relationship between antifungal diene
levels and fungal inhibition during quiescent infection of unripe avocado fruits by C.
gloesporioides. Plant Pathology 40:45-52.

Prusky, D., and Plumbley, RA. 1992. Quiescent Infections of Colletotrichum in
Tropical and Subtropical Fruits. In: Bailey, J .A. and Jeger, M.J. (eds), Colletotrichum:
Biology, Pathology and Control, CAB International, UK, pp. 289-307.

Swinburne, TR. 1976. Stimulants of germination and appressoria formation by
C olletotrichum musae (Berk. and Crut.) Arx. in banana leachate. Phytopathologische
Zeitschrift 87, 74-90.

Tu, J .C . 1980. The ontogeny of the sclerotia of Colletotrichum coccodes. Canadian
Journal of Botany 58:631-636.

Young, DH. and Kauss, H. 1984 Adhesion of Colletotrichum lindemuthianum spores to
Phaselus vulgaris hypocotyls and to polystyrene. Applied Environmental Microbiology
47, 616-619.

Younkin, SB. and Dimock, A.W. 1944. Foliage infections of Lycopersicon esculentum
by Colletotrichum phomoides. Phytopathology 34:976-977.

   

nrcurcnu sran UNIV. LIBRQRIES
llllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
31293014057511